Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli.

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

共表达烟酸转磷酸核糖激酶和丙酮酸羧化酶对大肠杆菌NZN111产丁二酸的影响

构建了共表达烟酸转磷酸核糖激酶(NAPRTase)和丙酮酸羧化酶(PYC)的重组质粒pTrc99a-pncB-pyc,并考察了重组菌E.coli NZN111/pTrc99a-pncB-pyc生产丁二酸的能力。结果表明:重组菌NZN111/pTrc99a-pncB-pyc的NAPRTase和PYC的比酶活达到最高,分别为20.75和1.04 U/mg,同时,辅酶NADH、NAD+及NAD(H)总量达到最高。厌氧摇瓶发酵结果:48 h能够消耗17.5 g/L的葡萄糖生成14.08 g/L的丁二酸,而丙酮酸的产量大幅度降低,仅为0.11 g/L。本研究为基因工程菌大肠杆菌厌氧条件下发酵生产丁二酸提供了一定的基础。     

Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant.

NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

High-level succinic acid production and yield by lactose-induced expression of phosphoenolpyruvate carboxylase in ptsG mutant Escherichia coli

Escherichia coli strains with foreign genes under the isopropyl-β-d-thiogalactopyranoside-inducible promoters such as lac, tac, and trc were engineered and considered as the promising succinic acid-producing bacteria in many reports. The promoters mentioned above could also be induced by lactose, which had not been attempted for succinic acid production before. Here, the efficient utilization of lactose as inducer was demonstrated in cultures of the ptsG, ldhA, and pflB mutant strain DC1515 with ppc overexpression. A fermentative process for succinic acid production at high level by this strain was developed. In flask anaerobic culture, 14.86 g l⁻¹ succinic acid was produced from 15 g l⁻¹ glucose with a yield of 1.51 mol mol⁻¹ glucose. In two-stage culture carried out in a 3-l bioreactor, the overall yield and concentration of succinic acid reached to 1.67 mol mol⁻¹ glucose and 99.7 g l⁻¹, respectively, with a productivity of 1.7 g l⁻¹ h⁻¹ in the anaerobic stage. The efficient utilization of lactose as inducer made recombinant E. coli a more capable strain for succinic acid production at large scale.     

烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc,构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。     

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.     

Metabolic engineering of Escherichia coli for the production of succinic acid from glucose

Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.     

Alanine production in an H+-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H⁺-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164–2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H⁺-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H⁺-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.     

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.     

过量表达烟酸单核苷酸腺苷酰转移酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因(ldhA)和丙酮酸-甲酸裂解酶的编码基因(pflB)的发酵生产丁二酸的潜力菌株。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。nadD为催化NAD(H)合成途径中烟酸单核苷酸(NaMN)生成烟酸腺嘌呤二核苷酸(NaAD)的烟酸单核苷酸腺苷酰转移酶(Nicotinic acid mononucleotide adenylyltransferase,NAMNAT)的编码基因,通过过量表达nadD基因能够提高NAD(H)总量与维持合适的NADH/NAD+比例。文中构建了重组菌E.coli NZN111/pTrc99a-nadD,在厌氧摇瓶发酵过程中通过添加终浓度为1.0 mmol/L的IPTG诱导表达,重组菌E.coli NZN111/pTrc99a-nadD中NAD+和NADH的浓度分别比宿主菌E.coli NZN111提高了3.21倍和1.67倍,NAD(H)总量提高了2.63倍,NADH/NAD+从0.64降低为0.41,使重组菌株恢复了厌氧条件下生长和代谢葡萄糖的能力。重组菌与对照菌相比,72 h内可以消耗14.0 g/L的葡萄糖产6.23 g/L的丁二酸,丁二酸产量增加了19倍。     

过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111厌氧发酵的主要产物为丁二酸,是发酵生产丁二酸的潜力菌株。但是由于敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸甲酸裂解酶的编码基因 (pflB),导致辅酶NADH/NAD+不平衡,厌氧条件下不能利用葡萄糖生长代谢。构建烟酸转磷酸核糖激酶的重组菌Escherichia coli NZN111/pTrc99a-pncB,在厌氧摇瓶发酵过程中通过添加0.5 mmol/L的烟酸、0.3 mmol/L的IPTG诱导后重组菌的烟酸转磷酸核糖激酶 (Nicotinic acid phosphor     

进化代谢选育高浓度NH_4~+耐受型产丁二酸大肠杆菌

利用大肠杆菌厌氧制备丁二酸过程中,采用氨水作为p H调节剂不仅可以中和酸性产物还可提供无机氮,被菌体利用,然而高浓度NH_4~+的积累会抑制菌体生长及代谢产酸的能力。为增强大肠杆菌对高浓度NH_4~+的耐受性,以(NH4)2HPO4为NH_4~+供体,通过在连续培养装置中不断提高(NH_4)_2HPO4浓度,以获得可耐受0.53 mol/L NH_4~+的产丁二酸大肠杆菌。结果表明:突变株在0.53 mol/L NH_4~+胁迫下,摇瓶厌氧发酵72 h,细胞干质量浓度(DCW)可达1.82 g/L,丁二酸产量为11.72 g/L,分别比出发菌株提高了1.6和4.6倍。进一步地,在5 L发酵罐上考察其利用氨水调节p H生产丁二酸的能力,厌氧发酵90 h,丁二酸质量浓度达到27.32 g/L,生产强度为0.30g/(L·h),比出发菌株分别提高88.1%和87.5%。     

Anaerobic synthesis of succinic acid by Escherichia coli strains with activated NAD+ reducing pyruvate dehydrogenase complex

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.     

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.     

Expression of an anaplerotic enzyme,pyruvate carboxylase,improves recombinant protein production in Escherichia coli

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, beta-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.     

Efficient production of heat-labile enterotoxin mutant proteins by overexpression of dsbA in a degP-deficient Escherichia coli strain

Escherichia coli heat-labile enterotoxin (LT) mutants containing Val⁶⁰→Gly or Ser¹¹⁴→Lys substitutions in the A subunit do not produce the A subunit efficiently in E. coli. These mutants accumulate mostly the B pentamer devoid of the A subunit in the periplasmic space. Here we show that overproduction of the periplasmic chaperone DsbA, which is involved in disulfide bond formation, in a strain deficient in the periplasmic protease DegP allows efficient production of the mutant LT molecules. Our results suggest that the formation of the oligomeric toxin is influenced by DsbA, which helps protein folding, and by DegP, which removes the folded intermediates that can be untoxic for the cell. Received: 30 October 1996 / Accepted: 8 January 1997