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Letters in Applied Microbiology 2004, 39, 199–206 doi:10.1111/j.1472-765X.2004.01563.x Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production L.-M. Liu1, Y. Li1,2, H.-Z. Li1 and J. Chen1 1 The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Southern Yangtze University, Wuxi, and 2State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China 2004/0209: received 25 February 2004, revised 2 May 2004 and accepted 11 May 2004 ABSTRACT L . - M . L I U , Y . L I , H . - Z . L I A N D J . C H E N . 2004. Aims: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. Methods and Results: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680–685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103Æ5 or 57Æ4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68Æ7 g l)1 at 62 h (yield on glucose of 0Æ651 g g)1) in a 7-l jar fermentor. Conclusions: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. Significance and Impact of the Study: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites. Keywords: acetate assimilation, acetyl-CoA synthetase, pyruvate decarboxylase, pyruvate dehydrogenase bypass, pyruvate production, Torulopsis glabrata. INTRODUCTION Pyruvate, an important intermediate in hexose catabolism, is widely used in chemicals, drugs, and agrochemicals industries as a starting material (Li et al. 2001a). A multi-vitamin auxotrophic yeast, Torulopsis glabrata IFO 0005, is able to accumulate large amounts of pyruvate extracellularly (Miyata et al. 1989, 2000; Hua et al. 1999; Yonehara et al. 2000). The principle is that the activities of enzymes that are responsible Correspondence to: J. Chen, School of Biotechnology, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, P.R. China (e-mail: [email protected]). ª 2004 The Society for Applied Microbiology for further conversion of pyruvate, are limited by minimizing the concentrations of thiamine, nicotinic acid, pyridoxine, and biotin (cofactors of those enzymes) in the medium (Miyata and Yonehara 1996; Hua and Shimizu 1999). Among those vitamins, thiamine was found to be the most important vitamin affecting both cell growth and pyruvate production (Hua et al. 2001; Li et al. 2001b). This conclusion was solidly demonstrated in a defined medium using T. glabrata WSHIP303 as a model strain, which could assimilate ammonium as the sole nitrogen source. Thiamine serves as cofactor for pyruvate dehydrogenase (PDH) complex and pyruvate 200 L . - M . L I U ET AL. decarboxylase (PDC). Therefore, addition of a suboptimal concentration of thiamine is a prerequisite for achieving a high-level production of pyruvate (Li et al. 2001b). Thiamine must be added to the defined media of T. glabrata WSH-IP303, as maintaining the activity of PDH complex is essential for the strain to grow aerobically (Liu et al. 2004). However, the presence of thiamine activated PDC as well, and, as a consequence, ethanol was produced as a by-product. The production of ethanol might be retarded upon decreasing the activity of PDC of T. glabrata constitutively. Recently, a mutant of T. glabrata IFO 0005, ACII-3, was reported with a 54Æ5% reduced activity of PDC (Miyata and Yonehara 1999). The yield of pyruvate produced by strain ACII-3 was 15% higher than the parent strain. However, the mutant grew poorly on glucose (the sole carbon source) minimum medium. In Saccharomyces cerevisiae, PDC, acetaldehyde dehydrogenase (ALDH) and acetyl-CoA synthetase (ACS) are involved in a so-called PDH bypass (Holzer and Goedde 1957; Pronk et al. 1994, 1996) to provide additional cytoplasmic acetyl-CoA required for lipid synthesis (Flikweert et al. 1996). The poor growth pattern of T. glabrata mutant ACII-3 on glucose minimum medium (Miyata and Yonehara 1999) was similar to that of the PDC-deficient mutant of S. cerevisiae (Flikweert et al. 1996), indicating the physiological role of PDC in T. glabrata might be similar to that of in S. cerevisiae. In our previous studies, pyruvate production by T. glabrata WSH-IP303 had been enhanced in two aspects: (i) high yield was achieved by optimizing the vitamins concentrations (Li et al. 2001b); (ii) high productivity was reached by applying a two-stage dissolved oxygen control strategy (Li et al. 2002). In this study, we aim to manipulate the PDH bypass of T. glabrata WSH-IP303 to further increase the yield of pyruvate on glucose. To this aim, the selection of a mutant with decreased PDC activity and increased ACS activity was pursued. Such a mutant would have an increased capability of assimilating acetate, by which the decreased availability of cytoplasmic acetyl-CoA caused by the decreased activity of PDC may be compensated. In that way, cell growth would not be affected while pyruvate production can be further enhanced, upon addition of acetate. Our studies show that manipulation of the PDH bypass of T. glabrata could be accomplished by using traditional chemical mutation approach. Furthermore, the strategy of manipulating PDH bypass in T. glabrata may be applied to the production of other useful metabolites in glycolysis. M A T E R I A LS A N D M E T H O D S Micro-organism A multi-vitamin-auxotrophic yeast T. glabrata WSH-IP303, screened by our laboratory in previous studies that could use NH4Cl as a sole nitrogen source (Li et al. 2001b), was used as a parent strain. Media Medium composition for slant and seed cultures was (per litre): 30 g glucose, 10 g peptone (Biochemical grade, SinoAmerican Biotechnology Co., Shanghai, China), 1 g KH2PO4 and 0Æ5 g MgSO4Æ7H2O. Agar (20 g l)1) was added to slant cultures. The complete medium (CM) consisted of (per litre): 100 g glucose, 6 g sodium acetate, 7 g NH4Cl, 5 g KH2PO4, 0Æ8 g MgSO4Æ7H2O, 4 mg nicotinic acid, 15 lg thiamine HCl, 100 lg pyridoxine HCl, 10 lg biotin, 50 lg riboflavin. The minimal medium (MM) composition was the same as CM except that MM was free of sodium acetate. In plate cultures, 5 g l)1 CaCO3 was added. The initial pH of all media was adjusted to 5Æ0. All vitamins were filter-sterilized prior to addition to the medium. CaCO3 was sterilized by dry-heat sterilization at 160C for 30 min before being added to the medium. Cultivation The culture inoculated from a slant was incubated in a 500-ml flask with 50 ml seed medium and cultivated for 24 h on a reciprocal shaker. The seed culture was then inoculated either into 500-ml flasks containing 50 ml CM (or MM), or into a 7-l jar fermentor (KF-7 l, Korea Fermentor Co., Inchon, Korea) with 4 l CM for fermentation. The inoculum size was 10% (v/v). In flask cultures, the medium was buffered by 40 g l)1 CaCO3, while in fermentor cultures, the pH was automatically controlled at 5Æ0 with 8 mol l)1 NaOH solution. The flask cultures were grown for 48 h and the rotation rate was controlled at 200 rev min)1. The fermentor cultures were stirred at 300 rev min)1 with an aeration rate of 4 l min)1. All cultivations were made at 30C. Mutagenesis of T. glabrata WSH-IP303 and selection Cells grown in the seed medium for 12 h were harvested by centrifugation (6000 g) and washed twice with 0Æ1 mol l)1 potassium phosphate buffer (pH 7Æ5). One gram (wet weight) of washed cells was added to 100 ml of 0Æ1 mol l)1 potassium phosphate buffer (pH 7Æ5) containing 10 mg nitrosoguanidine (NTG) per litre and shaken at 30C for 1 h. The NTG-treated cells were centrifuged at 6000 g, followed by a wash in sterile saline (0Æ85% NaCl, w/v) and a re-centrifugation. The pelleted cells were diluted to 10)4, 10)5 and 10)6 and spread onto CM plates, followed by an incubation at 30C for 48 h. Colonies that appeared on CM plates were replicated on MM plates to select acetate requiring mutants, which were small colonies on MM but ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 39, 199–206, doi:10.1111/j.1472-765X.2004.01563.x MANIPULATING THE PYRUVATE DEHYDROGENASE BYPASS normal colonies on CM. The pyruvate-producing ability and genetic stability of those mutants were examined subsequently. Analytical methods Pyruvate concentrations were determined by the enzymatic assay described previously (Li et al. 2001b). Glucose concentration was measured by the dinitrosalicylic acid spectrometric method (Miller 1959). Cell concentrations were measured using a spectrophotometer (Biospec-1601; Shimadzu Co., Kyoto, Japan) at 660 nm after an appropriate dilution. The optical density (O.D.660) value was converted to dry cell weight (DCW) using the equation 1 O.D.660 ¼ 0Æ23 g DCW l)1. Ethanol concentration in the fermentor culture was detected using an on-line ethanol detector (Katakura et al. 1998). The concentration of sodium acetate was measured by HPLC (Miwa et al. 1985), under the following conditions: 4-ml culture was centrifuged at 6000 g for 10 min, following which the supernatant was syringe-filtered (0Æ45 lm, Agilent Technology, Palo Alto, CA, USA) and transferred to an auto-sampling HPLC system (Agilent Technology). Sample injections (10 ll) were made from samples kept at 10C onto a reverse phase C18 cartridge column at 45C. The mobile phase was 25% (v/v) acetonitrile: water solution at a rate of 1 ml min)1, with u.v. detection at 400 nm and peak area integration used for quantification. At the end of each sample elution a 15-min stabilization period was maintained before the injection of subsequent samples. Enzyme assays Cells were cultivated aerobically in 500-ml flasks containing 50 ml MM or CM at 30C for 24 h. Cells harvested from 4 ml culture were washed with ice-cold saline (0Æ85% NaCl, w/v) and re-suspended in 4 ml ice-cold 0Æ1 mol l)1 potassium phosphate buffer (pH 7Æ5). Four millilitres of cell suspension containing two grams of glass-beads with 0Æ7 mm diameter was ultrasonically disrupted at 0C for four cycles of 30 s (ACX 400 sonicator, 20 kHz, Sonic and Materials Inc., Newton, MA, USA). Cell debris was removed by centrifugation (6000 g, 4C, 10 min). The clear supernatant, typically containing 2–4 mg of protein per ml, was used as cell-free extract (CFE). Protein concentrations were measured by the Lowry method. All values of enzyme assay are the mean of results of at least three independent measurements. The reaction mixture (1 ml) for ACS assay contained 100 mmol l)1 of Tris–HCl (pH 7Æ7), 10 mmol l)1 of )1 L-malate (pH 7Æ7), 0Æ2 mmol l of CoA, 8 mmol l)1 of )1 ATP (pH 7Æ5), 1 mmol l of NAD+, 10 mmol l)1 of MgCl2, 3 U of malate dehydrogenase, 0Æ4 U of citrate 201 synthase, and CFE. The reactions were started by the addition of 100 mmol l)1 potassium acetate (Van den Berg et al. 1996). The reaction mixture (2 ml) for alcohol dehydrogenase (ADH) assay comprised 60 mmol l)1 sodium pyrophosphate buffer (pH 8Æ5), 0Æ7 mol l)1 ethanol, 0Æ5 mmol l)1 NAD+, and CFE (Miyata and Yonehara 1999). The solutions were allowed to stand for 10 min in the cuvettes until they reached 30C. The assay was initiated by the addition of 0Æ2 ml CFE and terminated after 60 s by the addition of 0Æ3 ml of 1 mol l)1 KOH. Both NAD+-dependent and NADP+-dependent ALDH activities were assayed as follows (Miyata and Yonehara 1999). The assay mixture (2Æ2 ml) comprised 50 mmol l)1 Tris–HCl (pH 8Æ0), 13 mmol l)1 p-mercaptoethanol, 100 mmol l)1 KCl, 3 mmol l)1 pyrazol, 1Æ0 mmol l)1 NAD+ or NADP+, and CFE. Enzyme assays for ACS, ADH and ALDH were performed at 30C, and the increase in absorbance at 340 nm was monitored. The activity was calculated from the linear slope of increasing absorption of NAD(P)H by using e ¼ 6220 (mol l)1))1 cm)1. The PDC activity was assayed as follows (Flikweert 1999). The reactions mixture (1Æ5 ml) comprised 50 mmol l)1 citrate buffer (pH 6Æ0), 0Æ07 mmol l)1 thiamine pyrophosphate, 12 mmol l)1 sodium pyruvate and CFE. The reaction mixture was incubated at 30C for 20 min, afterwards the reaction was stopped by the addition of 1 ml of 0Æ8 mmol l)1 2,4-dinitrophenyl-hydrazine dissolved in 2 mol l)1 hydrochloric acid. The reaction product was left for further 15 min to convert the produced acetaldehyde into its hydrazone. Upon addition of 2 ml of methanol, the 2,4dinitrophenyl-hydrazone of acetaldehyde and its derivatives were dissolved and determined by high-performance liquid chromatography (HPLC, Agilent Ltd 1100 Series) under the following conditions: column, Capcel pack C18 (Interactive Chromatography, San Jose, CA, USA); mobile phase, 30 g l)1 acetate aqueous solution : methanol (1 : 2); flow rate, 1Æ0 ml min)1; injection volume, 5 ll; detection, u.v. (365 nm); temperature, 30C. A reaction mixture excluding pyruvic acid was used as a control. Pyruvate carboxylase (PC) activity was determined as follows (Dunn et al. 1996). The reaction mixture (3Æ5 ml) contained: PBS buffer (pH 7Æ8), 0Æ5 mol l)1 NaHCO3, 0Æ1 mol l)1 MgCl2, 1Æ0 mmol l)1 acetyl-CoA, 0Æ1 mol l)1 pyruvate, 0Æ1 mol l)1 ATP, 0Æ15 mmol l)1 DTNB (5,5¢dithiobis-2-nitrobenzoic acid), and 1000 U citrate synthase, in 10 ml 100% ethanol. The assay was initiated by addition of pyruvate and terminated by addition of 0Æ3 ml of 1 mol l)1 KOH. The reaction was performed at 30C for 60 s and the absorption at 412 nm was measured. One units of PC activity is defined as the amount of enzyme required to produce 1Æ0 lmol of oxaloacetate per minute. ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 39, 199–206, doi:10.1111/j.1472-765X.2004.01563.x 202 L . - M . L I U ET AL. The PDH complex activity was determined as follows (Hinman and Blass 1981). The reaction mixture (1 ml) contained 2Æ5 mmol l)1 NAD+, 0Æ2 mmol l)1 thiamine pyrophosphate, 0Æ1 mmol l)1 coenzyme A, 0Æ3 mmol l)1 dithiothreitol, 0Æ5 mmol l)1 pyruvate, 1 mmol l)1 magnesium chloride, CFE, 1 mg ml)1 bovine serum albumin, 0Æ6 mmol l)1 p-iodonitrotetrazolium violet (INT), and lipoamide dehydrogenase (0Æ1 mg ml)1) in 0Æ05 mmol l)1 potasium phosphate buffer (pH7Æ8). The assay was initiated by addition of pyruvate and terminated by 0Æ5 ml 1 mol l)1 H2SO4. The absorption was measured at 500 nm and 30C. One unit was defined as the amount of enzyme required to reduce 1 lmol of INT per minute under the conditions specified. The extinction coefficient of INT is 15Æ01 mg)1 cm)1 at 500 nm and 30C. RESULTS Mutagenesis to generate mutants that require acetate for complete growth The PDC null mutant of S. cerevisiae grew poorly when glucose was used as the sole carbon source in a chemically defined medium. However, upon addition of acetate, the growth of the mutant restored to the wild-type level (Flikweert et al. 1996; Pronk et al. 1996). These results indicated that manipulating the activity of PDC (the major constituent of the PDH bypass pathway) was very likely correlated with the metabolism of acetate. Therefore, in the present study, the capability of assimilating acetate was used as a model to screen mutants of T. glabrata that require acetate for complete growth. Mutants with altered activity of PDH bypass, preferably decreased activity of PDC but increased activity of ACS, are expected to be obtained by using this approach. Such a mutant is anticipated to produce more pyruvate when acetate is supplemented as a cosubstrate. The parent strain, T. glabrara WSH-IP303, grew equally well either in MM or in CM. T. glabrara WSH-IP303 was treated by NTG, after which 45 mutants that exhibited poor growth on MM plates but grew better than the parent strain on CM plates, were obtained. Table 1 shows the amount of pyruvate produced by these mutants in CM, from which, 26 mutants produced pyruvate higher than the parent strain. For the 14 mutants that produced more than 43 g l)1 pyruvate, the specific PDC activity was decreased by roughly 11–41%. One of the mutants, WSH-LQ307, produced the highest concentration of pyruvate and exhibited the strongest genetic stability within generations (Table 2). This mutant was chosen as an ideal working strain for further study. It is preserved at the China Center for Type Culture Collection (CCTCC, Wuhan, China), and designated as CCTCC M202019. Table 1 Pyruvate producing ability of mutants with sodium acetate as carbon source* Pyruvate production (g l)1) No. of mutants 19–29 30–34 35–39 40–42 43–45 ‡46 2 7 10 12 11 3 *Pyruvate production by the parent strain, WSH-IP303, is 38Æ3 ± 0Æ5 g l)1. Growth comparison of T. glabrata WSH-IP303 and T. glabrata CCTCC M202019 The effect of acetate on the growth of T. glabrata WSHIP303 (parent) and T. glabrata CCTCC M202019 (mutant) was examined. With acetate as the sole carbon source, none or very poor growth (only 0Æ28 g DCW l)1) was observed in the culture of the parent strain WSH-IP303, while 2Æ34 g DCW l)1 was achieved in that of the mutant strain CCTCC M202019, conceiving that hardly could the parent strain use acetate as the sole carbon source to grow. Subsequently, the effect of acetate concentration on the growth of parent and mutant strain was tested. As shown in Fig. 1, the cell concentration of strain CCTCC M202019 grown on MM was approx. 28% lower than that of the parent strain. However, the growth of strain CCTCC M202019 on CM was approx. 21Æ7% higher than that of the parent strain, suggesting that acetate plays an important role in the growth of strain CCTCC M202019. Activities of PDH, PDC, ALDH, ACS, ADH and PC of mutant CCTCC M202019 which requires acetate for complete growth Compared with the parent strain, the mutant strain CCTCC M202019 exhibited a decreased growth on MM but an increased growth on CM. This indicates some metabolic changes might have occurred in pyruvate metabolism pathways, as pyruvate metabolism is normally correlated with the growth of yeast cells on a defined medium. To characterize the metabolic changes, the activities of pyruvate metabolism-related enzymes (i.e. PDH, PDC, ALDH, ACS, ADH and PC, shown in Fig. 2) of the mutant strain CCTCC M202019 and the parent strain WSH-IP303 grown on MM and CM were examined. As shown in Table 3, the specific activities of PDH complex and PC of the mutant strain CCTCC M202019 were identical to that of parent strain, independent of the medium used. However, significant changes of enzyme activities were observed in PDH bypass, where ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 39, 199–206, doi:10.1111/j.1472-765X.2004.01563.x MANIPULATING THE PYRUVATE DEHYDROGENASE BYPASS 203 Table 2 Genetic stability of selected mutants grown on CM* First generation Third generation Eighth generation Strain Pyruvate (g l)1) PDC activity (nmol min)1 mg)1) Pyruvate (g l)1) PDC activity (nmol min)1 mg)1) Pyruvate (g l)1) PDC activity (nmol min)1 mg)1) WSH-IP303 WSH-LQ204 WSH-LQ208 WSH-LQ214 WSH-LQ303 WSH-LQ306 WSH-LQ307 WSH-LQ312 38Æ3 44Æ7 44Æ4 43Æ2 45Æ7 44Æ4 46Æ2 43Æ8 15Æ4 12Æ0 11Æ9 13Æ7 9Æ8 13Æ1 9Æ1 10Æ1 37Æ5 39Æ7 40Æ2 38Æ6 44Æ9 41Æ5 46Æ7 39Æ1 15Æ3 13Æ1 14Æ6 16Æ2 10Æ1 13Æ5 9Æ2 12Æ3 38Æ7 36Æ7 38Æ4 37Æ3 45Æ2 36Æ7 46Æ8 35Æ4 15Æ4 15Æ8 15Æ0 16Æ8 9Æ8 15Æ4 9Æ2 14Æ9 ± ± ± ± ± ± ± ± 0Æ4 0Æ2 0Æ3 0Æ4 0Æ2 0Æ1 0Æ1 0 ± ± ± ± ± ± ± ± 0Æ2 0Æ3 0Æ4 0Æ2 0Æ2 0Æ3 0Æ1 0Æ2 ± ± ± ± ± ± ± ± 0Æ2 0Æ4 0Æ2 0Æ3 0Æ2 0Æ3 0Æ2 0Æ2 ± ± ± ± ± ± ± ± 0Æ3 0Æ2 0Æ2 0Æ1 0Æ4 0Æ2 0Æ2 0Æ0 ± ± ± ± ± ± ± ± 0Æ2 0Æ3 0Æ4 0Æ2 0Æ3 0Æ2 0Æ2 0Æ3 ± ± ± ± ± ± ± ± 0Æ3 0Æ2 0Æ3 0Æ3 0Æ2 0Æ3 0Æ3 0Æ2 *Mean ± S.D. (n ¼ 3). Strain WSH-IP303 is the parent strain. Glucose (a) 16 PEP DCW (g l–1) 12 ¢ Pyruvate 8 Bio ¢ Acetaldehyde B1 ¢ ¢ Ethanol ¢ NA 4 ¢ Acetyl-CoA Acetate Oxalacetate 0 0 10 20 30 40 50 60 Citrate Time (h) TCA Cycle (b) 16 α -KG DCW (g l–1) 12 Fig. 2 The metabolism of pyruvate in Torulopsis glabrata. B1: thiamine; NA: nicotine acid; Bio: biotin; I: pyruvate decarboxylase (PDC); II: alcohol dehydrogenase (ADH); III: acetaldehyde dehydrogenase (ALDH); IV: acetyl-CoA synthetases (ACS); V: pyruvate dehydrogenase (PDH) complex; VI: pyruvate carboxylase (PC) 8 4 0 0 10 20 30 40 50 60 Time (h) Fig. 1 Effect of acetate (6 g l)1) on cell growth. Growth media: (a) MM, (b) CM. e, Parent strain WSH-IP303; r, mutant strain CCTCC M202019 (i) the specific activity of PDC of the mutant strain CCTCC M202019 grown either on CM or MM reduced by roughly 40% compared with that of the parent strain; (ii) the specific activities of ACS of the mutant strain CCTCC M202019 grown on CM or MM were 103Æ5 or 57Æ4% higher than that of the parent strain, respectively. The specific activities of ALDH and ADH of the parent strain were almost unaltered upon mutation (Table 3). Most interestingly, the specific ACS activity of the mutant strain CCTCC M202019 grown on CM was 31% higher than that grown on MM, while the specific activities of PDH, PC, PDC, ALDH and ADH of the mutant strain CCTCC M202019 were not affected upon acetate addition. ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 39, 199–206, doi:10.1111/j.1472-765X.2004.01563.x Enzyme (lmol min)1 mg)1) Parent strain IP 303 Mutant CCTCC M202019 CM CM MM PDH (10)2) 9Æ86 ± 0Æ12 9Æ74 ± 0Æ1 15Æ24 ± 0Æ2 15Æ04 ± 0Æ1 PDC (10)2) ALDH (10)2) 7Æ12 ± 0Æ1 8Æ04 ± 0Æ07 3Æ69 ± 0Æ1 3Æ64 ± 0Æ2 ACS (10)2) 1Æ41 ± 0Æ05 1Æ36 ± 0Æ04 ADH (10)1) 9Æ52 ± 0Æ1 9Æ83 ± 0Æ1 PC (10)1) *Mean ± S.D. 9Æ79 9Æ07 7Æ92 7Æ51 1Æ38 9Æ34 Glucose(g l–1), Pyruvate(g l–1) Table 3 Comparison of PDH, PDC, AIDH, ACS, ADH, and PC activities of parent and mutant strains* MM ± ± ± ± ± ± 0Æ2 0Æ3 0Æ2 0Æ15 0Æ08 0Æ06 9Æ81 9Æ13 8Æ12 5Æ73 1Æ41 9Æ86 ± ± ± ± ± ± 0Æ1 0Æ2 0Æ1 0Æ10 0Æ04 0Æ07 (n ¼ 3). 11 2 14 96 12 80 10 64 8 48 6 32 4 16 2 Effect of acetate concentration on growth of T. glabrata and pyruvate production 0 Pyruvate fermentation by strain CCTCC M202019 in 7-l jar fermentor The time course of pyruvate fermentation under optimal conditions, using the MM supplemented with 6 g l)1 sodium acetate is presented in Fig. 4. Pyruvate concentra16 0 0 As the mutant strain CCTCC M202019 produced 21% higher pyruvate than the parent strain (eighth generation, Table 2) in a glucose medium supplemented with acetate, the effect of acetate concentration (the carbon content of 1 g l)1 sodium acetate equals to approx. 0Æ7 g l)1 glucose) on cell growth and pyruvate production was studied to determine the optimal acetate concentration (Fig. 3). A linear relationship was observed between DCW and acetate concentration, while the maximum yield and concentration of pyruvate, 0Æ586 g g)1 and 46Æ8 g l)1 respectively, were achieved when 6 g l)1 sodium acetate was added to MM. However, the concentration and yield of pyruvate decreased when sodium acetate higher than 6 g l)1 was added to MM. DCW(g l–1), Acetate(g l–1) 204 L . - M . L I U ET AL. 14 28 42 Time (h) 56 70 Fig. 4 Time-course of pyruvate production by strain CCTCC M202019. , glucose concentration; e, DCW; m, pyruvate concentration; ·, acetate concentration tion reached 68Æ7 g l)1 at 62 h, achieving a yield on glucose of 0Æ651 g g)1. In the process, the ethanol concentration was <0Æ8 g l)1, in contrast to 5Æ7 g l)1 carried by parent strain. In this study, strain CCTCC M202019 proved to be a superior strain to produce pyruvate. DISCUSSION In yeast, respiratory dissimilation of pyruvate is initiated by its conversion into acetyl-CoA, the precursor metabolite of the TCA cycle (Pronk et al. 1996). This can occur in two ways: via a direct reaction catalysed by the mitochondrial PDH complex or via an indirect route involving PDC, ALDH and ACS, which is frequently referred to the PDH 50 90 0·7 8 0·3 0·2 4 80 45 70 40 60 Pyruvate (g l–1) 0·4 Glucose (g l–1) 0·5 Yield (g g–1) DCW (g l–1) 0·6 12 0·1 0 0 2 4 6 8 Concentration of acetate (g l–1) 10 0 50 0 2 4 6 8 35 10 Concentration of acetate (g l–1) Fig. 3 Effect of acetate concentration on the production of pyruvate and growth of strain CCTCC M202019. Culture time was 48 h as indicated in the Materials and methods. , DCW; s, pyruvate yield on glucose; m, pyruvate concentration; n, consumption of glucose ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 39, 199–206, doi:10.1111/j.1472-765X.2004.01563.x MANIPULATING THE PYRUVATE DEHYDROGENASE BYPASS bypass (Pronk et al. 1996). During glucose respiratory growth, the PDH complex is primarily responsible for the conversion of pyruvate into acetyl-CoA. However, a small flux through the PDH bypass is essential during growth on a minimum medium, the role of which is to provide cytosolic acetyl-CoA for lipid synthesis (Pronk et al. 1996). In a previous study (Liu et al. 2004), the further degradation of pyruvate was prevented by, preferably, the limited addition of thiamine, which is a cofactor of the PDH complex and the PDC in T. glabrata (Fig. 2). However, a certain amount of ethanol was still produced at the expense of pyruvate, due to the presence of thiamine-activated PDC. Constitutive disruption of PDC biosynthesis is anticipated to contribute to the further increase of pyruvate yield, but the activity of PDH bypass might also be affected, leading to a shortage of cytosolic acetyl-CoA and a decrease of cell growth on a glucose minimum medium afterwards. The disruption of PDC biosynthesis would decrease cell growth unless the activity of ACS, converting acetate into acetyl-CoA, could be enhanced (Fig. 2). In that case, the cytosolic acetyl-CoA might be compensated by assimilating acetate, which can be provided by its addition to the medium. In the present study, a mutant strain CCTCC M202019, which required acetate for complete growth and produced higher concentration of pyruvate, was isolated. The observation that the addition of acetate enhanced the growth of the mutant strain CCTCC M202019 (the activity of PDC decreased by 40%) suggests that PDC might play an important role in supplying cytosolic acetyl-CoA in T. glabrata cells, which cannot be fulfilled by the mitochondrial PDH complex. Further enzyme analysis of pathways of the mutant strain CCTCC M202019 revealed that the activity of ACS, the key enzyme in PDH bypass, increased 103Æ5% than that of the parent strain when grown on CM, whereas the activity of PDH complex, PC, ALDH and ADH were not affected. It is proposed that the enhanced activity of ACS in T. glabrata facilitated the assimilation of acetate to acetyl-CoA. Part of the cytosolic acetyl-CoA could be transported into the mitochondrion via carnitine-acetyl transferase shuttle (Chase et al. 1965) to channel into the TCA cycle. On the other hand, as the PDC biosynthesis of the mutant strain CCTCC M202019 was not completely disrupted, the mutant strain could still grow on MM, mainly as a consequence of that the activity of PDH bypass was not completely lost but only decreased. A 40% decrease on the activity of PDC in the mutant strain CCTCC M202019 led to a decrease of ethanol production from 5Æ7 g l)1 to <0Æ8 g l)1. As a consequence, a yield of pyruvate on glucose of 0Æ651 g g)1, was achieved after cultivating the mutant strain CCTCC M202019 in a 7-l fermentor for 62 h, with 6 g l)1 acetate as the supplemented carbon source (Fig. 4). These results validate the original idea of further increasing pyruvate yield by manipulating the 205 activity of PDH bypass, namely, decreasing the activity of PDC and increasing the activity of ACS. 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