溶氧量与谷氨酸棒杆菌代谢

正自从1957年Kinoshita等首次描述谷氨酸棒杆菌(Corynebacterium glutamicum)为谷氨酸产生菌[1]以来,其已成为用于氨基酸生产的主要菌株。目前,全世界每年利用谷氨酸棒杆菌生产约100万t L-谷氨酸用于食品调味剂和约45万t L-赖氨酸用作食品添加剂[2]。通过谷氨酸棒状杆菌发酵获得谷氨酸的发酵水平已较高,通过进一步优化工艺来提高产量具有较大困难[3]。     

Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry

The 2-methylcitrate cycle as the primary way to metabolize propionate was investigated using metabolic profiling. For this purpose, a fast harvesting procedure was applied in which cells growing in liquid minimal medium were harvested by a short centrifugation and freeze-dried. Subsequently, gas chromatography–mass spectrometry of polar extracts derivatized by MSTFA was employed for metabolite characterization. Routinely more than 300 different peaks were obtained in the chromatograms, and 74 substances were identified unequivocally by using pure standards. The procedure provided reliable data which closely relate to prior knowledge on flux distributions during growth on glucose and acetate as carbon sources.

Propionate degradation via the 2-methylcitrate cycle was demonstrated on the metabolite level by the detection of the intermediates 2-methylcitrate and 2-methylisocitrate. Further characterization of the 2-methylcitrate cycle was carried out by comparing different mutant strains of this pathway. The growth deficit of a prpD2-mutant strain observed when propionate is added to a culture growing on acetate indicates that the toxic effect of propionate is based on the accumulation of 2-methylcitrate. It could also be shown that the 2-methylcitrate cycle is active in the absence of propionate and might fulfill house-keeping functions in the degradation of fatty acids or branched-chain amino acids.     

The cloning and nucleotide sequence of a Corynebacterium glutamicum 3-deoxy-d- arabinoheptulosonate-7-phosphate synthase gene

Abstract The aro gene of Corynebacterium glutamucum CCRC 18310 encoding 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase was isolated by complementation of a DAHP synthase-deficient mutant of Escherichia coli AB3257. The specific activity of DAHP synthase was increased four-fold in a C. glutamicum strain harboring the cloned aro gene. The complete nucleotide sequence of the aro gene and 5' and 3' flanking regions has been determined. The sequence contained an open reading frame of 368 codons, from which a protein with a molecular mass of 39 340 Da could be predicted. The deduced amino acid sequence shows high identity with the aro gene products of E. coli and Salmonella typhimurium .     

Molecular Analysis of the Corynebacterium glutamicum Transketolase Gene

Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

不同溶氧对谷氨酸棒杆菌代谢的影响

【目的】以谷氨酸棒杆菌为研究对象,分别控制在0、30%、50%3种溶氧水平下进行发酵,分析不同溶氧水平下代谢的变化。【方法】通过检测发酵代谢物中有机酸、氨基酸的含量,以及测定代谢途径中关键酶活性及其编码基因的表达情况来考察不同溶氧水平下物质代谢发生的变化。通过检测胞内还原力和ATP的含量来分析不同溶氧水平对能量代谢产生的影响。【结果】谷氨酸棒杆菌代谢支路受溶氧的影响而发生改变,氨基酸、有机酸的产量也随之改变。特别是在低溶氧(0)情况下,细胞内氧化磷酸化减弱,导致维持生命活动所必需的ATP供应减少,因此细胞通过增强底物水平磷酸化来产生ATP以满足生命活动的需求。在此情况下,胞内NADH得到较多积累,TCA循环代谢流量减小,而转向糖酵解、乙醛酸循环等,并且这个过程伴随多种杂酸包括乳酸、缬氨酸、亮氨酸等的产生,必将影响目的产物的产量。【结论】研究结果对于进一步采取措施优化溶氧的控制策略,提高目的产物的产量具有指导意义。     

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。     

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.     

Mapping the membrane proteome of Corynebacterium glutamicum

In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.     

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.     

Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is sensitive to feedback inhibition by tyrosine. One feedback-insensitive mutant was obtained by in vitro chemical mutagenesis and the mutation was identified as a C-->G mutation at nucleotide 560 causing a Ser(187) to Cys(187) substitution. Replacing Ser(187) with cysteine, tyrosine or phenylalanine by site-directed mutagenesis not only reduced the enzymatic activity but also relieved its feedback inhibition by tyrosine, while Ser(187)Ala exhibited a comparable activity to that of wild-type enzyme and sensitized to allosteric regulation. The His(6)-tagged enzymes were expressed in Escherichia coli and purified to homogeneity by immobilized nickel-ion affinity chromatography. Kinetic analysis showed that tyrosine is a competitive inhibitor of phosphoenol pyruvate, one of the precursors for DAHP biosynthesis.     

谷氨酸棒杆菌metX、dapA基因敲除对苏氨酸合成的影响

谷氨酸棒杆菌中metX基因编码蛋氨酸合成途径关键酶高丝氨酸乙酰转移酶,dapA基因编码赖氨酸合成途径关键酶二氢吡啶二羧酸合成酶。为研究这两个基因缺失对苏氨酸积累的影响,以谷氨酸棒杆菌R102(AHVr)为出发菌株,通过重叠延伸PCR及同源重组技术分别构建了metX、dapA单基因缺失突变株R102ΔmetX、R102ΔdapA以及双基因缺失的突变株R102ΔmetXΔdapA。对出发菌以及上述3株重组菌进行初步摇瓶发酵试验,用HPLC法测定发酵液中苏氨酸含量。结果表明,发酵72 h后,3株重组菌的苏氨酸产量分别为2.58、2.38和3.01 g/L,比原始菌株分别提高了42.5%、31.5%和66.3%。     

谷氨酸棒状杆菌技术研发态势分析

利用美国Thomson公司旗下的Thomson Data Analyzer和微软公司的Excel等分析工具,从时间、地域、专利权人、技术领域等方面,对德温特专利数据库中全球谷氨酸棒状杆菌领域专利的生命周期、区域布局、竞争机构和技术趋势进行了分析。以期为谷氨酸棒状杆菌技术发展态势提供情报支持,辅助企业、高校制定和调整技术发展战略、完善技术发展布局,在当今激烈的市场竞争环境下保持较高的技术优势。     

谷氨酸棒杆菌YILW苏氨酸脱水酶基因的克隆表达及酶学性质

将L-异亮氨酸生产菌谷氨酸棒杆菌(Corynebacterium glutamicum YILW)苏氨酸脱水酶(threonine de-hydratase,TD)的编码基因ilvA在大肠杆菌中进行异源表达及进行初步的酶学性质研究。分别以C.glutamicum ATCC13032、YILW的基因组DNA为模板,利用PCR技术扩增出苏氨酸脱水酶的编码基因ilvA,测序获得编码序列。利用质粒PET-His将该基因在大肠杆菌BL21(DE3)中进行重组表达、金属螯合纯化,对其酶学性质进行初步研究。结果显示C.glutamicum YILW编码基因序列与已报道的ilvA序列相差5个碱基,相似度为99.6%,第383位氨基酸由苯丙氨酸突变为缬氨酸。酶学性质研究表明:重组酶YilwTD最适反应温度为32℃,在20~55℃范围内该酶较稳定,最适pH为6.7,该酶底物专一性强,对最适底物苏氨酸的米氏常数Km=8.32 mmol/L,最大反应速度Vmax=3.18×104U/mg,与野生型酶相比,突变(F383V)后可显著降低终产物对酶的反馈抑制作用。为揭示突变对苏氨酸脱水酶活性的影响及进一步利用基因工程技术改造L-异亮氨酸生产菌,提高L-异亮氨酸产量奠定了基础。