The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.     

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.     

赖氨酸-尸胺反向转运蛋白cadB基因谷氨酸棒杆菌表达载体的构建及转化

旨在提高谷氨酸棒杆菌合成尸胺的能力,将CadB克隆至谷氨酸棒杆菌中,与LDC共表达,在谷氨酸棒杆菌合成尸胺的同时,帮助尸胺转运至细胞外,解除尸胺的反馈抑制作用。谷氨酸棒杆菌能够高产赖氨酸脱羧酶的底物L-赖氨酸,但不含ldc和cadB基因,因而不能够直接合成尸胺。从E.coliK12中克隆出赖氨酸-尸胺反向转运蛋白基因,与绿色荧光蛋白基因gfp融合构建成融合表达载体pXBG,并转化至谷氨酸棒杆菌进行诱导表达,结果表明表达的CadB蛋白可以正确的定位于谷氨酸棒杆菌的细胞膜上。将基因cadB连接到含有赖氨酸脱羧酶基因的pXMJ19-ldc上,构建成能够共表达赖氨酸脱羧酶和赖氨酸-尸胺反向转运蛋白的重组质粒pXLB,并转化到谷氨酸棒杆菌中。     

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.     

Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1

The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE). The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3). Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3). If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.     

谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除

本研究以谷氨酸棒杆菌（Corynebacterium glutamicum）标准菌株ATCC 13032染色体为模板,设计引物PCR扩增高丝氨酸脱氢酶编码基因（hom）,在hom基因内部插入一段来源于质粒pET28a的卡那霉素抗性基因（Km）,得到基因元件hom::Km;通过电击转化法将hom::Km转入出发菌株替换原菌株的hom,在含卡那霉素的平板上挑取阳性转化子,通过PCR验证得到高丝氨酸脱氢酶缺陷的重组菌。发酵结果表明重组菌C.g- hom::Km -8发酵60小时赖氨酸产量达到4.7 g／L,是出发菌株谷氨酸棒杆菌ATCC 13032（0.7 g／L）的6.7倍。     

Molecular analysis of the cytochrome bc 1-aa 3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c 1

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.     

谷氨酸棒杆菌H+-ATPase基因失活提高谷氨酸产生量

为了证实在谷氨酸棒杆菌中,利用H⁺-ATPase基因失活构建高产谷氨酸基因工程菌的应用可行性,通过重组PCR技术部分缺失H⁺-ATPaseγ亚基基因序列,采用插入失活方法构建H⁺-ATPase失活的谷氨酸棒杆菌。考察了其谷氨酸产生能力及对生长速率的影响。实验结果表明,H⁺-ATPase失活的谷氨酸棒杆菌在含有100g/L的葡萄糖培养基中摇瓶发酵,其谷氨酸最大累积量为51.6g/L, 比野生菌株提高了42.9%。生长速率研究结果表明,H⁺-ATPase失活的谷氨酸棒杆菌生长速率略低于野生谷氨酸棒杆菌。证实了H⁺-ATPase基因失活对提高谷氨酸产量的作用,为利用H⁺-ATPase基因构建高产谷氨酸基因工程菌株提供了科学依据。     

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.     

Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth

The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa(3) is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa(3) are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa(3) or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa(3) is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa(3) or cytochrome aa(3), is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa(3) makes the strain sporulation deficient.     

Detection of D-ornithine extracellularly produced by Corynebacterium glutamicum ATCC 13032::argF

We found that Corynebacterium glutamicum ATCC 13032::argF extracellularly produced a large amount of D-ornithine when cultivated in a CGXII medium containing 1 mM L-arginine. This is the first report that C. glutamicum ATCC 13032 or its mutant produces a D-amino acid extracellularly. C. glutamicum ATCC 13032::argF produced 13 mM D-ornithine in 45 h of cultivation.     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

Identification and characterization of the last two unknown genes,dapC and dapF,in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum

The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.     

谷氨酸棒杆菌1dh基因的敲除

谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸.利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk 18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞.分别在卡那霉素抗性平板及10％蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-(4)ldh.应用荧光定量PCR检测,ATCC 13032-(z)ldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为O.ldh基因的敲除对菌株的生长造成了一定的影响.     

谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响

以谷氨酸棒杆菌(Corynebacterium glutamicum) SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062 的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(Y_P/X)提高了15.13%。     

The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic, Gram-positive microorganism, well known as a pro-ducer of several amino acids. Amino acid products are used on a large scale for food industry flavouring, feed additive, pharmaceutical and cosmetic purpose[1,2]. The organism is able to grow not only on glucose, fructose and lactose, but also on acetate, lactate as its sole carbon source. The growth on acetate requires its activation to acetyl-CoA. In C. glutamicum, acetate is activated in a two-step …     

In silico genome-scale reconstruction and validation of the Corynebacterium glutamicum metabolic network

A genome-scale metabolic model of the Gram-positive bacteria Corynebacterium glutamicum ATCC 13032 was constructed comprising 446 reactions and 411 metabolites, based on the annotated genome and available biochemical information. The network was analyzed using constraint based methods. The model was extensively validated against published flux data, and flux distribution values were found to correlate well between simulations and experiments. The split pathway of the lysine synthesis pathway of C. glutamicum was investigated, and it was found that the direct dehydrogenase variant gave a higher lysine yield than the alternative succinyl pathway at high lysine production rates. The NADPH demand of the network was not found to be critical for lysine production until lysine yields exceeded 55% (mmol lysine (mmol glucose)(-1)). The model was validated during growth on the organic acids acetate and lactate. Comparable flux values between in silico model and experimental values were seen, although some differences in the phenotypic behavior between the model and the experimental data were observed.