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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. Most importantly,
this is the first report showing that the increased activity of
ACS in T. glabrata facilitates the assimilation of acetate,
which provides an alternative pathway for the supply of
cytosolic acetyl-CoA. Previously we have reported the
crucial role of thiamine in pyruvate production (Li et al.
2001b), presumably due to that the thiamine-controlled
PDH complex and PDC are normally considered as the
exclusive origin of acetyl-CoA. The increased supply of
cytosolic acetyl-CoA (and probably part of the mitochondrial acetyl-CoA) by the enhanced activity of ACS may
permit to reduce the dependence of pyruvate production on
a critical thiamine concentration, which is of industrial
importance.
ACKNOWLEDGEMENTS
This research was partially supported by the Natural
Science Foundation of Jiangsu Province of China (contract
No. BK2002072). Y. Li was supported by the Open Project
Program of the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology of
China. L.-M. Liu was supported by the Post-graduate
Innovative Program of Jiangsu Province of China. The
authors thank Prof. G.-C. Du, for his suggestions within the
research process.
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