Gene lmrB of Corynebacterium glutamicum confers efflux-mediated resistance to lincomycin

The lmrB gene of Corynebacterium glutamicum, which confers specific resistance to lincosamides, such as lincomycin and clindamycin, was isolated. C. glutamicum cells, carrying the lmrB gene in a multicopy plasmid, showed increased resistance to lincomycin with a MIC of 230 microg/ml, which is a 9-fold increase compared to that of the wild type. The lmrB-disrupted mutant became sensitive to the compound. No difference in sensitivity to erythromycin, penicillin G, tetracycline, chloramphenicol, spectinomycin, nalidixic acid, gentamicin, streptomycin, ethidium bromide, and sodium dodecyl sulfate was observed. The protonophore carbonyl cyanide m-chlorophenylhydrazone abolished the lincomycin-resistance of lmrB-carrying cells. The putative protein product of the gene contained 14-transmembrane regions and showed high amino acid-sequence homology to the drug efflux pumps of other organisms. In addition, the putative protein contained a motif for major facilitators, suggesting a role in efflux-mediated resistance to lincomycin.     

Characteristics of methionine production by an engineered Corynebacterium glutamicum strain

A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene (hom(FBR)) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (delta thrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/hom(FBR)/delta thrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity.     

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1). A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

DivS, a novel SOS-inducible cell-division suppressor in Corynebacterium glutamicum

DNA damage-induced SOS response elicits the induction of cell-division suppressor as well as DNA repair genes. In Gram-positive bacteria, cell-division suppressor genes, so far characterized from Bacillus subtilis (yneA) and Mycobacterium tuberculosis (rv2719c), share limited homology, but are both located in the vicinity of lexA on their respective genomes. Using this proximity to lexA, Corynebacterium glutamicum R divS (cgR1759) was identified as an SOS-inducible cell-division suppressor in this study. The amino acid sequence of DivS showed no homology to that of YneA and Rv2719c. divS expression was markedly induced by DNA-damaging mitomycin C treatment in wild-type cells, but not in its DeltarecA mutant cells, which are unable to induce the SOS response. Wild-type C. glutamicum R cells exposed to DNA-damaging mitomycin C exhibited elongated morphology that, using green fluorescent protein-FtsZ fusion protein, was attributed to defects in FtsZ ring assembly. Cells defective in FtsZ ring assembly were subsequently incapable of septum wall synthesis. In the presence of mitomycin C, divS mutant cells did not exhibit this elongated morphology, whereas cells overexpressing divS were elongated and abnormally branched.     

Biochemical analysis on the parallel pathways of methionine biosynthesis in Corynebacterium glutamicum

Two alternative pathways for methionine biosynthesis are known in Corynebacterium glutamicum: one involving transsulfuration (mediated by metB and metC) and the other involving direct sulthydrylation (mediated by metY). In this study, MetB (cystathionine gamma-synthase) and MetY (O-acetylhomoserine sulfhydrylase) from C. glutamicum were purified to homogeneity and the biochemical parameters were compared to assess the functional and evolutionary importance of each pathway. The molecular masses of the native MetB and MetY proteins were measured to be approximately 170 and 280 kDa, respectively, showing that MetB was a homotetramer of 40-kDa subunits and MetY was a homohexamer of 45-kDa subunits. The Km values for the O-acetylhomoserine catalysis effected by MetB and MetY were 3.9 and 6.4 mM, and the maximum catalysis rates were 7.4 (kcat = 21 s(-1)) and 6.0 (kcat=28 s(-1)) micromol mg(-1) min(-1), respectively. This suggests that both MetB and MetY can be comparably active in vivo. Nevertheless, the Km value for sulfide ions by MetY was 8.6 mM, which was too high, considering the physiological condition. Moreover, MetB was active at a broad range of temperatures (30 and 65 degrees C) and pH (6.5 and 10.0), as compared with MetY, which was active in a range from 30 to 45 degrees C and at pH values from 7.0 to 8.5. In addition, MetY was inhibited by methionine, but MetB was not. These biochemical data may provide insight on the role of the parallel pathways of methionine biosynthesis in C. glutamicum with regard to cell physiology and evolution.     

Characterization of a novel plasmid pXZ608 from Corynebacterium glutamicum

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20-40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA-GtfJ fusion could not be removed from the cell by 5 M LiCl wash.     

Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg-matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.     

Efficient production of glutathione with multi-pathway engineering in Corynebacterium glutamicum

Journal of Industrial Microbiology & Biotechnology - Glutathione is a bioactive tripeptide composed of glycine, l-cysteine, and l-glutamate, and has been widely used in pharmaceutical, food,...     

谷氨酸棒杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种具有重要应用价值的四碳平台化合物。微生物法发酵生产琥珀酸以其社会、环境和经济优势展现出良好的发展前景。谷氨酸棒杆菌被广泛应用于氨基酸、核苷酸等高附加值化学品的工业化生产,在厌氧条件下细胞处于生长停滞状态,但仍能高效利用碳源合成有机酸,通过代谢工程改造的谷氨酸棒杆菌有望成为理想的琥珀酸生产菌株。结合近年来谷氨酸棒杆菌生产琥珀酸取得的最新成果,本文综述了构建高产琥珀酸工程菌株的代谢工程策略、底物的扩展利用,并展望了将来的研究方向。     

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD^Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.     

Metabolic engineering of l-leucine production in Escherichia coli and Corynebacterium glutamicum: a review

l-Leucine, as an essential branched-chain amino acid for humans and animals, has recently been attracting much attention because of its potential for a fast-growing market demand. The applicability ranges from flavor enhancers, animal feed additives and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. Microbial fermentation is the major method for producing l-leucine by using Escherichia coli and Corynebacterium glutamicum as host bacteria. This review gives an overview of the metabolic pathway of l-leucine (i.e. production, import and export systems) and highlights the main regulatory mechanisms of operons in E. coli and C. glutamicum l-leucine biosynthesis. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating l-leucine producing strains. Finally, future perspectives to construct industrially advantageous strains are considered with respect to recent advances in biology.     

谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较

【背景】谷氨酸棒状杆菌的基因敲除系统较为匮乏且效率不高,难以对其进行代谢工程改造,不利于高性能工业菌株的构建及规模生产。【目的】分别采用CRISPR-Cpf1和Cre/loxP基因敲除系统对谷氨酸棒状杆菌ATCC13032(CorynebacteriumglutamicumATCC13032)基因组上的argR和argF基因进行敲除,比较两种敲除方法的优缺点,为合理选择敲除系统提供依据。【方法】特异性重组的Cre/loxP敲除系统是首先利用同源重组将基因组上的靶基因替换为两端带有重组位点loxP的kanR片段,然后由重组酶Cre识别loxP位点并发生重组反应,从而去除替换到基因组上的kanR片段,进一步利用质粒的温敏特性将其消除,从而实现靶基因的敲除。CRISPR-Cpf1敲除系统是利用Cpf1对pre-crRNA进行加工,形成的成熟crRNA引导Cpf1识别和结合到靶DNA的特定序列上并切割双链DNA分子,通过同源重组作用去除靶基因,基于质粒自身的温敏特性将其消除,从而完成基因敲除的整个过程。【结果】Cre/lox P系统可在8N+2 d内完成N轮迭代基因敲除,而CRISPR-Cpf1系统可在5N+2d内完成N轮迭代基因无痕敲除,理论上还可以一次对多个靶位点进行编辑,效率更高,但存在同源重组效率较低、假阳性率高等缺点。【结论】与Cre/loxP系统相比,CRISPR-Cpf1辅助的同源重组基因敲除方法可省时、省力地实现基因的无痕敲除,理论上还可实现多个基因的同时敲除、总体效率更高,然而编辑效率还有提高的空间。     

Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilissacB with the PlacM fused at the 5′-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.