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`APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 159–166
`0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.1.159–166.2004
`Copyright © 2004, American Society for Microbiology. All Rights Reserved.
`
`Vol. 70, No. 1
`
`Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces
`cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and
`Pyruvate-Hyperproducing Yeast
`Antonius J. A. van Maris,1 Jan-Maarten A. Geertman,1 Alexander Vermeulen,1
`Matthijs K. Groothuizen,1 Aaron A. Winkler,2 Matthew D. W. Piper,1
`Johannes P. van Dijken,1,2 and Jack T. Pronk1*
`Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft,1 and BIRD
`Engineering B.V., NL-3044 CK Rotterdam,2 The Netherlands
`
`Received 23 September 2003/Accepted 26 September 2003
`
`The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdcⴚ) strains of Saccharo-
`myces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However,
`Pdcⴚ S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat
`cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C2
`compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two
`subsequent phenotypic selection strategies to obtain a Pdcⴚ strain without these growth defects. An acetate-
`independent Pdcⴚ mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively
`lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C2
`independence. Further selection for glucose tolerance in shake flasks resulted in a Pdcⴚ S. cerevisiae mutant
`(TAM) that could grow in batch cultures (␮max ⴝ 0.20 hⴚ1) on synthetic medium, with glucose as the sole
`carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not
`resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-
`repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose.
`In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated
`glucose feeding, a pyruvate concentration of 135 g literⴚ1 was obtained, with a specific pyruvate production rate
`of 6 to 7 mmol g of biomassⴚ1 hⴚ1 during the exponential-growth phase and an overall yield of 0.54 g of
`pyruvate g of glucoseⴚ1.
`
`Traditionally, Saccharomyces cerevisiae has been used to rap-
`idly ferment sugars to ethanol and carbon dioxide. More re-
`cently, developments in molecular biology have led to the
`application of S. cerevisiae as a host for therapeutic protein
`production (13) and for the production of chemicals with com-
`mercial value via metabolic engineering (28, 29, 31). In view of
`the process economy of manufacturing bulk products, the yield
`of the desired product should be maximized. In the case of
`yeasts as production organisms, this necessitates the redirec-
`tion of carbon fluxes away from alcoholic fermentation towards
`the desired product (1, 4, 7, 8, 19).
`Pyruvate decarboxylase (EC 4.1.1.1) is located at the branch
`point between fermentative and respiratory sugar catabolism
`and catalyzes the first step in the fermentative branch. S. cer-
`evisiae contains three structural genes (PDC1, PDC5, and
`PDC6) that encode active pyruvate decarboxylase isoenzymes
`(18). Pyruvate decarboxylase was long considered to be a
`strictly catabolic enzyme, but recently a biosynthetic function
`of the enzyme was discovered (8). Growth of pyruvate decar-
`boxylase-negative (Pdc⫺) S. cerevisiae in aerobic glucose-lim-
`ited chemostat cultures on synthetic media required a supply of
`acetate or ethanol corresponding to ca. 5% of the carbon fed
`
`* Corresponding author. Mailing address: Department of Biotech-
`nology, Delft University of Technology, Julianalaan 67, NL-2628 BC
`Delft, The Netherlands. Phone: 31 15 278 3214. Fax: 31 15 213 3141.
`E-mail: J.T.Pronk@tnw.tudelft.nl.
`
`to the cultures (6, 8). This requirement for a C2 compound
`probably reflects an essential function of pyruvate decarboxyl-
`ase in the synthesis of cytosolic acetyl-coenzyme A (CoA) (Fig.
`1), which is required for lysine and fatty acid synthesis (6).
`The overproduction of threonine aldolase, catalyzing the
`cleavage of threonine to glycine and acetaldehyde, can circum-
`vent the essential biosynthetic role of pyruvate decarboxylase
`(46). Even when the C2 requirement of Pdc⫺ strains is met by
`overexpression of threonine aldolase or by inclusion of ethanol
`or acetate in the medium, Pdc⫺ strains can only grow on
`glucose when the glucose supply is growth limiting. When Pdc⫺
`strains are exposed to the glucose concentrations normally
`applied in batch cultures, they excrete significant amounts of
`pyruvate but are completely unable to grow (9). This glucose
`sensitivity is a general characteristic of Pdc⫺ strains (6, 46).
`The exact cause of the glucose sensitivity of Pdc⫺ strains
`remains unknown. In the absence of alcoholic fermentation,
`which is blocked in Pdc⫺ S. cerevisiae, cells rely on respiration
`(Fig. 1) for the reoxidation of cytosolic NADH (33). However,
`respiration of wild-type S. cerevisiae in batch cultures on glu-
`cose is repressed but not blocked, judging from the significant
`oxygen consumption rate under these conditions (1, 4). It is
`therefore unlikely that glucose repression of respiration is the
`sole cause of the glucose sensitivity of Pdc⫺ S. cerevisiae.
`From the time of their invention, chemostats have been
`associated with selection of spontaneous mutants (25, 26). The
`first chemostat studies had already described the selection of
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`selection was performed in batch cultures to select for C2-
`independent Pdc⫺ S. cerevisiae that could grow on high con-
`centrations of glucose. A second goal was to physiologically
`characterize the selected strain and to gain insight into the
`molecular mechanisms underlying the selected phenotype. To
`this end, biomass and product yields were analyzed in batch
`and chemostat cultures and genome-wide transcriptome anal-
`ysis was performed, using nitrogen-limited chemostat cultures
`grown under conditions of excess glucose.
`
`MATERIALS AND METHODS
`
`Strains and maintenance. All S. cerevisiae strains used for this study (Table 1)
`were derived from the congenic CEN.PK family (41). Stock cultures were pre-
`pared from shake flask or chemostat cultures by the addition of 20% (vol/vol)
`glycerol to cultures and storage of 2-ml aliquots in sterile vials at ⫺80°C.
`Strain construction. RWB837 was obtained from a cross between CEN.PK182
`and CEN.PK111-61A (constructed by P. Ko¨tter, Frankfurt, Germany, and ob-
`tained from Jefferson C. Lievense, Tate and Lyle North America). The resulting
`diploid was sporulated and the asci were heated for 15 min at 56°C. This random
`dissection mix was then plated on YP medium, with 0.2% acetate as the carbon
`source. The resulting colonies were tested for growth on YP medium with
`glucose or ethanol. Colonies that could not grow on glucose were subsequently
`checked by PCR for the presence of a disrupted PDC6 gene and for mating type.
`Determination of the auxotrophic markers present, in this case ura3-52, then
`gave RWB837. The final strain (designated TAM), selected as described below,
`was transformed with YEplac195 (14) according to the high-efficiency protocol
`described by Gietz and Woods (15), resulting in the prototrophic (ura⫹)
`TAM⫹YEplac195 strain.
`Chemostat cultivation. Aerobic carbon-limited or nitrogen-limited chemostat
`cultivation reactions were performed as described previously (45). To comple-
`ment auxotrophy, 0.15 g of uracil liter⫺1 (32) was added to the media. The
`synthetic medium for the glucose-limited chemostat cultures contained 250 mM
`substrate carbon. When acetate was present, this was added in addition to the
`250 mM carbon from glucose, at concentrations corresponding to 0 to 5%
`acetate on a substrate-carbon basis. For nitrogen-limited cultures, the glucose
`concentration in the synthetic medium was adjusted during a trial run to result
`in a residual glucose concentration in the culture broth of approximately 100
`mM. Afterwards, reproducible duplicate cultures were obtained at this glucose
`concentration.
`Shake flask cultivation. The 500-ml shake flasks, containing 100 ml of syn-
`thetic medium (49), were incubated at 30°C in a rotary shaker (200 rpm). To
`rescue auxotrophy, 0.15 g of uracil liter⫺1 (32) was added to the media. Precul-
`tures of RWB837 were grown in 2% ethanol. For all other shake flask cultures,
`glucose was used as the carbon source, with concentrations ranging from 2 to
`10% (wt/vol). The selected strain was routinely checked for uracil auxotrophy to
`verify culture purity.
`Fermenter batch cultivation. Aerobic batch cultivation was performed at 30°C
`in 2-liter fermenters (Applikon, Schiedam, The Netherlands) with a working
`volume of 1 liter. The pH was controlled at 5.0 via automated addition of 10 M
`KOH (Applikon ADI 1030 biocontroller). The dissolved-oxygen concentration
`was maintained above 10% air saturation at all times by adjusting the stirrer
`speed between 800 and 1,000 rpm and the airflow between 0.50 and 0.75 liter
`min⫺1. A synthetic medium with twice the concentrations described by Verduyn
`et al. (49) was used for cultures. The initial glucose concentration was 100 g
`liter⫺1. During the repeated batch cultivation, 100 g of nonsterile solid glucose
`was added twice, at 32 and 48 h after inoculation. Antifoam (BDH) product was
`
`FIG. 1. Schematic representation of the metabolism of pyruvate
`decarboxylase-negative S. cerevisiae growing on glucose. By deletion of
`all genes encoding pyruvate decarboxylase (reaction a), two important
`processes (dotted lines) are impaired as follows. First, reoxidation of
`cytosolic NADH via alcohol dehydrogenase (reaction b) is blocked.
`Cytosolic NADH must therefore be oxidized by the mitochondria via
`external NADH dehydrogenase (reaction c) or redox shuttle systems.
`Second, the formation of cytosolic acetyl-CoA from acetaldehyde is
`blocked. Instead, the C2 compounds required for cytosolic acetyl-CoA
`for lysine and fatty acid biosynthesis (reaction d) must be taken up
`from the environment. When oxygen consumption exceeds the amount
`of oxygen necessary for oxidation of glucose to pyruvate, mitochondrial
`oxidation of pyruvate, via pyruvate dehydrogenase (reaction e) and the
`tricarboxylic acid cycle (TCA cycle), can occur, resulting in CO2 for-
`mation and the oxidation of NADH via internal NADH dehydroge-
`nase (reaction f).
`
`an Escherichia coli strain with a higher affinity for the growth-
`limiting nutrient (26). Subsequent review articles cited a vari-
`ety of other examples of selection in chemostats (5, 37) and
`elaborated on the theory of selection during chemostat culti-
`vation (17). Similarly, extended cultivation of microorganisms
`in shake flasks can be used to select for spontaneous mutants
`that grow under conditions in which the original strain would
`not grow (10, 37).
`The first goal of this study was to apply selection pressure in
`batch and chemostat cultures to obtain a Pdc⫺ S. cerevisiae
`strain capable of growth in batch cultures on synthetic medium
`containing high concentrations of glucose as the sole carbon
`and energy source. Prolonged chemostat cultivation on glucose
`with progressively decreasing acetate feeds was used to select
`for C2-independent Pdc⫺ S. cerevisiae. A subsequent round of
`
`Strain
`
`TABLE 1. S. cerevisiae strains used in this study
`Genotype
`
`CEN.PK 113-7D............................................................MATa URA3 PDC1 PDC5 PDC6
`CEN.PK 182...................................................................MATa pdc1(⫺6,⫺2)::loxP pdc5(⫺6,⫺2)::loxP pdc6(⫺6,⫺2)::loxP
`CEN.PK 111-61A ..........................................................MAT␣ ura3–52 leu2–112 his3-⌬1
`RWB837 .........................................................................MATa pdc1(⫺6,⫺2)::loxP pdc5(⫺6,⫺2)::loxP pdc6(⫺6,⫺2)::loxP ura3–52
`RWB837ⴱ .......................................................................MATa pdc1(⫺6,⫺2)::loxP pdc5(⫺6,⫺2)::loxP pdc6(⫺6,⫺2)::loxP ura3–52, selected for C2
`independence in glucose-limited chemostats
`TAM ...............................................................................MATa pdc1(⫺6,⫺2)::loxP pdc5(⫺6,⫺2)::loxP pdc6(⫺6,⫺2)::loxP ura3–52, selected for C2
`independence in glucose-limited chemostats and glucose-tolerant growth in batch culture
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`FIG. 2. Growth of three Pdc⫺ S. cerevisiae strains and wild-type S. cerevisiae on synthetic medium agar plates with ethanol (left plate) or glucose
`(right plate) as the carbon source. Both plates were supplemented with uracil to alleviate the auxotrophy of the Pdc⫺ S. cerevisiae strains. Ethanol
`plates were incubated for 7 days, and glucose plates were incubated for 3 days. Strains: a, RWB837 (Pdc⫺ S. cerevisiae); b, RWB837* (selected
`C2-independent S. cerevisiae); c, TAM (selected C2-independent and glucose-tolerant Pdc⫺ S. cerevisiae); d, CEN.PK 113-7D (wild type).
`
`added to the fermenters when required. Culture purity was checked microscop-
`ically at the end of the fermentation and no contaminants were observed.
`Microarray analysis. Sampling of cells from chemostats, probe preparation,
`and hybridization to Affymetrix GeneChip microarrays were performed as de-
`scribed previously (30). The results were derived from two independent replicate
`cultures for the selected Pdc⫺ strain and from three independent replicate
`cultures for the wild type.
`Microarray data acquisition and analysis. Acquisition and quantification of
`array images and data filtering were performed with the Affymetrix software
`packages Microarray Suite, v. 5.0, MicroDB, v. 3.0, and Data Mining Tool, v. 3.0.
`For further statistical analysis, Microsoft Excel running the Significance Analysis
`of Microarrays (v. 1.12) add-in was used, with a delta value that corresponded to
`the minimum expected median false-positive rate and a minimum change of
`twofold (40). In our experience, these criteria establish a data set that is able to
`be reproduced by an independent laboratory (30).
`Before comparison, all arrays were globally scaled to a target value of 150,
`using the average signal from all gene features, with Microarray Suite, v. 5.0. For
`the 9,335 transcript features on the YG-S98 arrays, a filter was applied to extract
`6,383 yeast open reading frames, of which 6,084 were different genes. This
`discrepancy was due to several genes being represented more than once when
`suboptimal probe sets were used in the array design. Since the 900 transcripts
`with the lowest transcription level could not be reliably measured, their level was
`set to a value of 12 for the comparison analyses.
`Promoter analyses were performed with the web-based software Regulatory
`Sequence Analysis Tools (42), as described previously (2).
`Analytical procedures. Dry weight determination, glucose, acetate, and me-
`tabolite analysis, off-gas analysis, and pyruvate decarboxylase and threonine
`aldolase assays were performed as described previously (46). The protein content
`of whole cells was determined by a modified biuret method (48).
`
`RESULTS
`Selection of C2-independent Pdcⴚ S. cerevisiae in chemostat
`cultures. For this study, the power of chemostat cultivation as
`a tool for the selection of microorganisms (5, 17) was used in
`an attempt to eliminate the C2 compound requirement of Pdc⫺
`S. cerevisiae (6, 8). A pdc1,5,6⌬ ura3⌬ S. cerevisiae strain
`(RWB837) was used for selection. The ura3⌬ auxotrophic
`marker was used to facilitate controls for culture purity. First,
`a steady state of Pdc⫺ S. cerevisiae on a mixture of 5% acetate
`and 95% (on the basis of carbon) glucose was established. The
`metabolism of this culture was fully respiratory, as indicated by
`a respiratory quotient of just over one carbon dioxide molecule
`produced per oxygen molecule consumed. The biomass yield
`on carbon was 14.6 g of biomass mol of carbon⫺1, and all
`
`glucose and acetate were consumed. The acetate content of the
`synthetic medium was then reduced in five consecutive steps,
`from 5% of the total carbon content to zero. Each step lasted
`five volume changes. During this slow transition, RWB837
`adapted to growth in aerobic carbon-limited chemostat cul-
`tures, with glucose as the sole carbon source, at a dilution rate
`of 0.10 h⫺1. The biomass yield on substrate (14.7 g of biomass
`mol of carbon⫺1), oxygen consumption rate, and carbon diox-
`ide production rate (both around 2.9 mmol g of biomass⫺1
`h⫺1) of this glucose-limited culture indicated respiratory car-
`bon metabolism of the C2-independent Pdc⫺ S. cerevisiae cul-
`ture, as was observed with wild-type S. cerevisiae under these
`conditions (43).
`Transcriptome analysis of the C2-independent Pdcⴚ S. cer-
`evisiae strain. Transcriptome analysis of the glucose-limited
`chemostat culture of the C2-independent S. cerevisiae strain
`was performed to study the genetic changes responsible for C2
`independence. The C2-independent Pdc⫺ strain was compared
`to glucose-limited chemostat cultures of the wild type (30). Of
`the genes with a known function, only 18 were upregulated and
`only 16 were downregulated in the selected strain. These up-
`regulated genes included 11 that were involved in meiosis or
`sporulation (HOP2, IME2, REC102, REC104, RED1, SLZ1,
`SPO13, SPO16, SPR1, YER179W, and ZIP1). The other seven
`upregulated genes were CAR1, ECM1, HXT3, HXT4, IRE1,
`NUF1, and NUF2. The downregulated genes included four
`expected genes (PDC1, PDC5, PDC6, and URA3) and, in ad-
`dition, ALP1, AQY1, GND2, FUI1, HSP30, HXT5, MEP2,
`MLS1, PDR12, PHO4, SSA3, and SSA4. None of these genes
`had a clear link to the C2 independence of the selected mutant.
`Transcript levels of the GLY1 gene, overexpression of which
`alleviates the C2 requirement of Pdc⫺ S. cerevisiae (46), were
`not significantly changed in the selected strain.
`Selection for glucose tolerance in shake flask cultures. After
`selection for C2 independence, a small aliquot of the chemostat
`culture was transferred to a shake flask with synthetic medium
`containing uracil and 20 g of glucose liter⫺1. As was expected
`from previous results, neither the original Pdc⫺ S. cerevisiae
`nor the C2-independent Pdc⫺ strain grew on agar plates with
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`FIG. 3. Growth and pyruvate production during an aerobic re-
`peated batch culture on glucose of the selected TAM strain. The
`results shown are from one representative batch experiment. Biomass
`and pyruvate concentrations in independent replicate experiments var-
`ied by ⬍5%. Closed squares, pyruvate concentration; open symbols,
`glucose concentration (diamonds) or OD660 (circles).
`
`produce pyruvate, the fermentation was continued as a re-
`peated batch by the addition of solid glucose to the fermenter
`(Fig. 3). During this repeated batch phase, the specific growth
`rate gradually decreased and growth ultimately ceased, prob-
`ably due to nutrient limitations in the medium. The pyruvate
`concentration in the supernatant exceeded 100 g liter⫺1 within
`60 h of inoculation of the fermenter. The final concentration of
`pyruvate obtained after 100 h was 135 g liter⫺1, with an overall
`yield of 0.54 g of pyruvate g of glucose⫺1.
`Glucose-limited chemostat cultivation of the TAM strain.
`The obtained maximum specific growth rate of the TAM strain
`in batch culture with glucose was 0.20 h⫺1. Under the same
`conditions, wild-type S. cerevisiae CEN.PK 113-7D grew with a
`higher maximum specific growth rate of 0.37 h⫺1 (data not
`shown). For further study of this deviation in growth, glucose-
`limited chemostat cultures of the TAM strain were performed
`at increasing dilution rates. At a dilution rate of 0.10 h⫺1,
`glucose dissimilation by the TAM strain was fully respiratory,
`without the accumulation of metabolites. Except for a lower
`biomass yield of the selected strain (0.43 g of biomass g of
`glucose⫺1, compared to 0.48 g of biomass g of glucose⫺1 for
`the wild type), physiological parameters were comparable to
`those of the wild type (43). At a dilution rate of 0.15 h⫺1, the
`biomass yield of the TAM strain had increased to 0.47 g of
`biomass g of glucose⫺1, which is still lower than the 0.50 g of
`biomass g of glucose⫺1 for the wild type at this dilution rate.
`The TAM strain was capable of growth at a dilution rate of
`0.20 h⫺1 in glucose-limited chemostat cultures, but this growth
`was accompanied by a variable pyruvate production rate (0.25
`to 0.45 mmol of pyruvate g of biomass⫺1 h⫺1). At a dilution
`rate of 0.23 h⫺1, the TAM strain washed out of the chemostats,
`indicating a maximum specific growth rate of this strain be-
`tween 0.20 and 0.23 h⫺1 in glucose-limited chemostat cultures.
`After this prolonged glucose-limited chemostat cultivation, an
`
`synthetic medium, uracil, and 2% glucose (Fig. 2, right panel),
`whereas both strains did grow on agar plates with synthetic
`medium, uracil, and 2% ethanol (Fig. 2, left panels). In agree-
`ment with this, no growth was observed during the first 7 days
`of the initial shake flask culture of the C2-independent Pdc⫺
`strain on 2% glucose. Prolonged cultivation of the C2-indepen-
`dent Pdc⫺ strain, however, resulted in significant biomass for-
`mation, indicating an accumulation of spontaneous glucose-
`tolerant mutants. The observed growth rate was well below
`0.01 h⫺1. After growth had ceased, which occurred at a rela-
`tively low biomass density due to acidification of the culture by
`pyruvic acid accumulation, 1 ml of the culture was transferred
`to a new shake flask with identical synthetic medium.
`The process of serial transfer was repeated 27 times in total.
`The specific growth rate of the Pdc⫺ strain after the sixth
`transfer was already 0.10 h⫺1 on 20 g of glucose liter⫺1. After
`14 shake flask cultivations and an obtained specific growth rate
`of 0.18 h⫺1, the glucose content of the medium was raised to
`32, 54, 69, and 100 g liter⫺1 in consecutive cultures. At 100 g of
`glucose liter⫺1, the finally obtained C2-independent, glucose-
`tolerant Pdc⫺ S. cerevisiae culture grew at a specific growth
`rate of 0.20 h⫺1.
`The culture, possibly consisting of a mixture of different
`spontaneous mutants, was streaked onto agar plates containing
`synthetic medium, glucose, and uracil. Four of the resulting
`colonies were tested for growth in shake flasks on glucose, and
`no significant differences in specific growth rate were observed.
`One of these cultures was chosen for further study, and this
`C2-independent glucose-tolerant Pdc⫺ S. cerevisiae strain will
`be referred to as TAM in this and future work.
`The differences in growth among the original Pdc⫺ strain
`(RWB837), the C2-independent Pdc⫺ strain, the TAM strain,
`and the isogenic wild-type strain on synthetic medium, with
`glucose or ethanol as the sole carbon source, were clearly
`demonstrated by agar plate growth, as depicted in Fig. 2. Al-
`though the TAM strain displayed a 3-day-longer lag phase, all
`four strains grew on plates with ethanol as the carbon source.
`As described above, when glucose was the carbon source, the
`original Pdc⫺ strain (RWB837) and the C2-independent Pdc⫺
`strain did not grow. Consistent with the growth in shake flasks
`with glucose, the selected TAM strain, and of course the wild
`type, proliferated well on the agar plates with glucose (Fig. 2).
`Pyruvate production by the selected TAM strain in fer-
`menter cultures. During selection for glucose tolerance in
`shake flasks, rapid acidification of the culture due to pyruvate
`excretion was observed. To study the growth and pyruvate
`production of the TAM strain under controlled conditions,
`aerobic batch cultivations with 100 g of glucose liter⫺1 were
`performed in fermenters at a constant pH of 5.0. During the
`exponential-growth phase (Fig. 3),
`the maximum specific
`growth rate of the TAM strain was 0.20 h⫺1, which was equal
`to the maximum specific growth rate in shake flasks. Consistent
`with the observations with shake flasks, large amounts of pyru-
`vate were produced in fermenter cultures. The rate of pyruvate
`production during the exponential-growth phase was 6 to 7
`mmol g of biomass⫺1 h⫺1. In the first 40 h of this batch,
`starting with a low biomass concentration (optical density at
`660 nm [OD660], 0.1), a pyruvate concentration of 50 g liter⫺1
`was obtained, with a yield of 0.55 g of pyruvate g of glucose⫺1.
`For further assessment of the potential of the TAM strain to
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`type. The respiratory oxidation of the NADH formed during
`pyruvate formation lowered the respiratory quotient to 0.70
`mmol of carbon dioxide produced per mmol of oxygen con-
`sumed. The protein content of the biomass was slightly higher
`for the TAM strain (0.33 g of protein g of biomass⫺1) than for
`the wild type (0.29 g of protein g of biomass⫺1). This higher
`protein content of the cells partially explains the significantly
`lower yield on nitrogen of the TAM strain (14.7 g of biomass
`g of nitrogen⫺1) than of the wild type (18.8 g of biomass g of
`nitrogen⫺1).
`Transcriptome analysis of the TAM strain. Central in tran-
`scriptome analysis is the choice of adequate culture conditions
`for the comparison. In the case of the selected TAM strain, the
`absence of C2 compounds and the presence of high levels of
`glucose in the broth are typical for uncovering its phenotype.
`To combine the benefit of chemostat cultures in microarray
`studies (30) and the requirement for excess glucose, the nitro-
`gen-limited chemostat cultures of the TAM strain and the
`isogenic wild-type strain CEN.PK 113-7D were chosen for the
`transcriptome analysis.
`The comparison of the nitrogen-limited chemostat cultures
`revealed 305 genes of which the mRNA levels were signifi-
`cantly changed and at least twofold higher in the TAM strain
`than in the wild type. The mRNA abundance of 168 genes was
`significantly changed and at least twofold lower in the TAM
`strain than in the wild type. In total, these changed genes
`comprise almost 8% of the total S. cerevisiae genome. Of these
`changed genes, 273 (58%) have an unknown function, which is
`higher than the percentage of not fully annotated genes in the
`whole S. cerevisiae genome (47%).
`Sequence analysis of the upstream regions of genes that are
`upregulated in the selected strain showed an overrepresenta-
`tion of possible Mig1p-binding sites among these genes, indi-
`cating an (partial) alleviation of Mig1p-mediated repression.
`Although the transcript level of the primarily posttranscrip-
`tionally (12) regulated MIG1 was not changed, the transcript
`level of its close homologue MIG2 was downregulated almost
`11-fold. Many genes required for growth on carbon sources
`other than glucose were upregulated in the TAM strain. This
`included genes involved in gluconeogenesis and ethanol utili-
`zation (ACS1, ADH2, ADR1, CAT8, FBP1, and SIP4), fatty
`acid metabolism (CAT2, CRC1, ECI1, FAA2, FOX2, PEX11,
`POT1, POT1, and YAT2), galactose metabolism (GAL2,
`GAL3, and GAL4), maltose metabolism (MPH2, MPH3, and
`YFL052W), and pyruvate and lactate metabolism (DLD1 and
`JEN1).
`A striking observation was the change in expression of the
`genes coding for the hexose transporters. Despite the high
`glucose concentrations under nitrogen limitation, the low-af-
`finity transporters (HXT1 and HXT3) were downregulated 50-
`fold in the TAM strain compared to in the wild type (Fig. 4).
`The known high-affinity transporters (HXT6 and HXT7) were
`also downregulated (fourfold) in this strain (Fig. 4). As a re-
`sult, the summed transcript abundance of all HXT genes rep-
`resented on the arrays (HXT1 to HXT10, HXT12, HXT14, and
`HXT16) was four times lower in the TAM strain under nitro-
`gen limitation than in the wild type. In the glucose-responsive
`regulatory network of the HXT genes (35), the only significant
`transcriptional change was the 12-fold downregulation of
`
`TABLE 2. Physiology of strain TAM (C2-independent, glucose-
`tolerant Pdc⫺ S. cerevisiae) and the isogenic wild-type strain
`CEN.PK 113-7D in aerobic nitrogen-limited chemostat culture at a
`dilution rate of 0.10 h⫺1a
`
`Characteristicb
`
`⫺1)
`
`⫺1)
`
`Reservoir glucose concentration
`(g liter⫺1)
`Residual glucose concentration
`(g liter⫺1)
`Ysx (gbiomass gglucose
`⫺1)
`Ynx (gbiomass gN
`Protein content (gprotein gbiomass
`Respiratory quotient
`⫺1 h⫺1)
`qglucose (mmol gbiomass
`⫺1 h⫺1)
`qethanol (mmol gbiomass
`⫺1 h⫺1)
`qpyruvate (mmol gbiomass
`⫺1 h⫺1)
`qglycerol (mmol gbiomass
`⫺1 h⫺1)
`qacetate (mmol gbiomass
`⫺1 h⫺1)
`qCO2 (mmol gbiomass
`⫺1 h⫺1)
`qO2 (mmol gbiomass
`Recovery of consumed carbon (%)
`Total carbon recovery (%)
`
`Wild type
`
`58.8 ⫾ 0.1
`
`TAM
`
`35.1 ⫾ 0.1
`
`16.7 ⫾ 0.7
`
`20.4 ⫾ 0.1
`
`0.09 ⫾ 0.00
`18.8 ⫾ 0.1
`0.29 ⫾ 0.01
`4.5 ⫾ 0.2
`5.8 ⫾ 0.1
`8.0 ⫾ 0.1
`0.1 ⫾ 0.0
`0.08 ⫾ 0.00
`0.06 ⫾ 0.02
`12.1 ⫾ 0.2
`2.7 ⫾ 0.1
`94.0 ⫾ 1.0
`95.6 ⫾ 0.7
`
`0.21 ⫾ 0.00
`14.7 ⫾ 0.1
`0.33 ⫾ 0.01
`0.70 ⫾ 0.01
`2.6 ⫾ 0.1
`⬍0.01
`2.8 ⫾ 0.0
`⬍0.01
`⬍0.01
`2.8 ⫾ 0.0
`4.0 ⫾ 0.1
`97.4 ⫾ 0.7
`98.6 ⫾ 0.4
`
`a The wild-type data were obtained from the same cultures as those used by
`Boer et al. (2).
`b Averages and mean deviations were obtained from duplicate (TAM) and
`triplicate (wild type) experiments with independent steady-state cultures. Calcu-
`lations of carbon recovery were based on a 48% (wt/vol) carbon content of the
`biomass. Ysx and Ynx are the biomass yields on glucose and nitrogen, respectively.
`Specific production or consumption rates are indicated with a q.
`
`aliquot of the culture was transferred to a shake flask with
`100 g of glucose liter⫺1. With this shake flask, rapid growth was
`observed, indicating that the culture had maintained its glu-
`cose-tolerant phenotype.
`Nitrogen-limited chemostat cultivation of the TAM strain.
`Comparison of the selected TAM strain with wild-type S. cer-
`evisiae CEN.PK 113-7D is best performed at high glucose con-
`centrations and in the absence of C2 compounds in the me-
`dium. Nitrogen-limited chemostat cultivation at the same
`dilution rate, with glucose as the sole carbon source, combines
`these conditions with the advantages of chemostat cultivation
`for reproducible physiological studies. The glucose concentra-
`tions in the synthetic medium were chosen such that approxi-
`mately the same residual glucose concentration was obtained
`in the cultivations of both strains (Table 2).
`The wild type showed alcoholic fermentation, as is charac-
`teristic for S. cerevisiae under conditions of excess glucose. This
`resulted in a low biomass yield on glucose (0.09 g of biomass g
`of glucose⫺1), an ethanol production rate of 8.0 mmol g of
`biomass⫺1 h⫺1, and a respiratory quotient of 4.5 mmol of
`carbon dioxide produced per mmol of oxygen consumed. The
`protein content (0.29 g of protein g of biomass⫺1) and the
`biomass yield on nitrogen (18.8 g of biomass g of nitrogen⫺1)
`were in good agreement with previously published values (44,
`45) for nitrogen-limited chemostat cultures of wild-type strain
`CEN.PK 113-7D.
`Under the same conditions, the TAM strain, which fully
`depends on respiration in the absence of alcoholic fermenta-
`tion, had a higher biomass yield on glucose (0.21 g of biomass
`g of glucose⫺1) and produced pyruvate as the only major by-
`product, at a rate of 2.8 mmol g of biomass⫺1 h⫺1 (Table 2).
`The oxygen consumption rate was 4.0 mmol g of biomass⫺1
`h⫺1, compared to 2.7 mmol g of biomass⫺1 h⫺1 for the wild
`
`

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`VAN MARIS ET AL.
`
`APPL. ENVIRON. MICROBIOL.
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`
`mostat cultivation resulted in selection of a Pdc⫺ strain that
`did not require the addition of C2 compounds to the growth
`environment (Fig. 1). Transcriptome analysis did not reveal the
`mechanism underlying the physiological changes in this strain.
`However, based on physiology, some possible sources of cyto-
`solic acetyl-CoA can be excluded.
`Massive overexpression of GLY1, encoding threonine aldo-
`lase, has been shown to circumvent the essential biosynthetic
`role of pyruvate decarboxylase in Pdc⫺ S. cerevisiae (45). De-
`spite the higher GLY1 transcript levels in the TAM strain, the
`low affinity of Gly1p for threonine (Km, 55 mM [22]) and the
`relatively low intracellular threonine concentration (5 to 10
`mM [16, 23]), combined with the low in vitro activity (⬍0.005
`U mg of protein⫺1), make it unlikely that threonine aldolase is
`responsible for its C2-independent phenotype. In addition,