Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains

Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for sucrose uptake and catalyzation. Succinic acid production by this modified strain and the SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic acid efficiently from all combinations of different carbon sources tested with minimal byproduct formation and with high molar succinate yields close to that of the maximum theoretic values. The molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a 1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of succinic acid by our metabolic engineered E. coli strain.     

Succinate production from sucrose by metabolic engineered Escherichia coli strains under aerobic conditions

Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.     

Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains

This study presents an in-depth analysis of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase (PYC) for the production of succinate. Previously, a metabolic network design that includes an active glyoxylate pathway implemented in vivo increased succinate yield from glucose in an E. coli mutant to 1.6 mol/mol under fully anaerobic conditions. The design consists of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). Mutant strains previously constructed during the development of high-yield succinate-producing strains were selected for further characterization to understand their metabolic response as a result of several genetic manipulations and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate. Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios to the glyoxylate pathway and the fermentative pathway at the oxaloacetate (OAA) node as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The most favorable split ratio to obtain the highest succinate yield was the fractional partition of OAA to glyoxylate of 0.32 and 0.68 to the fermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG (pHL413). The succinate yields achieved in these two strains were 1.6 and 1.7 mol/mol, respectively. In addition, an active glyoxylate pathway in an ldhA, adhE, ack-pta mutant strain is shown to be responsible for the high succinate yields achieved anaerobically. Furthermore, in vitro activity measurements of seven crucial enzymes involved in the pathways studied and intracellular measurements of key intermediate metabolite pools provided additional insights on the physiological perturbations caused by these mutations. The characterization of these recombinant mutant strains in terms of flux distribution pattern, in vitro enzyme activity and intracellular metabolite pools provides useful information for the rational modification of metabolic fluxes to improve succinate production.     

Effect of culture operating conditions on succinate production in a multiphase fed-batch bioreactor using an engineered <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> strain

A metabolically engineered Escherichia coli strain SBS550MG (pHL413) was used in this study to investigate the impact of various culture operating conditions for improving the specific succinate production rate for better final titer while maintaining the theoretical succinate yield on glucose in multiphase fed-batch cultures. Previously, we reported that changes in the level of aeration during the cell growth phase significantly modified gene expression profiles and metabolic fluxes in this system (Martinez et al. 2010). Based on these observations, the examination of culture conditions was mainly focused on the aerobic growth phase. It was found that 2–5 h of low dissolved oxygen culture during the aerobic phase improves cell productivity, but pH control during the aerobic phase was not favorable for the system. Cell viability has been identified as a major limiting factor for succinate production. Supplementing LB medium and betaine, an anti-osmotic stress reagent, did not improve cell activity. A higher succinate titer (537.8 mM) using the current metabolic engineering E. coli strain was achieved, which can potentially be improved further by increasing cell viability.     

Metabolic Analysis of Escherichia coli in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase

Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc⁺), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc⁺) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc⁺ strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc⁺ strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.     

Identification of Pyruvate Carboxylase Genes in Pseudomonas aeruginosa PAO1 and Development of a P. aeruginosa-Based Overexpression System for α4- and α4β4-Type Pyruvate Carboxylases

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the α₄ enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis α₄ PYC and Pseudomonas aeruginosa α₄β₄ PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa Δpyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The α₄β₄ PYC of this organism was encoded by the ORFs PA5436 and PA5435.     

Expression of Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli without Affecting Glucose Uptake

Plasmids carrying the pyc gene from Rhizobium etli were used to express pyruvate carboxylase in Escherichia coli. Results of batch fermentations of a wild-type E. coli (MG1655), this wild-type with the pUC18 cloning/expression vector (MG1655/pUC18) and this wild-type carrying the pyc gene (MG1655/pUC18-pyc) were compared in glucose-limited medium. The results indicate that the final succinate concentration upon complete glucose utilization was increased from 1.18 g/L to 1.77 g/L by the expression of pyc, while the final succinate concentration in MG1655/pUC18 was slightly lower than in the parent strain. This increased succinate concentration came at the expense of lactate synthesis, whose final concentration decreased from 2.33 g/L to 1.88 g/L. The expression of pyc did not affect the maximum glucose uptake (2.17 g/Lh for MG1655 versus 2.47 g/Lh for MG1655/pUC18-pyc), but did decrease the maximum rate of cell mass production (0.213 g/Lh for MG1655, 0.169 g/Lh for MG1655/pUC18 and 0.199 g/Lh for MG1655/pUC18-pyc).     

Culture conditions' impact on succinate production by a high succinate producing Escherichia coli strain

This work aimed to identify the key operational factors that significantly affect succinate production by the high succinate producing Escherichia coli strain SBS550MG (pHL413), which bears mutations inactivating genes adhE ldhA iclR ackpta::Cm(R) and overexpresses the pyruvate carboxylase from Lactococcus lactis. The considered factors included glucose concentration, cell density, CO(2) concentration in the gas stream, pH, and temperature. The results showed that high glucose concentrations inhibited succinate production and that there is a compromise between the total succinate productivity and succinate specific productivity, where the total productivity increased with the increase in cell density and the specific productivity decreased with cell density, probably due to mass transfer limitation. On the other hand, a CO(2) concentration of 100% in the gas stream showed the highest specific succinate productivity, probably by favoring pyruvate carboxylation, increasing the OAA pool that later is converted into succinate. A full factorial design of experiments was applied to analyze the pH and temperature effects on succinate production in batch bioreactors, where succinate yield was not significantly affected by either temperature (37 to 43°C) or pH (6.5 to 7.5). Additionally, the temperature effect on succinate productivity and titer was not significant, in the range tested. On the other hand, a pH of 6.5 showed very low productivity, whereas pH values of 7.0 and 7.5 resulted in significantly higher specific productivities and higher titers. The increase on pH value from 7.0 to 7.5 did not show significant improvement. Then, pH 7.0 should be chosen because it involves a lower cost in base addition.     

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E_550-535) of 19.7 ± 6.3 mM⁻¹ cm⁻¹ and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 ± 122 μmol min⁻¹ mg⁻¹ protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.     

Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD⁺. Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD⁺ regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD⁺ as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA‐pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25°C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by Metabolically Engineered Strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.     

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38 ± 0.64 × 10^?5. The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75 ± 2.7% and 5–6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45 mM of NaAsO₂. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.     

Anaerobic fermentation of Salmonella typhimurium with and without pyruvate carboxylase

A plasmid that expressed pyruvate carboxylase (PYC) from Rhizobium etli was introduced into Salmonella typhimurium LT2. Anaerobic fermentations of S. typhimurium with and without PYC were compared with glucose as a carbon source. The presence of PYC increased the succinate yield from glucose from 0.044 g g^–1 to 0.22 g g^–1, while the lactate yield decreased from 0.31 g g^–1 to 0.16 g g^–1. Metabolic flux calculations during the early growth phase indicate that under these growth conditions in the presence of PYC more carbon flows to oxaloacetate via pyruvate carboxylase than via phosphoenolpyruvate carboxylase. Also, under these growth and induction conditions, the presence of PYC diminished the cell growth rate from 0.34 h^–1 to 0.28 h^–1, the specific rate of ATP formation from 45 mmol l^–1 h^–1 to 27 mmol l^–1 h^–1, and the specific rate of glucose consumption from 17 mmol l^–1 h^–1 to 10 mmol l^–1 h^–1.     

Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli

Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.