as_vuralhan_20060411.

as_vuralhan_20060411.
Engineering of Aromatic Amino Acid
Metabolism in Saccharomyces cerevisiae
Zeynep Vuralhan
Engineering of Aromatic Amino Acid
Metabolism in Saccharomyces cerevisiae
PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus Prof. dr. ir. J.T. Fokkema
voorzitter van het College voor Promoties,
in het openbaar te verdedigen op dinsdag 11 april 2006 te 10.30 uur
door
Zeynep VURALHAN
Technical University of Denmark (DTU), Master of Food Science and Technology,
Lyngby, Denmark
Geboren te Istanbul, Turkije
Dit proefschrift is goedgekeurd door de promotoren:
Prof. dr. J. T. Pronk
Prof. dr. J. P. van Dijken
Samenstelling promotiecommissie:
Rector Magnificus
voorzitter
Prof. dr. J. T. Pronk
Prof. dr. J. P. van Dijken
Prof. dr. ir. J. A. M. de Bont
Prof. dr. I. J. van der Klei
Prof. dr. ir. J. J. Heijnen
Dr. J. R. Dickinson
Dr. J. M. Daran
Technische Universiteit Delft, promotor
Technische Universiteit Delft, promotor
Technische Universiteit Delft
Rijksuniversiteit Groningen
Technische Universiteit Delft
Cardiff University, UK
Technische Universiteit Delft
Dr. J. M. Daran heeft als begeleider in hoge mate bijgedragen aan het totstandkomen
van dit proefschrift.
The studies presented in this thesis were performed at the Industrial Microbiology section,
Department of Biotechnology, Delft University of Technology. The research was financially
supported by the Board of the Delft University of Technology (Beloning Excellent Onderzoek
program), DSM and the Dutch Ministry of Economic Affairs (NWO-CW project 99601). The
Industrial Microbiology section is part of the Kluyver Center for Genomics of Industrial
Fermentation, which is supported by the Netherlands Genomics Initiative.
Printed by:
ISBN:
Contents
Chapter 1 ..................................................................................................................................7
General Introduction
Chapter 2 ................................................................................................................................37
Identification and Characterization of Phenylpyruvate Decarboxylase Genes in
Saccharomyces cerevisiae
Chapter 3 ................................................................................................................................57
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae
Chapter 4 ................................................................................................................................77
Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid
biosynthesis: quantification of metabolic impact
Summary ................................................................................................................................99
Samenvatting........................................................................................................................101
Publications ..........................................................................................................................103
Curriculum Vitae ................................................................................................................105
Acknowledgements ..............................................................................................................107
1
GENERAL INTRODUCTION
1.0 Amino acids as building blocks for biomass constituents
Amino acids are α-substituted carboxylic acids, the building blocks of proteins. Asparagine was the first
amino acid discovered in 1806, whereas threonine was the last one to be identified in 1938. Amino acids carry
common names and have sometimes trivial names. The names of the amino acids are given mostly according to
the sources they were originally isolated from. For example; glutamate was for the first time isolated from wheat
gluten, whereas asparagine was first found in asparagus (66).
The general structure of an amino acid is presented in Fig. 1. All 20 common amino acids have a carboxyl
group and an amino group, which are attached to the same carbon atom. The difference between the various
amino acids lies in the side chains, which is called “R group” (Fig. 1). The R-groups vary in size, structure and
electric charge (66).
R
Amino group
H2N
Cα
carboxylic acid group
COOH
α- carbon
H
FIG. 1. General structure of an amino acid.
An important property of naturally occurring amino acids is their chirality. Chirality is defined as the ability
of a molecule to rotate the plane of polarized light to the right (dextrorotatory) or to the left (levorotatory).
These two mirror-image forms of amino acids are called D-isomer and L-isomer respectively (66). Life has
developed in a way to favour the use of the L-form instead of D-form in case of proteins. On the other hand, Dforms of amino acids are also natural compounds. They are found in the cell walls of bacteria (58). D-amino
acids are important building blocks in the production of pharmaceuticals, food additives, herbicides and other
agrochemicals (66).
Apart from 20 common amino acids, there are also amino acids that are modified after the protein is
synthesized. For example 4-hydroxyproline and 5-hydroxylysine, which are the derivatives of proline and lysine
Chapter 1
respectively, are abundant in collagen, a fibrous protein of connective tissue. In addition to the amino acids
synthesized by posttranslational modifications in proteins, there are some other amino acids such as ornithine
and citrulline, which are the intermediates in the biosynthesis of arginine and the urea cycle (66).
When a few amino acids are covalently connected through a condensation reaction, the product of this
reaction is called a peptide. When more than three or four amino acids make bonds, the product is named as
polypeptide. Peptides also have industrial importance such as aspartame, an artificial sweetener, which is Laspartyl-L-phenylalanine methyl ester. Polypeptides can act as hormones such as oxytocin, which is a vertebrate
hormone and secreted by the pituitary gland and glucagon, which is a pancreas hormone are examples (66).
Another one is insulin that is nowadays produced with genetically modified Saccharomyces (22).
Amino acids have a broad variety of characteristics with respect to nutritional value, taste, and medicinal
action and therefore have many potential uses. A summary of the application areas is presented in the following
section.
Human Food
50%
Pharmaceuticals
20%
Animal Feed
30%
FIG. 2. Representation of amino acid application areas in percentages of amounts used (64)
2.0 Application areas of amino acids
Amino acids are used in a variety of ways and they have extensive industrial applications (12,45) (Table 1).
They find applications as feed and food additives, taste and aroma enhancers, pharmaceuticals, components of
drugs, dietary formulas, nutraceuticals and as ingredients in cosmetics (7). In addition, fusel alcohols derived
from amino acids by fermentation such as 3-methyl-1-butanol, 2-methyl-1-butanol and 2-methyl-1-propanol are
compounds which result from the degradation of L-leucine, L-isoleucine and L-valine respectively. They
contribute to the characteristic bread flavour (30). Phenylethanol which exhibits a rose-like aroma is formed
8
General Introduction
from phenylalanine. It has significant industrial value not only in cosmetics but also in the formulation of
beverages and food products (27,85). Below the application areas of amino acids are discussed as two
categories:
2.1 Food & Feed
The main application field of amino acids nowadays is in food. About fifty percent of amino acids
manufactured worldwide are applied in human food production (Fig. 2). The amino acids with the largest
market volumes are glutamic acid, lysine and methionine (64) (Table 1).
The microbial production of amino acids dates back to 1957, when a group of scientists isolated a soil
bacterium (a Corynebacterium sp.) capable of excreting large amounts of glutamic acid. The main application of
glutamic acid in the food industry is in the form of a salt; the flavour enhancer mono sodium glutamate (MSG).
A Japanese scientist first discovered MSG in 1908, He observed that MSG is responsible for the characteristic
flavour of kelp, which is a traditional Japanese dish; this taste is known as “Umami” in Japan (48). In recent
years, special receptor proteins have been identified in the human tongue for sensing the taste of MSG (67).
L-Phenylalanine is a key amino acid in the manufacturing of aspartame, an artificial sweetener. Aspartame is
150-200 times sweeter than sucrose. Corynebacterium glutamicum and E. coli are the preferred organisms for
the microbial production of phenylalanine by fermentation (53). The main producers of L-phenylalanine are
Nutrasweet Kelco (USA), Ajinomoto Co (Japan), Amino GmbH(Germany) and Degussa (Germany) (7).
Amino acids such as alanine, arginine and glycine have great significance in the traditional food applications
in the Far East. L-Alanine has found application as flavour for the traditional Japanese rice wine Sake and in
alitame (Pfizer, Inc., alitame (brand name Aclame™)) as sweetener for cakes and pastries. L-arginine and Lglycine serve as a flavour enhancer in many products (green tea, meat, fish and soy sauce) (64).
About 30% of the worldwide production of amino acids is used for animal feed applications (Fig. 2) because
livestock diets, which mainly consist of corn and soy products, are deficient in various essential amino acids.
Therefore supplementation of amino acids is an absolute necessity in pig and poultry farming. Methionine,
which is the third largest in market volume (Table 1), not only serves as a food additive but also and unlike
glutamic acid, it is used as animal feed especially for poultry and pigs (64).
Lysine is also an essential amino acid that is required in the diets of animals. It shows a yearly increase of 710 % in production. The supplementation of animal diet is done by adding feed ingredients such as soybean
meal, which is very rich in lysine, or by direct addition of lysine. Kyowa Hakko (Japan) and ADM (USA) are
the market leaders in lysine production (64). L-threonine is also used for balancing the diets of livestock. In
contrast to the other amino acids produced, E.coli is exclusively used in the production of threonine (15). Main
producers of L-threonine are Ajinomoto, Kyowa Hakko, ADM and Degussa.
9
Chapter 1
TABLE 1. Market, use and production method of some amino acids (Modified from (53))
Amino acid
Market volume*
Major use
Production method
(tons)
Arginine
1.000
Pharmaceutical
Aspartic Acid
10.000
Sweetener
Cysteine
3.000
Food Additive
1000.000
Flavour Enhancer
Glycine
22.000
Food Additive
Leucine
500
Pharmaceutical
Lysine
600.000
Feed Additive
Methionine
400.000
Feed Additive
Phenylalanine
10.000
Sweetener
Threonine
20.000
Feed Additive
Tryptophan
500
Pharmaceutical
Valine
500
Pharmaceutical
Glutamic acid
In vivo production via microbial
fermentation.
In vitro biocatalysis
Extraction
In vitro biocatalysis
In vivo production via microbial
fermentation.
Chemical Synthesis
In vivo production via microbial
fermentation.
In vivo production via microbial
fermentation.
Chemical Synthesis
In vivo production
fermentation.
In vivo production
fermentation.
In vivo production
fermentation.
In vivo production
fermentation.
via microbial
via microbial
via microbial
via microbial
*
(Data are average value of 2002-2003)
2.2 Pharmaceuticals
20 % of the amino acids produced are used in medical applications. Amino acids have found applications in
the formulation of post and pre-operative nutrition. Standard infusion liquid contains at least eight amino acids,
which are essential for the human body.
L-Serine is used in the formulations of antibiotics. L-tyrosine, which has an important biological function as
a precursor of thyroid hormone is used for the treatment of the diseases of thyroid gland
(http://www.standardvitanet.com/ioandty120ve.html). L-cysteine, a sulphur containing amino acid, is essential
for glutathione and taurine synthesis (antioxidants for the liver's detoxification process)
(http://www.aminoacidpower.com/osc/product_info.php?cPath=3_23&products_id=180). It is important for
10
General Introduction
skin, hair and collagen formation, detoxification of toxic compounds & heavy metals, and immune support.
Therefore it has found applications in the cosmetics, specifically in hair care products (57,64).
3.0 Production of amino acids
The mode of production of amino acids varies from full chemical synthesis to a microbial fermentation
process (Table 1). Three main types of production processes are carried out in the industrial practice: amino
acid extraction from protein hydroylsates, chemical synthesis and biochemical synthesis (12,45).
3.1. Extraction
This method is based on the availability, in large quantities of proteinaceous material, from which the amino
acid can be obtained via hydrolysis. In the past, this method was prevailing. It is still being used for some amino
acids such as L-cysteine, L-leucine, and L-tyrosine. Human hair and chicken feather which are rich in cysteine
but also blood meal or soybeans are some of the sources from which these amino acids are extracted (45). First
protein hydrolysates are obtained by acid-hydrolysis from the sources mentioned above, active charcoal is used
for neutralization and decolourization. Finally electrodialysis is applied to separate amino acids into fractions
(73). The combination of extraction with other methods can increase the efficiency and productivity of the
production. In the case of L-cysteine for instance, commercial production is performed by enzymatic synthesis
in addition to the extraction (45).
3.2 Chemical synthesis
Four out of twenty amino acids are still produced via chemical synthesis (Table 1). Via chemical synthesis,
a mixture of D- and L- forms of amino acids is produced. Additional steps are required to get the required Lisomers (45).
3.3 Biochemical methods
Biochemical methods can be summarized under two categories; 1- ) in vitro biocatalysis and 2- ) in vivo
production via microbial fermentation.
3.3.1 In vitro biocatalysis
Enzymes are very helpful catalysts for the enantioselective synthesis of amino acids and are used to obtain
optically pure D- and L-amino acids. As compared to chemical synthesis, fewer by products are formed which
eases the downstream processing. The enzymes used for this purpose are obtained from microorganisms.
However, industrial application of enzymes is limited by thermal and mechanical stability. In order to overcome
this hurdle, immobilized enzyme systems have been developed (57). The cost of starting substrates is a very
important additional limitation for the applicability of this method.
Enzymatic synthesis of L-aspartic acid can be given as a good example for the enzymatic method. In the
case of L-aspartic acid, immobilized E. coli cells that express aspartase convert ammonia and fumarate to
11
Chapter 1
aspartic acid in a continuous process. Other amino acids such as L-alanine, L-lysine and L-phenylalanine have
also been produced commercially by enzymatic methods. In the case of L-alanine, a decarboxylase from
Pseudomonas dacunhae is used to convert L-aspartate to L-alanine with a release of carbon dioxide. In this
process, immobilized cells are used and a titer of 400 g.l-1 L-alanine has been reported (57).
3.3.2 In vivo production via microbial fermentation
Most of the amino acids are produced by fermentation. The carbon source, fermentation yield, purification
method and productivity in the overall process determine the economy of this method. This method has been
proven successful for the bulk production of amino acids such as monosodium glutamate, L-lysine-HCl and Lthreonine (45). In the fermentation processes, the production strains are fed with low cost carbon sources such as
sucrose or glucose. Batch or fed-batch cultivation is the preferable type of process for amino acid production. Lphenylalanine production by E. coli by fed-batch process which resulted in a titer of 34 g.l-1 can be given as an
example (31).
The strains used in amino acid fermentations are a very important factor for overall process economy.
Therefore much emphasis is given on the selection of suitable production organisms and their improvement.
Selection of strains with a high yield on the carbon source is never an ending story (45).
The classification of production strains falls into three categories: (1) wild type strains having the ability to
produce specific amino acids (2) auxotrophic or regulatory mutants which lost their feed-back inhibition
mechanisms, and (3) strains which have gained the ability to overproduce amino acids by overexpression of
genes that are responsible for the synthesis of enzymes in rate-limiting steps (45).
Commonly, improvement of amino acid producing microorganisms has been achieved by combining the
desired genetic and physiological changes in one host. This results in higher production and lowered by-product
formation (45). In the following section, subjects will be discussed in more detail.
4.0 Engineering of amino acid production
Since there is a growing interest in amino acid production, strain and process improvement are of
considerable importance. Although strains isolated from nature may produce significant amounts of amino
acids, the concentrations, yields and rates are generally much too low for a commercial process. Before the birth
of recombinant DNA technology in the 1980s, “classical strain improvement” was the only approach available.
4.1 Classical strain improvement
4.1.1 Selection
Charles Darwin postulated in 1859; "The key is man's power of accumulative selection: nature gives
successive variations; man adds them up in certain directions useful to him”. This has been exploited by several
researchers to obtain microbial strains with the desired characteristics for amino acid production.
In 1957, a soil bacterium “Corynebacterium” that excreted a large amount of glutamate into the medium was
discovered (49). Soon similar bacteria were isolated from nature that produced D or L-alanine or L-valine
12
General Introduction
(Brevibacterium flavum, B. lactofermentum, B. thiogenitalis, Microbacterium ammoniaphilum). These
discoveries opened the door for a new era in industry: microbial production of amino acids by fermentation (32).
Subsequently the application of a large variety of amino acid producing strains commenced. When
Corynebacterium spp. were started to be used for the large-scale production of glutamic acid, MSG prices
dropped from $ 8 to $ 2 worldwide (45).
The optimisation of the production of L-amino acids with Corynebacterium spp. has become one of the most
popular targets of industrial microbiology. L-glutamic acid and L-lysine are the bulk products but also L-valine,
L-isoleucine, L-threonine, L-aspartic acid and L-alanine are commercially produced by fermentation (35).
Corynebacterium spp. are superior compared to Enterobacteriaceae because of the simplicity of their
metabolism (52) For example: there is only one aspartokinase enzyme in C. glutamicum, whereas in E. coli,
there are three aspartokinase isoenzymes which are subject to feed back inhibition by lysine, or threonine or
isoleucine which very much complicates the overproduction of amino acids with E.coli (53).
4.1.2 Mutagenesis and selection
Random mutagenesis and screening
The fundamentals of this method rest on inducing mutations and randomly picking the survivors from the
population. A variety of chemical or physical agents, called mutagens, are used to obtain changes in genotype.
The goal of the mutagenesis is to get the highest possible number of desired mutations whilst avoiding
undesired additional mutations caused by mutagens.
Chemical mutagens can be grouped into three categories depending on their mode of action:
(1) Mutagens which affect non-replicating DNA, (2) analogs showing structural similarity to the bases that
are incorporated into replicating DNA and (3) frameshift mutagens.
Mutagens such as HNO3, hydroxylamine (NH2OH) and alkylating agents (EMS, NTG, and mustard gas)
affect non-replicating DNA. Besides deaminating adenine to hypoxanthine and cytosine to uracil, HNO3 also
induces crosslinks between two DNA strands. EMS and NTG form alkylated bases in DNA. These agents
change the DNA through transversions, deletions and frame-shift mutations (12). In several cases, the mutagen
nitrosoguanidine (NTG) has been used to provoke the highest mutations per survivor (4). Several examples can
be given for overproducing mutants resulting from the use of NTG as a chemical mutagen. Base analogs are
another type of mutagens which can be incorporated into the replicating DNA such as 5-bromouracil or 2-amino
purine. They incorporate into the replicating DNA by replacing the bases thymine and adenine. Sometimes
acridine dyes are used. Their mode of action lies in causing frameshift mutations (12).
The success of random mutagenesis and selection is based on screening strategies. The population obtained
after mutagenic treatment is heterogeneous. Therefore individual clones from such populations must be
examined to identify the most desired phenotype. The most fundamental techniques for screening are spatial
separation and an assay system to characterize the phenotype of interest. High throughput screening is an
efficient method to achieve this. An example of high throughput screening is a combination of flow cytometry
and cell sorting. This is a very rapid technique to analyse single cells. The analysis and detection is done as cells
13
Chapter 1
flow in a liquid medium through the focus of a laser beam surrounded by an array of detectors. With the help of
different fluorescent stains this technique makes it possible to distinguish between different cells in large cell
populations. Automated screening is of course a very handy tool to minimize labour cost (74).
In several studies, the aim was to obtain mutants, resistant to feedback inhibition and as a result
overproduction of the desired amino acid. The selection was generally done by using amino acid analogues. For
example, phenylalanine producers have been constructed as tyrosine auxotrophs of C. glutamicum and as
mutants of B. flavum resistant to phenylalanine analogues such as p-aminophenylalanine and pfluorophenylalanine. The highest production was 25 g. l-1 with a B. lactofermentum mutant, which was resistant
to PFP, 5- methyltryptophan and sensitive to decoynin, an analogue of purine (79).
Although yeasts are not generally regarded as amino acid producing organisms, S. cerevisiae has been
studied for amino acid production. In one study (16), S. cerevisiae mutants were obtained by applying NTG and
EMS mutagenesis. These mutants did not have feedback inhibition of aspartokinase (catalyzing the first reaction
of the threonine-methionine biosynthetic route). Two other genes, responsible for the excretion of threonine
were mutated as well. As a result of these multiple mutations intracellular threonine concentrations increased up
to 20-fold.
4.2 Metabolic engineering of amino acid production
In contrast to classical strain improvement, the metabolic engineering approach tries to understand the
metabolic network and use this knowledge for improving strain properties. This is evident from the definition of
Bailey; “Metabolic engineering is a field of scientific inquiry aiming at the directed improvement of cellular
properties through the modification of specific biochemical reactions or introduction of new ones using
recombinant DNA technology”(2). Application of recombinant DNA techniques makes it possible to gain
knowledge of the relevant steps and regulation mechanisms in microbial metabolism and to (subsequently) use
this knowledge to improve the productivity of conventional strains or to develop new strains (25).
Metabolic engineering has many application areas. Recombinant DNA technology and traditional
mutagenesis plus selection have been key factors in the construction of strains that can produce high levels of
amino acids. For example, final concentrations (kg. m-3) have been reported for: L-threonine, 100; L-isoleucine,
40, L-leucine 34; L-valine 31, L-phenylalanine 28; L-tryptophan, 55; L-tyrosine 26; L-proline, 100, L-arginine,
100 and L-histidine, 40 (17).
Before developing strategies to improve amino acid production, it is essential to understand the biochemical
reactions involved and how these work in cooperation. Key parameters are: substrate uptake, central metabolic
pathways, amino acid excretion, global regulatory networks, energy & redox balance, amino acid catabolism
pathways etc. (53) (Fig. 3).
14
General Introduction
Substrate
Substrate uptake
Substrate
Catabolis m
Redo x & energy balance
By -products
Global
Regulatory
Networks
Bio mass
Anabolism
Degradation
Specific
feedback
regulation
Amino acid
Product
export
Product re-uptake
Amino acid
FIG. 3. Basic steps in amino acid production (53)
15
Chapter 1
Several strategies have been followed for improving microbial amino acid production via metabolic
engineering:
a-) Modification of product pathways
i) Increasing the flux through the steps in the biosynthetic pathway that exert a high degree of flux control.
ii) Amplification of a branch point enzyme in order to shift the metabolic flow towards the desired direction
of amino acid production.
iii) Introduction of heterologous enzymes which have a different catalytic mechanism.
b-) Engineering of the central metabolism
Since the central metabolism provides the precursors for amino acid production, it is essential to engineer
central metabolism for channelling the precursors required for amino acid production into the biosynthetic
pathways. Therefore calculation of flux distribution, theoretical yield, ATP and redox balances should be
determined. Thereafter the redirection of the fluxes can be attempted for constructing strains with desired
properties.
c-) Engineering of transport processes
Both the uptake of nutrients and product excretion are possible limitations in amino acid production.
Detailed knowledge of these processes is a necessity to tackle these limitations (25,52).
4.3 “OMICS” as a tool for the improvement of amino acid production
In the last decades, straightforward approaches such as deletion and overexpression of genes have been used
to improve strains. Nowadays with the availability of complete sequence of more than 80 microbial genomes,
powerful analytical tools can be applied to keep track of the results of the genotype changes induced in the
organism (68). These tools are: DNA microarrays by which quantification of all mRNA levels is achieved,
Proteomics, in which the amount of proteins is analysed by 2D-gel electrophoresis followed by identification of
the proteins via MALDI-TOF MS (matrix assisted laser desorption ionization-time of flight mass spectrometry),
Metabolomics makes it possible to detect and quantify relevant metabolites by GC-MS or LC-MS (68).
In the field of amino acid production; focus was initially on Corynebacterium glutamicum. DNA
microarrays were used for analysing the expression of the genes of selected transport systems, central carbon
metabolism, product formation and regulatory mechanisms (44).
Via MALDI-TOF-MS-based peptide finger printing, a proteomic map of 200 cytoplasmic and membrane
associated proteins of C. glutamicum has been obtained (75).
4.4 Optimization of process conditions
The cultivation of microorganisms used in the production of amino acids is performed in a constant
environment where parameters are carefully controlled. Parameters like medium composition, pH of the culture,
16
General Introduction
oxygen supply, feed rate of carbon source, and process temperature are main targets for control and optimisation
(35). Three basic methods are available for industrial amino acid fermentations: batch, fed-batch and continuous
fermentations.
In batch fermentations, a short fermentation time is applied to avoid accumulation of unwanted metabolites
in the stationary phase (35). In fed-batch fermentation, which is the standard process for the production of
biochemicals, the parameters that require attention are maintenance of limiting substrate concentration and
carefully controlled oxygen supply. When the oxygen supply becomes insufficient, production of unwanted byproducts such as lactate or acetate may occur in bacterial cultures (35).
In repeated fed batch, 60-95% of the culture is replaced by a fresh medium. The advantage of this method is
that no new inoculum has to be prepared and prior sterilization of the bioreactor is by-passed (70). Another
advantage of repeated fed-batch processes is the increased reactor productivity as a result of the shortening of
the lag phase and reduction of the preparation times between the batches, thus reducing the variable costs. (36).
Although continuous fermentations have superiority over other methods by offering the highest average
volumetric productivity, they have one major disadvantage: the occurrence of variants of the parent production
strain by back mutation or loss of expression cassettes (53). Additionally, contaminations and phage infections
can be very serious problems. Use of fresh starting material for each run, and the use of stable mutants are
required to overcome this hurdle.
In order to improve the process conditions in continuous cultures, sometimes two or more reactors are used
in a cascade bioprocess. This involves a growth phase in the first reactor whereas in the second reactor
production takes place. Apart from the mode of cultivation, the composition of the medium is a crucial factor for
success. For example carbon and phosphate double limitation instead of single limitation increased the
production of L-lysine from 3.18 to 3.75 g lysine-HCl .h-1 (13).
Separation of amino acids from the culture broth is also an important issue. Centrifugation and filtration are
the preferred methods. Poor centrifugation behaviour of cells may be improved by the application of ionic
charges on the surfaces of microorganisms. In filtration, parameters such as the properties of the filtrate,
characteristics of the solid particles, pressure applied and effects of antifoaming agents on filtration should be
taken into consideration. The filtration efficiency may be increased also by the use of filter-aids. These filteraids improve the porosity of a resulting filter cake leading to a higher flow rate.
For further purification, chromatographic separation and concentration-crystallization methods are applied
(45). When the amino acid is used to supplement cattle feed, no further purification is required and the cell-free
culture broth can be spray-dried.
5.0 Aromatic amino acid metabolism and its regulation in bacteria
Early studies about the aromatic amino acid biosynthesis generally focused on prokaryotic organisms,
mainly on the tryptophan branch. The prokaryotic operon concept of regulatory mechanisms does not apply to
eukaryotes (8). There are two major differences between eukaryotic and prokaryotic regulation of amino acid
17
Chapter 1
synthesis, those are: the more complex arrangement of the genetic material and compartmentation of the cellular
space.
In bacteria such as E .coli, three unlinked genes aroF, aroG and aroH (Fig. 4) are in charge of the regulation
of the common aromatic pathway. Those three genes encode isozymes of the first enzyme, DAHP synthase.
Feedback inhibition is the major control mechanism in vivo (62). In E. coli, all the genes of the tryptophan
branch of the pathway, are arranged in the tryptophan operon (Fig. 4) (8). In all eukaryotic organisms, however,
the tryptophan genes are scattered over the genome. Every one of them requires its own regulatory signal
sequences (42). In the next section, aromatic amino acid biosynthesis and its regulation in S.cerevisiae will be
discussed in more detail.
In Cyanobacteria, coryneform bacteria (Brevibacterium spp. and Corynebacterium spp.), and some spore
forming Actinomycetes, phenylalanine is produced via phenylpyruvate and tyrosine from arogenate and not
from hydroxyphenyl pyruvic acid as in E. coli and S. cerevisiae (10,79). DAHP synthase enzyme is strongly
inhibited by phenylalanine and tyrosine. Furthermore, chorismate mutase also shows sensitivity towards
phenylalanine and tyrosine. Prephenate dehydratase and anthranilate synthase are also further targets. They are
feedback inhibited by the end products of the pathway (43).
6.0 Aromatic amino acid Metabolism and its regulation in S. cerevisiae
6.1 Chorismate pathway (Common genes in phenylalanine, tyrosine and tryptophan pathway)
6.1.1 ARO3 and ARO4
The first reaction of the shikimate pathway is performed by 3-deoxy-D-arabino-heptulosonate (DAHP)
synthase that catalysed the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) (Fig.
5). Two genes (ARO3, ARO4) encoding differentially feedback inhibited DAHP synthase are found in S.
cerevisiae. On one hand, Aro3p is reported as inhibited by phenylalanine and on the other Aro4p is feedback
inhibited by tyrosine (8,78).
6.1.2 ARO1 and ARO2
The five reactions leading from DAHP to 5-enolpyruvoylshikimate 3-phosphate (EPSP) are catalyzed by a
pentafunctional enzyme, which is encoded by ARO1 (55,63). ARO1 presents similarities with the aroA, aroB,
aroD and aroE from E. coli that catalyzed the individual step of the EPSP synthesis (8,23,24). The first reaction
catalysed by the product of the ARO1 gene converts DAHP into 3-dehydroquinate (DHQ). This step is
performed by the DHQ synthase. Sequentially, the DHQ is converted into 3-dehydroshikimate, shikimate, and
shikimate-3-phosphate, 5-enoylpyruvyl shikimate-3-phosphate by the 3-dehydroquinate dehydratase, the
shikimate dehydrogenase, shikimate kinase and the 5-enolpyruvylshikimate-3-phosphate synthase respectively.
This enzyme has been reported to have applications such as target of herbicide action (56). Finally, the last
reaction is catalyzed by the chorismate synthase encoded by ARO2 that converts EPSP to chorismate (8,55).
18
General Introduction
E-4P
PEP
aroG
aroF
aroH
DAHP
trpE
Chorismate
ANTH
PPA
pheA
tyrA
trpD
PRA
trpC
CDRP
PPY
HPP
Phe
Tyr
trpC
InGP
trpA
Indole
trpB
Trp
FIG. 4. Aromatic amino acid biosynthesis in E. coli. Full circles represent feed-back inhibition. Abbreviations
in italics denote the genes. E-4P: erythrose 4-phosphate, PEP: phosphoenol pyruvate, DAHP: 3-deoxy-Darabino-hetulosonate,
ANTH:
anthranilate,
PRA:phosphoribosyl
anthranilate,
CDRP:
1-(ocarboxyphenylamino-1-deoxyribulose 5-phosphate, InGP: indole 3-glycerol-phosphate, PPA: prephenate, PPY:
phenylpyruvate, HPP: 4-hydroxyphenylpyruvate, Trp: tryptophan, Phe: phenylalanine, Tyr: tyrosine. Modified
from (5).
19
Chapter 1
E-4P
PEP
ARO3
ARO4
DAHP
DHQ
ARO1
EPSP
ARO2
Chorismate
ARO7
TRP2/3C
ANTH
PPA
PHA2
TRP4
PRA
TRP1
CDRP
TYR1
PPY
HPP
Phe
Tyr
TRP3B
TRP5
InGP
Trp
FIG. 5. Aromatic amino acid biosynthesis in S. cerevisiae. Full circles represent feed-back inhibition, whereas open
circles display activation. Abbreviations in italics denote the genes. E-4P: erythrose 4-phosphate, PEP: phosphoenol
pyruvate, DAHP: 3-deoxy-D-arabino-hetulosonate, DHQ: 3-dehydroquinate, EPSP: 5 enolpyruvoylshikimate 3-phosphate,
ANTH: anthranilate, PRA:phosphoribosyl anthranilate, CDRP: 1-(o-carboxyphenylamino-1-deoxyribulose 5-phosphate,
InGP: indole 3-glycerol-phosphate, PPA: prephenate, PPY: phenylpyruvate, HPP: 4-hydroxyphenylpyruvate, Trp:
tryptophan, Phe: phenylalanine, Tyr: tyrosine.Modified from (8).
20
General Introduction
6.2 Genes common for phenylalanine and tyrosine pathway
ARO7
The first branching point in the pathway for the three aromatic amino acids is chorismate. Chorismate is also
of importance for the synthesis of other aromatic compounds such as vitamin K, ubiquinone or p-amino
benzoate. Aro7p catalyzes the first step in the phenylalanine-tyrosine branch (Fig. 5). The enzyme is feedback
inhibited by tyrosine and strongly activated by tryptophan. Interestingly, in contrast to the other enzymes of the
pathway, there is no similarity between yeast chorismate mutase and bacterial (E. coli) enzymes (3,8,9,77).
6.3 Genes unique for the phenylalanine pathway
PHA2
Prephenic acid is the branching point in the synthesis of phenylalanine and tyrosine. Pha2p catalyzes the
conversion of phenylpyruvic acid (8) (Fig. 5). There is only limited information on the gene product of PHA2
(prephenate dehydratase), which converts prephenate to phenylpyruvate (8)
6.4 Genes unique for the tyrosine pathway
TYRI
Prephenate dehydrogenase (Tyr1p) catalyzes the oxidative decarboxylation and dehydratation of prephenate
to p-hydroxyphenylpyruvic acid. The TYR1 gene contains an ORF of 441 codons with a molecular weight of 52
kDa. It encodes for prephenate dehydrogenase. There is a consensus sequence for a NAD+-binding site, which is
unique for dehydrogenases (60).
Transaminase ARO8 and ARO9
The ARO8 and ARO9 genes of S. cerevisiae encode aromatic aminotransferases I and II respectively (51)
that catalyze the final step in tyrosine and phenylalanine pathway by transferring the amine group from a donor
amino acid onto phenylpyruvate and p-hydroxyphenylpyruvate: acceptor of 2-oxo acids. (See section 6.6.2 for
the detailed information).
6.5 Genes unique for the tryptophan pathway
TRP1, TRP2, TRP3, TRP3B, TRP4, TRP5
In S. cerevisiae, there are five steps from chorismate to tryptophan (Fig. 5). The first step of the tryptophan
branch is the conversion of chorismate to anthranilate by anthranilate synthase. This enzyme is subject to
feedback inhibition by tyrosine. Two genes: TRP2 and TRP3 encode this enzyme. TRP2 encodes anthranilate
synthase, which shows a decrease in activity when ammonia is the nitrogen source instead of glutamine (71).
The TRP3 gene encodes for two enzyme activities: the first one supplies nitrogen from glutamine for the
synthesis of anthranilate. The second enzyme activity will be discussed below as the gene product of TRP3B.
TRP4 is the phosphoribosyl transferase that catalyzes the transfer of a 5-phosphoribosyl moiety from 521
Chapter 1
phosphoribosylpyrophosphate to the amino group of anthranilate, resulting in phosphoribosylanthranilate
(PRA). TRP1 is known as PRA isomerase. The amino glycoside PRA goes through an internal redox reaction, in
which carboxyphenylamino-l-deoxyribulose 5-phosphate (CDRP) is formed. The InGP synthase catalyzing the
fourth reaction in the pathway is encoded by TRP3B the second domain of the bifunctional enzyme which is
encoded by the 3’ half of the TRP3 gene. TRP5 encodes the enzyme, which catalyzes the last step of the
tryptophan synthesis. In this reaction, InPG is degraded and tryptophan is produced by tryptophan synthase as a
condensation reaction with serine. The enzyme has two active sites: one for the aldol cleavage of InGP to yield
indole and glyceraldehyde-3-phosphate, and the other for the synthesis of L-tryptophan from indole and serine
(61).
6.6 Regulation of aromatic amino acid metabolism in S. cerevisiae
6.6.1 General control
Cells can respond to environmental changes via transcriptional activation and repression. Schematically,
transcriptional activators bind to specific promoters DNA sequences controlling expression of the corresponding
genes (38). In Saccharomyces cerevisiae amino acid biosynthesis is under the control of GCN4. Upon starvation
of some amino acids or even purines, Gcn4p activates the expression of a large set of genes involved in amino
acid synthesis. Indeed the starvation is sensed through level of uncharged t-RNA. Accumulation of uncharged
tRNA stimulates Gcn2p kinase activity which in return activates GCN4 translation. The synthesized factor will
activate gene expression by targeting promoter sequences. Besides the activation of the translation of GCN4
transcript, degradation of the factor itself is an important point of regulation (47).
Historically, GCN4 has been implicated in the control of 36 genes: all implicated in amino acids metabolism
(37). If this list is still recognized as primary markers for GCN4 response, more recent transcriptomics studies
revealed that about 10% of the yeast genome might be controlled by Gcn4p (65). As indicated in Fig. 6, genes
involved in pathways as diverse as storage carbohydrates metabolism, vitamin and co-factor biosynthesis,
signaling pathway were influenced by the GCN4 response (Fig. 6). Now the number of genes implicated in
amino acid biosynthesis increase up to 73 genes (65). Among these genes, genes of the aromatic amino acid
family biosynthesis such as ARO1, ARO2, ARO3 and ARO4 which are in charge of chorismic acid synthesis as
well as tryptophan synthesis genes such as TRP2, TRP3, TRP 4 and TRP5 are in control of GCN4 (8).
It is interesting to notice here that for instance ARO7 is not controlled by GCN4 while the branch leading to
tryptophan is under GCN4 control. As a result of this, a simple overexpression of ARO7 may lead to a complete
deregulation of the pathway and result to partial tryptophan auxotrophy (9) as chorismate is preferentially
channeled towards phenylalanine and tyrosine biosynthesis (see Chapter 4).
As will be shown in the following paragraphs in detail, Aro9p is an aromatic aminotransferase, which is
induced by tryptophan. Aro10p; a broad substrate specificity decarboxylase, has high transcriptional levels in
the presence of leucine, phenylalanine and methionine (82). These two genes (ARO10 and ARO9) are also the
targets of GCN4 as well as Aro8p, which is known as the aromatic aminotransferase I.
22
General Introduction
6.6.2 Specific control
As shown in the biosynthesis of other amino acids, the general transcriptional control supervised by GCN4 is
not the only regulatory mechanism controlling the transcription. In the case of aromatic amino acid metabolism
the activator encoded by ARO80 was shown to participate in the transcriptional activation of ARO9 and ARO10
encoding aromatic amino acid aminotransferase II and a broad substrate 2-oxo acid decarboxylase respectively.
In the case of ARO9, the expression was triggered when aromatic amino acids were added to the growth
medium. Furthermore as shown by (46), ARO9 expression would be correlated to the quality of the nitrogen
source. The effect of ammonia on ARO9 expression would be linked to the exclusion of the inducer by lack of
import system in presence of a good nitrogen source. Finally the zinc finger transcriptional activator of the
Zn2Cys6 family Aro80p acts through upstream activating sequence: (UASaro) that has been reported in only two
genes ARO9 and ARO10 (46). Aromatic aminotransferase II is active with phenylalanine, tyrosine and
tryptophan as amino donors and with phenylpyruvate, hydroxyphenylpyruvate, pyruvate as amino acceptors
(51,81). Interestingly, it was reported that the donor acceptor couple formed by methionine and 3methylthio 2oxobutanoate could be used by Aro9p.
6.7 Regulation of enzyme activity
The two DAHP synthases are regulated by feedback inhibition: phenylalanine feedback inhibits the ARO3
encoded DAHP synthase, and tyrosine feedback inhibits the ARO4 encoded DAHP synthase (Fig. 5). For the
ARO3 encoded isoenzyme of DAHP synthase, phenylalanine inhibition is competitive with respect to E4P and
non-competitive with PEP, whereas for the ARO4 encoded isoenzyme of DAHP synthase, tyrosine inhibition is
competitive with respect to PEP (8,78).
From the studies which focused on the evolution of two differently regulated DAHP synthases (Aro3p and
Aro4p), interesting findings were reported: The exchange of a single amino acid showed different regulatory
patterns. Aro3p with (Serine)S219G(Glycine) showed an Aro4p like behaviour and regulation by tyrosine
whereas Aro4p with G226S substitution indicated an Aro3p like phenylalanine regulation pattern (34).
DAHP synthases are metal ion-dependent enzymes. In a recent study (50): the crystal structures of several
complexes between the tyrosine-regulated form of DAHP synthases from Saccharomyces cerevisiae and E. coli
different metal ions and ligands have been determined. The crystal structures indicated that simultaneous
presence of a metal ion (Co+2, Zn+2, Cu+2, Fe+2, Ni+2) and PEP triggers an ordering of the protein into a
conformation that allowed the binding of the second substrate erythrose-4-phosphate (E4P) (8,50).
Chorismate mutases that are encoded by ARO7 are unique enzymatic activities that are found only in
microorganisms and plants but not in animals (72). In S. cerevisiae, the enzyme competes for a common
substrate with anthranilate synthase that catalyses the first step of the tryptophan-specific branch (Fig. 5). The
yeast chorismate mutase, encoded by the ARO7 gene, serves as simple model for an allosteric enzyme (76).
Aro7p is a dimer composed of two identical subunits of 30 kDa, each of them can bind one substrate and one
23
24
Amino acid transporter genes: 6
Amino acid
Biosynthetic genes: 73
FIG. 6. Target Genes of Gcn4p (modified from (65)).
Repressed genes,
translation factor and
other genes: 539
Gcn4p
Amino acid Precursor
Biosynthetic genes: 4
Peroxisomal genes: 6
Activation
Regulatory M olecules
Protein kinase genes :11
Phosphatase regulatory subunit genes:4
Transcription factor genes: 26
Salvage Pathway
Autophagy genes: 3
Vacuolar Protease genes: 2
Purine
Biosynthetic genes: 5
M itochondrial Carrier
Family Proteins
Other genes :363
Glycogen and Trehalose
M etabolism genes: 10
Chapter 1
General Introduction
activator molecule. Additionally each subunit can also bind one inhibitor molecule (76). ARO7 is known to be
allosterically regulated by tyrosine (inhibition) and tryptophan (activation) (Fig. 5) (54).
According to a study by Krapmann et al. (54): Construction of a strain carrying the ARO7T226I (ARO7c) allele
results in the unregulated chorismate mutase, which depletes the chorismate pool and causes growth defects due
to tryptophan limitation. This situation is recovered by the induction of TRP2 that encodes anthranilate synthase.
As mentioned in the previous paragraphs, anthranilate synthase competes with chorismate mutase for the same
substrate. It was concluded that: ARO7 is not transcriptionally regulated (not regulated by GCN4) and that the
transcription of anthranilate synthase-encoding gene is regulated by the availability of the amino acids.
7.0 S. cerevisiae
7.1 Applications in biotechnology
For the history of yeast biotechnology, one has to go back to the beginning of human civilization, because
yeast applications already started with ancient Egyptians (14,84). Mankind has exploited S. cerevisiae for
several centuries for the production of food and alcoholic drinks. Being non-pathogenic, having a long history of
application and furthermore holding GRAS (Generally Regarded As Safe) status makes this yeast a very
attractive organism (69). Today, the application of Saccharomyces spp. is not only limited to bread and beer
production but also has found its way in the pharmaceutical industry. With the development of metabolic
engineering, it has also become a key organism for biotechnology (Fig. 7).
7.2 Fusel alcohols and genetics of fusel alcohol production by S. cerevisiae
In addition to the production of ethanol and carbon dioxide during fermentation, S. cerevisiae also produces
a large variety of low molecular weight flavour compounds. For example, in the case of beer, the chemical
components are not only the aromas derived from the barley and hops, but also originate from branched chain
amino acid catabolism. These are called higher or fusel alcohols and their acetate esters (33). The fusel alcohols
and their acetate esters coming from the branched chain amino acid catabolism (leucine, isoleucine and valine
catabolism) such as isoamyl acetate or 3-methlybutyl acetate contribute to beer with characteristic banana and
pear flavour whereas 3-methylbutanol is also a very important aroma compounds for beer (13) and other
alcoholic beverages (86); including Japanese drink: sake (1).
Ehrlich (26) in 1908 postulated the first principles of fusel alcohol formation (Fig. 8). According to his
theory, fusel alcohol formation from amino acids proceeds via three enzyme-catalysed reactions. First, amino
acids are transaminated to the corresponding 2-oxo acids, then these are decarboxylated to their corresponding
aldehydes and as the last step, the aldehyde is reduced to an alcohol. Some of the fusel alcohols and the amino
acids they are derived from are presented in Table 2.
25
Chapter 1
Fermentation
Industries
Food & Chemical
Industries
Environmental
Technology
Yeast Biotechnology
Biomedical
Research
Fundamental Research
Health Care Industries
FIG. 7. Application areas of S. cerevisiae (Modified from (14)).
The genetics and regulation of Ehrlich Pathway is poorly understood until now. There is very limited
information especially on the effects of the expression levels of the decarboxylase genes in fusel alcohol
production (82).
The first irreversible step in Ehrlich pathway is the decarboxylation of the 2-oxo acid. Therefore more
emphasis was given to this step by scientists. Before looking into the details of those studies, it is important to
introduce the candidate decarboxylase genes. There are five TPP dependent decarboxylases that share the same
sequence characteristics (39-41). PDC1, PDC5, and PDC6 genes are known to encode pyruvate decarboxylases.
PDC1 and PDC5 generally have the highest expression levels (29), whereas PDC6 is specifically expressed
under low-sulfur conditions. It encodes a pyruvate decarboxylase that has a low content of sulfur-containing
amino acids (6,28).
The other members of the decarboxylases family are ARO10 (YDR380w) and THI3 (YDL080c). They are
thought to encode TPP-dependent decarboxylases (41). Thi3p is a positive regulator of the thiamin biosynthetic
pathway. When it was deleted the transcription of all the genes of thiamin biosynthesis were negatively affected
(41).
The genetics of the branched chain amino acid catabolism and specifically decarboxylase genes were
investigated in detail by Dickinson and co-workers. They reported that in leucine catabolism, YDL080c (THI3,
KID1) is the major decarboxylase (94 % of the decarboxylation), whereas YDR380w (ARO10) was found to be
the minor decarboxylase. In valine catabolism, one of the three isoenzymes of pyruvate decarboxylase appeared
26
General Introduction
to be sufficient enough to convert α-ketoisovalerate to 2-methylpropanol whereas in isoleucine catabolism any
of the five decarboxylases can decarboxylase α-ketomethylvalerate (19-21). In an other study by (80), it was
presented that pyruvate decarboxylase (PDC1 and PDC5) can catalyse the decarboxylation of branched chain 2oxo acids. However, it is not essential for the production of fusel alcohols. Experiments performed with wild
type and Pdc- strains provided results such as the lower rate of fusel alcohol production in Pdc- strain compared
to the wild type strain only in the case of 2-methylpropanol pointing to other genes or mechanisms involved in
fusel alcohol production other than PDC complex (80).
TABLE 2. Amino acids and derivatives that are metabolized via the Ehrlich pathway
Amino Acid
2-Oxo acid
Acid
Alcohol
Leucine
α-ketoisocaproate
3-methyl 1-butanoate
3-methyl 1- butanol
Valine
α-ketoisovalerate
2-methyl propanoate
2-methyl 1-propanol
Isoleucine
α-ketomethyl valerate
2-methyl butanoate
2-methyl 1-butanol
Methionine
4-methyl thiooxobutanoate
3-methyl thiopropanoate
3-methyl thiopropanol
Phenylalanine
phenylpyruvate
phenylacetate
2-phenylethanol
In case of aromatic amino acids such as phenylalanine and tryptophan; (18) indicated that any of the gene
products of PDC1, PDC5, PDC6, or YDR380w can catalyse the decarboxylation reaction whereas YDL080c has
no part in the catabolism of either of these amino acids.
The present literature on the decarboxylation step of the Ehrlich pathway pictured a rather intricate
mechanism involving the five members of the decarboxylase family, and that is partially dependent on the
nature of the nitrogen source. Indeed, it seems that still two redundant mechanisms could be operate i) a route
involving THI3 and ARO10 as shown for leucine catabolism, ii) a route dependent of Pdcs . This pictured that
the complexity of this single reaction step is not yet resolved.
27
Chapter 1
Amino acid
α-ketoglutarate
transamination
glutamate
α-Keto Acid
decarboxylation
CO2
oxidation
Aldehyde
Organic Acid
NAD+
NADH+ H
reduction
NAD+
Primary Alcohol
FIG. 8. Schematic representation of Ehrlich Pathway
28
NADH+ H
General Introduction
8.0 The Scope and Outline of the Thesis
The research described in this thesis addresses critical processes in the metabolism of aromatic amino acids
by Saccharomyces cerevisiae, with the aim to enable metabolic engineering of aromatic compound formation by
this yeast. Two research lines were followed.
The first research line focused on the molecular identity and substrate specificity of 2-oxo- acid
decarboxylase in S. cerevisiae. In Chapter 2, formation of biomass and metabolic products was quantified in
aerobic, glucose-limited chemostat cultures of S. cerevisiae grown with phenylalanine as the sole carbon source.
Phenylacetic acid and 2-phenylethanol were produced in large quantities, indicating that the Ehrlich pathway for
phenylalanine catabolism was active under these conditions. To identify candidate structural genes for
phenylpyruvate decarboxylase, a key enzyme in the Ehrlich pathway, transcriptome data for these cultures were
compared with those of cultures grown with ammonia as the sole carbon source. After identifying ARO10 as a
strong candidate gene, this work was followed up by physiological characterization of deletion mutants. This
confirmed that the ARO10 gene, which exhibits sequence similarity with thiamine pyrophosphate dependent
decarboxylases, is indeed involved in phenylpyruvate decarboxylation.
In Chapter 3, the substrate specificity of the Aro10p-dependent decarboxylase and its role in the production
of fusel alcohols and acids was studied in more detail. Transcriptome analysis of chemostat cultures grown with
different nitrogen sources (leucine, methionine, phenylalanine, asparagine and proline) suggested that the
Aro10p-dependent decarboxylase activity may have broad substrate specificity. This was further investigated by
overexpression of ARO10 in an S. cerevisiae strain in which all five known and putative structural genes for
thiamine pyrophosphate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3) had been deleted.
Chapter 4 describes a second line of research: the elimination of feedback inhibition steps in the
phenylalanine biosynthetic pathway in S. cerevisiae. The reactions focused on were those catalyzed by DAHP
synthase (Aro3p and Aro4p, which are subject to feed-back inhibition by tyrosine and phenylalanine,
respectively) and chorismate mutase (Aro7p, which is feed-back inhibited by tyrosine). Engineered strains were
constructed that exhibited single and combined expression of feedback-insensitive forms of these key enzymes.
Subsequently, the impact of feedback inhibition on the aromatic biosynthesis pathway was quantified by
analyzing intra- and extracellular concentrations of relevant aromatic compounds in glucose-limited chemostat
cultures of wild-type and engineered strains. Moreover, the effect of this deregulation on the intracellular pools
of other amino acids was quantified.
29
Chapter 1
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35
2
IDENTIFICATION AND CHARACTERIZATION OF
PHENYLPYRUVATE DECARBOXYLASE GENES IN
SACCHAROMYCES CEREVISIAE
ABSTRACT
Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding α-keto acids,
followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may
be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucoselimited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were
produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the
structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific
step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes
encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ ARO10,
YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures
grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures.
Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced
ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate
decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase
activity in the absence of the four other candidate genes. There was also an alternative activity with a higher
capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active
phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1,
PDC5, and PDC6) for the alternative activity. The Km and Vmax values of the two activities differed, showing
that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to
this gene could therefore be important for metabolic engineering of the Ehrlich pathway.
This chapter has been published as: Z. Vuralhan, M.A. Morais, S.L. Tai, M.D.W. Piper and J.T. Pronk. Applied
and Environmental Microbiology. 2003, 69 (8), 4534-4541.
Chapter 2
INTRODUCTION
The yeast Saccharomyces cerevisiae can use a variety of amino acids as sole nitrogen sources, including
three aromatic amino acids, L-tryptophan, L-phenylalanine, and L-tyrosine (10). The primary catabolic products
are tryptophol, phenylethanol, and tyrosol, respectively, which are collectively known as fusel oils (32, 34, 40).
Fusel oil formation from amino acids is assumed to proceed via the Ehrlich pathway by means of three enzymecatalyzed reactions. In the case of phenylalanine, the amino acid is deaminated to phenylpyruvic acid and then
decarboxylated to phenylacetaldehyde and reduced to phenylethanol (Fig. 1) (16). Phenylethanol, which has a
rose-like aroma, is an important fragrance in the cosmetic industry (9, 19) and possesses organoleptic
characteristics that contribute to the quality of beverages and foods (19, 22, 52). While chemically synthesized
phenylethanol is a valuable compound, phenylethanol that is synthesized biologically is 250- to 300-fold more
expensive (17). Various organisms, including S. cerevisiae, can produce phenylethanol (2, 18, 50), and
optimization of production in S. cerevisiae has been the subject of recent research (41). Despite this interest, the
production of phenylethanol by S. cerevisiae is poorly characterized both genetically and biochemically.
FIG. 1. Catabolism of phenylalanine via the Ehrlich pathway.
A critical step in phenylethanol production is the decarboxylase reaction, which is the first specific step in
phenylalanine catabolism (Fig. 1). The S. cerevisiae genome contains five candidate genes that could encode
phenylpyruvate decarboxylase activity. These are PDC1, PDC5, and PDC6, as well as two open reading frames,
YDR380w and YDL080c, which are also thought to encode thiamine diphosphate-dependent decarboxylases
(28). PDC1, PDC5, and PDC6 encode the major activity for pyruvate decarboxylation (27). Both the activity
38
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
and nature of this enzyme activity in yeast have been extensively studied (for reviews see references 21 and 39).
In the catabolism of branched-chain amino acids, the PDC genes contribute to fusel alcohol production, but a
PDC-independent activity also exists (42). The PDC homologs Ydl080cp and Ydr380wp contribute to the
catabolism of isoleucine (12), and the protein encoded by YDL080c is important for leucine catabolism, while
valine catabolism involves several pyruvate decarboxylase isozymes (13, 14). For the aromatic amino acids,
decarboxylases for the derived α-keto acids have not been described. However, Iraqui et al. (30) found that the
YDR380w/ARO10 open reading frame was transcriptionally induced when cells were grown in the presence of
tryptophan with urea as a nitrogen source.
In this study, our objective was to identify the gene(s) that encodes phenylpyruvate decarboxylase(s) in S.
cerevisiae. We hypothesized that one or more of the five S. cerevisiae genes for thiamine diphosphatedependent decarboxylases encode phenylpyruvate decarboxylation activity. By using a combination of genetic,
genomic, physiological, and biochemical approaches, we found that YDR380w/ARO10 encodes the main
physiologically relevant phenylpyruvate decarboxylase activity in wild-type S. cerevisiae. Additionally, we
partially characterized an alternative activity that requires the presence of both YDL080c and one of the pyruvate
decarboxylase genes.
MATERIALS AND METHODS
Strains
The S. cerevisiae strains used in this study are listed in Table 1. Strains were constructed by using standard
yeast media and genetic techniques (3, 51). The kanamycin resistance cassette was amplified by using the pUG
vector as the template (24).
Chemostat cultivation
Aerobic chemostat cultivation was performed at 30°C in 1-liter (working volume) laboratory fermentors
(Applikon, Schiedam, The Netherlands) at a stirrer speed of 800 rpm and pH 5.0 with a dilution rate of 0.10 h-1,
as described by Van den Berg et al. (44). The pH was kept constant by using an ADI 1030 biocontroller
(Applikon) and automatic addition of 2 M KOH. The fermentor was flushed with air at a flow rate of 0.5 liter
min-1 by using a Brooks 5876 mass flow controller (Brooks Instruments, Veenendaal, The Netherlands). The
dissolved oxygen concentration was continuously monitored with an Ingold model 34 100 3002 probe (MettlerToledo, Greifensee, Switzerland) and was more than 50% of air saturation. Carbon-limited steady-state
chemostat cultures of both wild-type and mutant strains were grown on the mineral medium described by
Verduyn et al. (47) containing 7.5 g of glucose liter-1 as carbon source and either 5.0 g of (NH4)2SO4 liter-1 or 5.0
g of phenylalanine liter-1 as the sole nitrogen source. When phenylalanine was the sole nitrogen source, the
amino acid solution was sterilized separately by autoclaving it before addition to the medium, and the absence
of (NH4)2SO4 was compensated for by addition of equimolar amounts of K2SO4. For chemostat cultivation of
pyruvate decarboxylase-negative strains, 7.1 g of glucose liter-1 and 0.38 g of acetate liter-1 (5% acetate on a
39
Chapter 2
carbon basis) were used as carbon sources to overcome the C2 requirement of PDC-negative strains (20). For
anaerobic cultivation, media were supplemented with the anaerobic growth factors ergosterol and Tween 80 (10
and 420 mg liter-1, respectively), and the glucose concentration was increased to 25 g liter-1 (49). To maintain
anaerobic conditions, both the culture vessel and inflowing media were sparged with nitrogen gas at a flow rate
of 0.5 liter min-1, and the fermentors were equipped with Norprene tubing and butyl rubber septa to prevent O2
diffusion into the cultures.
TABLE 1. Strains used in this study
a
Strain
Genotype
Source/
reference
CEN.PK113-7D
MATa MAL2-8c SUC2
P.Köttera
CEN.PK555-4A
MATa MAL2-8c SUC2 ydr380w:: loxP-Kan-loxP
this study
CEN.PK632-3B
MATα MAL2-8c SUC2 ydl080c::loxP-Kan-loxP ydr380w::loxP-Kan-loxP
this study
CEN.PK 608-4B
MATa MAL2-8c SUC2 pdc1::loxP pdc5::loxP pdc6::loxP ydl080c::loxP-Kan-loxP
this study
CEN.PK609-11A
MATa MAL2-8c SUC2 pdc1::loxP pdc5::loxP pdc6::loxP ydr380w::loxP-Kan-loxP
this study
CEN.PK 689-6C
MATa MAL2-8c SUC2 pdc5::loxP-Kan-loxP ydr380w::loxP-Kan-loxP
this study
Institut für Mikrobiologie der J.W. Goethe Universität, Marie-Curie-Strasse 9, Biozentrum N250, 60439 Frankfurt,
Germany
Shake flask cultivation
Growth rate experiments were performed in 500-ml flasks containing 100 ml of medium, which were
incubated at 30°C on an orbital shaker set at 200 rpm. When growth rates on phenylalanine were determined,
mineral medium (47) with 5.0 g of phenylalanine liter-1 as the sole nitrogen source was used. The pH was
adjusted to 6.0 with 2 M KOH, and then the medium was filter-sterilized with a MediaKap-5 filter (Spectrum
Europe, Breda, The Netherlands) with a pore size of 0.2 µm. Sterile glucose was added to a final concentration
of 2% as the carbon source.
Preparation of cell extracts
For preparation of cell extracts, culture samples were harvested by centrifugation, washed twice with 10 mM
potassium phosphate buffer (pH 7.5) containing 2 mM EDTA, concentrated fourfold, and stored at -20°C.
Before cell breakage, the samples were thawed at room temperature, washed, and resuspended in 100 mM
potassium phosphate buffer (pH 7.5) containing 2 mM MgCl2 and 2 mM dithiothreitol. Extracts were prepared
by sonicating preparations with 0.7-mm-diameter glass beads at 0°C for 2 min at 0.5-min intervals with an MSE
sonicator (150 W output, 7-µm peak-to-peak amplitude). Unbroken cells and debris were removed by
40
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
centrifugation at 4°C for 20 min at 36,000 x g. The purified cell extracts were used for enzyme assays.
Enzyme assays
Pyruvate decarboxylase activity was measured as described by Flikweert et al. (20). Phenylpyruvate
decarboxylase activity was measured at 30°C immediately after preparation of cell extracts by using a coupled
reaction. Activity was measured by monitoring the reduction of NAD+ at 340 nm in the presence of excess
aldehyde dehydrogenase from yeast. The reaction mixtures (total volume, 1ml) contained 70 mM KH2PO4/
K2HPO4 buffer (pH 7.0), 2 mM NAD+, 0.2 mM thiamine diphosphate, 0.35 U of yeast aldehyde dehydrogenase
(Sigma-Aldrich, Zwijndrecht, The Netherlands) (dissolved in 1 mM dithiothreitol), and 2 mM phenylpyruvic
acid to initiate the reaction. The reaction rates were linearly proportional to the amount of cell extract added. For
determination of Km and Vmax, the reaction mixture remained the same while the substrate concentration was
adjusted from 0.125 to 5 mM.
Analytical procedures
Measurements of biomass, metabolites from culture supernatants, and gasses were obtained as previously
described (5). The metabolites of phenylalanine catabolism were analyzed with a high-performance liquid
chromatograph fitted with an Alltech Platinum EPS C18 column (pore size, 0.01 µm; particle size, 5 µm; Alltech
Nederland, Breda, The Netherlands). The mobile phase was phosphate buffer (pH 2.7) with a 5 to 40%
acetonitrile gradient at a flow rate of 1 ml min-1 at room temperature. The error introduced by the measurement
technique was less than 5%.
Microarrays
DNA microarray analyses were performed with S98 Yeast GeneChip arrays from Affymetrix (Santa Clara,
Calif.) as previously described (37). Cells were transferred directly from chemostats into liquid nitrogen and
processed according to the manufacturer’s instructions (Affymetrix technical manual). Data analyses were
performed with the following Affymetrix software packages: Microarray Suite v5.0, MicroDB v3.0, and Data
Mining Tool v3.0. Microsoft Excel with the Significance Analysis of Microarrays (SAM v1.12) (43) plug-in
was used for further statistical analyses.
RESULTS
Physiology of S. cerevisiae grown with phenylalanine as the sole nitrogen source
Phenylpyruvate, phenylacetate, and phenylethanol were all detected in the supernatants of aerobic, glucoselimited chemostat cultures grown with phenylalanine as the sole nitrogen source but not in cultures grown with
ammonia as the sole nitrogen source (Table 2). These three catabolites and the residual phenylalanine in the
41
42
0.07±0.00 8.1±0.2
Phenylalanine
Anaerobic
(30 mM )
NA
NA
0.30±0.01 1.7±0.2 7.1±0.3
7.0±0.4
2.8±0.3
qCO2e
12.4±0.3 15.2±0.4
8.5±0.0 8.7±0.6
NA
NAh
qethanold
21.7±1.5
NA
19.2±4.6
NA
0.12±0
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
8.3±0.2
<0.1
11.0±3.1 1.7±0.2
<0.1
Metabolite profiles of culture supernatants
Residual
Phenyl- Phenyl- Phenylphenylpyruvate acetate ethanol
alanine
concn
concn
concn
concn
(mM)
(mM)
(mM)
(mM)
100±5%
NA
106± 7%
NA
Recovery of
phenyl C
skeleton (%)f
b
All values are means ± maximal deviations derived from two independent experiments.
Rate of glucose consumption, expressed in millimoles of glucose consumed per gram of biomass per hour.
c
Rate of O2 consumption, expressed in millimoles of O2 consumed per gram of biomass per hour.
d
Rate of ethanol production, expressed in millimoles of ethanol produced per gram of biomass per hour.
e
Rate of CO2 production, expressed in millimoles of CO2 produced per gram of biomass per hour.
f
Amount of phenylalanine consumed (sum of phenylalanine catabolites in spent medium plus 0.17 mM per biomass for protein synthesis [33])
expressed as a percentage of the phenylalanine in the feed.
g
Data from reference 5.
h
NA, not applicable.
i
Data are from reference 46.
a
qO2c
0.49±0.01 1.1±0.0 2.8±0.3
0.10±0.00 5.6±0.2
Aerobic
Aerobicg
Aeration
Biomass
yield
qglucoseb
(g g of
glucose-1)
Physiological characteristics
Anaerobici
(NH4)2SO4
(38 mM)
Phenylalanine
(30 mM )
(NH4)2SO4
(38 mM)
N source
Culture
TABLE 2. Characteristics of aerobic and anaerobic glucose-limited chemostat cultures (D=0.1 h-1) of wild-type S. cerevisiae CEN.PK 113-7D
with phenylalanine or ammonia as the sole nitrogen sourcea
Chapter 2
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
culture medium could account for all the phenylalanine supplied in the feed, indicating that no other metabolites
were formed from phenylalanine (Table 2). During anaerobic growth the metabolite profile of the supernatant
was different from that during aerobic growth. No phenylacetate was detected, and the phenylalanine supplied
could be accounted for by the amount of residual phenylalanine, phenylpyruvate, and phenylethanol (Table 2).
Phenylpyruvate decarboxylase activity (53 ± 4 nmol mg of protein-1 min-1) was detected in cell extracts of
aerobic, glucose-limited chemostat cultures grown with phenylalanine as the sole nitrogen source but not in
extracts of cultures grown with ammonia as the sole nitrogen source. Thus, we concluded that phenylpyruvate
decarboxylase was regulated and was induced in cultures with phenylalanine as the sole nitrogen source.
Identification of a putative phenylpyruvate decarboxylase gene
We used DNA microarrays to compare the transcriptomes of wild-type cells grown in glucose-limited
chemostats with phenylalanine as the sole nitrogen source and the transcriptomes of cells grown with ammonia
as the sole nitrogen source. (The entire data set is available at http://www.phepdc.bt.tudelft.nl.) We identified 89
transcripts that were expressed at a significantly higher level when phenylalanine was the sole nitrogen source
and 146 transcripts that were expressed at a significantly higher level when ammonia was the sole nitrogen
source. Of the five thiamine diphosphate-dependent decarboxylases, YDR380w/ARO10 was the only transcript
whose level increased (it increased 30-fold) when cells were grown on phenylalanine (Fig. 2). YDR380w/ARO10
was therefore considered a strong candidate to encode phenylpyruvate decarboxylase activity.
To test this hypothesis, we grew an aro10 deletion strain (CEN.PK 555-4A) in shake flasks with
phenylalanine as the sole nitrogen source. The maximum specific growth rate of this strain was three- to
fourfold lower than that of the wild-type strain in the same medium (data not shown). In contrast, the growth
rates of the two strains were similar when ammonia was the sole nitrogen source, indicating that the reduced
growth rate of the mutant was related to phenylalanine catabolism. In cell extracts of the aro10 knock-out strain
grown with phenylalanine as the sole nitrogen source, there was no detectable phenylpyruvate decarboxylase
activity. In contrast, there was measurable activity (22 ± 1 nmol mg of protein-1 min-1) in wild-type extracts
grown under the same conditions. These data suggest that ARO10 is both necessary and sufficient for
phenylpyruvate decarboxylase activity in shake flask cultures of S. cerevisiae.
Identification of an alternative phenylpyruvate decarboxylase activity
The supernatant profile and enzyme activities of the aro10 mutant strain were determined when cells were
grown in aerobic, glucose-limited chemostat cultures at a dilution rate of 0.10 h-1 with phenylalanine as the sole
nitrogen source. The profiles of phenylalanine catabolites in the culture supernatants were similar for wild-type
strain CEN.PK113-7D and mutant strain CEN.PK555-4A (Table 3). However, in contrast to the situation in
shake flask cultures, the measured phenylpyruvate decarboxylase activity of the aro10 mutant grown with
phenylalanine as the sole nitrogen source was fourfold higher than that of the wild-type strain. Thus, there was
an alternative phenylpyruvate decarboxylase activity in chemostat-grown cells that was not expressed during
growth in shake flasks. Indeed, a strain in which all thiamine diphosphate dependent decarboxylase genes except
43
Chapter 2
ARO10 were deleted (CEN.PK 608-4B) still exhibited phenylpyruvate decarboxylase activity and had
phenylpyruvate catabolites in the culture supernatants when it was grown in chemostat cultures with
phenylalanine as the sole nitrogen source (Table 3).
FIG. 2. Genome-wide transcription levels for wild-type S. cerevisiae grown on ammonia as the sole nitrogen source plotted
versus genome-wide transcription levels for wild-type S. cerevisiae grown on phenylalanine as the sole nitrogen source:
average signals in microarrays from glucose-limited chemostat cultures grown with ammonia (n = 4) and phenylalanine (n =
3) as the sole nitrogen sources for all 6,383 yeast open reading frames. The open circles represent genes whose expression
was significantly different for the two conditions, while the solid dots represent transcripts whose expression was not
significantly altered. The diagonal lines are the boundaries for two-and fivefold differences in signals between conditions.
The full data set is available at http://www.phepdc.bt.tudelft.nl.
The S.cerevisiae genome contains four PDC-like open reading frames (PDC1, PDC5, PDC6, and YDL080c).
Potentially, each of these could encode an enzyme that could decarboxylate phenylpyruvate in the absence of
ARO10. The PDC1, PDC5, and PDC6 gene products are extremely similar (79 to 86% sequence identity for all
44
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
pairwise comparisons), and each product has pyruvate decarboxylase activity (25, 26). The fourth homologous
open reading frame, YDL080c, is less well characterized but has been implicated in the decarboxylation of the
branched-chain 2-oxo acids and the regulation of genes involved in thiamine metabolism (12–14, 36).
Both a double-deletion strain (aro10 ydl080c; CEN.PK 632-3B) and a quadruple-deletion strain (pdc1 pdc5
pdc6 aro10; CEN.PK 609-11A) were grown in aerobic, glucose-limited chemostat cultures with phenylalanine
as the sole nitrogen source. Cell extracts from these strains contained no measurable phenylpyruvate
decarboxylase activity (Table 3). These assay data were supported by metabolite profile analyses of the culture
supernatants, since phenylacetate was undetectable in both cultures and only small amounts of phenylethanol
were found in the culture of the double-deletion strain (aro10 ydl080c) (Table 3). The quadruple-deletion strain
containing only YDL080c (CEN.PK 609-11A) showed no evidence of the decarboxylase activities examined
(Table 3), demonstrating that Ydl080cp alone could not decarboxylate phenylpyruvate or pyruvate. This result
also indicated that it is unlikely that there are any other genes that encode the alternative decarboxylase activity.
Instead, the phenotypes of the multiple-deletion strains indicate that the ARO10-independent phenylpyruvate
decarboxylase activity requires the presence of both YDL080c and at least one of the PDC genes (Table 3).
Physiological relevance of S. cerevisiae phenylpyruvate decarboxylase activities
We determined the Km and Vmax values for Aro10p and the alternative phenylpyruvate decarboxylase
activity. Cell extracts from a strain containing only the ARO10-encoded activity (CEN.PK 608-4B) were
compared with extracts from a strain containing only the alternative activity involving Ydl080cp and one or
more of the pyruvate decarboxylases (CEN.PK 555-4A). The phenylpyruvate decarboxylase activities of
extracts of the wild-type strain and the quadruple-deletion strain (containing only Aro10p) displayed MichaelisMenten saturation kinetics. Reduction of the substrate concentration in the assay mixture to less than 0.5 mM
resulted in altered enzyme activity, from which the Km and Vmax values were estimated (Table 4). The Vmax
values for the activities of these two extracts were similar (Table 3). However, the extract from the quadruple
mutant (CEN.PK 608-4B) had a slightly higher affinity (Km, 0.062 ± 0.005 mM) for the substrate
phenylpyruvate than the wild type had (Km, 0.10 ± 0.001 mM). Extracts from the strain lacking ARO10 alone
(CEN.PK 555-4A) resulted in a sigmoidal curve in a plot of substrate concentration versus velocity. This result
is consistent with previous observations of cooperativity of pyruvate decarboxylase in the presence of phosphate
(6). This PDC-like behavior is consistent with the genetic data, according to which at least one PDC gene is
required for the Aro10p-independent phenylpyruvate decarboxylase activity. In supernatants from wild-type
cultures grown with phenylalanine as the sole nitrogen source, all of the phenylalanine consumed was recovered
as either phenylacetate or phenylethanol (Table 2). By using the sum of the concentrations of phenylalanine
catabolites in the supernatant and the biomass concentration in the culture vessel (2.23 g liter -1), the specific rate
of catabolite production was calculated to be 0.57 mmol g of biomass -1 h -1. Since the catabolites were derived
from phenylpyruvate decarboxylation, the steady-state flux through the decarboxylase enzyme must also have
been 0.57 mmol g of biomass -1 h -1 (or 9.5 nmol mg of biomass -1 h -1). Since the estimated soluble protein
content is 0.33 g g of biomass -1 for yeast (38), this rate was converted to a specific activity of 29 nmol mg of
45
Chapter 2
protein-1 min-1. If values were substituted into the Michaelis-Menten equation and the Vmax and Km values
obtained for wild-type cell extracts were used, the substrate concentration inside the cells was 0.11 mM. This
value is the same as the Km determined for wild-type extracts and is slightly higher than the Km found for the
mutant containing Aro10p only (CEN.PK 608-4B) (Table 4). The Km of the activity from cells lacking ARO10
was five- to eightfold higher than that from strains that contained a wild-type ARO10 allele (Table 4). Therefore,
at deduced intracellular phenylpyruvate concentrations of ca. 0.1 mM, this compound is preferentially
catabolized by the ARO10-encoded activity.
Involvement of PDC5 in ARO10-independent phenylpyruvate decarboxylation
We used a recently compiled transcriptome database for cells grown under four different nutrient limitation
regimens (5) to evaluate the correlation between the expression of thiamine diphosphate-dependent
decarboxylase genes and phenylpyruvate decarboxylase activity in wild-type S. cerevisiae (Table 5) (for the
complete data sets accompanying these arrays see reference 5). Phenylpyruvate decarboxylase was detected
only during aerobic growth under nitrogen limitation conditions with ammonia as the nitrogen source and
glucose as the carbon source and when there was phosphate-limited growth with ammonia as the nitrogen
source and glucose as the carbon source (Table 5). Under these conditions, the levels of the ARO10 transcript
were negligible, indicating that the observed phenylpyruvate decarboxylase activity was due to the ARO10independent activity discussed above. In all four cultures, low but significant levels of the YDL080c transcript
were detected. However, phenylpyruvate decarboxylase was detected in cell extracts when PDC5 was
transcribed at high levels but not when PDC1 (glucose limitation) or PDC6 (sulfur limitation) was the
predominantly transcribed PDC gene. Furthermore, the levels of the PDC5 transcript in glucose-limited
chemostat cultures of the aro10 strain grown with phenylalanine as the sole nitrogen source were over eightfold
higher than the levels in similar cultures of the wild type, while the levels of the PDC1, PDC6, and YDL080c
transcripts differed by less than twofold (data not shown).
To test the hypothesis that PDC5 but not PDC6 or PDC1 contributes to the alternative phenylpyruvate
decarboxylase activity, we grew a pdc5 aro10 double-deletion strain (CEN.PK 689-6C) in aerobic, glucoselimited chemostat cultures with phenylalanine as the sole nitrogen source. Cell extracts of steady-state
chemostat cultures did not exhibit significant phenylpyruvate decarboxylase activity (<2.5 nmol mg of protein-1
min-1), and <10% of the phenylpyruvate formed was converted to phenylacetate. The pyruvate decarboxylase
activity in cell extracts of these cultures was 565 ± 45 nmol mg of protein-1 min-1, and preliminary experiments
indicated that PDC1 but not PDC6 was expressed transcriptionally (as determined with a single measurement)
46
140 ± 2.0
PDC1 PDC5 PDC6 YDR380w YDL080cb
PDC1 PDC5 PDC6 ydr380w YDL080c
PDC1 PDC5 PDC6 ydr380w ydl080c
pdc1 pdc5 pdc6 ydr380w YDL080c
pdc1 pdc5 pdc6 YDR380w ydl080c
CEN.PK 113-7D
CEN.PK 555-4A
CEN.PK 632-3B
CEN.PK 609-11A
CEN.PK 608-4B
47
b
a
YDR380w is ARO10, and YDL080c is THI3.
40 ± 1.5
< 2.5
< 2.5
220 ± 17
52.5 ± 3.5
Phenylpyruvate
decarboxylase
All values are means ± maximal deviations derived from two independent experiments.
< 10
< 10
540 ± 55
180 ± 2.0
Pyruvate
decarboxylase
Strain
Relevant genotype
Enzyme activities
(nmol.mg of protein-1.min-1)
0.41 ± 0.1
6.5 ± 1.0
7.37 ± 0.12
0.75 ± 0.40
< 0.1
Phenylpyruvate
7.4 ± 2
< 0.1
< 0.1
8.93 ± 0.18
11.0 ± 3.1
Phenylacetate
3.5 ± 0.3
< 0.1
0.81 ± 0.0
2.5 ± 0.0
1.7 ± 0.2
Phenylethanol
Conc. of phenylalanine catabolites in
culture supernatants (mM)
TABLE 3. Enzyme activities and metabolite profiles of aerobic glucose-limited chemostat culture of S. cerevisiae strains grown with phenylalanine
as the sole nitrogen source (Dilution rate= 0.1 h-1)a
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
Chapter 2
TABLE 4. Km and Vmax valuesa determined for phenylpyruvate decarboxylase activity in cell extracts from three S.
cerevisiae strains grown in aerobic glucose-limited chemostats with phenylalanine as the sole nitrogen source (dilution
rate, 0.1 h-1)
Strain
a
Km
Vmax
(mM)
(nmol.mg of protein-1.min-1)
Relevant genotype
CEN.PK 113-7D
PDC1 PDC5 PDC6 YDR380w YDL080c
0.10 ± 0.001
54 ± 6
CEN.PK 608-4B
pdc1 pdc5 pdc6 YDR380w ydl080c
0.062 ± 0.005
38 ± 3
CEN.PK 555-4A
PDC1 PDC5 PDC6 ydr380w YDL080c
0.48 ± 0.08
236 ± 28
The values are means ± maximal deviations derived from two independent experiments for CEN.PK 113-7D and
CEN.PK 608-4B and from three independent experiments for CEN.PK 555-4A.
(data not shown). However, when the cultures were grown for more than eight generations, the phenylpyruvate
decarboxylase activity gradually increased and, after approximately 15 generations, reached specific activities in
vitro that exceeded those of wild-type cultures and were similar to that of the aro10 deletion strain (265 ± 10
nmol mg of protein-1 min-1). This increase was reflected in the metabolite profile of the growth medium, which
was similar to that of the aro10 deletion strain growth medium (data not shown).
TABLE 5. Enzyme activities and transcript levels of the thiamine diphosphate-dependent decarboxylases of S. cerevisiae under
four nutrient limitation regimens in chemostat culturesa
Transcript levelb
Enzyme activities
-1
(nmol.mg of protein .min)
Growth
limitation
a
b
Pyruvate
decarboxylase
Phenylpyruvate
decarboxylase
PDC1
PDC5
PDC6
YDR380w
YDL080c
Carbon
580 ± 0.0
<2.5
1946 ± 449
95 ± 5
66 ± 44
67 ± 4
92 ± 9
Nitrogen
Phosphorous
Sulfur
2265 ± 25
1830 ± 50
520 ± 30
10.15 ± 0.05
4.37 ± 0.35
<2.5
2524 ± 188
2366 ± 354
1969 ± 238
1708 ± 87
836 ± 33
169 ± 36
161 ± 30
37 ± 7
1874 ± 236
< 12
14 ± 1
< 12
249 ± 34
120 ± 27
105 ± 16
The cultivation conditions and transcript data are from reference 5.
The transcript levels are data for triplicate arrays sampled from three independent chemostats.
48
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
DISCUSSION
Products of phenylalanine catabolism in S. cerevisiae
When wild-type S. cerevisiae was grown in glucose-limited chemostats with phenylalanine as the sole
nitrogen source, phenylethanol and phenylacetate could account for all of the phenylalanine consumed from the
feed (Table 2). This result is consistent with the involvement of the Ehrlich pathway in phenylalanine
catabolism (Fig. 1). In anaerobic chemostat cultures there was an almost stoichiometric conversion of
phenylalanine to phenylethanol (Table 2) that probably reflected the altered redox state of the cells to favor the
reductive branch of the Ehrlich pathway over the oxidative, phenylacetate-yielding branch (Fig. 1). The
presence of these catabolites in the medium was reflected in the physiological growth parameters measured.
When phenylalanine was used instead of ammonia as the sole nitrogen source, the biomass yield on glucose
(expressed in grams [dry weight] of biomass per gram of glucose consumed) of wild-type S. cerevisiae was
substantially lower (Table 2). In addition, higher rates of carbon dioxide production and, in the aerobic cultures,
oxygen consumption accompanied the reduced biomass yield. It has been hypothesized that these changes are
indicative of uncoupling caused by enhanced proton cycling via the weak acid phenylacetate (48) and by the
stimulating effect of phenylethanol on membrane fluidity (29, 41).
Decarboxylation of phenylpyruvate is not a prerequisite for the transamination of phenylalanine and, hence,
utilization of this compound as sole nitrogen source (Fig. 1). Nevertheless, growth with phenylalanine as the
sole nitrogen source in shake flask cultures was substantially slower for an aro10 mutant, which lacked
detectable phenylpyruvate decarboxylase activity, than for the wild type. This difference suggests that the physiological role of phenylpyruvate decarboxylation may be to prevent the accumulation of growth-inhibiting
concentrations of phenylpyruvate.
Transcriptional regulation of phenylalanine catabolism
Transcriptional regulation of genes in response to phenylalanine occurs via at least two routes in S.
cerevisiae. The first route involves an intracellular sensor of aromatic amino acids that regulates genes through
the transcriptional activator Aro80p (30), and the second route involves a sensor of external amino acids (31).
We identified the binding site for Stp1p (a transcriptional regulator downstream of extracellular amino acid
sensing [35]), which was overrepresented in the promoters of genes whose transcription was greater when
phenylalanine was the nitrogen source (45). An additional element known to bind the GATA family of
transcriptional regulators (8) also was found, indicating that there was control via the general response to the use
of a nonpreferred nitrogen source (nitrogen catabolite repression [NCR]). Only nine gene promoters in the
genome contain an exact match with the proposed binding site for Aro80p (direct repeat of 5’T[A/T][A/G]CCG-3’ separated by four nucleotides) (30). Among the nine genes, there are four pairs of
divergently transcribed genes and one gene without a shared promoter. There were three genes with
significantly higher transcript levels in chemostat cultures containing phenylalanine instead of ammonia as the
sole nitrogen source (ARO9, ARO10, and ESPB6, exhibiting 6-, 30-, and 2.5-fold changes in transcript levels,
49
Chapter 2
respectively). The promoters of these three genes contained the binding site repeat in the forward direction. If
this promoter element operates unidirectionally, it could explain why five of the remaining genes, which have
the reverse complement sequence in their promoters, were not regulated in a similar manner. Based on
phenotypic analysis of an aro80 deletion strain, it is not surprising that the domain of Aro80p’s control is
limited to the degradation of aromatic amino acids (1, 30). Similar regulatory events have also been reported in
other microorganisms (4, 11, 23) and probably result from the need to separate phenylalanine biosynthesis from
phenylalanine catabolism since the two pathways share phenylpyruvate as an intermediate and are thought to be
colocalized in the cytosol.
The wider effects on the transcriptome could be caused by the activity of NCR due to the presence of an
aromatic amino acid as the sole nitrogen source. Many genes (including a number of regulated genes according
to our data that are required for nutrient transport) are under the control of this regulon. However, previous
results have shown that NCR does not directly regulate expression of ARO9 or ARO10 (30). Rather, NCR
modulates the expression of these genes indirectly by preventing Aro80p-dependent induction by inducer
exclusion. Thus, cells cofed ammonia and an aromatic amino acid should preferentially catabolize ammonia by
preventing uptake of the aromatic amino acid. Conversely, when ammonia is limiting for growth (or absent),
this general repression is relieved, which allows uptake and assimilation of amino acids for use as nitrogen
sources (See supplementary material at http://www.phepdc.bt.tudelft.nl for all gene changes).
Substrate specificity of thiamine diphosphate-dependent decarboxylases
Of the five thiamine diphosphate-dependent decarboxylase genes in S. cerevisiae, the ARO10 transcript was
the only transcript changed, and the level was 30-fold higher during growth on phenylalanine as the sole
nitrogen source than during growth on ammonia as the sole nitrogen source. Two lines of evidence confirm that
ARO10 encodes an active phenylpyruvate decarboxylase: (i) the clear phenotype of an aro10 null mutant in
shake flask cultures grown with phenylalanine as the sole nitrogen source and (ii) the phenotype of a quadruple
pdc1 pdc5 pdc6 ydl080c mutant in chemostat cultures grown with phenylalanine as the sole nitrogen source.
An alternative, ARO10-independent phenylpyruvate decarboxylase activity also was observed in chemostat
cultures of the aro10 null mutant. This activity was not detectable in an aro10 ydl080c double mutant. The
YDL080c product exhibits strong sequence similarity with known thiamine diphosphate-dependent decarboxylases and has a regulatory role in thiamine metabolism (7). Our data show that Ydl080cp cannot
decarboxylate phenylpyruvate by itself. However, the combined presence of Ydl080cp and a pyruvate
decarboxylase is required for the ARO10-independent phenylpyruvate decarboxylase activity. We have recently
obtained evidence that a similar situation exists for the branched-chain 2-oxo acids that are formed during the
catabolism of leucine, valine, and isoleucine (M. A. Morais and Z. Vuralhan, unpublished data). Analysis of
transcript levels in wild-type cultures, as well as physiological analysis of a pdc5 aro10 strain, indicated that
Pdc5p is primarily involved in the ARO10-independent, YDL080c-dependent phenylpyruvate decarboxylase
activity. However the reappearance of phenylpyruvate decarboxylase activity of a pdc5 aro10 double mutant
after prolonged chemostat cultivation suggests that only minor genetic changes allow another PDC gene to take
50
Identification of Phenylpyruvate Decarboxylase Genes in S. cerevisiae
over this role. We have not yet identified the nature of these mutations.
Part of the regulation of the different decarboxylases may occur at the level of transcription through a
mechanism in which Thi3p acts as a sensor of intracellular thiamine diphosphate (7). In the presence of
phenylalanine, the levels of the ARO10 transcript were among the highest 3% of the levels of transcripts of
transcribed genes in wild-type cells. Deletion of this gene would probably alter the levels of intracellular thiamine diphosphate and trigger a signal via Thi3p to control transcription. However, the poor correlation between
the transcript level and enzyme activity (Table 5) indicates that posttranscriptional regulation of the
decarboxylase activities also occurs. An attractive model for this posttranscriptional regulation depends upon the
in vivo tetrameric form of pyruvate decarboxylase (21). If the Pdc-like proteins can form heterotetramers, the
resulting decarboxylase activities may have different substrate specificities. In this model, Aro10p and Thi3p
could combine with one or more of the PDC-encoded proteins to produce enzymes that decarboxylate the αketo acids produced during catabolism of the aromatic and branched-chain amino acids. This hypothesis can be
tested with reconstitution experiments performed with different amounts and combinations of the purified
proteins.
This study increased our understanding of phenylalanine catabolism in S. cerevisiae and illustrated the
power of combining genome-wide transcript analyses with biochemical and genetic techniques to untangle
functionally redundant enzyme activities. To date, analyses of single and multiple knockout mutants have
proven to be insufficient to identify singular roles for the thiamine diphosphate-dependent decarboxylases in
amino acid catabolism (12–15, 20). Our results provide new insight into the complexity of the regulation of
substrate specificity of these decarboxylases, and they also provide a good basis for targeted metabolic
engineering of phenylalanine catabolism.
ACKNOWLEDGMENTS
This work was financially supported by the Board of the Delft University of Technology (BEO program),
the Dutch Ministry of Economic Affairs, and the Kluyver Centre for Genomics of Industrial Fermentation.
M.A.M. received support from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq). We
thank Hans van Dijken for helpful comments during preparation of the manuscript and Peter Kötter for
providing strains. The pdc1pdc5 pdc6 triple-deletion strain was used with the permission of J. Lievense, Tate &
Lyle/A.E. Staley Manufacturing Company.
ADDENDUM
While this manuscript was under review, Dickinson et al. (15) reported on the catabolism of phenylalanine
to phenylethanol and the catabolism of tryptophan to tryptophol in S. cerevisiae. Using 13C nuclear magnetic
resonance spectroscopy, gas chromatography-mass spectrometry, and a range of mutants, these authors showed
that Aro10p can catalyse the decarboxylation of phenylpyruvate to phenylacetaldehyde and the decarboxylation
of indolepyruvate to indolacetaldehyde and that, in the absence of an active aro10 gene, pyruvate
decarboxylases are involved in phenylpyruvate decarboxylation.
51
Chapter 2
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55
3
PHYSIOLOGICAL CHARACTERIZATION OF THE ARO10DEPENDENT, BROAD-SUBSTRATE-SPECIFICITY 2-OXO
ACID DECARBOXYLASE ACTIVITY OF SACCHAROMYCES
CEREVISIAE
ABSTRACT
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with
different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source
contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich
pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant,
suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five
thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was
transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source
compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity
measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed
similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited
chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with
ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and
methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced
significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in
an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid
decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with
phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results
reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10dependent, broad-substrate specificity decarboxylase activity.
This chapter has been published as: Z. Vuralhan, M.A.H. Luttik, S.L. Tai, V.M. Boer, M.A. Morais, D. Schipper,
M.J.H. Almering, P. Kötter, J.R. Dickinson, J.M. Daran, J.T. Pronk. Applied and Environmental Microbiology,
2005, 71 (6), 3276-3284.
Chapter 3
INTRODUCTION
Saccharomyces cerevisiae has a narrow range of carbon sources that support growth (1) but is considerably
more flexible with respect to the utilization of nitrogen sources (2). Most amino acids can be utilized as sole
nitrogen sources but not as sole carbon sources for growth (28). The most common mechanism for utilizing
amino acids as nitrogen sources is transamination, using 2-oxoglutarate or other 2-oxo acids as amino acceptors.
This process leaves the carbon skeleton of the amino acid intact, in the form of a 2-oxo acid. For some amino
acids (e.g., alanine), the resulting 2-oxo acid, pyruvate, can be readily co-metabolized in central metabolism. In
other cases, such as for the aromatic and branched-chain amino acids, the 2-oxo acids resulting from
transamination are not intermediates of central metabolism. Even though they cannot be used as auxiliary
carbon sources, these compounds are often transformed by the yeast cells before they are excreted into the
growth medium.
An important and common pathway for catabolism of amino acids by yeasts is called the Ehrlich pathway
(7–12, 37). This pathway is initiated by transamination of the amino acid to the corresponding 2-oxo acid. This
2-oxo acid is then decarboxylated to the corresponding aldehyde. Depending on the redox status of the cells
(44), the aldehydes can then be reduced by alcohol dehydrogenases (yielding a group of compounds commonly
referred to as fusel alcohols) or be oxidized to the corresponding organic acid (“fusel acids”) by aldehyde dehydrogenases (Fig. 1). The fusel alcohols and their esters are especially important contributors to the flavor and
aroma of fermented beverages (6, 16, 45). Phenylethanol, which has a typical rose-like flavor, can be produced
by biotransformation of phenylalanine with S.cerevisiae cell suspensions (38, 39).
The identity of the decarboxylase(s) that catalyzes the initial step of the Ehrlich pathway has recently been
investigated in our laboratories (7, 9–11, 44). The S.cerevisiae genome contains five genes that share sequence
similarities with genes encoding thiamine pyrophosphate (TPP)-dependent decarboxylases (19, 20, 27) (for a
review, see reference 21). Three of these genes (PDC1, PDC5, and PDC6) encode pyruvate decarboxylases.
PDC1 and PDC5 encode the major pyruvate decarboxylases under most cultivation conditions (15, 20); PDC6
is specifically expressed under low-sulfur conditions and encodes a pyruvate decarboxylase that has a low
content of sulfur-containing amino acids (4, 14). Mutants in which all three PDC genes have been inactivated,
and which completely lack pyruvate decarboxylase activity, still express branched-chain and aromatic 2-oxo
acid decarboxylase activities (7, 40, 44). The other two members of this gene family are ARO10 and THI3.
Based on studies with deletion mutants, both have been implicated in the decarboxylation of branched-chain and
aromatic 2-oxo acids (7, 10, 44). In addition, Thi3p has been assumed to be a positive regulator of the thiamine
biosynthetic pathway. Upon its deletion, the transcription of all the genes of thiamine biosynthesis was
negatively affected (13, 21). An aro10 thi3 double-deletion mutant completely lacks phenylpyruvate decarboxylase activity, whereas the single-deletion mutants in these genes retain this enzyme activity (44). This
might lead to the simple conclusion that both genes encode active phenylpyruvate decarboxylases. However, the
situation is more complicated, as pdc1 pdc5 pdc6 thi3 quadruple-deletion mutants, but not pdc1 pdc5 pdc6
aro10 mutants, express phenylpyruvate decarboxylase activity (7, 10, 44). These and other observations have
led to the proposal that THI3 may not by itself encode an active phenylpyruvate decarboxylase but requires the
58
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
simultaneous expression of one of the PDC genes to contribute to phenylpyruvate decarboxylase activity (44).
This provided a first indication that the regulation and substrate specificities of the TPP-dependent
decarboxylases in S.cerevisiae may be more complicated than a simple situation in which substrate specificity is
determined by a mixture of five decarboxylases with defined—if overlapping—substrate specificities and
kinetics.
With the exception of the transcriptional regulation of ARO10 by aromatic amino acids modulated by the
positive transcription factor ARO80 (24), comparatively little is known about the regulation of fusel alcohol
production in S.cerevisiae and the impact of the expression levels of the decarboxylase genes on the rates of
production of the different decarboxylases.
FIG. 1. Formation of fusel alcohols and fusel organic acids during the catabolism of the amino acids leucine, phenylalanine,
and methionine
The aim of the present study was to analyze the substrate specificity of the ARO10-dependent decarboxylase
activity in S.cerevisiae, its impact on the production of fusel alcohols and acids, and the importance of
transcriptional regulation in controlling its in vivo activity. To this end, we correlated the expression of ARO10
(as well as that of the other decarboxylase genes) with the levels of fusel alcohols and acids in chemostat
cultures of S.cerevisiae grown with different nitrogen sources. Subsequently, we investigated the substrate
59
Chapter 3
specificity of the ARO10-dependent decarboxylase activity and the impact of transcriptional regulation of
ARO10 on this activity by constitutively expressing ARO10 in a wild-type S.cerevisiae strain, as well as in a
pdc1 pdc5 pdc6 aro10 thi3 quintuple-null mutant.
MATERIALS AND METHODS
Strains
The Saccharomyces cerevisiae strains used in this study are listed in Table 1.
Recombinant-DNA techniques
Standard protocols were followed for plasmid isolation, restriction, ligation, transformation, and gel
electrophoresis (30). Yeast chromosomal DNA was isolated by a method described previously (22). S.cerevisiae
strains were transformed using the lithium acetate–single-stranded carrier DNA–polyethylene glycol method
(17).
TABLE 1. S. cerevisiae strains used in this study
a
Strain
Genotype
Source or
reference
CEN.PK113-7D
MATa MAL2-8c SUC2 isogenic prototrophic strain
P. Köttera
CEN.PK 113-5D
MATa MAL2-8c SUC2 ura3
P. Köttera
CEN.PK 555-4D
MATa MAL2-8c SUC2 aro10∆
(44)
CEN.PK 711-7C
MATa MAL2-8c SUC2 ura3 pdc1∆pdc5∆pcd6∆aro10∆thi3∆
This study
IMZ001
MATa MAL2-8c SUC2 ura3 pdc1∆pdc5∆pcd6∆aro10∆thi3∆ p426GPD (URA3)
This study
IMZ002
This study
IME003
MATa MAL2-8c SUC2 ura3 pdc1∆pdc5∆pcd6∆aro10∆ thi3∆ pUDe001
(URA3 TDH3p-ARO10)
MATa MAL2-8c SUC2 ura3 pUDe001 (URA3 TDH3p-ARO10)
IME004
MATa MAL2-8c SUC2 ura3 p426GPD (URA3)
This study
Institut fur Mikrobiologie der J. W. Goethe Universitat, Frankfurt, Germany.
60
This study
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
Overexpression of ARO10
The ARO10 (YDR380W) open reading frame was PCR amplified from CEN.PK113-7D genomic DNA using
primers ARO10-fwd(GGTCTAGAATGGCACCTGTTACAATTGAAAAG) and ARO10-rev (GGCTCG
AGCTATTTTTTATTTCTTTTAAGTGCCGC), designed to introduce restriction sites (underlined) for
endonuclease XbaI upstream of the ATG and XhoI downstream of the stop codon, respectively. The PCR
product and the vector p426GPD (31) were digested by XbaI and XhoI. The XbaI-XhoI PCR fragment was
directionally cloned behind the glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p) into p426GPD,
resulting in plasmid pUDe001. The ARO10 open reading frame sequence was confirmed by sequencing. The
plasmid pUDe001 was transformed by the lithium acetate–single-stranded carrier DNA–polyethylene glycol
method (17) into the S.cerevisiae CEN.PK 113-5D strain, resulting in strain IME003, and into strain CEN.PK
711-7C, resulting in strain IMZ002. Similarly, CEN.PK 113-5D and CEN.PK 711-7C were transformed with
p426GPD (31), resulting in IME002 and IMZ001, respectively.
Chemostat cultivation
Aerobic chemostat cultivation was performed at 30°C in 1-liter working volume laboratory fermentors
(Applikon, Schiedam, The Netherlands) at a stirrer speed of 800 rpm, pH 5.0, with a dilution rate (D) of 0.10 h-1
as described previously (42), with the exception of the strains IMZ001 and IMZ002, which were grown at a
dilution rate of 0.05 h-1 . The pH was kept constant, using an ADI 1030 biocontroller (Applikon, Schiedam, The
Netherlands), via the automatic addition of 2 M KOH. The fermentor was flushed with air at a flow rate of 0.5
liter min-1 using a Brooks 5876 mass-flow controller (Brooks Instruments, Veenendaal, The Netherlands). The
dissolved-oxygen concentration was continuously monitored with an Ingold model 34 100 3002 probe (MettlerToledo, Greifensee, Switzerland) and was above 50% of air saturation.
Carbon-limited steady-state chemostat cultures of S.cerevisiae strains were grown as described previously
(43) on synthetic medium containing 7.5 g of glucose liter-1 or 5.7 g liter-1 of ethanol, keeping molar carbon
equivalence constant at 0.25 M, and either 5.0 g liter-1 (NH4)2SO4, 5.0 g liter-1 of L-phenylalanine (44), 10 g
liter-1 L-leucine, 11.3 g liter-1 L-methionine, 5 g liter-1 L-asparagine, or 8.8 g liter-1 L-proline as the sole
nitrogen source. The absence of (NH4)2SO4, was compensated for by the addition of equimolar amounts of
K2SO4 when phenylalanine, leucine, methionine, proline, or asparagine was used as the only nitrogen source.
Culture dry weight
Culture dry weights were determined via filtration as described previously (35).
Extracellular-metabolite analysis
For the determination of phenylalanine, leucine, and methionine catabolism products and carbon recovery,
culture supernatants and media were analyzed by high-performance liquid chromatography (HPLC), fitted with
an AMINEX HPX-87H ion-exchange column (300 by 7.8 mm; Bio-Rad) mounted in a Waters Alliance 2690
HPLC apparatus, at 60°C using H2SO4 as the mobile phase with a flow rate of 0.6 ml · min-1. Metabolites were
61
Chapter 3
detected by a dual-wavelength absorbance detector (Waters 2487) and a refractive-index detector (Waters 2410)
and integrated with Chrompack Maitre 2.5 software.
Identification of metabolites by NMR spectroscopy
After lyophilization, samples of culture supernatants were dissolved in D2O. 1H, 1H-1H TOCSY, and 1H-13C
correlation spectra (direct and long range) were measured at 300 K on a Bruker Avance 600 nuclear magnetic
resonance (NMR) spectrometer equipped with an inverse triple-resonance probe and a pulse field gradient
system. Quantitative 1H-NMR experiments were also performed at 600 MHz. To 0.5 ml of supernatant, an equal
amount of a standard solution containing maleic acid and EDTA was added. After lyophilization, the residue
was dissolved in D2O and the 1H -NMR spectrum was measured using a relaxation delay of 30 seconds, ensuring full relaxation of all the hydrogen atoms between pulses. The integrals of the characteristic resonance for
each component and the internal standard (singlet at 6.1 ppm) were measured, and the contents of the individual
components were calculated.
Preparation of cell extracts
For the preparation of cell extracts, culture samples were harvested by centrifugation; washed twice with 10
mM potassium-phosphate buffer, pH 7.5, containing 2 mM EDTA; concentrated fourfold; and stored at -20°C.
Before cell breakage, the samples were thawed at room temperature, washed, and resuspended in 100 mM
potassium phosphate buffer, pH 7.5, containing 2 mM MgCl2 and 2 mM dithiothreitol. Extracts were prepared
by sonication with 0.7 mm glass beads at 0°C for 2 min at 0.5-min intervals with an MSE sonicator (150-W
output; 8-µm peak-to-peak amplitude). Unbroken cells and debris were removed by centrifugation at 4°C (20
min; 36,000 x g). The purified cell extract was used for enzyme assays.
2-Oxoacid decarboxylase assays
2-Oxo acid decarboxylase activity was measured at 30°C immediately after preparation of cell extracts using
a coupled reaction. Activity was measured by following the reduction of NAD+ at 340 nm in the presence of
excess aldehyde dehydrogenase from yeast. The reaction mixtures contained, in a total volume of 1 ml, 100 mM
KH2PO4 / K2HPO4 buffer, pH 7.0; 2 mM NAD+ ; 5 mM MgCl2; 15 mM pyrazole; 0.2 mM thiamine diphosphate;
1.75 U of yeast aldehyde dehydrogenase (Sigma-Aldrich, Zwijndrecht, The Netherlands) (dissolved in 1 mM
dithiothreitol); and 2 mM phenylpyruvic acid, α-ketoisocaproate, α-ketoisovalerate, α-ketomethylvalerate, 3methylthio-α-ketobutyrate, or pyruvate to initiate the reaction. Reaction rates were linearly proportional to the
amount of cell extract added.
Activity data normalization
The per-strain normalization accounts for the difference in detection efficiency between 2-oxo acid
decarboxylase activities. It also allows comparison of the relative change in activity levels, as well as displaying
62
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
these levels in similar scales on the same graph. GeneSpring (Silicon Genetics, Redwood City, CA) uses the
following formula to normalize to the median for each strain: (activity of strain X on substrate Y) / (median of
every measurement of strain X).
Protein determination
Protein concentrations in cell extracts were determined by the Lowry method (29). Bovine serum albumin
(fatty acid free; Sigma, St. Louis, Mo.) was used as a standard.
Microarray analysis
DNA microarray analyses were performed with the S98 Yeast GeneChip arrays from Affymetrix as
previously described (34). Cells were transferred directly from chemostats into liquid nitrogen and processed
according to the manufacturer’s instructions (Affymetrix technical manual; Affymetrix, Santa Clara, CA.). Data
analyses were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0, and
Data Mining Tool v3.0. The Significance Analysis of Microarrays (SAM version 1.12) (41) add-in to Microsoft
Excel was used for comparisons of replicate array experiments.
RESULTS
Measurement of phenylalanine and phenylethanol in chemostat cultures of S.cerevisiae grown on
various nitrogen sources
The S.cerevisiae reference strain CEN.PK113-7D was grown on synthetic medium in aerobic, glucoselimited chemostat cultures with different nitrogen sources: ammonium sulfate, phenylalanine, leucine,
methionine, proline, or asparagine. During growth on phenylalanine as the nitrogen source, HPLC analysis of
culture supernatants revealed the presence of high concentrations of phenylethanol and phenylacetate, consistent
with the operation of the Ehrlich pathway. Surprisingly, low but significant concentrations of these metabolites
were also observed when leucine or methionine was the sole nitrogen source (Table 2). The concentrations of
phenylethanol and phenylacetate in leucine-and methionine-grown cultures were 20- to 50-fold higher than in
cultures grown with ammonium sulfate as the nitrogen source (Table 2). Similarly, 2-methylpropanoate and phydroxyphenylacetate, which are Ehrlich pathway-derived catabolites of valine and tyrosine, respectively, were
also detectable when phenylalanine, leucine, or methionine was used as the sole nitrogen source. Although 3methylbutanol, an expected product of leucine catabolism, could not be detected in our HPLC setup, the
compound was detected by 1H-NMR in leucine, phenylalanine, and methionine cultures (data not shown).
Conversely, none of these metabolites were detected in cultures grown with ammonium sulfate, proline, or
asparagine as the nitrogen source (Table 2).
These results can be explained in two different ways. First, growth on amino acids whose catabolism
involves the Ehrlich pathway may lead to coordinate induction of Ehrlich pathway enzymes with different
substrate specificities. Alternatively, these amino acids may induce Ehrlich pathway enzymes with broad
63
b
a
ND
9.915 ± 0.681
ND
0.137 ± 0.036
0.180 ± 0.045
ND
ND
ND
4.538 ± 0.351
ND
ND
b
[mM]
3-Methylbutanoate
ND
0.161 ± 0.021
ND
0.310 ± 0.000
0.204 ± 0.000
ND
[mM]
2-Methylpropanoate
0.026 ± 0.004
ND
0.037 ± 0.007
ND
0.757 ± 0.309
ND
[mM]
3-Methylthio
propanol
ND
ND
ND
ND
ND
ND
[mM]
3-Methyl-thiopropanoate
ND
0.076 ± 0.020
ND
0.135 ± 0.031
0.190 ± 0.017
ND
[mM]
p-Hydroxyphenylacetate
p-Hydroxy-
ND
ND
ND
ND
0.054 ± 0.025
ND
[mM]
phenylethanol
(derived from valine), 2-methylbutanol, and 2-methylbutanoate (derived from isoleucine) were not detected by the HPLC setup used in the present study.
ND, not detected.
In aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) of S. cerevisiae CEN.PK 113-7D grown with different nitrogen sources. Data are presented as
average ± mean deviation of metabolite quantification from two independent chemostat cultures. 3-Methylbutanol (derived from leucine), 2-methylpropanol
ND
1.261 ± 0.141
ND
Phenylalanine
Proline
Asparagine
0.059 ± 0.013
0.225 ± 0.071
Leucine
Methionine
0.003 ± 0.000
[mM]
[mM]
0.003 ± 0.000
Phenylacetate
Phenylethanol
Ammonia
N source
TABLE 2. Concentrations of fusel alcohols and corresponding organic acidsa
Chapter 3
64
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
substrate specificities. To further investigate this phenomenon, we focused on the irreversible decarboxylase
reaction.
Decarboxylation of 2-oxoacids by cell extracts of wild-type S.cerevisiae grown on various nitrogen
sources
2-Oxo-acid-decarboxylase activities involved in the Ehrlich pathway were analyzed in cell extracts of
S.cerevisiae CEN.PK113-7D grown in aerobic carbon-limited chemostat cultures with different amino acids as
the sole nitrogen source (Table 3). Phenylpyruvate, α-ketoisovalerate, α-ketoisocaproate, α-ketomethylvalerate, and 3-methylthio-α-ketobutyrate were selected as substrates based on the observed metabolite profiles
(Table 2). Significant activities with all five substrates were detected in cultures grown with leucine, methionine,
or phenylalanine as the nitrogen source (Table 3). Conversely, no activity was measured in cell extracts from
cultures grown on ammonium, asparagine, or proline, in good agreement with the absence of alcohols and acids
in the corresponding culture supernatants (Table 2). When activities were expressed relative to the activity with
phenylpyruvate, the substrate specificity did not differ markedly as a function of the nitrogen source for growth.
This suggested involvement of a single common decarboxylase activity in the catabolism of leucine,
methionine, and phenylalanine (Fig. 2).
Transcript levels of TPP-dependent decarboxylase genes in wild-type S.cerevisiae grown on various
nitrogen sources
The pyruvate-decarboxylase genes PDC1, PDC5, and PDC6 and the related genes THI3 and ARO10 have all
been implicated in the production of fusel alcohols and fusel acids by S.cerevisiae in the literature (7, 9–11, 44),
but their substrate specificities and catalytic contributions remain unknown. To check whether the induction of a
“broad-substrate-specificity decarboxylase activity” observed in cell extracts could be correlated with the transcriptional induction of a single gene, expression of the five decarboxylase genes was analyzed.
The levels of the ACT1 transcript, a commonly used “loading standard” for mRNA analysis (32), were the
same for all six nitrogen sources (Table 4). PDC5, PDC6, and THI3 were transcribed at a constant, very low
level. PDC1 showed much higher transcript levels, but they did not significantly differ for the six nitrogen
sources (t test analysis at P < 0.01). Only ARO10 was differentially transcribed for the different nitrogen
sources (Table 4). In cultures grown with leucine, phenylalanine, or methionine as the nitrogen source, the
transcript level was at least 15-fold higher than in cultures grown with ammonium sulfate as the nitrogen source.
Moreover, cultures grown with proline or asparagine as the nitrogen source yielded the same very low ARO10
transcript levels as ammonium sulfate-grown cultures (Table 4).
ARO10 encodes a broad-substrate-specificity 2-oxo-acid-decarboxylase in S.cerevisiae
The transcriptional regulation of ARO10; the similar substrate specificities of decarboxylase activities in cell
extracts of leucine-, methionine-, and phenylalanine-grown cultures; and the metabolite profiles in these cultures
all suggested that Aro10p is responsible for a broad-substrate-specificity decarboxylase activity involved in the
65
Chapter 3
production of fusel alcohols and acids. To test this hypothesis, an S.cerevisiae strain lacking all five TPPdependent decarboxylase genes (CEN-PK711-7C pdc1∆ pdc5∆ pdc6∆ thi3∆ aro10∆ ura3∆) was constructed.
The ura3 genotype was complemented by transformation either with the empty expression vector p426GPD
(strain IMZ001) or with the same vector carrying ARO10 under the control of the constitutive TDH3 promoter
(strain IMZ002). Strains IMZ001 and IMZ002 could not grow on glucose synthetic media as a result of the
pdc1∆ pdc5∆ pdc6∆ genotype (15). Therefore, ethanol was used as a carbon source.
TABLE 3. Specific activities of 2-oxo acid decarboxylation by cell extractsa
Specific decarboxylase activity [nmol.min-1.(mg protein)-1]
Nitrogen
source
Phenylpyruvate
[phenylalanine]
Ammonia
BD
13.5 ± 0.7 (100%)
22.25 ± 1.8 (100%)
67.5 ± 0.7 (100%)
BD
BD
Leucine
Methionine
Phenylalanine
Proline
Asparagine
a
b
α-Ketoisovalerate
[valine]
α-Ketoisocaproate
[leucine]
α-Ketomethylvalerate
[isoleucine]
3-Methylthio αketobutyrate
[methionine]
BD
BD
BD
BD
4 ± 0.01 (29%)
8.5 ± 0.5 (38%)
19 ± 0 (28%)
BD
BD
6.5 ± 0.7 (48%)
9.25 ± 0.5 (42%)
29.5 ± 0.7 (43%)
BD
BD
4.5 ± 0.7 (33%)
5.5 ± 0.9 (25%)
25.7 ± 3.8 (38%)
BD
BD
5.5 ± 0.6 (41%)
9 ± 0.01 (40%)
22 ± 0 (32%)
BD
BD
Prepared from aerobic, glucose-limited chemostat cultures of S. cerevisiae CEN.PK 113-7D grown with different amino
acids as the sole nitrogen source. Data are the average ± mean deviation of assays from two independent chemostat
b
cultures. The relative 2-oxo acid activities, expressed as a percentage of phenylpyruvate activity, are in parentheses. The
column headings include in parentheses the amino acid which the 2-oxo acid used as a substrate is derived from.
BD, below detection limit.
Cell extracts of the quintuple-deletion strain IMZ001, grown in aerobic, ethanol-limited chemostat cultures
at a dilution rate of 0.05 h-1 and with ammonium sulfate as the nitrogen source, did not exhibit any
decarboxylase activity (Table 5). Constitutive expression of ARO10 in this genetic background (strain IMZ002)
restored decarboxylase activity with the 2-oxo acids derived from leucine, phenylalanine, and methionine.
Interestingly, no activity could be measured with pyruvate as a decarboxylase substrate (Table 5). The relative
specific activities with the non-pyruvate substrates were similar to those observed in cell extracts of the
reference strain CEN.PK 113-7D grown with phenylalanine, leucine, or methionine as the nitrogen source (Fig.
2 and Tables 3 and 5).
The quintuple-deletion strain IMZ001 was unable to grow in ethanol-limited chemostat cultures (D = 0.05 h1
) when phenylalanine was the sole nitrogen source. This ability was recovered in strain IMZ002, which
constitutively expresses ARO10 from the TDH3 promoter. Unexpectedly, decarboxylase activities in cell
extracts of strain IMZ002 grown with phenylalanine as the nitrogen source were 4.5-fold higher than in cultures
66
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
grown with ammonium sulfate as the nitrogen source (Table 5). Reintroduction of ARO10 in the quintupledeletion strain also restored the production of fusel alcohols and acids. HPLC analysis of ethanol-limited,
ammonium-grown chemostat cultures of IMZ002 revealed low but significant concentrations of phenylethanol
(0.11 ± 0.01 mM) and phenylacetate (0.20 ± 0.03 mM). The concentrations of these compounds were below the
HPLC detection limit (0.003 mM) in chemostat cultures of the quintuple-deletion strain IMZ001. When the
ARO10-expressing strain was grown with phenylalanine as the nitrogen source, high concentrations of
phenylethanol (2.69 ± 0.06 mM) and phenylacetate (7.29 ± 0.34 mM) were observed in culture supernatants.
Furthermore, low concentrations of 3-methylthiopropanol (0.67 mM) and p-hydroxyphenylacetate (0.16 mM)
were identified, confirming the involvement of Aro10p in the synthesis of a broad range of fusel alcohols and
acids in vivo.
FIG. 2. Eisen representation of relative 2-oxo acid decarboxylase activity. Cell extracts of CEN.PK113-7D, CEN.PK 5554D, IME003, IMZ001, and IMZ002 grown in aerobic carbon-limited (glucose or ethanol) chemostat cultures with different
nitrogen sources were measured for 2-oxo-acid decarboxylase activity. Each cell extract was tested for conversion of
phenylpyruvate, α-ketoisovalerate, α-ketoisocaproate, α-ketomethylvalerate, and 3-methylthio-α-ketobutyrate. The activity
data were normalized to the mean and clustered by hierarchical clustering using Genespring (Silicon Genetics, Redwood
City, CA). The so-called normalized data were displayed on a scale from 0 to 5 (see Materials and Methods).
67
Chapter 3
Overexpression of ARO10 in the isogenic reference strain CEN.PK 113-5D
To investigate whether overexpression of ARO10 can be used to modify fusel alcohol production by wildtype S.cerevisiae strains, the expression vector carrying ARO10 under the control of the TDH3 promoter was
introduced into the reference strain CEN.PK113-5D (resulting in strain IME003 [Table 1]). Surprisingly, except
for pyruvate decarboxylase, no 2-oxo acid decarboxylase activity was detectable in cell extracts of this strain
when it was grown in glucose-limited chemostat cultures with ammonium sulfate as the nitrogen source (Table
6). Monitoring of the ARO10 transcript level by quantitative PCR in strain IME003 grown in a glucose-limited
chemostat with ammonium sulfate as the nitrogen source revealed expression of the TDH3-driven construct.
TABLE 4. Transcript levels of genes with sequence similarity to thiamin-pyrophosphate-dependent decarboxylases in
aerobic, glucose-limited chemostat cultures of S. cerevisiae CEN.PK113-7D grown with different amino acids as the sole
nitrogen sourcea
Nitrogen
source
Ammonia
Leucine
Methionine
Phenylalanine
Proline
Asparagine
a
ACT1
ARO10
PDC1
PDC5
PDC6
THI3
2488 ± 81
2149 ±204
2831 ±624
2917 ±575
2294 ±127
2416 ±122
67 ± 3
1045 ± 167
1335 ± 130
1996 ± 201
37 ± 6
61 ± 11
1946 ± 449
1311 ± 90
1459 ±226
894 ±319
1505 ± 173
1170 ± 109
95 ± 4
73 ± 7
87 ± 14
87 ± 32
81 ± 8
64 ± 6
66 ± 4
31 ± 8
17 ± 3
25 ± 5
76 ± 8
46 ± 8
92 ± 9
128 ± 11
126 ± 22
109 ± 17
115 ± 12
105 ± 47
Transcript levels were determined with Affymetrix Gene Chips. Data are the average ± standard deviation of three
independent chemostat cultures. The ACT1 transcript is included as a reference
The level of expression was equivalent to half of the ACT1 reference transcript signal. In the meantime, no
ARO10 transcript was detected in strain IME004 grown under similar conditions. Furthermore, decarboxylase
activities in glucose-limited chemostat cultures grown with phenylalanine as the nitrogen source were the same
as those of the empty-vector reference strain IME004 (Table 6). When ethanol instead of glucose was used as
the carbon source, the presence of the ARO10 expression vector did result in increased decarboxylase activities
relative to an empty-vector reference strain (Table 6). These results contradict the simple view that ARO10
encodes a fully functional decarboxylase whose expression is primarily regulated at the level of transcription.
DISCUSSION
Formation of fusel alcohols by S.cerevisiae
In brewery and wine fermentations, S.cerevisiae is responsible for the production of a variety of metabolites
that contribute to flavor and aroma. Among the volatile flavor compounds, an important class consists of higher
alcohols that are less volatile than ethanol (18). These higher alcohols are derived from the carbon skeletons of
amino acids, which can in theory be synthesized de novo but in brewery and wine fermentations are generally
68
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
taken up from the wort or grape must. It is commonly accepted that branched-chain (9–11) and aromatic (7, 44)
amino acid-derived alcohols originate from the Ehrlich pathway (12) (Fig. 1). Our results support the notion that
this pathway is also involved in the production of 3-methylthiopropanol (methionol) and 3methylthiopropanoate from methionine. These sulfur-containing compounds are relevant to the production of
alcoholic beverages. Methionol, which has a raw-potato odor, is commonly measured in wine and is known to
negatively affect white wine and red wine aroma above 0.6 mg/liter and 2 to 3 mg/liter, respectively (3) (Table
1).
TABLE 5. Substrate specificity of the ARO10-dependent 2-oxo-acid-decarboxylase activity in S. cerevisiaea
-1
-1
Enzyme activity nmol.min .(mg protein)
Substrate
IMZ001
[(NH4)2 SO4c]
IMZ002
[(NH4)2 SO4c]
IMZ002
[ Phenylalaninec ]
Ratio
Phenyl-pyruvate
BDd
61.75±1.71 (100%)
270 ± 6.98 (100%)
4.37
α-Keto isovalerate
α-Keto isocaproate
α-Keto methylvalerate
3-Methylthio α-ketobutyrate
Pyruvate
BD
BD
BD
BD
BD
16.75 ±2.21 (27%)
25±0.82 (40%)
21±1.63 (34%)
18.5±1.29 (30%)
BD
78.25 ± 4.1 (29%)
118 ± 7.53 (44%)
97 ± 6.68 (36%)
87.7 ± 7.69 (32%)
BD
4.67
4.72
4.62
4.74
-
a
b
Strain IMZ001 is pdc1∆ pdc5∆ pdc6∆ aro10∆ thi3∆ carrying the empty expression vector p426GPD (2µ URA3 TDH3p).
Strain IMZ002 is the same strain carrying the plasmid pUDe001 (2µ URA3 TDH3p-ARO10). Both strains were grown in
aerobic, ethanol-limited chemostat cultures with ammonia as the nitrogen source. Enzyme activities were assayed in cell
extracts. Data are the average ± average deviation of the mean from assays of two independent chemostat cultures. The
relative 2-oxo acid activities, expressed as a percentage of phenylpyruvate activity, are in parentheses.
b
c
d
Ratio of phenylalanine versus (NH4)2SO4.
N source.
BD, below detection limit.
Our results indicated that induction of an Ehrlich pathway for catabolism of one amino acid leads to the
formation of significant amounts of fusel alcohols and acids from other amino acids. This suggested that
conversion of branched-chain, aromatic, and sulfur-containing amino acids to the corresponding fusel alcohols
and acids, via an Ehrlich pathway, involves common broad-substrate-specificity enzyme activities. Furthermore,
as our experiments were performed with synthetic media to which only single amino acids were added, these
results indicated that the decarboxylase activity involved in the Ehrlich pathway could compete for 2-oxo acids
with the transaminases involved in de novo amino acid biosynthesis. The chemostat conditions used in this
study were designed to reveal the molecular nature of the decarboxylase step of the Ehrlich pathway. Although
these conditions are different from typical alcoholic fermentation processes, the conclusion drawn about the role
of ARO10 is relevant for interpreting the patterns of flavor production in wine and beer fermentation.
69
Chapter 3
Aro10p is involved in a broad-substrate-specificity Ehrlich pathway decarboxylase activity
Transcript analysis demonstrated that the induction of Ehrlich pathway activity by the amino acids leucine,
phenylalanine, and methionine coincided with the transcriptional up-regulation of ARO10, but not with that of
the other four genes encoding (putative) thiamine pyrophosphate-dependent decarboxylases. Indeed,
overexpression of ARO10 in a strain in which the five chromosomal decarboxylase genes had been deleted was
sufficient to restore a broad-substrate-specificity decarboxylase activity. The substrate specificity profile of this
strictly ARO10-dependent activity was the same as those of the activities induced by leucine, phenylalanine, and
methionine in wild-type cells.
Previous research in S.cerevisiae with aro10 null mutants has indicated the presence of an ARO10independent decarboxylase activity (44). This alternative activity has been reported to require the simultaneous
expression of at least one of the three pyruvate decarboxylase genes (PDC1, -5, and -6) and the putative
decarboxylase gene THI3. Based on previous work by Dickinson and coworkers, the last gene has also been
implicated in the decarboxylation of the 2-oxo acids derived mainly from leucine (11) and to a lesser extent
isoleucine (10). This ARO10-independent 2-oxo acid decarboxylase activity exhibited a completely different
substrate specificity profile. In particular, the decarboxylase activity observed in cultures of an aro10 null
mutant grown with phenylalanine as the nitrogen source showed no activity with α-ketoisovalerate and α-ketomethylvalerate as the substrate (Fig. 2, strain CEN.PK 555-4D).
The results described here support the notion that the ARO10-dependent, broad-substrate-specificity
decarboxylase is primarily responsible for the Ehrlich pathway decarboxylation reaction in wild-type
S.cerevisiae. The molecular basis and substrate specificity of the ARO10-independent activity that is detected in
aro10 null mutants (44) (Table 5), as well as its possible involvement in (off-) flavor production by wild-type
strains, require further research.
Transcriptional regulation of ARO10
Previous work has shown that transcription of ARO10 is induced by tryptophan (24) and phenylalanine (44)
and is dependent on the transcriptional regulator Aro80p (24). Other studies (7, 9–11) suggested that expression
of ARO10 might also be up-regulated by valine and isoleucine, based on metabolite profiling; however, this
conclusion was not backed up by expression analysis. Our results clearly show that ARO10 expression was
strongly up-regulated in the presence of leucine and methionine (Table 3), consistent with its proposed role as a
broad-substrate-specificity decarboxylase.
Further analysis of the transcriptome data revealed that ARO9 (aromatic amino transferase II) (23) was coexpressed with ARO10 in cultures grown with different nitrogen sources (data not shown). This suggested that
the transaminase activity of Aro9p might not be restricted to aromatic amino acids (24) but, similar to the
Aro10p-dependent decarboxylase activity, might have a broad substrate specificity. It remains to be in
vestigated whether and to what extent Aro80p is involved in the transcriptional up-regulation of ARO9 and
ARO10 by the non aromatic amino acids leucine and methionine. This question could not be resolved by the
microarray analyses, since ARO80 transcript levels were extremely low and did not differ significantly for the
70
71
BD
BD
α-ketomethylvalerate
3-methylthio-α-ketobutyrate
b
a
64 ± 1.2
17 ± 2.2
28 ± 3.1
BDb
BD
BD
BD
BD
BD
BD
BD
(NH4)2SO4
25.7± 3.8
22 ± 0
67.5 ± 0.7
19 ± 0
29.5 ± 0.7
Phenylalanine
Glucose
12.5 ± 0.7
11 ± 0
35 ±5.7
19 ± 0
15 ± 1.4
(NH4)2SO4
28 ± 4.0
23.5 ± 1.2
76.5 ± 1.5
21.75 ± 1.7
30.5 ± 3
Phenylalanine
Ethanol
BD
BD
BD
BD
BD
(NH4)2SO4
28.3 ± 13.6
24.25 ± 9.1
82.75 ± 26.73
22.4 ± 6.4
31.5 ± 9.8
Phenylalanine
Glucose
BD, below detection limit.
protein)-1. Data are presented as average ± average deviation of the mean of two chemostat cultures.
Both strains were grown in aerobic, carbon-limited chemostat cultures with glucose or ethanol as a carbon source and ammonia or phenylalanine as a carbon source and
ammonia or phenylalanine as a nitrogen source. Enzyme activities were assayed in cell extracts of independent duplicate cultures and are expressed as nmol . min-1 (mg
23 ± 0.7
20.5 ± 1.2
Phenylalanine
(NH4)2SO4
Ethanol
Phenylpyruvate
α-ketoisovalerate
α-ketoisocaproate
Substrate for decarboxylase
Assay
IME004 [ura3 p426GPD (URA3)]
IME003 [ura3 pUDe001 (URA3 TDH3p-ARO10)]
TABLE 6. Regulation of decarboxylase activities in the reference S. cerevisiae strain IME004 (CEN.PK113-5D, p426GPD) and in an isogenic strain expressing a
multicopy plasmid-borne ARO10 gene from a constitutive TDH3 promoter, strain IME003a
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
Chapter 3
nitrogen sources studied (data not shown). Further research with aro80 null strains is required to investigate
whether the regulatory role of Aro80p extends beyond aromatic amino acid metabolism expression control or,
alternatively, another regulatory protein or proteins control the upregulation of ARO9 and ARO10 in leucine and
methionine-grown cultures. A comprehensive discussion of the genome-wide transcriptional responses of
S.cerevisiae to the six nitrogen sources used in this study will be published elsewhere (V. M. Boer, S. L. Tai, Z.
Vuralhan, Y. Arifin, M. C. Walsh, M. D. W. Piper, J. H. de Winde, J.-M. Daran, and J. T. Pronk, unpublished
data).
Involvement of other factors in the activity and regulation of Aro10p
Earlier works (7, 9–11, 15, 40, 44) on the decarboxylation of branched-chain and aromatic 2-oxo acid
decarboxylation were based on the implicit assumption that single proteins (e.g., Aro10p and/or Thi3p) would
act as thiamine pyrophosphate-dependent decarboxylase enzymes. While the present study proves that Aro10p
plays a key role in broad-substrate-specificity decarboxylase activity, it also provides several clear indications
that additional factors are involved in this activity and its regulation.
Our attempt to overexpress ARO10 under the control of the TDH3 promoter in order to uncouple its
expression from environmental parameters, such as the presence of phenylalanine, yielded unexpected results.
In cultures grown with ammonium sulfate as the nitrogen source, the TDH3p-ARO10 construct yielded activity
in ethanol-grown cultures but, surprisingly, not when glucose was the carbon source. This unexpected
dependency on the carbon source was independent of the expression of the other four decarboxylase genes. As
the TDH3 promoter is known to give very high transcript levels in glucose as well as ethanol-grown cultures,
this observation suggests that transcription of the ARO10 gene is not sufficient to yield an active broadsubstrate-specificity decarboxylase activity. Furthermore, in ethanol-grown cultures of the “ARO10 constitutive”
strains, addition of phenylalanine to culture media caused a strong increase in the broad-substrate-specificity
decarboxylase activities in cell extracts.
These observations may indicate that the functional expression of the ARO10 gene is regulated at a
posttranscriptional level in a carbon and nitrogen source-dependent manner. Alternatively, the catalytic activity
and/or stability of Aro10p may require the presence of one or more additional proteins whose expression is
carbon and nitrogen source dependent.
Recent protein interactome studies based on the two-hybrid approach (25) identified two potential Aro10p
interaction partners. Fit2p is possibly involved in iron uptake (33, 36), and Ena5p is an ATP-driven sodium
transporter, a member of the Na+ transporting ATPase family in the superfamily of P-type ATPases (5, 26).
Taking into account the subcellular localization of Fit2p and Ena5p (the cell wall and plasma membrane,
respectively), it is difficult to envision them as key factors in controlling the activity or stability of Aro10p. Of
these two putative partners, only FIT2 would show an expression profile that would corroborate our assumption
(data not shown).
The present study has clearly established the importance of Aro10p in the key decarboxylation step of the
Ehrlich pathway. At the same time, it has raised new and important questions about the additional factors
72
Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity Decarboxylase Activity
involved in the molecular composition, posttranscriptional regulation, and/or stability of the Aro10p-dependent
decarboxylase activity. These questions need to be resolved before strategies can be devised to rationally modify
the production of volatile flavor compounds by S.cerevisiae in beverages and fine-chemical production, e.g., via
genetic modification of in vivo decarboxylase activity. Purification and characterization of the broad-substratespecificity decarboxylase from cell extracts is likely to be essential to resolve the outstanding issues.
ACKNOWLEDGMENTS
This work was financially supported by the Board of the Delft University of Technology (“Beloning
Excellent Onderzoek” Program), the Dutch government (CW-NOW program “Transition towards Sustainable
Technology”), and the Kluyver Center for Genomics of Industrial Fermentation. Z.V. and V.M.B. were
financially supported by DSM.
We thank Pascale Daran-Lapujade and the DSM discussion group for helpful comments on the manuscript
and Matthew Piper for his contribution in the initial phase of the project.
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76
4
ALLEVIATION
OF
FEEDBACK
INHIBITION
IN
SACCHAROMYCES CEREVISIAE AROMATIC AMINO ACID
BIOSYNTHESIS: QUANTIFICATION OF METABOLIC
IMPACT
ABSTRACT
A quantitative analysis of the impact of feedback inhibition on aromatic amino-acid biosynthesis was performed
in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive ARO4 allele (encoding
DAHP synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These
amino acids were not detected extracellularly. However, an over 50-fold increase of the extracellular levels of
phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux
through the aromatic pathway was estimated to be 1.9 fold. Overexpression of a tyrosine and tryptophan
insensitive allele of ARO7, either alone or in combination with the Tyr-insensitive ARO4 allele, had little impact
on the synthesis of aromatic compounds. Elimination of allosteric control on these two key reactions in aromatic
amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids.
This broader impact of amino-acid biosynthesis presents a challenge in rational optimisation of the production
of specific amino acids and derived flavour compounds.
This chapter is in preparation for publication: Z. Vuralhan, M.A.H. Luttik, G.H. Braus, J.M. Daran and J.T.
Pronk
Chapter 4
INTRODUCTION
The aromatic amino acids phenylalanine and tryptophan are mainly used in food and feed applications. They
are produced on an industrial scale via bacterial fermentation processes using Escherichia coli and
Corynebacterium glutamicum (16,29). Interest in phenylalanine has increased proportionally with the increased
demand for low caloric food and soft drinks, as phenylalanine is a precursor for the low caloric sweetener
aspartame (12). While Saccharomyces cerevisiae is not under consideration for the industrial production of
amino acids, aromatic amino acid metabolism by this yeast is of interest for other industrial applications.
In Saccharomyces cerevisiae and other yeasts (7, 45) the phenylalanine biosynthesis pathway is involved in
the synthesis of phenylethanol. This molecule has interesting sensory properties (including a rose-like aroma)
and is of increasing economical interest. Phenylethanol can be produced via bio-transformation of phenylalanine
with S. cerevisiae (6). This conversion involves the reactions of the Ehrlich pathway (5), which is involved in
the catabolism of several amino acids by S. cerevisiae (Fig. 1). In the case of phenylalanine, the Ehrlich pathway
is initiated by its transamination to phenylpyruvate. This 2-oxo acid is then decarboxylated to
phenylacetaldehyde (42). Depending on the redox status of the cells, phenylacetaldehyde is then reduced by
alcohol dehydrogenases (yielding phenylethanol) or oxidized to phenylacetic acid by (43). Phenylpyruvate, the
precursor for phenylethanol production by S. cerevisiae, is also an intermediate in the de novo biosynthesis of
phenylalanine from sugars.
In bacteria, extensive strain improvement programmes, involving a combination of random mutagenesis and
targeted metabolic engineering, have been applied to improve aromatic amino acid biosynthesis (17, 18). In
these strain improvement programmes, a first and essential step is invariably the elimination of feedback
inhibition on key enzymes in the biosynthetic pathway. In S. cerevisiae, two reactions in the phenylalanine
biosynthesis pathway are known to be subject to feedback inhibition (Fig. 1). The first committed step in
aromatic amino acid metabolism is catalysed by 3-deoxy-D-arabinoheptulonate-7-phosphate (DAHP) synthase,
for which two isoenzymes exist in S. cerevisiae, encoded by the ARO3 and ARO4 genes (21, 36). Aro3p and
Aro4p are feedback inhibited by phenylalanine and tyrosine, respectively. Besides, both DAHP synthases show
considerable tryptophan regulation as recently reported (15). A single lysine-to-leucine substitution in Aro4p at
position 229 results in a deregulated enzyme that is no longer feedback inhibited by tyrosine (13). Chorismate
mutase, encoded by ARO7, has been identified as a second reaction subject to allosteric regulation (2, 3), being
subject to feedback inhibition by tyrosine as well as activation by tryptophan. Substitution of the serine residue
141 by a glycine abolished effects of both tyrosine and tryptophan, thus leading to a non-allosterically regulated
chorismate mutase (20, 33). Although considerable knowledge is available on the molecular basis for feedback
inhibition of aromatic amino acid metabolism (14), no quantitative studies have yet been performed on its
impact on product formation by growing S. cerevisiae cultures.
The aim of this study is to quantify the combinatorial effects of deregulation of DAHP synthase and
chorismate synthase in S. cerevisiae. To this end, a tyrosine- feedback insensitive DAHP synthase and a nonallosterically regulated chorismate mutase were expressed in an aro3∆ genetic background. Subsequently, the
production of aromatic amino acids and the corresponding fusel alcohols and ‘fusel acids’ were quantified in
78
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
PEP
E-4P
Aro3p
Aro4p
DAHP
DHQ
DHS
Shikimate
Aro1p
SHP
EPSP
Aro2p
Trp3p
Chorismate
Trp2p
ANTH
Trp4p
Trp1p
Aro7p
Pha2p
PPY
PRA
Phe
CDRP
InGP
Tyr
HPP
Aro10p
PAC
pPAC
IPY
Aro10p
PAA
IAC
IAA
HPP
Aro8p
Aro9p
PPY
Trp
Aro8p
Aro9p
Tyr1p
Aro8p
Aro9p
Trp3p
Trp5p
PPA
PET
pPAA
pPET
IET
FIG. 1. Pathways of aromatic amino acid biosynthesis and catabolism in S. cerevisiae. The dashed lines indicate feedback
inhibition of Aro4p and Aro7p by tyrosine and feedback inhibition of Aro3p by phenylalanine. The solid line indicates
activation of Aro7p by tryptophan. E-4P: erythrose 4-phosphate, PEP: phosphoenol pyruvate, DAHP: 3-deoxy-D-arabinoheptulosonate, DHQ: 3-dehydroquinate, DHS: SHP: EPSP: 5 enolpyruvoylshikimate 3-phosphate, ANTH: anthranilate,
PRA:phosphoribosyl anthranilate, CDRP: 1-(o-carboxyphenylamino-1-deoxyribulose 5-phosphate, InGP: indole 3-glycerolphosphate, PPA: prephenate, PPY: phenylpyruvate, HPP: para-hydroxy-phenylpyruvate, PAC: phenylacetaldehyde, PAA:
phenylacetate, PET: phenylethanol, pPAC: para-hydroxy-acetaldehyde pPAA:para-hydroxy-acetate, pPET: para-hydroxyphenylethanol, IAC: indole-acetaldehyde, IAA: indole-acetate, IET: indole-ethanol, Trp: tryptophan, Phe: phenylalanine,
Tyr: tyrosine.
79
Chapter 4
aerobic, glucose-limited chemostat cultures. To assess the specificity of this approach to deregulate aromatic
amino acid metabolism, we also analysed intracellular concentrations of other amino acids in reference and
engineered strains.
MATERIALS AND METHODS
Strain construction and maintenance
The Saccharomyces cerevisiae strains used in this study are listed in Table 1. Stock cultures were grown at
o
30 C in shake flasks on synthetic medium (26) supplemented with 20 g of glucose l-1. When stationary phase
was reached, sterile glycerol was added to 30% (vol/vol), and 2-ml aliquots were stored in sterile vials at –80oC.
The construction of the ARO3 and ARO4 null mutants was done using the PCR based method using short
flanking homologies (SFH) (26). SFH deletion cassettes were obtained by PCR using the primers ARO3-S1
(GAATCTCCAATGTTCGCTGCCAACGGCATGCCAAAGGTAACAGCTGAAGCTTCGTACGC)/ARO3-S
2 (TCAAGGCCTTTCTTCTGTTTCTAACACCTTCTGCCAATAGGCATAGGCCACTAGTGGATCTG) and
ARO4-S1(GAATCTCCAATGTTCGCTGCCAACGGCATGCCAAAGGTAACAGCTGAAGCTTCGTACGC)
/ARO4-S2 (TTGTTAACTTCTCTTCTTTGTCTGACAGCAGCAGCCAATTGCATAGGCCACTAGTGGAT
CTG) and pUG6 as template (11). The deletion cassettes were transformed in the prototrophic diploid yeast
strain CEN.PK122. After sporulation and tetrad analysis the corresponding segregants were isolated resulting in
strains CEN.PK532-1A and CEN.PK437-1C. Both strains were crossed to obtain after tetrad analysis the aro3
aro4 double deletion strain CEN.PK557-5A.
The plasmid pME2027 harbouring the ARO4K229L (13) was PCR-amplified using the primers ARO4-A8
(TTGTTAACTTCTCTTCTTTGTCTG)/ARO4-A9 (GAATCTCCAATGTTCGCTGCC) yielding a fragment
starting at nucleotide 7 to 1106 of the ARO4 coding region. The resulting PCR product was transformed in the
aro3∆ aro4∆ double deletion strain CEN.PK557-5A and transformants were selected on SM plates. Sequencing
using primer ARO4-A11 (CGAATCTCAACTGCA CAGAG) proofed the ARO4 point mutation in the obtained
strain CEN.PK718-5A.
The addition of the ura3-52 allele in the aro3∆ ARO4K229L strain CEN.PK718-5A was achieved by crossing
it with the strain CEN.PK113-13D (ura3-52). After tetrad analysis the corresponding segregants were identified
on the respective media (YEPD, YEPD+G418, SCD-ura, SCD+trp+tyr+phe, SCD+trp+tyr, SCD+ura+trp+tyr
and SCD+ura+trp+tyr+phe) and the ARO4K229L point mutation of resulting strain CEN.PK790-2D was
confirmed again by sequencing using primer ARO4-A11.
To express the ARO4 gene under the control of the TPI1 promoter the TPI1promter-loxP-KanMX-loxP cassette
was amplified using primers ARO4-T1 (TGGCATGCCGTTGGCAGCGAACATTGGAGATTCACTCATTCT
AGTTTATGTATGTGTTTTTTGTAG) and ARO4-P2 (GTAACGGTCTCACGGAACACTGTGTAGTTGCAT
TACTGTCGCATAGGCCACTAGTGGATCTG) and plasmid pPK261 that carries theTPI1promoter-loxPKanMX4-loxP integration cassette as template.
80
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
TABLE 1. Saccharomyces cerevisiae strains used in this study.
Strain
Genotype
Reference
CEN.PK122
MATa / MATα prototrophic reference diploid strain
P. Köttera
CEN.PK113-7D
CEN.PK113-5D
CEN.PK113-13D
MATa Prototrophic reference haploid strain, ARO3 ARO4 ARO7
MATa ura3 ARO3 ARO4 ARO7
MATα ura3 ARO3 ARO4 ARO7
P. Köttera
P. Köttera
P. Köttera
CEN.PK532-1A
MATa aro3∆ ARO4
This study
CEN.PK532-1C
MATα aro3∆ ARO4
This study
This study
CEN.PK437-1C
MATα ARO3 aro4∆
CEN.PK557-5A
MATa aro3∆ aro4∆
CEN.PK790-2D
MATa ura3 aro3∆ ARO4
This study
K229L
This study
K229L
CEN.PK718-5A
MATa aro3∆ ARO4
This study
CEN.PK621-1B
CEN.PK661-1C
MATa TPI1p-ARO4
MATa aro3∆ TPI1p-ARO4
This study
This study
IMZ014
MATa ura3 aro3∆ ARO4K229L pUDe003 (2µ URA3 TDH3p-ARO7)
K229L
pUDe004(2µ URA3 TDH3p-ARO7
MATa ura3 aro3∆ ARO4
IMZ016
MATa ura3 ARO3 ARO4 pUDe003 (2µ URA3 TDH3p-ARO7)
IMZ017
MATa ura3 ARO3 ARO4 pUDe004 (2µ URA3 TDH3p-ARO7
a
This study
G141S
IMZ018
)
This study
This study
G141S
)
This study
Institut fur Mikrobiologie der J. W. Goethe Universitat, Frankfurt, Germany.
The resulting PCR product was transformed in the diploid strain CEN.PK122 and transformants were selected
on YPD+G418 plates. After sporulation and tetrad analysis the corresponding segregants were isolated resulting
in strain CEN.PK621-1B. Correct integration of the TPI1promoter-loxP-KanMX-loxP cassette was confirmed by
diagnostic PCR. For the construction of the aro3 TPI1pro-ARO4 strain CEN.PK661-1C, the two strains
CEN.PK532-1C and CEN.PK621-1B were crossed and segregants were isolated showing in tetrad analysis a 2:2
segregation of the kan-marker.
The ARO7 wild type and mutant ARO7G141S open reading frame were PCR-amplified from CEN.PK113-7D
genomic DNA and pME1463 plasmid DNA (33) respectively using primers ARO7-fwd (GGGCTAGCATGG
ATTTCACAAAACCAGAAACTG) and ARO7-rev (GGCTCGACTTACTCTTCCAACCTTCTTAGCAAG)
designed to introduce restriction sites (underlined) for endonuclease NheI, upstream of the ATG and XhoI,
downstream of the stop codon respectively. The PCR product and the 2µm-based expression vector p426GPD
(27) were digested by XbaI and XhoI. The NheI-XhoI PCR fragments were directionally cloned behind the
glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p) into p426GPD resulting in plasmids pUDe003
(WT ARO7) and pUDe004 (ARO7G141S). The ARO7 open reading frame sequences were confirmed by
sequencing. The plasmids pUDe003 and pUDe004 were transformed by the LiAc/SS/PEG method (10) into the
S. cerevisiae CEN.PK 113-5D strain resulting in strain IMZ016 and IMZ17 respectively. Similarly, the
81
Chapter 4
plasmids pUDe003 and pUDe004 were transformed into the S. cerevisiae CEN.PK 790-2D strain resulting in
strains IMZ014 and IMZ18 respectively.
Growth conditions
Shake flask cultures were performed as described previously (39). The synthetic medium (SM, reference)
contained 20 g.l-1 glucose as carbon source and 5 g.l-1 ammonium sulphate as nitrogen source. Where
mentioned, filter-sterilized L-tryptophan was added to a concentration of 50 mg.l-1. To test growth inhibition by
L-tyrosine, 24 h SM-shake flask cultures were streaked out on SM-agar plates with 2% glucose and L-tyrosine
concentrations ranging from 0 to 1 mM. After inoculation, the plates were incubated at 30oC for 52 h.
Steady-state chemostat cultures were grown in Applikon laboratory fermenter of 1-liter working volume
(Applikon, Schiedam, The Netherlands), as described in detail elsewhere (38). The cultures were fed with a
defined synthetic medium containing glucose as the growth-limiting nutrient (41). The dilution rate (which
equals the specific growth rate) in the steady-state cultures was 0.10 h-1, the temperature was 30°C, and the
culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter.min-1). The
dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe
(Mettler-Toledo, Greifensee, Switzerland), remained above 50% of air saturation.
Culture dry weight
Culture dry weight was determined via filtration as described previously (30).
Extracellular metabolite analysis
Culture supernatants and media were analysed by high-performance liquid chromatography (HPLC), fitted
with an AMINEX HPX-87H ion-exchange column (300 by 7.8 mm; Bio-Rad) mounted in a Waters Alliance
2690 HPLC apparatus, at 60°C using H2SO4 as the mobile phase having a flow rate of 0.6 ml · min–1. Aromatic
amino acid metabolites were analysed by a dual-wavelength absorbance detector (Waters 2487) and integrated
with Chrompack Maitre 2.5 software.
Intracellular metabolite Analysis
30 ml of cell broth was collected at steady state. The cells were spun down at 4oC, 5000 rpm for 5 min. The
pellet was resuspended in 10 ml of boiling water and incubated above 90oC in a thermo-bath for 10 min.
Subsequently, cells were spun down. The supernatant was collected and then evaporated in a Speedvac® under
full vacuum conditions. The sample was resuspended in 300 µl of H2O before further processing.
Phenomenex amino acid kit for GC-MS (Phenomenex Inc, Torrence, CA, USA) was used to measure
intracellular amino acid concentrations. The amino acids were derivatized according the supplier’s
recommendations after a solid phase extraction on a strong cation exchanger to remove proteins and other
interfering sample components; the amino group was alkylated with alkyl chloroformate while the carboxylic
group was simultaneously esterified. The volatile alkoxycarbonyl alkyl esters of amino acids were injected into
82
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
a GC-MS TRACE DSQ™ (Dual-Stage Quadrupole) (ThermoFinnigan, San Jose, CA, USA). The GC
temperature program as proposed by Phenomenex was: initial temp 110oC, ramped to 320oC at 30oC.min-1 (hold
1 min) with a column flow of 1.1 ml.min-1. To compare genotype-dependent concentration profiles for different
amino acids, concentrations were normalized relative to the concentration in the reference strain CEN.PK1137D:
Relative [AAj]strain i = ([AAj]strain i /[AAj]CEN.PK113-7D) x 100 %
This normalized data set was submitted to hierarchical data clustering and the data presented as a heat map
using GeneSpring® software (Silicon Genetics, Redwood City, CA).
Enzyme Assays
Chorismate mutase: Cell extract was prepared as described previously (43) Chorismate mutase (EC 5.4.99.5)
was assayed according to Schmidheini et al. (31) with minor modifications. A 1 ml incubation mix with TrisHCl pH 7.6 (50 mM), dithiothreitol (DTT, 1mM), and EDTA (0.1 mM) was incubated with cell extract (20-200
µl) at 30˚C. At t=0 min, 1 mM (final concentration) of barium chorismate was added. In order to check allosteric
control of the various alleles of ARO7 used in this study, either 0.5 mM of tyrosine or 0.5 mM of tryptophan
were used to test inhibition or activation respectively. For the duration of 5 minutes, a 100 µl sample was taken
every minute, and added to 100 µl of 1 M HCl. These samples were each incubated at 30˚C for 10 minutes, after
which 800 µl of 1 M NaOH was added. Absorbance was measured at 320 nm, an experimentally determined
extinction coefficient of 13.165 mM-1 ·cm-1 was used to calculate the phenylpyruvate concentration.
Blank measurements and controls with prephenic acid or phenylpyruvic acid showed no detectable substrate
decay or chemical decay of the product formed. Specific activities were linearly proportional to the amount of
cell extract added.
DAHP synthase: Biomass samples from chemostat cultures (ca. 3.4 mg dry weight) were washed and resuspended in 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM MgCl2 and 1 mM dithiothreitol
(DTT). Cell extracts were prepared using a French pressure cell operated at 2 kbar. After disruption, cell
extracts were centrifuged as described previously (43). Prior to DAHP-synthase assays, the cell extract was
desalted using Pharmacia PD-10 Sephadex G-25 M columns (9.1 ml bed volume, 5 cm bed height). The DAHP
synthase assay was modified from (35). The assay mixture of 1 ml contained 100 mM KPB buffer, 0.5 mM PEP
and E-4P and the reaction was started with the addition of cell extract (volume). After 5, 10, 15 minutes of
reaction times, 200 µl of the reaction mixture was transferred to 280 µl of 2.85 % w/v trichloroacetic acid
solution. The resulting mixture was centrifuged for 5 min in an eppendorf table centrifuge to remove proteins.
125 µl of the supernatant was removed into eppendorf tubes for assaying DAHP. DAHP (0.01 to 0.05 µmole in
0.125 ml of solution) was treated with 0.125 ml of periodic acid having a final concentration of 2 mM in 0.125
N H2SO4. This was then left in room temperature for 45 minutes. 0.25 ml of 1.33 % (w/v) sodium arsenite in 0.5
N HCl was added to destroy excess periodate (2 min at room temperature). 1 ml of thiobarbituric acid (2%, w/v,
pH 2.0) was added to the tubes, which were subsequently placed in a boiling-water bath for 5 min. The mixture
83
Chapter 4
was cooled in a water bath for 10 min at 40oC and the pink colour developed was measured immediately at 549
nm in an Amersham Pharmacia Novaspec II spectrophotometer against a blank run with water under the same
conditions. One µmole of DAHP gives an OD549 of 13.6 in the above assay.
Protein Determination
Protein concentrations in cell extracts were determined by the Lowry method (25). Bovine serum albumin
(fatty acid free; Sigma, St. Louis, Mo.) was used as a standard.
RESULTS
Elimination of allosteric control on DAHP synthase and chorismate mutase
To confirm that the expression of the ARO4 K229L and ARO7G141S alleles resulted in the elimination of
allosteric control on DAHP synthase and chorismate mutase, respectively, plate assays and in vitro enzyme
activity measurements were performed. Plate assays in the presence of 1 mM tyrosine completely inhibited
growth of aro3∆ strain CEN.PK532-1A (Fig. 2), thus confirming the tyrosine-feedback sensitivity of the Aro4pencoded DAHP synthase in the CEN.PK strain background. This tyrosine sensitivity could not be overcome by
overexpression of the wild-type ARO4 allele (strain CEN.PK666-1C, Fig. 2). However, introduction of the
ARO4 K229L allele completely eliminated the tyrosine sensitivity (Fig. 2), thus confirming the lack of tyrosine
sensitivity of the DAHP synthase encoded by this mutant allele (13). Allosteric control of DAHP synthase in
the strains used in this study was further analysed by measuring enzyme activities in cell extracts, prepared
from glucose-limited chemostat cultures. Although levels of DAHP synthase activity in cell extracts varied
among replicate experiments, they confirmed the complete absence of tyrosine inhibition in the strains carrying
the ARO4 K229L allele (data not shown).
Allosteric control of chorismate mutase was analysed in cell extracts of glucose-limited chemostat cultures.
In strains carrying a single chromosomal copy of the wild-type ARO7 allele, activities of chorismate mutase
were close to the detection limit (Fig. 3), which made it difficult to quantify allosteric effects of tyrosine and
tryptophan, the two known allosteric regulators of Aro7p (31, 32). Overexpression of the wild type ARO7 allele
led to an over 50-fold increase of chorismate mutase activities relative to reference strains carrying a single
chromosomal copy and confirmed both activation by tryptophan and inhibition by tyrosine (Fig. 3). Finally,
overexpression of the ARO7G141S allele led to the complete elimination of both tryptophan activation and
tyrosine inhibition. Although the chorismate mutase activities in strains overexpressing the ARO7G141S allele
were lower than those in strains overexpressing the wild type ARO7 allele, they were still over ten-fold higher
than in strains that carried a single chromosomal copy of ARO7 (Fig. 3). Plate assays showed that feedback
inhibition of Aro7p by tyrosine was less critical than for Aro4p DAHP, as no growth defect was recorded for
strains IMZ014 and IMZ016. This suggests that the inhibition constant of Aro7p for tyrosine is lower than the
Ki reported for Aro4p (Ki = 0.9 µM tyrosine) (31).
84
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
1
1
8
8
2
2
7
7
1 3
3
6
6
4
4
0.1 mM
0 mM
5
0.5 mM
1
5
1
1 mM
8
8
2
2
7
7
3
3
6
6
4
5
4
5
FIG. 2. Growth inhibition by L-tyrosine of the reference S. cerevisiae strain CEN.PK113-7D and strains engineered in key
steps of aromatic amino acid biosynthesis. The yeast strains were streak on SM medium with 2 % glucose containing either 0
(left top panel), 0.1 mM (right top panel), 0.5 mM (left bottom panel) or 1 mM (right bottom panel) L-tyrosine. Plates were
incubated at 30°C for 52 h. 1: CEN.PK 113-7D (prototrophic reference strain) 2: CEN.PK532-1A (aro3∆ ARO4) 3:
CEN.PK666-1C (aro3∆ TPI1p-ARO4) 4: CEN.PK718-5A (aro3∆ ARO4K229L ) 5: IMZ014 (aro3∆ ARO4K229L TDH3-ARO7)
6: IMZ018 (aro3∆ ARO4K229L TDH3- ARO7G141S ) 7: IMZ016 (TDH3-ARO7) 8: IMZ017 (TDH3- ARO7G141S)
85
Chapter 4
CEN.PK 113-7D
IMZ016
ARO7↑
CEN.PK 718-5A
ARO4 K229L
IMZ014
ARO4 K229L ARO7↑
+ Tyrosine 0.5 mM
IMZ017
ARO7 G141S↑
Tryptophan 0.5 mM
Chorismate
+ Tyrosine 0.5 mM
IMZ018
ARO4 K229L -ARO7 G141S↑
Tryptophan 0.5 mM
Chorismate
0.00
0.25
0.50 1
10
µmol.min-1.mg protein-1
20 0.00
0.25
0.50 1
10
µmol.min-1.mg protein-1
20
0.00
0.25
0.50 1
10
20
µmol.min-1.mg protein-1
FIG. 3. Activities of chorismate mutase in the presence and absence of L-tryptophan and L-tyrosine in the reference S.
cerevisiae strain CEN.PK113-7D and in strains engineered in key steps of aromatic amino acid biosynthesis. Enzyme
activities were assayed in cell extracts of aerobic, glucose-limited chemostat cultures. Data represent the average ± standard
deviation of results from two independent chemostat cultures.
Partial tryptophan auxotrophy of strains overexpressing different ARO7 alleles
It has previously been reported that overexpression of ARO7 leads to tryptophan auxotrophy by
preferentially channelling chorismate towards phenylalanine and tyrosine biosynthesis (20). Although CEN.PK
strains that overexpressed the wild-type ARO7 allele (strains IMZ016, IMZ014, Table 1) exhibited a reduction
of their specific growth rate, this could not be restored by adding tryptophan (Table 2). Strains overexpressing
the tryptophan-insensitive allele ARO7G141S (IMZ017, IMZ018) (33) grew even slower than strains
overexpressing the wild-type ARO7 allele (Table 2). In this case, a partial restoration of growth was observed
upon the addition of tryptophan. This partial tryptophan auxotrophy was most pronounced in the strain that
expressed both ARO4 K229L and ARO7G141S (Table 2). Even in the latter strain, the specific growth rate in the
absence of tryptophan was 0.15 h-1. This enabled us to quantitatively analyse product formation by the
engineered strains in chemostat cultures grown at a dilution rate of 0.10 h-1 without the need for tryptophan
supplementation.
Analysis of extracellular product formation in chemostat cultures
The impact of the expression of the ARO4 K229L and the overexpression of the ARO7G141S alleles on biomass
yields and on the production of extracellular products was analysed in aerobic, glucose-limited chemostat
cultures grown at a dilution rate of 0.10 h-1. In all of the chemostat cultures, the extracellular concentrations of
86
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
phenylalanine and tyrosine were below the detection limit of the analysis method (10 µM for both amino acids).
However, significant concentrations of the fusel alcohols phenylethanol and p-hydroxyphenylethanol, as well as
of the corresponding organic acids were detected in several of the cultures (Table 3). The expression of the
ARO4 K229L allele resulted in an over 50-fold increase of the total extracellular concentration of these compounds
relative to the isogenic reference strain CEN.PK113-7D, in which concentrations of these compounds were
close to or below the detection limit (Table 3). Overexpression of the ARO7G141S allele did not by itself have a
significant impact on the production of the aromatic fusel alcohol and corresponding acids (Table 3). When both
the ARO4 K229L and ARO7G141S alleles were expressed, the resulting extracellular concentrations of aromatic
metabolites were only marginally higher than in the strain that expresses only the ARO4 K229L allele (Table 3).
The strains that combined the expression constructs for ARO4 K229L, ARO7 or ARO7G141S all exhibited a ca. 10
% lower biomass yield than the isogenic reference strain CEN.PK113-7D (Table 3). This reduction of the
biomass yield was not clearly correlated to the concentrations of aromatic metabolites in the cultures and may
therefore reflect an effect of protein burden (44) rather than a specific metabolic effect related to aromatic amino
acid metabolism.
TABLE 2. Specific growth rate µ (h-1) determination of chorismate-mutase-overexpressing strains mutants in presence and
absence of 50 mg.l-1 tryptophan in SM medium with 2% glucose. Data are presented as average ± mean deviation of
metabolite quantification from two independent shake flask cultures.
Maximum specific growth rate
µmax (h-1)
Strain
Genotype
-Trp
+Trp
CEN.PK113-7D
ARO3 ARO4
0.46 ± 0.01
0.45 ± 0.01
IMZ016
ARO3 ARO4 TDH3p-ARO7
0.25 ± 0.01
0.26 ± 0.01
IMZ017
ARO3 ARO4 TDH3p-ARO7G141S
0.22 ± 0.01
0.27 ± 0.00
IMZ014
aro3∆ ARO4K229LTDH3p-ARO7
0.31 ± 0.01
0.33 ± 0.01
IMZ018
aro3∆ ARO4K229LTDH3p-ARO7G141S
0.15 ± 0.00
0.39 ± 0.01
Intracellular amino acid concentrations in chemostat cultures
As the absence of significant extracellular concentrations of aromatic amino acids may reflect a limitation in
their export from the cells, intracellular amino acid concentrations were analysed in the reference and
engineered strains. The Expression of the ARO4 K229L allele led to a circa 3-fold increase of the intracellular
concentrations of phenylalanine and tyrosine (Fig. 4). As observed for the extracellular aromatic metabolites, the
simultaneous overexpression of the ARO7G141S allele did not cause a clear further increase of the intracellular
phenylalanine and tyrosine concentrations. However, in contrast to the effects observed for the extracellular
87
88
aro3∆ ARO4
IMZ018
3.27 ± 0.03
3.45 ± 0.03
205 ± 5
170 ± 20
<2
<2
220 ± 20
210 ± 0
220 ± 60
<2
<2
110 ± 10
200 ± 0
160 ± 50
<2
<2
150 ± 10
<2
(µM)
p-hydroxyphenylethanol
<2
<2
<2
<2
17 ± 2
<2
(µM)
p-hydroxyphenylacetate
615
<8
<8
550
497
< 10
(µM)
[metabolites]
Σa
corresponds to the sum of the phenylethanol, phenylacetate, p-hydroxyphenylethanol and p-hydroxyphenylacetate concentration measured in each
culture
K229L
TDH3pr-ARO7
aro3∆ ARO4K229LTDH3pr-ARO7
IMZ014
a
3.43 ± 0.14
ARO3 ARO4 TDH3pr-ARO7
ARO3 ARO4 TDH3pr-ARO7G141S
G141S
3.35 ± 0.05
3.33 ± 0.03
aro3∆ ARO4
IMZ016
IMZ017
3±0
3±0
CENPK718-5A
K229L
(µM)
(µM)
3.60 ± 0.02
ARO3 ARO4
CEN.PK 113-7D
g (g glucose)-1
Genotype
Strain
Phenylacetate
Phenylethanol
Biomass yield
TABLE 3. Biomass yields on glucose and extracellular concentrations of fusel alcohols and corresponding organic acids in aerobic, glucose-limited
chemostat cultures (D = 0.10 h-1) of different S. cerevisiae strains. Data are presented as average ± mean deviation of results from two independent
chemostat cultures for each strain.
Chapter 4
89
ARO3 ARO4 TDH3pr-ARO7
IMZ016
aro3 ∆ ARO4
aro3∆ -ARO4
IMZ014
IMZ018
TDH3pr-ARO7
K229L
K229L
TDH3pr-ARO7
ARO3 ARO4 TDH3pr-ARO7
IMZ017
G141S
0.019
0.016
<0.001
<0.001
0.014
<0.001
Aromatic
metabolitesa
0.024
0.024
0.024
0.024
0.024
0.024
Phe and Tyr
in proteinb
0.004
0.004
0.002
0.002
0.004
0.001
Phe and Tyr
intracellularc
Fluxes (mmol.g-1.h-1)
0.047
0.044
0.026
0.026
0.042
0.025
Total
188
176
104
104
168
100
Normalized
flux
%
sum of the phenylethanol, phenylacetate, p-hydroxyphenylethanol and p-hydroxyphenylacetate concentrations measured multiplied by the
dilution rate and divided by the biomass concentration in each culture
b
concentrations of phenylalanine and tyrosine in protein were calculated from published data on protein content and amino acid composition of S.
cerevisiae (22, 29), multiplied the dilution rate and divided by the biomass concentration in each culture
c
sum of the free intracellular phenylalanine and tyrosine concentrations in the cultures were calculated based on an intracellular volume of 2 ml.g
biomass-1 (23), multiplied by the dilution rate and divided by the biomass concentration in each culture
a
aro3∆ ARO4
CENPK718-5A
G141S
ARO3 ARO4
CEN.PK 113-7D
K229L
Genotype
Strain
TABLE 4. Specific fluxes through the aromatic amino acid biosynthesis pathway after the chorismate branch. Total fluxes were calculated by
combining (i) specific rates of production of aromatic metabolites (fusel alcohols and acids) derived from phenylalanine and tyrosine, (ii) specific
rates of production of phenylalanine and tyrosine in cellular protein and (iii) specific rates of production of intracellular free phenylalanine and
tyrosine.
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
Chapter 4
metabolites, the individual overexpression of either the wild type ARO7 allele or the ARO7G141S alleles caused a
significant, ca. 50 % increase of the intracellular concentrations of phenylalanine and tyrosine relative to the
reference strain CEN.PK113-7D.
Intracellular concentrations of tryptophan were low in the reference strain CEN.PK113-7D (ca. 0.2 mM, Fig.
4). Elimination of feedback inhibition on DAHP synthase caused a ca. 3-fold increase of the intracellular
tryptophan concentration. Interestingly, this increase was still present when a tryptophan- and tyrosine
insensitive chorismate mutase (encoded by ARO7G141S) was simultaneously overexpressed. Conversely,
overexpression of the wild-type ARO7 allele reduced the intracellular tryptophan in a strain expressing a
feedback insensitive DAHP synthase to the level observed in the reference strain. This difference may be related
to the observation that the maximum chorismate mutase activity in strains overexpressing the wild-type ARO7
allele (measured in the presence of the activator tryptophan) was higher than those in strains overexpressing the
tryptophan-insensitive ARO7G141S allele (Fig. 3).
The effects of the expression of the ARO4 K229L and overexpression of ARO7G141S alleles on intracellular
amino acids were not confined to the aromatic amino acids (Fig. 4). Based on their intracellular concentration
profiles, two classes of non-aromatic amino acids could be identified. A first group of amino acids did not show
a clear correlation with the genotype of the strains and included valine, alanine, glycine, leucine, serine,
glutamate, isoleucine, proline and aspartate (Fig. 4). Concentration changes between the different strains were
below 50 % for this group of ‘non-responsive’ amino acids. Interestingly, a second class of amino acids was
responsive to the genotype of the strains. This class included asparagine, histidine, ornithine, threonine, lysine
and methionine, which consistently showed a genotype-dependent concentration profile that closely mirrored
that of phenylalanine and tyrosine (Fig. 4). This result demonstrates that deregulation of the amino acid
biosynthesis pathway has more widespread repercussions on amino acid biosynthesis.
Quantification of metabolic fluxes through the phenylalanine-tyrosine branch of aromatic amino acid
biosynthesis
In order to quantify the overall effect of the expression of the ARO4K229L and ARO7G141S alleles on the
metabolic fluxes through the phenylalanine-tyrosine branch of amino acids (i.e. excluding the flux towards
tryptophan and related metabolites), three components need to be taken into account:
(i) The specific production rates of the aromatic metabolites phenylethanol, phenylacetate and their phydroxy derivatives. In calculating the specific production rates mentioned in Table 4, it was assumed
that these metabolites do not accumulate inside the cells.
(ii) The specific production rate of phenylalanine and tyrosine incorporated into cellular protein. Based on
the assumption that the protein content in all strains is identical to that of the reference strain
CEN.PK113-7D grown at a dilution rate of 0.10 h-1 (22) and that the amino acid composition of the
cellular protein is equal to that reported by Oura (1972) (28), this rate is identical for all strains (Table
4).
90
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
(iii) The specific production rates of free intracellular phenylalanine and tyrosine, which can be calculated
from the data presented in Fig. 4 by assuming an intracellular volume of 2 ml (g biomass)-1 (Table 4).
4.2 ± 0.3
5.2 ± 0.5
3.5 ± 0.1
4.3 ± 0.5
3.8 ± 0.3
19.8 ± 0.6
20.3 ± 1.4
20.6 ± 1.4
15.8 ± 0.2
19.2 ± 0.2
18.5 ± 0.5
2.4 ± 0.1
1.9 ± 0.3
1.9 ± 0.3
1.9 ± 0.1
2.3 ± 0.5
2.1 ± 0.0
1.6 ± 0.1
1.6 ± 0.1
1.3 ± 0.1
1.6 ± 0.1
1.7 ± 0.5
1.8 ± 0.1
1.8 ± 0.1
1.5 ± 0.1
1.8 ± 0.1
1.7 ± 0.1
1.9 ± 0.5
1.7 ± 0.1
69.8 ± 2.1
66.9 ± 1.1
74.0 ± 6.6
70.9 ± 2.0
79.2 ± 1.6
64.6 ± 3.2
1.36 ± 0.0
1.4 ± 0.1
1.4 ± 0.1
15.8 ± 0.2
1.4 ± 0.0
1.4 ± 0.2
3.0 ± 0.1
2.2 ± 0.1
2.2 ± 0.1
3.4 ± 0.1
3.2 ± 0.2
2.8 ± 0.1
0.2 ± 0.0
0.1 ± 0.0
0.6 ± 0.0
0.5 ± 0.0
0.2 ± 0.0
5.4 ± 0.9
6.7 ± 0.1
8.1 ± 0.6
7.0 ± 0.2
4.6± 0.2
2.8 ± 0.1
2.9 ± 0.1
3.6 ± 0.3
3.3 ± 0.2
4.0 ± 0.1
4.6 ± 0.0
1.2 ± 0.0
1.9 ± 0.1
3.0 ± 0.3
5.5 ± 0.1
3.1 ± 0.3
3.0 ± 0.2
1.4 ± 0.0
2.0 ± 0.2
3.52 ± 0.3
4.8 ± 0.1
4.0 ± 0.0
3.6 ± 1.0
21.1 ± 0.6
32.1 ± 0.6
32.2 ± 0.6
57.7 ± 1.7
67.5 ± 0.2
66.5 ± 1.0
0.7 ± 0.0
1.0 ± 0.1
1.3 ± 0.2
2.3 ± 0.1
2.8 ± 0.2
2.4 ± 0.2
2.1 ± 0.1
2.3 ± 0.0
3.2 ± 0.2
2.74 ± 0.1
3.1 ± 0.2
2.7 ± 0.1
1.7 ± 0.1
2.6 ± 0.1
6.2 ± 0.5
5.5 ± 0.1
3.0± 0.5
3.0 ± 0.3
0.1 ± 0.0
0.2 ± 0.0
0.2 ± 0.0
0.2 ± 0.0
0.2 ± 0.1
0.3 ± 0.0
5
3
1
0.5
0
O
7½
41
S
IM
AR Z 0
O 14
4K
22
9L
½
AR
O
7 G1
½
AR
K2
29
L
½
C
AR EN
O .PK
4K 1
22 1
9L 8½ 5A
IM
Z
AR 0
O 18
4
14
1S
½
IM
AR Z 0
O 17
7G
IM
AR Z0
O 16
7
CE
N.
PK
11
37D
½
0.2 ± 0.0
6.0 ± 0.2
Val
Ala
Gly
Leu
Ser
Glu
Ile
Pro
Trp
Asp
Asn
His
Orn
Phe
Tyr
Thr
Lys
Met
Normalised concentration
5.6 ± 0.2
FIG. 4. Intracellular amino acid concentrations in the reference S. cerevisiae strain CEN.PK113-7D and in strains
engineered in key steps of aromatic amino acid biosynthesis. Numbers indicated the average ± mean deviation of results
from two independent chemostat cultures. Colors represent a concentration heat map normalized for the concentration of
each amino acid in cultures of the reference strain CEN.PK113-7D.
In the reference strain CEN.PK113-7D, the flux towards phenylalanine and tyrosine was virtually
completely incorporated into cellular protein. In the strains that expressed the ARO4 K229L allele, the overall flux
through this branch of aromatic amino acid metabolism increased by almost two-fold (Table 4). This was
91
Chapter 4
primarily due to the increased synthesis of the fusel alcohols and corresponding acids. The increased
concentrations of free intracellular phenylalanine and tyrosine had little impact on the overall fluxes.
DISCUSSION
This study presents a quantitative analysis of the impact of allosteric control of two key enzymes in aromatic
amino acid metabolism on product formation by S. cerevisiae. The results demonstrate that DAHP synthase
exerts a strong degree of control on the synthesis of aromatic compounds by S. cerevisiae. This result is not
surprising: it has previously been demonstrated that non-defined feedback insensitive mutants of S. cerevisiae
obtained by ethyl methyl-sulfonate mutagenesis and screening on phenylalanine analogues produce increased
levels of phenylethanol (8, 9). Moreover, elimination of feedback regulation is a classical first step in improving
amino acid production by prokaryotes. In stark contrast with the situation in bacteria such as E. coli and C.
glutamicum (1, 23, 24), in S.cerevisiae the alleviation of feed-back inhibition on DAHP synthase did not lead to
measurable extracellular concentrations of phenylalanine or tyrosine. Apparently, even in situations where the
intracellular concentration of phenylalanine reaches levels of over 60 mM (Fig. 4), export across the plasma
membrane does not occur. So far the only system reported to catalyse the export of amino acids from
S.cerevisiae cells involved the AQR1 gene product (40), which mediates the export of several amino acids via an
exocytosis-like mechanisms. However, phenylalanine and tyrosine are not among the reported substrates for this
export system. If S. cerevisiae is ever to be considered for the production of amino acids, engineering of product
export is clearly a priority target. The steep gradient of phenylalanine that exists across the plasma membrane of
S. cerevisiae strains that express the ARO4K229L and ARO7G141S alleles make them ideally suited as a model
system to explore strategies for introducing amino acid export into S. cerevisiae.
In S. cerevisiae, chorismate mutase (Aro7p) is known to be subject to allosteric regulation by tyrosine
(inhibition) and tryptophan (activation). Our hypothesis that, especially in strains expressing a feed-back
insensitive DAHP synthase, this allosteric regulation would have a strong effect on aromatic amino acid
biosynthesis was not borne out by the experimental results. In fact, the product concentrations and specific
aromatic product formation rates observed in strains with a combined expression of the feed-back insensitive
ARO4K229L and the non-allosterically controlled ARO7G141S alleles showed only small differences in the
formation of intracellular and extracellular aromatic products (Fig. 4, Table 3). This indicates that, in strains
with a deregulated DAHP synthase, control of the synthesis of aromatic compounds resides elsewhere. Since we
have not analysed levels of all intermediates of the aromatic amino acid biosynthesis pathway, a discussion on
the actual rate-controlling step(s) in these strains is by necessity speculative. Still, three possibilities may be of
special interest in further work.
Firstly in the strains that express the ARO4K229L allele the activity of DAHP synthase may exert a high degree
of flux control. In that case, overexpression of ARO4K229L might lead to increased fluxes toward aromatic
metabolites (Table 4). Secondly, formation of these compounds requires the conversion of phenylpyruvate and
p-hydroxylphenylpyruvate, the penultimate compounds in phenylalanine and tyrosine biosynthesis, respectively,
92
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
by a TPP-dependent decarboxylase activity (4, 42, 43). Measurements of the activity of this decarboxylase
activity in cell extracts of the chemostat cultures of the wild-type and engineered strains yielded only very low
activities of this key enzyme activity. We are currently attempting to overexpress phenylpyruvate decarboxylase
in S. cerevisiae. However, these studies are complicated by the fact that the molecular nature of this
decarboxylase activity is not yet fully understood (42). A final possibility involves a reaction outside the main
pathway for synthesis of aromatic amino acids. The pentose phosphate pathway is essential for aromatic amino
acid biosynthesis as it provides erythrose-4-phosphate, an essential precursor for the shikimate pathway (Fig. 1).
It has been demonstrated that transketolase, the enzyme that catalyses erythrose-4-phosphate formation, is
inhibited by p-hydroxyphenylpyruvate, the penultimate intermediate in tyrosine biosynthesis (34). Although no
p-hydroxyphenylpyruvate-insensitive mutant forms of yeast transketolase have been described, it may be of
interest to exchange the yeast TKL1 / TKL2 genes for a heterologous transketolase gene.
The deregulation of the aromatic amino acid biosynthesis pathway had a strong impact on the intracellular
concentrations of several non-aromatic amino acids (Fig. 4). We are not aware of regulatory networks that might
account for the observation that intracellular concentrations of asparagine, histidine, ornithine, threonine, lysine
and methionine all showed genotype-dependent concentration profiles similar to those of phenylalanine and
tyrosine (Fig. 4). Involvement of transaminases with overlapping substrate specificities in the synthesis of these
amino acids may contribute to this phenomenon by connecting a number of intracellular amino acid 2-oxo acid
equilibrium reactions. For example, it is known that the aromatic amino transferase encoded by ARO8 can also
transaminate methionine (19, 37). However, a complete overview of the substrate specificities of the S.
cerevisiae transaminases is currently not available. The perturbance of non-aromatic amino acid biosynthesis
pathways upon the deregulation of the shikimate pathway represents an important challenge for further research
on metabolic engineering of aromatic metabolism in S. cerevisiae.
ACKNOWLEDGEMENTS
This work was financially supported by the Dutch government (CW-NWO program "Transition towards
Sustainable Technology") and the Kluyver Center for Genomics of Industrial Fermentation. Z.V. was financially
supported by DSM. We thank Pascale Daran-Lapujade and the DSM discussion group for helpful comments on
the manuscript. The research group of J.T.P. is part of the Kluyver Centre for Genomics of Industrial
Fermentation, which is supported by The Netherlands Genomics Initiative. G. H. B. was supported by the
Deutsche Forschungsgemeinschaft.
93
Chapter 4
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Van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. Van Dijken, J. T. Pronk, and H. Y.
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Saccharomyces cerevisiae, yielding a C(2)-independent, glucose-tolerant, and pyruvate-hyperproducing
yeast. Appl. Environ. Microbiol. 70:159-166.
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Vuralhan, Z., M. A. Luttik, S. L. Tai, V. M. Boer, M. A. Morais, D. Schipper, M. J. Almering, P.
Kötter, J. R. Dickinson, J. M. Daran, and J. T. Pronk. 2005. Physiological characterization of the
ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces
cerevisiae. App. Environ. Microbiol. 71:3276-3284.
Vuralhan, Z., M. A. Morais, S. L. Tai, M. D. Piper and J. T. Pronk. 2003. Identification and
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Microbiol. 69:4534-4541.
96
Alleviation of Feedback Inhibition in Saccharomyces cerevisiae: Quantification of Metabolic Impact
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97
SUMMARY
Saccharomyces cerevisiae is a popular industrial microorganism. It has since long been used in bread, beer
and wine making. More recently it is also being applied for heterologous protein production and as a target
organism for metabolic engineering. The work presented in this thesis describes how S. cerevisiae may be used
as a metabolic-engineering platform to produce aromatic compounds such as phenylalanine or its catabolites,
phenylethanol and phenylacetate.
S. cerevisiae is capable of using many amino acids as sole nitrogen source via deamination or
transamination. In case of transamination the amino group may be transferred to a variety of (acceptor) keto
acids, which then become amino acids. In this process the nitrogen-donating amino acid becomes a 2-oxo-acid
itself. When this 2-oxo-acid cannot be further converted into cell constituents, it is catabolized via the Ehrlich
pathway: upon transamination of the amino acid to the corresponding 2-oxo-acid, the latter compound is
decarboxylated to the corresponding aldehyde. The redox state of the cells (i.e. the availability of oxygen)
determines the fate of the aldehyde: it can be reduced by alcohol dehydrogenases (yielding a group of
compounds commonly referred to as fusel alcohols) or be oxidized to the corresponding organic acid (“fusel
acids”) by aldehyde dehydrogenases (Chapter 1). In the case of nitrogen assimilation from phenylalanine, the
product of phenylalanine transamination, phenylpyruvic acid, is decarboxylated to phenylacetaldehyde which, in
turn, is reduced to phenylethanol or oxidized to phenylacetic acid.
Chapter 2 of this thesis focuses on the identification of genes that code for phenylpyruvate decarboxylase.
According to the literature, S. cerevisiae has five candidate genes that may encode this activity. These are
PDC1, PDC5, PDC6 and ARO10, and THI3.
In this Chapter it is reported that phenylpyruvate decarboxylase activity is present in cells from glucoselimited chemostat cultures of S. cerevisiae CEN.PK 113-7D, growing with phenylalanine as N-source. This
activity could not be detected in cultures grown with ammonia as N-source. Transcriptome analysis
demonstrated that ARO10 transcript levels increased 30-fold when phenylalanine replaced ammonia as Nsource. Metabolite profile analysis and enzyme activity measurements on cells from chemostat cultures
indicated that ARO10 alone is sufficient for phenylpyruvate decarboxylase activity when other candidate genes
are absent. However, extracts of cells lacking Aro10p (PDC1 PDC5 PDC6 YDL080c aro10∆) still exhibited
phenylpyruvate decarboxylase activity. Therefore, it seems that one or more of the other candidate genes can
replace ARO10. One of the candidate genes; THI3 (YDL080c) seemed to require one of the other decarboxylase
genes (PDC1, PDC5, PDC6) for phenylpyruvate decarboxylase activity. In vivo Aro10p is probably the relevant
enzyme in phenylalanine catabolism. This conclusion is based on an analysis of the kinetic parameters of
phenylpyruvate decarboxylase activity in cells with or without Aro10p.
Summary
Although ARO10 is not the only gene encoding 2-oxoacid decarboxylase activity, it is crucial to the
formation of fusel alcohols and acids (Chapter 3). In aerobic glucose- limited chemostat cultures growing with
ammonia, proline, asparagine, leucine, methionine or phenylalanine as N-source, only with the latter three
amino acids formation of fusel alcohols and acids was observed. Transcriptome analysis revealed that under the
latter three growth conditions ARO10 was up-regulated, whereas the expression levels of the other four
candidate genes, encoding putative thiamine pyrophosphate-dependent decarboxylases, remained unchanged.
Measurements of decarboxylase activity in cell-free extracts confirmed that only when the yeast was grown with
phenylalanine, leucine or methionine as nitrogen source, phenylpyruvate decarboxylase activity was present.
Not only phenylpyruvate, but also α-ketoisovalerate, α-ketoisocaproate, α-ketomethylvalerate and 3-methylthio-α-ketobutyrate served as substrate for the decarboxylase activity. In a decarboxylase-negative background
(pdc1, pdc5, pdc6, thi3), overexpression of ARO10 resulted in decarboxylase activities with the 2-oxoacids
derived from phenylalanine, namely leucine and methionine. The ratio between these activities in the mutant
overexpressing ARO10 was the same as in the wild type. Therefore, Aro10p is probably a broad-substrate
decarboxylase. This enzyme is subject to posttranscriptional regulation. In spite of constitutive expression of
ARO10 under the control of the TDH3 promotor, enzyme levels were strongly affected by the nature of the
carbon and nitrogen sources for growth.
Microbial production of aromatic amino acids requires insight into the regulation of the synthesis of these
compounds and in the mechanism and regulation of (unwanted) catabolism to fusel alcohols and acids, as well
as into the mechanism of product export.
In the last chapter of this thesis initial attempts are described to overproduce phenylalanine and tyrosine with
S. cerevisiae. To this end the combination of deregulated DAHP synthase and chorismate mutase was
investigated. The introduction of a tyrosine-insensitive allele of ARO4 led to a three-fold increase in intracellular
levels of phenylalanine and tyrosine. However, these amino acids were not excreted into the culture broth. This
probably results from the absence of an adequate export system. On the other hand, extracellular concentrations
of phenylacetic acid, phenyl ethanol and their para-hydroxy-analogues increased fifty-fold, thus confirming the
necessity of preventing amino acid catabolism in a production process for aromatic amino acids.
Production of aromatic amino acids by S. cerevisiae will influence other parts of the metabolic network. For
example, ARO7 overexpression combined with the tyrosine-insensitive ARO4 allele did not only affect the
intracellular levels of aromatic amino acids, but also resulted in enhanced levels of other amino acids. Thus,
changing the concentration of non-aromatic amino acids, regulated via the shikimate pathway, might provide
new targets for engineering of the metabolism of aromatic compounds in S. cerevisiae.
100
SAMENVATTING
Saccharomyces cerevisiae wordt veel gebruikt voor industriële toepassingen. Deze gist wordt reeds lang
benut voor de bereiding van brood, bier en wijn. Meer recente toepassingen zijn productie van heterologe
eiwitten en gebruik van de gist voor metabolic engineering. Dit proefschrift beschrijft hoe S. cerevisiae kan
worden gebruikt als metabolic engineering platform voor de productie van aromatische verbindingen zoals
fenylalanine, of de afbraakproducten daarvan: fenylalcohol en fenylazijnzuur.
S. cerevisiae is in staat om vele aminozuren te gebruiken als enige stikstofbron, via deaminering of
transaminering. In het geval van transaminering kan de aminogroep aan diverse ketozuren overgedragen
worden. Deze ketozuren worden dan de overeenkomstige aminozuren. Het aminozuur dat de aminogroep afstaat
wordt daarbij zelf een ketozuur. Wanneer dit ketozuur niet verder omgezet kan worden tot celbestanddelen,
wordt dit gedeeltelijk afgebroken via de Ehrlich route: na transaminering van het aminozuur tot het
corresponderende ketozuur, wordt deze verbinding gedecarboxyleerd tot het overeenkomstige aldehyde. De
redox-status van de cel (de aan of afwezigheid van zuurstof) bepaalt de vervolgreactie. Het aldehyde kan
gereduceerd worden door alcohol dehydrogenasen (tot zogenaamde foezelalcoholen) of geoxideerd worden door
aldehyde dehydrogenasen tot het overeenkomstige zuur ( foezelzuren) (Hoofdstuk 1).
In het geval van fenylalanine wordt het transaminerings product, fenylpyruvaat, gedecarboxyleerd tot
fenylacetaldehyde dat vervolgens gereduceerd wordt tot fenylalcohol of geoxideerd tot fenylazijnzuur.
Hoofdstuk 2 van dit proefschrift richt zich op de genen die coderen voor fenylpyruvaat decarboxylase . In de
literatuur is beschreven dat vijf genen kunnen coderen voor deze activiteit te weten PDC1, PDC5, PDC6,
ARO10 en THI3.
Hoofdstuk 2 laat zien dat fenylpyruvaat decarboxylase activiteit aanwezig is in S. cerevisiae CEN.PK1137D, wanneer de gist gekweekt wordt in glucose- gelimiteerde chemostaat cultures met fenylalanine als
stikstofbron. Deze activiteit is afwezig wanneer ammonium als stikstof dient.
Transcriptoomanalyse toonde aan dat het ARO10 transcriptieniveau met een factor 30 toeneemt wanneer, in
plaats van ammonium, fenylalanine als stikstofbron dient. Analyse van extracellulaire metabolieten en metingen
van enzymactiviteiten met cellen van chemostaat cultures gaven aan dat ARO10 op zich voldoende is voor het
genereren van fenylpyruvaat decarboxylase wanneer de andere decarboxylase genen afwezig zijn.
Echter, extracten van cellen zonder Aro10p (PDC1,PDC5, PDC6, YDL080C, aro10∆) vertoonden toch
fenylpyruvaat decarboxylase activiteit. Dit wijst erop dat één of meerdere genen ARO10 kunnen vervangen. Een
van deze genen, THI3 (YDL080C) leek één van de andere genen (PDC1, PDC5, PDC6) nodig te hebben voor
het bewerkstellingen van fenylpyruvaat decarboxylase activiteit. Aro10p is waarschijnlijk het belangrijkste
Samenvatting
enzym in vivo voor de afbraak van fenylalanine. Deze conclusie is gebaseerd op een analyse van de kinetiek van
de fenylpuruvaat decarboxylase activiteit in cellen met en zonder Aro10p.
Hoewel ARO10 niet het enige gen is dat codeert voor 2-ketozuur decarboxylase activiteit is het wel cruciaal
voor de vorming van foezelalcoholen en -zuren. Dit wordt beschreven in hoofdstuk 3. In aërobe glucosegelimiteerde chemostaatcultures gekweekt met ammonium, proline, asparagine, leucine, methionine of
fenylalanine als stikstofbron werd alleen met de laatste drie aminozuren vorming van foezelalcoholen en -zuren
waargenomen. Transcriptoom analyse wees uit dat bij groei met deze stikstofbronnen de transcriptie van
ARO10 toenam, terwijl de expressie van de andere vier genen die coderen voor mogelijke thiamine pyrofosfaat
(TPP)-afhankelijke decarboxylasen niet veranderde. Metingen van decarboxylase-activiteit in celvrije extracten
bevestigde dat alleen bij groei met leucine, methionine of fenylalanine als stikstofbron fenylpuruvaat
decarboxylase activiteit aanwezig was. Niet alleen fenylpyruvaat maar ook α-ketoisovaleraat, α-ketoisocaproaat,
α-ketomethylvaleraat en 3-methylthio-α-ketobutyraat fungeerden als substraat voor de decarboxylase-activiteit.
In een decarboxylase-negatieve achtergrond (pdc1, pdc5, pdc6, thi3) veroorzaakte overexpressie van ARO10
decarboxylase activiteit met de van fenylanaline afgeleide 2-ketozuren , leucine en methionine. De verhouding
tussen de drie decarboxylase-activiteiten in de mutant met ARO10 overexpressie was gelijk aan die in het wild
type. Aro10p is dus waarschijnlijk een decarboxylase met een brede substraatspecificiteit. Dit enzym wordt
waarschijnlijk ook gereguleerd op post-transcriptioneel niveau. Niettegenstaande constitutieve expressie van
ARO10 onder controle van de TDH3 promotor werd toch een sterke invloed van de koolstof- en stikstofbron op
de enzymniveau’s waargenomen.
Microbiële productie van aromatische aminozuren vereist inzicht in de regulatie van de synthese van deze
verbindingen, in mechanisme en regulatie van de vorming van ongewenste bijproducten zoals foezelalcoholen
en –zuren, en ook in het mechanisme van productexport. In het laatste hoofdstuk van dit proefschrift worden
experimenten beschreven die tot doel hadden overproductie van phenylalanine en tyrosine te bewerkstellingen
in S. cerevisiae via een combinatie van gereguleerd DAHP synthase en chorismaat mutase. De introductie van
een tyrosine-ongevoelig allel van ARO4 had een drievoudige verhoging van de intracellulair concentratie van
fenylalanine en tyrosine tot gevolg. Deze aminozuren werden echter niet uitgescheiden, hetgeen waarschijnlijk
z’n oorzaak heeft in de afwezigheid van een adequaat export systeem. Wel werd een 50-voudige verhoging van
de extracellulaire concentraties van fenylalcohol, fenylazijnzuur en hun para-hydroxy-analogen waargenomen.
Deze waarneming bevestigt de noodzaak, vorming van bijprodukten in een productieproces voor
aromatische aminozuren te voorkomen.
Productie van aromatische aminozuren door S. cerevisiae beïnvloedt ook andere delen van het metabole
netwerk. Bijvoorbeeld, overexpressie van ARO7 in combinatie met de tyrosine-ongevoelige ARO4 allel
beïnvloedde niet alleen de intracellulaire concentraties van aromatische aminozuren, maar ook die van andere
aminozuren. Dit fenomeen, gevolg van de regulatie van de shikimaatroute, zal mogelijk nieuwe
aangrijpingspunten opleveren voor het manipuleren van het metabolisme van aromatische aminozuren.
102
PUBLICATIONS
1. Vuralhan Z., M. A. Morais, S. L. Tai, M. D. Piper, and J. T. Pronk. 2003. Identification and
characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae.
App.Environ.Microbiol. 69:4534-4541
2. Vuralhan Z., M. A. Luttik, S. L. Tai, V. M. Boer, M. A. Morais, D. Schipper, M. J. Almering, P.
Kötter, J. R. Dickinson, J. M. Daran, and J. T. Pronk. 2005. Physiological characterization of the ARO10dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.
App.Environ.Microbiol. 71:3276-3284.
3. Vuralhan Z., M. A. Luttik, G.H. Braus, J.M. Daran, and J.T. Pronk. 2005. Alleviation of feedback
inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic
impact. Manuscript in preparation
4. Boer, V. M., S. L. Tai, Z. Vuralhan, Y. Arifin, M. C. Walsh, M. D. Piper, J. H. de Winde, J. M.
Daran, and J.T. Pronk (2005). Transcriptional responses of Saccharomyces cerevisiae to growth on
preferred and non-preferred nitrogen sources in glucose-limited chemostat cultures. Submitted for
publication.
103
Curriculum Vitae
Zeynep Vuralhan obtained her diploma as Food Engineer in July 1995 from the Faculty of Chemical &
Metallurgy Engineering at Istanbul Technical University (ITU). In 1995-1996, she completed an Executive
MBA study on a joint program between Marmara University, Istanbul and the University of Maine-USA,
while working as a junior engineer for an international company in Istanbul. In the same year (1996), she
obtained a scholarship to continue with academical studies in Denmark. In 1999, she received her M.Sc.
degree in Food Science and Technology from the Technical University of Denmark (DTU). Her M.Sc
thesis focused on the molecular biology of lactic acid bacteria. In December 1999, she became research /
teaching assistant at the University of Arkansas, Food Microbiology Department, Fayetteville, USA. In
2000, she returned to Europe by joining Industrial Microbiology Group as a Ph.D. student at the
Department of Biotechnology, TU-Delft where she studied aromatic amino acid metabolism in S.
cerevisiae.
105
ACKNOWLEDGEMENTS
I would like to take the opportunity to thank some people who directly and indirectly contributed to this
thesis.
First of all, I would like to thank my mentor and supervisor: Prof. Dilek Boyacioglu in Food Engineering
Dept. Istanbul Technical University for encouraging me to see what I really wanted to do. Her belief in me
played a very important role in being where I am today. Her professionalism, objectivity, broad vision and
humanism will guide me through all my life. Thank you Prof. Boyacioglu !
Secondly, I owe much to those who gave me this opportunity: my promoters, Prof. J. T. Pronk and Prof. J.
P.van Dijken. Thank you very much. Many thanks will be addressed to Dr. J. M. Daran for close and careful
supervision. I also would like to thank Dr. M. D. Piper who took part in the initial phase of the project as my
supervisor.
A very special thank you goes to Dr. Marcos A. Morais. During our collaboration, his determination and
enthusiasm about TPP-dependent decarboxylases and trying new ideas, experiments resulted in my first
publication. I would like to thank him for not only being a wonderful colleague but also a great friend.
Furthermore, amino acid project members: Prof. H. de Winde, Prof. J. J. Heijnen, Dr. Walter van Gulik, R.
Bovenberg, and Dr. D. Schipper are acknowledged for the fruitful work discussions which took part at DSM or
Kluyver lab. I also want to express my appreciation to Prof. Lex Scheffers for his help in the Dutch translation
and check-up of my summary and also many thanks to Dr. Henk van Urk for the translation of my statements. I
would like to thank Marijke Luttik for helping with the enzyme measurements. My gratitude is addressed to
Arjen van Tuijl and Erik Hulster for teaching me almost everything about fermenters and fermentations. I would
like to thank Angela ten Pierick and Cor Ras for doing metabolite analysis, Ko Vinke for giving wonderful ideas
about phenylalanine fermentations. Thanks to Astrid and Apilena for technical things related to medium kitchen
and also for being wonderful friends. Arno and his team from workshop: Thank you very much for solving
technical problems related to fermenters and pumps and I am grateful for your outstanding efforts in the
maintenance of “French Press”. Mark Strampraad from enzymology group: thank you very much for assay
suggestions; you are definitely the “guru” of enzyme assays. Frieda is acknowledged for administrative help,
Sjaak and Jos Lispet, Wim Morien and Herman Frumau for providing me with the things I needed even at a very
short notice. Thank you so much! Marcel van den Broek, Hans Kemper for computer related matters and Bart
Kurpershoek for always helping me with the due-dates and also having a very friendly attitude.
Acknowledgements
Hereby I would like to acknowledge my friends and some of my colleagues; Dr. Susanne Rudolf: thank you
very much for your support, wisdom and being always a great friend. David Senica: I still can not forget our
camping adventure in Luxemburg, and crossing the ice-cold river with bare feet in Ardennes!. Thanks to God, I
am still alive. Thank you for being such a good and a crazy Slovenian!. Dr. Martijn Hoeben: our friendship will
last longer than five years for sure. I guess the best sentence to describe you is: a man of big heart. Juliana:
sunshine of our lives, I really miss you a lot and I am still keeping a bottle of cashasa for your visit. Prof. Maria
Gonzales from Venezuela: I still remember our chatting in Sebastiansbrug like yesterday, you are a true friend.
Emel and Esengul, thank you for being there (oglen yemegi icin hala bulusamadik ama mutlaka bir gun bu olayi
gerceklestirecegiz). I also would like to thank Boy family from Delft for always being around and helpful,
Gokhan, Arzu, Piruze teyze ve Aydin amca; Hersey icin cok tesekkur ediyorum sizlere. I would like to thank:
Raji, Horia, Dimitry, Shabir, Daniela, Uly, Penia, Mlawule, Cagri, Lin, Viorika, Christian, Alfred, Sirous,
Maryam, Jac Wilsenac, Febe deWet, Roelco, Frederick, Cyryla, Marcia Morais, Yamira, Ha, Frank, Heidi, Ivan,
Jasmina, Marijana, Branko, Sasa and Svetlana (Hvala !).
Last but not least, I would like to dedicate this thesis to the people who will be mentioned below: without
them, this thesis would not be possible
My dear parents: You made this thesis possible with unlimited support, love, and understanding. Thank you
very much for your belief and confidence in me. My dear husband, my best friend, my love, Aleksandar: you are
the one who cheered for my success more than yours and who cried with me in difficult times. I will always
remember you saying: “Keep on walking”. You are the reason I completed this study successfully, Thank you
very much for everything.
108
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