Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

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。     

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.     

The effect of AmtR on growth and amino acids production in Corynebacterium glutamicum

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less alpha-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, Ile, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val,and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.     

Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum

Transketolase is a key enzyme of the nonoxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of␣the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a␣protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%–20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains. Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998     

Carboxylic acid consumption and production by Corynebacterium glutamicum

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l -lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P_tuf-ldcC^opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability. Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase. Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or l -lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g_{cell dry weight}⁻¹ hr⁻¹, respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids.     

Modified carbon flux during oxygen limited growth of Corynebacterium glutamicum and the consequences for amino acid overproduction

Summary The amino acid producing bacterium Corynebacterium glutamicum accumulated lactate, succinate and acetate under oxygen-limited growth conditions. Significant restructuring of carbon flux through the central metabolic pathways occurred with a notable decrease in pentose pathway flux and the operation of the TCA cycle in a reductive mode. Simultaneous consumption of residual sugar and organic acids took place when oxygen sufficient conditions were restored though amino acids yields were significantly perturbed.     

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.     

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.     

Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (Delta malE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.     

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.     

Purification and Characterization of Fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K _eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K _eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production

Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1 -aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by approximately 45% and of the maximal biomass by approximately 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by approximately 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum.