ARTICLE
Fumaric Acid Production by Torulopsis glabrata:
Engineering the Urea Cycle and the Purine
Nucleotide Cycle
Xiulai Chen,1,2,3 Jing Wu,1,2,3 Wei Song,1,2,3 Limei Zhang,1,2,3 Hongjiang Wang,1,2,3
Liming Liu1,2,3
1
State Key Laboratory of Food Science and Technology, Jiangnan University,
Wuxi 214122, China
2
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University,
Wuxi 214122, China
3
Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University,
Wuxi 214122, China; telephone: þ86-510-85197875; fax: þ86-510-85197875;
e-mail: mingll@jiangnan.edu.cn
ABSTRACT: A multi-vitamin auxotrophic Torulopsis glabrata
strain, a pyruvate producer, was further engineered to
produce fumaric acid. Using the genome-scale metabolic
model iNX804 of T. glabrata, four fumaric acid biosynthetic
pathways, involving the four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL),
fumarylacetoacetase (FAA), and fumarase (FUM1), were
found. Athough single overexpression of each of the four
enzymes in the cytosol improved fumaric acid production,
the highest fumaric acid titer (5.62 g L1) was obtained with
strain T.G-ASL(H)-ADSL(L) by controlling the strength of ASL
at a high level and ADSL at a low level. In order to further
improve the production of fumaric acid, the SpMAE1 gene
encoding the C4-dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H)-ADSL(L)-SpMAE1 and the final
fumaric acid titer increased to 8.83 g L1. This study provides
a novel strategy for fumaric acid biosynthesis by utilizing the
urea cycle and the purine nucleotide cycle to enhance the
bridge between carbon metabolism and nitrogen metabolism.
Biotechnol. Bioeng. 2015;112: 156–167.
ß 2014 Wiley Periodicals, Inc.
Correspondence to: L. Liu
Contract grant sponsor: Major State Basic Research Development Program of China
Contract grant number: 2013CB733602
Contract grant sponsor: National Natural Science Foundation of China
Contract grant number: 31270079
Contract grant sponsor: Provincial outstanding youth foundation of Jiangsu Province
Contract grant number: BK2012002
Contract grant sponsor: Program for Innovative Research Team in University
Contract grant number: IRT1249
Contract grant sponsor: Fundamental Research Funds for the Central Universities
Contract grant number: JUDCF12027
Received 26 April 2014; Revision received 1 June 2014; Accepted 7 July 2014
Accepted manuscript online 24 July 2014;
Article first published online 10 October 2014 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.25334/abstract).
DOI 10.1002/bit.25334
156
Biotechnology and Bioengineering, Vol. 112, No. 1, January, 2015
KEYWORDS: fumaric acid; Torulopsis glabrata; argininosuccinate lyase; adenylosuccinate lyase; genome-scale
metabolic model
Introduction
Fumaric acid, a four-carbon dicarboxylic acid
(HO2CCHCHCO2H) that is widely used in the food,
pharmaceutical, and chemical industries, is a naturally
occurring organic acid (Roa Engel et al., 2008). The U.S.
Department of Energy has identified fumaric acid as one of
the top 12 biomass building block chemicals that can be
converted to high-volume products from renewable sources
of carbohydrate (Werpy and Petersen, 2004). Fumaric acid is
currently produced on a large scale by microbial fermentation, as this is an environment-friendly, inexpensive, and
sustainable development process. Naturally occurring fumaric acid producers, such as Rhizopus formosa, Rhizopus arrhizus,
and Rhizopus oryzae, have been isolated, evaluated, and
optimized as biocatalysts in the production of fumaric acid by
fermentation (Table I). R. formosa has the advantage of
requiring low-cost nutrients, but it can produce only
21.3 g L1 fumaric acid (Carta et al., 1999). R. arrhizus can
produce 38.0 g L1 fumaric acid with a low yield 0.33 g g1
(Riscaldati et al., 2000). R. oryzae is used as the main producer
of fumaric acid and has achieved a high titer and productivity
of 56.2 g L1 and 0.7 g L1 h1, respectively (Fu et al., 2010).
However, the fermentation process using a rotary reactor
proved too complicated for industrial applications (Xu
et al., 2012c).
Four metabolic engineering strategies have been investigated for producing fumaric acid, and they relate to three
ß 2014 Wiley Periodicals, Inc.
Table I. Comparison of fumaric acid production by natural and metabolically engineered microorganisms.
Strains
Fumaric acid
(g L1)
Yield
(g g1)
Productivity
(g L1 h1)
References
21.3
38.0
22.8
37.2
30.2
32.1
41.1
56.2
56.5
40.5
0.34
0.33
0.29
0.53
0.28
0.45
0.48
0.54
0.94
0.51
–
0.46
0.16
1.03
0.19
0.32
0.37
0.70
0.67
0.56
Carta et al. (1999)
Riscaldati et al. (2000)
Zhou et al. (2014)
Zhou et al. (2002)
Roa Engel (2010)
Kang et al. (2010)
Huang et al. (2010)
Fu et al. (2010)
Wang et al. (2013)
Gu et al. (2014)
28.2
25
3.18
1.67
5.64
8.83
0.39
0.78
0.05
0.03
0.11
0.15
0.45
0.26
0.03
0.02
0.06
0.12
Song et al. (2013)
Zhang et al. (2012)
Xu et al. (2012a)
Xu et al. (2012b)
Xu et al. (2013a)
This study
Natural producers
R. formosa
R. arrhizus
R. oryzae
Engineered strains
E. coli CWF812
R. oryzae ppc
S. cerevisiae FMME 001 "PYC2 þ "RoMDH
S. cerevisiae FMME 002 DFUM1 þ "RoPYC þ "SFC1
S. cerevisiae FMME 006 DFUM1 þ "RoPYC þ "RoMDH þ "RoFUM1
T. glabrata-ASL(H)-ADSL(L)-SpMAE1
microorganisms, Escherichia coli, R. oryzae, and Saccharomyces cerevisiae (Table I). First, fumaric acid has been
produced via the noncyclic glyoxylate route of strategy
(i) by combining deletion of the iclR, fumA, fumB and, fumC
genes to redirect the carbon flux through the glyoxylate
shunt, overexpression of the native ppc gene under the
strong tac promoter to enhance the reductive TCA
cycle flux and deletion of the arcA and ptsG genes to
reinforce the oxidative TCA cycle flux, the final E. coli strain
CWF812 allowed production of 28.2 g L1 fumaric acid
(Song et al., 2013). Next, strategy (ii) invokes a fumaric acid
biosynthetic pathway involving the reductive reactions of
the TCA. The carbon flux towards oxaloacetate may be
increased through overexpression of endogenous pyruvate
carboxylase and exogenous phosphoenolpyruvate carboxylase in R. oryzae. The resultant R. oryzae strain is capable of
producing about 25 g L1 fumaric acid (Zhang et al., 2012).
In addition, by overexpressing the exogenous RoMDH and
RoFUM1 genes and up-regulating the endogenous PYC2
gene, the final engineered S. cerevisiae strain produced
3.18 g L1 fumaric acid (Xu et al., 2012a). Then, strategy
(iii) relates fumaric acid production to the oxidation of
citrate via the TCA cycle. A final concentration of fumaric
acid of 1.67 g L1 was obtained by deletion of the FUM1
gene and overexpression of the RoPYC and SFC1 genes in S.
cerevisiae (Xu et al., 2012b). Finally, in strategy (iv), the
engineered S. cerevisiae FMME004-6 could produce up to
5.64 g L1 of fumaric acid through simultaneous use of
oxidative and reductive routes (Xu et al., 2013a).
The multi-vitamin auxotrophic Torulopsis glabrata is a wellestablished microorganism used for the industrial production of pyruvate as it affords three advantages: (i) higher
pyruvate production; (ii) higher glucose tolerance; and (iii)
higher acid tolerance than S. cerevisiae (Chen et al., 2013).
Additionally, the genome-scale metabolic model iNX804 of
Candida glabrata (or T. glabrata), consisting of 804 genes, 1287
reactions, and 1025 metabolites, has been reconstructed by
genome sequence annotation and biochemical data mining
(Xu et al., 2013b). Through model iNX804 a systematical
understanding of the physiology and cellular metabolism of
T. glabrata may be gained. Furthermore, it has been
successfully used to predict the bottleneck of malate
production (Chen et al., 2013). In this study, the fumaric
acid-related metabolic pathways were identified using model
iNX804 and the cytosolic fumaric acid biosynthetic pathways
were engineered to enhance fumaric acid production
(Fig. 1). The final engineered strain, T.G-ASL(H)-ADSL(L)SpMAE1, was able to produce > 8 g L1 of fumaric acid from
glucose.
Materials and Methods
Strains and Plasmids
The multi-vitamin auxotrophic T. glabrata CCTCC M202019
was screened for pyruvate production (Liu et al., 2004b). The
engineered yeast strains used in this study (Table II) were
derived from T. glabrata CCTCC M202019. T. glabrata
CCTCC M202019 Dura3 (T.GDura3) and T. glabrata CCTCC
M202019 Dura3Darg8 (T.GDura3Darg8), screened by our
laboratory, were used as host strains for gene overexpression
(Zhou et al., 2009). E. coli JM109 and shuttle plasmids
pYX212, pY26, pY16, and pY2X were used for plasmid
construction.
Isolation of the Fumarate Biosynthesis Genes
The argininosuccinate lyase (ASL, 1386 bp), adenylosuccinate
lyase (ADSL, 1449 bp), fumarylacetoacetase (FAA, 786 bp) and
fumarase (FUM1, 1452 bp) genes were amplified by PCR using
the chromosomal DNA of T. glabrata CCTCC M202019 as
template.
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
157
Figure 1. The fumaric acid-related metabolic pathways leading to the formation
of fumarate in the cytosol. PYR: pyruvate; OXAL: oxaloacetate; FUM: fumaric acid; MAL:
malate; GLU: glutamate; ASP: aspartate; CIT: citrulline; GTP: guanosine triphosphate;
ARGSUC: argininosuccinate; ARG: arginine; FUMACAC: fumarylacetoacetate; ACAC:
acetoacetate; ADESUC: adenylosuccinate; AMP: adenosine monophosphate; IMP:
hypoxanthine nucleotide; TTP Metabolism: tyrosine, tryptophan and phenylalanine
metabolism. (A) fumarase; (B) argininosuccinate lyase; (C) fumarylacetoacetase;
(D) adenylosuccinate lyase.
with the MiniBEST Plasmid Purification Kit Ver. 2.0
(Takara). E. coli JM109 transformations were performed as
described by (Inoue et al., 1990). T. glabrata transformations
were performed as described by (Zhou et al., 2009). After
transformation of the T.GDura3 strain with the plasmids, the
yeast strains were plated onto solid medium A. Plasmids
pYX212, pYX212(PTPI)-ASL, pYX212(PTPI)-ADSL, pYX212
(PTPI)-FAA, pYX212(PTPI)-FUM1, pY16(PTEF)-ASL, pY26
(PGPD)-ASL, pY26(PTEF)-ASL, pY16(PTEF)-ADSL, pY26
(PGPD)-ASDL, pY26(PTEF)-ASDL, pY16(PTEF)-ASL-(PTEF)ADSL, pY26(PGPD)-ASL-(PTEF)-ADSL, and pY26(PTEF)ASL-(PGPD)-ADSL were transformed into the T.GDura3
strain by electroporation and yielded strains T.G-212, T.G212ASL, T.G-212ADSL, T.G-212FAA, T.G-212FUM1, T.GASL(L), T.G-ASL(M), T.G-ASL(H), T.G-ADSL(L), T.G-ADSL(M),
T.G-ADSL(H), T.G-ASL(L)-ADSL(L), T.G-ASL(M)-ADSL(H), T.GASL(H)-ADSL(M), respectively. Furthermore, plasmids pY16
(PTEF)-ASL and pY2X(PGPD)-ADSL, pY16(PTEF)-ASL and
pY2X(PTEF)-ADSL, pY2X(PGPD)-ASL and pY16(PTEF)-ADSL,
pY26(PGPD)-ASL and pY2X(PGPD)-ADSL, pY2X(PTEF)-ASL and
pY16(PTEF)-ADSL, pY26(PTEF)-ASL and pY2X(PTEF)-ADSL,
pY2X(PTEF)-ASL-(PGPD)-SpMAE1 and pY16(PTEF)-ADSL,
were simultaneously introduced into T.GDura3Darg8, resulting in strains T.G-ASL(L)-ADSL(M), T.G-ASL(L)-ADSL(H),
T.G-ASL(M)-ADSL(L), T.G-ASL(M)-ADSL(M), T.G-ASL(H)ADSL(L),
T.G-ASL(H)-ADSL(H),
T.G-ASL(H)-ADSL(L)SpMAE1, respectively.
Media
Plasmid Construction and Transformation
The plasmids and primers used in this work were given in
Tables II and III, respectively. The ASL, ADSL, FAA, and FUM1
genes were amplified from the chromosomal DNA of
T. glabrata CCTCC M202019 using the primers BamHI5’ASL&3’ASL-HindIII, EcoRI-5’ADSL&3’ADSL-HindIII,
BamHI-5’FAA&3’FAA-HindIII, and BamHI-5’FUM1&3’FUM1-HindIII, respectively. First, the PCR fragments
(ASL, FAA, and FUM1 genes) were cut at the introduced
BamHI and HindIII sites and ligated to the multicopy
plasmid pYX212 that was digested with BamHI and
HindIII, resulting in pYX212(P TPI )-ASL, pYX212(P TPI )FAA, and pYX212(P TPI )-FUM1, respectively. In the same
way, the plasmids pYX212(P TPI )-ADSL, pY16(P TEF )ASL-(P TEF )-ADSL, pY16(P TEF )-ASL, pY16(P TEF )-ADSL,
pY26(P GPD )-ASL, pY26(P TEF )-ASL, pY26(P GPD )-ASDL,
pY26(P TEF )-ASDL, pY26(P GPD )-ASL-(P TEF )-ADSL, pY26
(P TEF )-ASL-(P GPD )-ADSL, pY2X(P GPD )-ADSL, pY2XpY2X(P GPD )-ASL,
pY2X(P TEF )-ASL,
(P TEF )-ADSL,
pY2X(P TEF )-ASL-(P GPD )-SpMAE1, were constructed.
Restriction endonucleases and the DNA Ligase Kit Ver. 2.0
were used according to the instructions supplied by the
manufacturer (Takara). DNA fragments were separated by
electrophoresis in a 1% (w/v) agarose gel in Tris-borate/
EDTA. Plasmids were amplified in E. coli JM109 and isolated
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Biotechnology and Bioengineering, Vol. 112, No. 1, January, 2015
Medium A: the minimal medium (pH 6.0) used for
screening contained (per liter) 20 g glucose, 7 g NH4Cl, 5 g
KH2PO4, 0.8 g MgSO4-7H2O, 3 g sodium acetate, 32 mg
thiamine-HCl, 80 mg biotin, 0.8 mg pyridoxine-HCl, and
16 mg nicotinic acid. Medium B: the medium used for
fermentation contained (per liter) 60 g glucose, 7 g NH4Cl,
5 g KH2PO4, 0.8 g MgSO4-7H2O, 6.6 g K2SO4, 3 g sodium
acetate, 12 mg thiamine-HCl, 30 mg biotin, 0.4 mg pyridoxine-HCl, and 8 mg nicotinic acid. All vitamins were filtersterilized before addition to the medium. CaCO3 was
sterilized by dry-heating sterilization at 160 C for 30 min
before use as a pH buffer.
Culture Conditions
The seed culture inoculated from a slant was cultivated on a
reciprocal shaker (200 rpm) at 30 C in a 250 mL flask
containing 25 mL medium A for 24 h. The broth was
centrifuged, the supernatant liquid was removed and
discarded, the pellet was suspended in demineralized water,
and the cell suspension was divided equally between flasks
containing 25 mL fresh medium B with an initial biomass dry
weight of 1 g L1. Fermentation was performed in a 500 mL
flask containing 50 mL medium B. The medium was buffered
by the addition of 60 g L1 CaCO3 followed by fermentation
at 30 C for 72 h with rotation at 200 rpm and all experiments
were performed in triplicate.
Table II. Torulopsis glabrata strains and plasmids used in this study.
Strains and plasmids
Strains
T.GDura3
T.GDura3Darg8
T.G-212
T.G-212ASL
T.G-212ADSL
T.G-212FAA
T.G-212FUM1
T.G-ASL(L)
T.G-ASL(M)
T.G-ASL(H)
T.G-ADSL(L)
T.G-ADSL(M)
T.G-ADSL(H)
T.G-ASL(L)-ADSL(L)
T.G-ASL(L)-ADSL(M)
T.G-ASL(L)-ADSL(H)
T.G-ASL(M)-ADSL(L)
T.G-ASL(M)-ADSL(M)
T.G-ASL(M)-ADSL(H)
T.G-ASL(H)-ADSL(L)
T.G-ASL(H)-ADSL(M)
T.G-ASL(H)-ADSL(H)
T.G-ASL(H)-ADSL(L)-SpMAE1
Plasmids
pYX212
pYX212(PTPI)-ASL
pYX212(PTPI)-ADSL
pYX212(PTPI)-FAA
pYX212(PTPI)-FUM1
pY16
pY16(PTEF)-ASL-(PTEF)-ADSL
pY16(PTEF)-ASL
pY16(PTEF)-ADSL
pY26
pY26(PGPD)-ASL
pY26(PTEF)-ASL
pY26(PGPD)-ADSL
pY26(PTEF)-ADSL
pY26(PGPD)-ASL-(PTEF)-ADSL
pY26(PTEF)-ASL-(PGPD)-ADSL
pY2X
pY2X(PGPD)-ADSL
pY2X(PTEF)-ADSL
pY2X(PGPD)-ASL
pY2X(PTEF)-ASL
pY2X(PTEF)-SpMAE1
pY2X(PTEF)-ASL-(PGPD)-SpMAE1
Relevant characteristics
References
CCTCC M202019Dura3
CCTCC M202019Dura3Darg8
CCTCC M202019Dura3 (pYX212)
CCTCC M202019Dura3 (pYX212(PTPI)-ASL)
CCTCC M202019Dura3 (pYX212(PTPI)-ADSL)
CCTCC M202019Dura3 (pYX212(PTPI)-FAA)
CCTCC M202019Dura3 (pYX212(PTPI)-FUM1)
CCTCC M202019Dura3 (pY16(PTEF)-ASL)
CCTCC M202019Dura3 (pY26(PGPD)-ASL)
CCTCC M202019Dura3 (pY26(PTEF)-ASL)
CCTCC M202019Dura3 (pY16(PTEF)-ADSL)
CCTCC M202019Dura3 (pY26(PGPD)-ADSL)
CCTCC M202019Dura3 (pY26(PTEF)-ADSL)
CCTCC M202019Dura3
(pY16(PTEF)-ASL-(PTEF)-ADSL)
CCTCC M202019Dura3Darg8
(pY16(PTEF)-ASL, pY2X(PGPD)-ADSL)
CCTCC M202019Dura3Darg8
(pY16(PTEF)-ASL, pY2X(PTEF)-ADSL)
CCTCC M202019Dura3Darg8
(pY2X(PGPD)-ASL, pY16(PTEF)-ADSL)
CCTCC M202019Dura3Darg8
(pY2X(PGPD)-ASL, pY26(PGPD)-ADSL)
CCTCC M202019Dura3
(pY26(PGPD)-ASL-(PTEF)-ADSL)
CCTCC M202019Dura3Darg8
(pY2X(PTEF)-ASL, pY16(PTEF)-ADSL)
CCTCC M202019Dura3
(pY26(PTEF)-ASL-(PGPD)-ADSL)
CCTCC M202019Dura3Darg8
(pY26(PTEF)-ASL, pY2X(PTEF)-ADSL)
CCTCC M202019Dura3Darg8
(pY2X(PTEF)-ASL-(PGPD)-SpMAE1, pY16(PTEF)-ADSL)
Zhou et al. (2009)
Zhou et al. (2009)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
2 mm, Amp, URA3, PTPI
2 mm, Amp, URA3, PTPI-ASL
2 mm, Amp, URA3, PTPI-ADSL
2 mm, Amp, URA3, PTPI-FAA
2 mm, Amp, URA3, PTPI-FUM1
CEN6/ARSH4, Amp, URA3, PTEF
CEN6/ARSH4, Amp, URA3, PTEF-ASL, PTEF-ADSL
CEN6/ARSH4, Amp, URA3, PTEF-ASL
CEN6/ARSH4, Amp, URA3, PTEF-ADSL
2 mm, Amp, URA3, PGPD, PTEF
2 mm, Amp, URA3, PGPD-ASL, PTEF
2 mm, Amp, URA3, PGPD, PTEF-ASL
2 mm, Amp, URA3, PGPD-ADSL, PTEF
2 mm, Amp, URA3, PGPD, PTEF-ADSL
2 mm, Amp, URA3, PGPD-ASL, PTEF-ADSL
2 mm, Amp, URA3, PGPD-ASDL, PTEF-ASL
2 mm, Amp, ARG8, PGPD, PTEF
2 mm, Amp, ARG8, PGPD-ADSL, PTEF
2 mm, Amp, ARG8, PGPD, PTEF-ADSL
2 mm, Amp, ARG8, PGPD-ASL, PTEF
2 mm, Amp, ARG8, PGPD, PTEF-ASL
2 mm, Amp, ARG8, PGPD, PTEF-SpMAE1
2 mm, Amp, ARG8, PGPD-SpMAE1, PTEF-ASL
Xu et al. (2010)
This study
This study
This study
This study
Lab collection
This study
This study
This study
Xu et al. (2012a)
This study
This study
This study
This study
This study
This study
Chen et al. (2013)
This study
This study
This study
This study
Chen et al. (2013)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
159
Table III. Primers used in this study for gene clone and plasmid construction.
Primers
Sequence
0
0
5 -CGCGGATCCATGTCAGGTCAAGCTAAAACAAAACTT-30
50 -CCCAAGCTTTTATAACTTCAAGCTCTTTAATTGAGCGAG-30
50 -GGAATTCATGTCTGAGTTTGATAAGTACGCT-30
50 -CCCAAGCTTTCAAACATTTAGTTTAACTTCAGC-30
50 -CGCGGATCCATGAGCTACACTTACCTGAAGGACG-30
50 -CCCAAGCTTCTAAGTTTCTTTGTATTCGTAAGGACC-30
50 -CGCGGATCCATGTTGAGGGCTAGCAGTAGG-30
50 -CCCAAGCTTTTACTTGGTTGGTCCAACCATG-30
50 -AAGGAAAAAAGCGGCCGCATGTCAGGTCAAGCTAAAACAAAACTT-30
50 -TCCCCGCGGTTATAACTTCAAGCTCTTTAATTGAGCGAG-30
50 -AAGGAAAAAAGCGGCCGCATGTCTGAGTTTGATAAGTACGCT-30
50 -TCCCCGCGGTCAAACATTTAGTTTAACTTCAGC-30
BamHI-5 ASL
30 ASL-HindIII
EcoRI-50 ADSL
30 ADSL-HindIII
BamHI-50 FAA
30 FAA-HindIII
BamHI-50 FUM1
30 FUM1-HindIII
NotI-50 ASL
30 ASL-SacII
NotI-50 ADSL
30 ADSL-SacII
Analytical Methods
The optical absorbance at 660 nm (A660) was converted to dry
cell weight (DCW) according to a predetermined calibration
curve (Liu et al., 2004b):
A660 : DCW ¼ 1 : 0:23ðgL1 Þ
The culture supernatant samples were filtered through
0.22 mm films. Extracellular concentrations of glucose,
pyruvate, and fumaric acid were determined by highperformance liquid chromatography (HPLC) as described
by (Xu et al., 2012a). The pellets of 10 mL culture broth
including cells (OD660 ¼ 9–10) were used to extract
intracellular metabolites. AMP was determined using
HPLC as described in previous reports (Gusarova
et al., 2011). Arginine was determined by HPLC as described
by (Xu et al., 2011).
GFP Expression Analysis
Strains were grown overnight in 25 mL medium B, diluted to
OD660 ¼ 0.1 in the same medium and grown for additional
hours. The GFP fluorescence intensity of 10,000 yeast cells
was measured as described by (Sadeh et al., 2012).
cycles, the samples were centrifuged (10,000 g, 15 min,
10 C); the supernatants were collected separately, and the
pellets were re-extracted with 2.5 mL precooled 80% (v/v)
methanol by vortex for 30 s. After centrifugation, the
supernatants were pooled with the first extracts, and the
combined extracts were stored at 80 C until further use.
Preparation of Cell Extracts and Enzyme Activity Assays
Cell extracts were prepared for the determination of enzyme
activity (Liu et al., 2004a). ASL activity was determined by the
rate of urea synthesis from argininosuccinic acid in the
presence of excess arginase as described by (Shih et al., 1969).
ADSL activity was determined by measurement of the
absorbance at 282 nm (A282) as described by (Lee et al., 1997).
FAA activity was determined by hydrolyzing fumarylacetoacetate as described previously (Kvittingen and
Brodtkorb, 1986). FUM1 activity produced with L-malate
as the substrate was determined by measuring L-malate
consumption at 250 nm (A250) as described by (Xu
et al., 2012a). Protein concentrations in cell extracts were
determined by the Lowry method using bovine serum
albumin as the standard (Lowry et al., 1951).
Results
General Computational Protocol for In silico Prediction of
Metabolic Pathways
The fumaric acid-related metabolic pathways were searched
using the genome-scale metabolic model iNX804 for C.
glabrata (or T. glabrata) reconstructed by our laboratory
(Xu et al., 2013b).
Intracellular Metabolite Extraction
Samples were taken with a specialized rapid-sampling setup
as described (Canelas et al., 2008). Intracellular metabolites
were extracted by freezing-thawing in methanol as described
(Canelas et al., 2009). Each sample was re-suspended in
2.5 mL of 80% (v/v) aqueous methanol precooled to 40 C;
then, the resulting solution was frozen in liquid nitrogen for
5 min and thawed on ice for 3–5 min. After five freeze-thaw
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Metabolic Pathways for Fumarate Production
Ten fumaric acid-related metabolic reactions are contained in
T. glabrata GSMM iNX804, which participate in five branches
(Table IV): (i) amino acid metabolism; (ii) nucleic acid
biosynthesis; (iii) oxidative phosphorylation; (iv) citric acid
cycle; and (v) mitochondrial transport reaction. The cytosolic
reactions that involve branches (i), (ii), and part of (iii) are
catalyzed by FUM1, ASL, ADSL, and FAA (Table IV). FUM1,
ASL, and ADSL belong to a superfamily of hydrolases, which
share a homotetrameric structure and a similar catalytic
mechanism (Trevisson et al., 2009). FUM1 participates in the
TCA cycle and catalyzes the reversible dehydration of Lmalate to fumaric acid (Fig. 1A) (Xu et al., 2012c). ASL
participates in the urea cycle and catalyzes the reversible
breakdown of argininosuccinate to arginine and fumaric acid
Arginine and Proline Metabolism
Purine and Pyrimidine Biosynthesis
Tyrosine, Tryptophan, and Phenylalanine Metabolism
Oxidative Phosphorylation
Transport, Mitochondrial
Transport, Mitochondrial
Oxidative Phosphorylation
Oxidative Phosphorylation
Oxidative Phosphorylation
Citric Acid Cycle
argsuc[c] < ¼ > arg-L[c] þ fum[c]
adesuc[c] < ¼ > amp[c] þ fum[c]
4fumacac[c] þ h2o[c] -> acac[c] þ fum[c] þ h[c]
mal-L[c] < ¼ > fum[c] þ h2o[c]
fadh2[m] þ fum[c] -> fad[m] þ succ[c]
fum[m] þ succ[c] -> fum[c] þ succ[m]
fum[m] þ h2o[m] < ¼ > mal-L[m]
fadh2[m] þ fum[m] -> fad[m] þ succ[m]
fad[m] þ succ[m] < ¼ > fadh2[m] þ fum[m]
q6[m] þ succ[m] < ¼ > fum[m] þ q6h2[m]
Effect of Single Gene Overexpression on Fumarate
Production
Abbreviations: c, cytosol; m, mitochondrion.
CAGL0I08987g
CAGL0B02794g
CAGL0J06204g
CAGL0A01045g
CAGL0I01320g
CAGL0M09020g
CAGL0A01045g
CAGL0I01320g
CAGL0L01177g
CAGL0C03223g; CAGL0D01958g;
CAGL0E03850g; CAGL0F05863g;
CAGL0J00847g
ASL
ADSL
FAA
FUM1
SYGP
SFC1
FUM1
OSM1
SDH
SDH
argininosuccinate lyase
adenylosuccinate lyase
fumarylacetoacetase
fumarase
fumarate reductase, cytosolic/mitochondrial
succinate-fumarate transport, mitochondrial
fumarase, mitochondrial
fumarate reductase
succinate dehydrogenase
succinate dehydrogenase
Subsystem
Formula
Reactions description
GO
Gene
Table IV. The fumaric acid-related metabolic reactions identified using T. glabrata model iNX804.
(Fig. 1B) (Trevisson et al., 2009). ADSL participates in the
purine nucleotide cycle and catalyzes two reactions: the
conversion of succinylaminoimidazole carboxamide ribotide
to aminoimidazole carboxamide ribotide and fumaric acid
and the conversion of adenylosuccinate to AMP and fumaric
acid (Fig. 1D) (Lee and Colman, 2007). However, FAA
belongs to the fumarylacetoacetase family, participates in the
catabolism of tyrosine, tryptophan, and phenylalanine in the
cytosol and catalyzes the hydrolysis of fumarylacetoacetate to
acetoacetate and fumaric acid (Fig. 1C) (Mizutani and
Kunishima, 2007).
Because of these highlights, ASL, ADSL, and FAA make a
bridge between carbon metabolism and nitrogen metabolism,
respectively. In addition, FUM1 has been used for fumaric acid
production (Xu et al., 2012a; Xu et al., 2012b), whereas ASL,
ADSL, and FAA have not. Therefore, the enzymes FUM1, ASL,
ADSL, and FAA, which are encoded by CAGL0A01045g,
CAGL0I08987g, CAGL0B02794g, and CAGL0J06204g, respectively,
were selected for study.
Overexpression of the ASL or ADSL gene in the cytosol
channeled more pyruvate flux to fumaric acid production.
The specific activities of ASL and ADSL in strains T.G212ASL and T.G-212ADSL were 0.124 0.041 U (mg
protein)1 and 2.107 0.500 U (mg protein)1, approximately 7-fold and 5-fold higher, respectively, than that of the
control strain T.G-212 (Fig. 3). These increases in specific
activities were associated with large decreases in the pyruvate
titer (Fig. 2A) and significant increases in the fumaric acid
titer (Fig. 2B). The final fumaric acid titers were elevated to
0.96 g L1 and 0.61 g L1 in the strains T.G-212ASL and T.G212ADSL, respectively (Fig. 2B). In addition, this single
modification in ASL or ADSL in the cytosol improved cell
growth and accelerated the glucose consumption rate. DCW
was increased by 32.8% and 36.2% for strains T.G-212ASL
and T.G-212ADSL, respectively, compared to the control
strain T.G-212 (Fig. 2C). The glucose consumption rates for
strains T.G-212ASL and T.G-212ADSL were 1.7-fold and 1.3fold higher, respectively, than that of the control strain T.G212 (Fig. 2D). These improvements indicated that the single
modification redirected more pyruvate flux to the TCA cycle
and improved fumaric acid production through the urea
cycle or the purine nucleotide cycle.
However, a single overexpression of FUM1 or FAA resulted
in an increase in fumaric acid titer (Fig. 2B) and a decrease
in pyruvate production (Fig. 2A) that was less than that of
either strain T.G-212ASL or T.G-212ADSL, respectively.
FUM1 and FAA activities of the strains T.G-212FUM1 and
T.G-212FAA increased by about 6.0-fold and 4.5-fold,
respectively, compared to the control strain T.G-212
(Fig. 3). The improved activity resulted in minor increases
in cell growth (Fig. 2C) and glucose consumption rate
(Fig. 2D). All of these slight improvements indicated that
the pyruvate flux could not be more effectively redirected to
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
161
Figure 2.
The concentrations of metabolites in shake flask cultures of T.G-212 (&), T.G-212FAA (), T.G-212ADSL (D), T.G-212ASL ( ), and T.G-212FUM1 ( ).
fumaric acid by overexpressing FUM1 or FAA compared to
overexpression of ASL or ADSL, suggesting that further
stepwise improvement could be made through overexpression of ASL and ADSL.
Figure 3.
The specific activity of ASL, ADSL, FAA, and FUM1 in different
engineered T. glabrata strains. T.G-212FAA (overexpressing FAA), T.G-212ADSL
(overexpressing ADSL), T.G-212ASL (overexpressing ASL), and T.G-212FUM1 (overexpressing FUM1).
162
Biotechnology and Bioengineering, Vol. 112, No. 1, January, 2015
Effect of ASL and ADSL Overexpression on Fumarate
Production
In order to further investigate the effect of different strengths
of a single gene on fumaric acid production, the gene
expression strengths were divided into three levels using the
GFP reporter, high level (H) under TEF promoter in plasmids
pY26 or pY2X, mediate level (M) under GPD promoter
in plasmids pY26 or pY2X, and low level (L) under TEF
promoter in plasmid pY16 (Fig. 5A). Therefore, the strength
of ASL and ADSL were controlled at various levels by different
combinations of promoter and plasmid. When the strength
of ASL was kept at a high level in all combinations, the
concentration of fumaric acid up to 1.23 g L1 was obtained
with strain T.G-ASL(H), approximately 28% higher than that
of the engineered strain T.G-212ASL (Fig. 4). In addition, the
strength of ADSL at a low level improved the fumaric acid titer
to 1.82 g L1 with strain T.G-ADSL(L), corresponding to a
4.3-fold increase compared to that of the engineered strain T.
G-212ADSL (Fig. 4). These results indicated that fumaric
acid production was significantly improved by optimizing the
gene expression strengths, suggesting that fumaric acid titers
could be further enhanced by simultaneously controlling the
ASL and ADSL at different levels.
A single modification in ASL or ADSL improved the fumaric
acid titer up to 1.23 g L1 or 1.82 g L1, respectively. To
further fine-tune fumaric acid synthesis pathway, ASL and
Finally, controlling the strength of ASL at a high level and
ADSL at a low level obtained a large increase in fumaric acid
reaching 5.62 g L1 in the engineered strain T.G-ASL(H)ADSL(L) (Fig. 5C). In addition, this overexpression led to a
drastic decrease in the pyruvate titer (Fig. 6), while the DCW
of strain T.G-ASL(H)-ADSL(L) corresponded to a 1.14-fold
increase compared to that of the control strain T.G-212 and a
16.7% decrease compared to that of strain T.G-ASL(H)
(Fig. 6). These results indicated that, upon overexpression of
ASL and ADSL, pyruvate flux traveled through the TCA cycle
and was effectively channeled to fumaric acid via the urea
cycle and the purine nucleotide cycle.
Figure 4.
The effect of different strengths of a single gene on fumaric acid
production. A series of ASL or ADSL expression cassettes were designed at different
expression level: high level (H), mediate level (M), and low level (L).
ADSL were cytosolically overexpressed in T. glabrata at
different expression levels. Based on the above-mentioned
division for gene expression levels, a series of ASL and ADSL
expression cassettes were designed and assembled in T.
glabrata with various strengths to obtain the best metabolic
distribution for enhanced fumaric acid production (Fig. 5B).
Effect of SpMAE1 Overexpression on Fumarate
Production
To further improve fumaric acid production, intracellular
metabolites were determined for strains T.G-212, T.GASL(H), T.G-ADSL(L), and T.G-ASL(H)-ADSL(L). The concentrations of intracellular fumaric acid, arginine, and AMP
in strain T.G-ASL(H)-ADSL(L), were increased by 112.5%,
100.0% and 64.7%, respectively, compared with T.G-212,
whereas pyruvate was decreased by 28.0% (Table V).
Moreover, these four metabolites in strain T.G-ASL(H)ADSL(L) were also higher than that of the engineered strains
T.G-ASL(H) and T.G-ADSL(L). For example, the level of
Figure 5. (A) The gene expression was divided into three levels with GFP as reporter: high level (H), mediate level (M), and low level (L). (B) A series of ASL and ADSL expression
cassettes were designed at different expression level. (C) The concentrations of fumaric acid were achieved by different ASL and ADSL expression cassettes.
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
163
(Fig. 6). Furthermore, the pyruvate titer was reduced to
3.7 g L1, which was lower than that of strains T.G-ASL(H), T.
G-ADSL(L), and T.G-ASL(H)-ADSL(L) (Fig. 6). The concentration of intracellular pyruvate in T.G-ASL(H)-ADSL(L)SpMAE1 was decreased by 25.2% compared with T.GASL(H)-ADSL(L) (Table V). These results indicated that the
constructed pathway allowed fumaric acid to be generated
out of the pyruvate node and the C4-dicarboxylic acids
transporter effectively enabled the export of fumaric acid.
Discussion
Figure 6.
Concentrations of fumaric acid obtained in shake flask cultivation of
different strains of T. glabrata overexpressing combinations of ASL, ADSL, and
SpMAE1.
intracellular fumaric acid in strain T.G-ASL(H)-ADSL(L) was
34.2% and 27.5% higher than that of the engineered strains T.
G-ASL(H) and T.G-ADSL(L), respectively (Table V). These
increases suggested that the transport capacity of C4dicarboxylic acids probably needed to be enhanced to
transport cytosolic fumaric acid out of T. glabrata.
The gene SpMAE1 encodes the C4-dicarboxylic acids
transporter from S. pombe, which is used to effectively export
malate, fumaric acid, and succinate (Zelle et al., 2008). Given
this observation, the highest concentration of fumaric acid
(up to 8.83 g L1) was obtained with strain T.G-ASL(H)ADSL(L)-SpMAE1 in which the ASL, ADSL and SpMAE1 genes
were simultaneously overexpressed (Fig. 6). This dramatic
result corresponded to a 67.9-fold increase in the fumaric
acid titer compared to that of the control strain T.G-212 and a
1.57-fold increase compared to that of strain T.G-ASL(H)ADSL(L) (Fig. 6). The concentration of intracellular fumaric
acid was 0.28 mg/gDCW in T.G-ASL(H)-ADSL(L)-SpMAE1,
which was almost concordant with that of the control strain
T.G-212 (0.24 mg/gDCW) (Table V). In addition, the DCW
of strain T.G-ASL(H)-ADSL(L)-SpMAE1 was reduced by
11.9% compared to that of strain T.G-ASL(H)-ADSL(L),
which was similar to that of the control strain T.G-212
In order to effectively produce fumaric acid, all of the fumaric
acid biosynthetic pathways were searched using the T. glabrata
genome-scale metabolic model iNX804. Among those
pathways, the cytosolic biosynthesis pathways were selected
to compute the flux distribution and engineered to
investigate their influence on fumaric acid production.
Finally, the urea cycle and the purine nucleotide cycle were
demonstrated to be the most effective pathways for
producing fumaric acid. A high fumaric acid titer
(5.62 g L1) was obtained with strain T.G-ASL(H)-ADSL(L),
in which ASL and ADSL genes were simultaneously overexpressed by controlling the strength of ASL at a high level and
ADSL at a low level, respectively. In order to further increase
the production of fumaric acid, the SpMAE1 gene was
overexpressed in strain T.G-ASL(H)-ADSL(L)-SpMAE1 and
the final fumaric acid titer increased to 8.83 g L1. These
results lay a good foundation for further study of the fumaric
acid metabolic pathways.
The most pragmatic systems-based tool for metabolic
engineering is the in silico genome-scale metabolic model.
This tool has been widely adopted for searching metabolic
pathways, modeling cell growth, and predicting flux
distribution (Blazeck and Alper, 2010). In this study, T.
glabrata model iNX804 was used to search the fumaric acidrelated biosynthetic pathways and revealed 10 fumaric acidrelated metabolic reactions. These reactions may be divided
into three categories: cytosolic reactions, mitochondrial
reactions, and transport reactions. The cytosolic biosynthesis
pathways were selected for further research, due to the fact
that they had been successfully used for malate production in
T. glabrata (Chen et al., 2013) and utilized to produce fumaric
acid in S. cerevisiae (Zelle et al., 2008). A constraint-based
genome-scale metabolic model iNX804 was applied to
compute the flux distribution in the cytosolic reactions, in
Table V. The concentrations of intracellular metabolites in the engineered strains.
Strains
T.G-212
T.G-ASL(H)
T.G-ADSL(L)
T.G-ASL(H)-ADSL(L)
T.G-ASL(H)-ADSL(L)-SpMAE1
164
Pyruvate (mg/gDCW)
Fumaric acid (mg/gDCW)
Arginine (mg/gDCW)
AMP (mg/gDCW)
7.82 1.04
6.05 1.21
5.92 0.77
6.11 0.74
4.88 0.15
0.24 0.04
0.38 0.01
0.40 0.03
0.51 0.05
0.28 0.02
0.31 0.04
0.55 0.03
0.35 0.04
0.62 0.02
0.38 0.06
0.17 0.01
0.12 0.04
0.24 0.05
0.28 0.02
0.20 0.01
Biotechnology and Bioengineering, Vol. 112, No. 1, January, 2015
particular for ASL, ADSL, FAA, and FUM1. According to
model iNX804 simulated flux distribution combined with the
metabolic engineering results for ASL, ADSL, FAA and
FUM1, ASL, and ADSL were confirmed to be the most
effective pathways for producing fumaric acid. Moreover,
ASL in the urea cycle and ADSL in the purine nucleotide cycle
serve as a bridge between carbon metabolism and nitrogen
metabolism, respectively. Therefore, such observations
enhance the prevalent understanding of biosynthetic pathways for fumaric acid.
The urea cycle implicates ornithine, citrulline, and
arginine as participants in the synthesis of urea from
aspartate and carbon dioxide (Krebs, 1982). This cycle has
been identified in a variety of species, including E. coli and S.
cerevisiae (Yu and Howell, 2000). The main functions of the
urea cycle are the detoxification of waste nitrogen into
excretable urea and the de novo biosynthesis of arginine
(Brusilow et al., 2001). ASL catalyzes the reversible
hydrolysis of argininosuccinate to arginine and fumaric
acid, which is important for the detoxification of ammonia
via the urea cycle and for arginine biosynthesis (Yu and
Howell, 2000). Therefore, overexpression of ASL results in
a significant increase in the fumaric acid titer, probably due
to two aspects: (i) arginine, as the precursor for the
synthesis of many biologically important compounds,
including urea, polyamines, proline, and glutamate
(Nagamani et al., 2012), plays roles in physiology and
metabolism, including as an immediate precursor in
protein synthesis, in the post-translational conjugation
of arginine with the N-termini of proteins bearing Nterminal aspartate or glutamate, and as an allosteric
activator of N-acetylglutamate synthase (Wu and
Morris, 1998); (ii) fumaric acid, as an available TCA cycle
intermediate, can tightly regulate cellular metabolism as an
immediate precursor for protein synthesis, electron
acceptor, and conserver of energy during electron
transportation (Lambden and Guest, 1976).
In most organisms, the purine biosynthetic pathway is
nearly ubiquitous and results in the conversion of phosphoribosyl pyrophosphate to inosine 5’-monophosphate (IMP)
(Kappock et al., 2000). ADSL plays a critical role in both
cellular replication and metabolism via its action in the de
novo purine biosynthetic pathway, in which it catalyzes two
separate reactions, enabling it to participate in the addition of
a nitrogen atom at two different positions in adenosine
monophosphate (Toth and Yeates, 2000). These two separate
reactions are the conversion of succinyladenosine monophosphate to adenosine monophosphate (AMP) and the
conversion of succinylaminoimidazolecarboxamide ribonucleotide to aminoimidazolecarboxamide ribonucleotide
(Spiegel et al., 2006). Both reactions catalyzed by ADSL
involve the b-elimination of fumaric acid. Therefore, the
fumaric acid titer is substantially increased by overexpressing
ADSL, probably because the purine biosynthesis pathway not
only provides the majority of purine nucleotides needed for
cellular replication but also supplies amounts of available
citric acid cycle intermediates. The purine nucleotide cycle
aids in the tight regulation of cellular metabolism by
controlling both the amounts of available citric acid cycle
intermediates and the amount of free AMP (Toth and
Yeates, 2000).
In conclusion, the simultaneous overexpression of genes
ASL and ADSL can effectively enhance the connection between
carbon metabolism and nitrogen metabolism, improve the
carbon flux distribution and increase fumaric acid production. The final concentration of fumaric acid increased to
5.62 g L1 in the strain T.G-ASL(H)-ADSL(L) by controlling
the strength of ASL at a high level and ADSL at a low level,
probably due to two aspects: (i) controlling ASL at a high level
in the urea cycle is conducive to the detoxification of waste
nitrogen into excretable urea and the de novo biosynthesis of
fumaric acid and arginine (Brusilow et al., 2001); (ii)
controlling ADSL at a low level in the purine biosynthesis
pathway not only decreases the majority of purine
nucleotides needed for cellular replication relatively but
also increases amounts of available TCA cycle intermediates
fumaric acid (Toth and Yeates, 2000). However, when FUM1
or FAA was overexpressed in T. glabrata, the pyruvate flux
could not be more effectively redirected to fumaric acid
compared to overexpression of ASL or ADSL. The reasons may
be attributed to two factors: (i) FUM1, a key enzyme
participating in the TCA cycle, catalyzes the reversible
dehydration of malate to fumaric acid and exhibits higher
affinity towards fumaric acid than towards malate (Xu
et al., 2013a); (ii) FAA, participating in the catabolism of
tyrosine, tryptophan, and phenylalanine in the cytosol,
catalyzes the hydrolysis of fumarylacetoacetate to acetoacetate
and fumaric acid and is thought to be genotoxic (Cassiman
et al., 2009).
Finally, further stepwise improvement was made by
metabolic engineering based on overexpressing the SpMAE1
gene, thus achieving a higher fumaric acid titer of 8.83 g L1.
However, the engineered strain T.G-ASL(H)-ADSL(L)SpMAE1 still produced 3.68 g L1 pyruvate, indicating that
fumaric acid production can be further improved. With the
aid of the ‘omic’ techniques, systems metabolic engineering
emerges as a conceptual and technological framework to
speed the modification of existing pathways for the optimal
production of desired products, and has opened a novel
avenue for engineering microorganisms to produce industrial
products (Lee et al., 2012). For example, the bottleneck of
producing fumaric acid with the urea cycle and the purine
nucleotide cycle can be analyzed using transcriptome,
proteome, and metabolomics profiling. After that, modular
pathway engineering, systematic assembly, and optimization
of metabolic modules is used for fine tuning existent
pathways and balancing the metabolism of production hosts
to debottleneck, debug, and improve cellular phenotypes
(Juminaga et al., 2012).
References
Blazeck J, Alper H. 2010. Systems metabolic engineering: Genome-scale
models and beyond. Biotechnol J 5(7):647–659.
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
165
Brusilow S, Horwich A, Scriver C, Beaudet A, Sly W, Valle D. 2001.
The metabolic and molecular bases of inherited disease. 8th edn.
McGraw-Hill. New York: 1909–1963.
Canelas AB, Ras C, ten Pierick A, van Dam JC, Heijnen JJ, van Gulik WM.
2008. Leakage-free rapid quenching technique for yeast metabolomics.
Metabolomics 4(3):226–239.
Canelas AB, ten Pierick A, Ras C, Seifar RM, van Dam JC, van Gulik WM,
Heijnen JJ. 2009. Quantitative evaluation of intracellular metabolite
extraction techniques for yeast metabolomics. Anal Chem 81(17):7379–
7389.
Carta FS, Soccol CR, Ramos LP, Fontana JD. 1999. Production of fumaric
acid by fermentation of enzymatic hydrolysates derived from cassava
bagasse. Bioresour Technol 68(1):23–28.
Cassiman D, Zeevaert R, Holme E, Kvittingen EA, Jaeken J. 2009.
A novel mutation causing mild, atypical fumarylacetoacetase
deficiency (Tyrosinemia type I): A case report. Orphanet J Rare Dis
4:28.
Chen X, Xu G, Xu N, Zou W, Zhu P, Liu L, Chen J. 2013. Metabolic
engineering of Torulopsis glabrata for malate production. Metab Eng
19:10–16.
Fu YQ, Xu Q, Li S, Chen Y, Huang H. 2010. Strain improvement of Rhizopus
oryzae for over-production of fumaric acid by reducing ethanol synthesis
pathway. Korean J Chem Eng 27(1):183–186.
Gu S, Xu Q, Huang H, Li S. 2014. Alternative respiration and fumaric acid
production of Rhizopus oryzae. Appl Microbiol Biotechnol 98(11):5145–
5152.
Gusarova GA, Trejo HE, Dada LA, Briva A, Welch LC, Hamanaka RB, Mutlu
GM, Chandel NS, Prakriya M, Sznajder JI. 2011. Hypoxia leads to Na,
K-ATPase downregulation via Ca2þ release-activated Ca2þ channels and
AMPK activation. Mol Cell Biol 31(17):3546–3556.
Huang L, Wei PL, Zang R, Xu ZN, Cen PL. 2010. High-throughput screening
of high-yield colonies of Rhizopus oryzae for enhanced production of
fumaric acid. Ann Microbiol 60(2):287–292.
Inoue H, Nojima H, Okayama H. 1990. High efficiency transformation of
Escherichia coli with plasmids. Gene 96(1):23–28.
Juminaga D, Baidoo EE, Redding-Johanson AM, Batth TS, Burd H,
Mukhopadhyay A, Petzold CJ, Keasling JD. 2012. Modular engineering
of L-tyrosine production in Escherichia coli. Appl Environ Microbiol
78(1):89–98.
Kang SW, Lee H, Kim D, Lee D, Kim S, Chun GT, Lee J, Kim SW, Park C.
2010. Strain development and medium optimization for fumaric acid
production. Biotechnol Bioproc E 15(5):761–769.
Kappock TJ, Ealick SE, Stubbe J. 2000. Modular evolution of the purine
biosynthetic pathway. Curr Opin Chem Biol 4(5):567–572.
Krebs HA. 1982. The discovery of the ornithine cycle of urea synthesis.
Trends Biochem Sci 7(2):76–78.
Kvittingen E, Brodtkorb E. 1986. The pre-and post-natal diagnosis of
tyrosinemia type I and the detection of the carrier state by assay of
fumarylacetoacetase. Scand J Clin Lab Inv. 184:35.
Lambden P, Guest J. 1976. Mutants of Escherichia coli K12 unable to use
fumarate as an anaerobic electron acceptor. J Gen Microbiol 97(2):
145–160.
Lee JW, Na D, Park JM, Lee J, Choi S, Lee SY. 2012. Systems metabolic
engineering of microorganisms for natural and non-natural chemicals.
Nat Chem Biol 8(6):536–546.
Lee P, Colman RF. 2007. Expression, purification, and characterization of
stable, recombinant human adenylosuccinate lyase. Protein Expr Purif
51(2):227–234.
Lee TT, Worby C, Dixon JE, Colman RF. 1997. Identification of His(141) in
the active site of Bacillus subtilis adenylosuccinate lyase by affinity labeling
with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5’-monophosphate. J
Biol Chem 272(1):458–465.
Liu LM, Li Y, Li HZ, Chen J. 2004a. Manipulating the pyruvate
dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis
glabrata enhanced pyruvate production. Lett Appl Microbiol 39(2):199–
206.
Liu LM, Li Y, Li HZ, Chen J. 2004b. Manipulating the pyruvate
dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis
166
Biotechnology and Bioengineering, Vol. 112, No. 1, January, 2015
glabrata enhanced pyruvate production. Lett Appl Microbiol 39(2):199–
206.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement
with the Folin phenol reagent. J Biol Chem 193(1):265–275.
Mizutani H, Kunishima N. 2007. Purification, crystallization and preliminary
X-ray analysis of the fumarylacetoacetase family member TTHA0809
from Thermus thermophilus HB8. Acta Cryst F63:792–794.
Nagamani SC, Erez A, Lee B. 2012. Argininosuccinate lyase deficiency.
Genet Med 14(5):501–507.
Riscaldati E, Moresi M, Federici F, Petruccioli M. 2000. Direct ammonium
fumarate production by Rhizopus arrhizus under phosphorous limitation.
Biotechnol Lett 22(13):1043–1047.
Roa Engel C. 2010. Integration of fermentation and cooling crystallisation to
produce organic acids. Delft University of Technology: 87–108.
Roa Engel CA, Straathof AJ, Zijlmans TW, van Gulik WM, van der Wielen LA.
2008. Fumaric acid production by fermentation. Appl Microbiol
Biotechnol 78(3):379–389.
Sadeh A, Baran D, Volokh M, Aharoni A. 2012. Conserved motifs in the
Msn2-activating domain are important for Msn2-mediated yeast stress
response. J Cell Sci 125:3333–3342.
Shih VE, Littlefield JW, Moser HW. 1969. Argininosuccinase deficiency
in fibroblasts cultured from patients with argininosuccinic aciduria.
Biochem Genet 3(1):81–83.
Song CW, Kim DI, Choi S, Jang JW, Lee SY. 2013. Metabolic engineering of
Escherichia coli for the production of fumaric acid. Biotechnol Bioeng
110:2025–2034.
Spiegel EK, Colman RF, Patterson D. 2006. Adenylosuccinate lyase deficiency.
Mol Genet Metab 89(1–2):19–31.
Toth EA, Yeates TO. 2000. The structure of adenylosuccinate lyase, an enzyme
with dual activity in the de novo purine biosynthetic pathway. Structure
8(2):163–174.
Trevisson E, Burlina A, Doimo M, Pertegato V, Casarin A, Cesaro L, Navas P,
Basso G, Sartori G, Salviati L. 2009. Functional complementation in
yeast allows molecular characterization of missense argininosuccinate
lyase mutations. J Biol Chem 284(42):28926–28934.
Wang G, Huang D, Qi H, Wen J, Jia X, Chen Y. 2013. Rational medium
optimization based on comparative metabolic profiling analysis to
improve fumaric acid production. Bioresour Technol 137:1–8.
Werpy T, Petersen G. 2004. Top value added chemicals from biomass:
Volume I Results of screening for potential candidates from sugars and
synthesis gas. University of Pennsylvania Law Review 54(3):477.
Wu G, Morris SM Jr. 1998. Arginine metabolism: Nitric oxide and beyond.
Biochem J 336(Pt1):1–17.
Xu G, Chen X, Liu L, Jiang L. 2013a. Fumaric acid production in
Saccharomyces cerevisiae by simultaneous use of oxidative and reductive
routes. Bioresour Technol 148:91–96.
Xu G, Liu L, Chen J. 2012a. Reconstruction of cytosolic fumaric acid
biosynthetic pathways in Saccharomyces cerevisiae. Microb Cell Fact 11:24.
Xu G, Zou W, Chen X, Xu N, Liu L, Chen J. 2012b. Fumaric acid production
in Saccharomyces cerevisiae by in silico aided metabolic engineering. PLoS
One 7(12):52086.
Xu M, Rao Z, Xu H, Lan C, Dou W, Zhang X, Xu H, Jin J, Xu Z. 2011.
Enhanced production of L-arginine by expression of Vitreoscilla
hemoglobin using a novel expression system in Corynebacterium crenatum.
Appl Biochem Biotechnol 163(6):707–719.
Xu N, Liu L, Zou W, Liu J, Hua Q, Chen J. 2013b. Reconstruction and analysis
of the genome-scale metabolic network of Candida glabrata. Mol Biosyst
9(2):205–216.
Xu Q, Li S, Huang H, Wen J. 2012c. Key technologies for the industrial
production of fumaric acid by fermentation. Biotechnol Adv
30(6):1685–1696.
Xu S, Zhou J, Qin Y, Liu L, Chen J. 2010. Water-forming NADH oxidase
protects Torulopsis glabrata against hyperosmotic stress. Yeast 27(4):207–
216.
Yu B, Howell PL. 2000. Intragenic complementation and the structure and
function of argininosuccinate lyase. Cell Mol Life Sci 57(11):1637–1651.
Zelle RM, de Hulster E, van Winden WA, de Waard P, Dijkema C, Winkler
AA, Geertman JM, van Dijken JP, Pronk JT, van Maris AJ. 2008. Malic
acid production by Saccharomyces cerevisiae: Engineering of pyruvate
carboxylation, oxaloacetate reduction, and malate export. Appl Environ
Microbiol 74(9):2766–2777.
Zhang B, Skory CD, Yang ST. 2012. Metabolic engineering of Rhizopus oryzae:
Effects of overexpressing pyc and pepc genes on fumaric acid
biosynthesis from glucose. Metab Eng 14(5):512–520.
Zhou J, Dong Z, Liu L, Du G, Chen J. 2009. A reusable method for
construction of non-marker large fragment deletion yeast auxotroph
strains: A practice in Torulopsis glabrata. J Microbiol Methods 76(1):
70–74.
Zhou Y, Du J, Tsao GT. 2002. Comparison of fumaric acid production by
Rhizopus oryzae using different neutralizing agents. Bioprocess Biosyst
Eng 25(3):179–181.
Zhou Y, Nie K, Zhang X, Liu S, Wang M, Deng L, Wang F, Tan T. 2014.
Production of fumaric acid from biodiesel-derived crude glycerol by
Rhizopus arrhizus. Bioresour Technol 163C:48–53.
Chen et al.: Fumaric Acid Production by Torulopsis glabrata
Biotechnology and Bioengineering
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