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.     

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     

基于辅因子调控对大肠杆菌两阶段发酵产丁二酸的影响

考察了E.coli NZN111及其重组菌株E.coli NZN111/pTrc99a-pncB发酵生产丁二酸的性能。E.coli NZN111两阶段发酵丁二酸的同时,会造成丙酮酸的大量积累。研究发现:通过过量表达烟酸转磷酸核糖激酶,两阶段发酵重组菌株E.coli NZN111/pTrc99a-pncB,减少丙酮酸的积累且无副产物乙酸生成,提高丁二酸的产量,丁二酸得率和耗糖速率分别提高了139%和20%。     

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.     

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     

产丁二酸工程菌的构建及其厌氧发酵

为了考察过量表达苹果酸酶对于E.coli NZN111(ldhA::Kan pfl::Cam)厌氧发酵产丁二酸的影响, 将连接有苹果酸酶基因sfcA的表达载体pTrc99a-sfcA转化进NZN111中, 构建了重组NZN111(pTrc99a-sfcA)。0.5 mmol/L IPTG诱导8 h后, 测定的苹果酸酶比酶活为30.67 u/mg, 比受体菌提高了140倍。采用两阶段发酵模式, 结果表明: 过量表达的苹果酸酶在NZN111体内催化了从丙酮酸到苹果酸的逆向反应, 丁二酸是发酵过程中积累的主要有机酸, 且当加入0.7 mmol/L IPTG诱导, 初始葡萄糖糖浓度为18.5 g/L时, 选择对数生长期后期的菌种以10%的接种量转入厌氧发酵, 发酵结束时发酵液中丁二酸的浓度为12.84 g/L, 对葡萄糖的收率为69.43%, 乙酸为0.58 g/L, 二者浓度比为22:1, 没有检测到甲酸和乳酸。构建的菌种具有高产丁二酸和副产物极少的优点, 在同类菌种中处于先进水平。     

碳源对重组大肠杆菌两阶段发酵产丁二酸的影响

研究了在好氧培养基中分别添加不同碳源对两阶段发酵菌体生长、酶活及代谢产物分布的影响,结果表明添加4mmol/L葡萄糖和12,54,80mmol/L乙酸钠均可以提高好氧阶段的菌体密度和相关酶活。将不同条件下培养的菌体转接厌氧发酵后,厌氧阶段的酶活和代谢产物分布也发生改变。进一步对酶活及代谢产物分析表明：Escherichia coli NZN111(sfcA)厌氧发酵过程中,磷酸烯醇式丙酮酸羧化激酶(PCK)是产丁二酸的关键酶,丙酮酸激酶(PYK)主要和副产物丙酮酸的积累有关,异柠檬酸裂解酶(ICL)对丁二酸产量也有一定影响。好氧培养基中添加80mmol/L乙酸钠,厌氧发酵结束时丁二酸的质量收率可达89.0%,相比对照提高了16.6%。     

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。     

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).     

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture

Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.     

Effect of aeration on the activity of gluconeogenetic enzymes in Saccharomyces cerevisiae growing under glucose limitation

1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.     

Enhanced anaerobic succinic acid production by Escherichia coli NZN111 aerobically grown on gluconeogenic carbon sources

Escherichia coli strain NZN111, a pflB and ldhA double mutant of E. coli W1485, is considered a candidate of succinic acid producer. However, it is reported that this strain fails to ferment glucose anaerobically. In this study, it was demonstrated that when a gluconeogenic carbon source was used to replace glucose in aerobic culture, the NZN111 cells restored the ability to ferment glucose in the subsequent anaerobic culture with succinic acid as the major product even though no further genetic manipulation had been carried out. Activities of enzymes including phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, isocitrate lyase, malate dehydrogenase, malic enzyme, and pyruvate kinase in the NZN111 cells aerobically grown on different carbon sources were measured, and enhanced anaplerotic and oxaloacetate-reducing activities were revealed. Furthermore, supply of MgCO₃ or NaHCO₃ greatly improved succinate production by the malate-grown NZN111 cells. At the same time, pyruvic acid production was significantly reduced. When the malate-grown cells were anaerobically cultured in a salt medium with high pH buffering capacity, succinic acid was produced at a specific productivity of 308 mg/(g DCW h) with a molar yield of 1.31 mol succinic acid/mol glucose.     

过量表达苹果酸酶对E.coli FMJ39厌氧混合酸发酵的影响

为了考察苹果酸酶对厌氧混合酸发酵影响,从E.coli DH5α中PCR扩增苹果酸酶（NAD+-dependent, E.C1.1.1.38）基因sfcA,插入质粒pTrc99a构建了表达质粒pTrc99a-sfcA,有氧和厌氧的条件下,IPTG诱导在E.coli FMJ39（ldh,pfl）中均获得大量表达,从而构建和加强了一条在厌氧混合酸发酵中微弱的代谢途径。厌氧发酵结果表明,过量表达苹果酸酶会影响混合酸发酵中甲酸、乙酸、丁二酸途径。重组FMJ39甲酸和乙酸的量分别比FMJ39提高了17.58%和15.27%,丁二酸的量降低了26.87%,柠檬酸的量变化不大。证实即使pfl基因缺陷,高浓度的L-Thr和L-Ser也会诱导Tdc 操纵元把丙酮酸转化为甲酸和乙酸。实验结果为进一步改造和利用FMJ39奠定了基础。     

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.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

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。     

过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.