Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.     

C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H¹³CO₃ ^–-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% ¹³C enrichment in C-4 and 3.5% ¹³C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C₃-carboxylation reaction that also takes place in the absence of PEPCx. Received: 27 December 1995 / Accepted: 20 March 1996     

Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production

Corynebacterium glutamicum possesses phosphoenolpyruvate (PEP) carboxykinase, oxaloacetate decarboxylase and malic enzyme, all three in principle being able to catalyze the first step in gluconeogenesis. To investigate the role of PEP carboxykinase for growth and amino acid production, the respective pck gene was isolated, characterized and used for construction and analysis of mutants and overexpressing strains. Sequence analysis of the pck gene predicts a polypeptide of 610 amino acids showing up to 64% identity with ITP-/GTP-dependent PEP carboxykinases from other organisms. C. glutamicum cells harbouring pck on plasmid showed about tenfold higher specific PEP carboxykinase activities than the wildtype. Inactivation of the chromosomal pck gene led to the absence of PEP carboxykinase activity and the inability to grow on acetate or lactate indicating that the enzyme is essential for growth on these carbon sources and thus, for gluconeogenesis. The growth on glucose was not affected. Examination of glutamate production by the recombinant C. glutamicum strains revealed that the PEP carboxykinase-deficient mutant showed about fourfold higher, the pck-overexpressing strain two- to threefold lower glutamate production than the parental strain. Inactivation and overexpression of pck in a lysine-producer of C. glutamicum led to an only 20% higher and lower lysine accumulation, respectively. The results show that PEP carboxykinase activity in C. glutamicum is counteractive to the production of glutamate and lysine and indicate that the enzyme is an important target in the development of strains producing amino acids derived from citric acid cycle intermediates.     

A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant

Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.     

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions

Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth. Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control. Furthermore, when small amounts of pyruvate were added to the reaction mixture, acid production rates and the glucose consumption rate were multiplied by a factor ranging from 2 to 3. These phenomena were paralleled by an increase in the NAD(+)/NADH ratio, thus corroborating the view that the efficient regeneration of NAD(+) could be triggered by the addition of either bicarbonate or pyruvate. To investigate the global metabolism of corynebacteria under oxygen deprivation conditions, we engineered several strains where the genes coding for key metabolic enzymes had been inactivated by gene disruption and replacement. A lactate dehydrogenase (LDH)-deficient mutant was not able to produce lactate, suggesting this enzyme has no other isozyme. Although a pyruvate carboxylase (pyc) mutant exhibited similar behavior to that of the wild type, phosphoenolpyruvate carboxylase (ppc) mutants were characterized by a dramatic decrease in succinate production, which was concomitant to decreased lactate production and glucose consumption rates. This set of observations corroborates the view that in coryneform bacteria under oxygen deprivation conditions the major anaplerotic reaction is driven by the ppc gene product rather than by the pyc gene product. Moreover, intracellular NADH concentrations in C. glutamicum were observed to correlate to oxygen-deprived metabolic flows.     

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.     

Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering

The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates.     

谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

Effect of increased glutamate availability on L-ornithine production in Corynebacterium glutamicum

Glutamate availability in the argF-argR-proBDelta strain of Corynebacterium glutamicum was increased by addition of glutamate to the cell or inactivation of the phosphoenolpyruvate carboxykinase activity and simultaneous overexpression of the pyruvate carboxylase activity to assess its effect on Lornithine production. When glutamate was increased in an Lornithine- producing strain, the production of L-ornithine was not changed. This unexpected result indicated that the intracellular concentration and supply of glutamate is not a rate-limiting step for the L-ornithine production in an L-ornithine-producing strain of C. glutamicum. In contrast, overexpression of the L-ornithine biosynthesis genes (argCJBD) resulted in approximately 30% increase of L-ornithine production, from 12.73 to 16.49 mg/g (dry cell weight). These results implied that downstream reactions converting glutamate to L-ornithine, but not the availability of glutamate, is the rate-limiting step for elevating L-ornithine production in the argF-argR-proBDelta strain of C. glutamicum.     

Intracellular pool of free amino acids in a wild strain of Corynebacterium glutamicum and its lysine-producing mutants

The intracellular content of free amino acids was measured in the wild-type strain of Corynebacterium glutamicum 13032 and its lysine producing mutants 410 and 133, resistant to the combined effect of threonine and S-2-aminoethyl cysteine, a lysine analog. After 18- and 48-hour cultivation of all strains the major components of the amino acid pool were glutamic acid, alanine and lysine, and those of the cell-free supernatant were alanine and lysine. After 18-hour cultivation the lysine content in mutants was 2-3 times higher than in the wild-type strain. After 48-hour cultivation the lysine content in mutants remained unchanged and in the wild-type strain increased. After 18- and 48-hour cultivation the lysine content in the supernatant of mutants was 15 and 33 times higher than in that of the parental strain. These findings are compared with the activities of aspartokinase from Cor. glutamicum 13032, 410 and 133.     

Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation

To give clues about the respective importance of phosphoenol-pyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4 mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4 microg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。