Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.     

L-鸟氨酸生产菌的选育及其发酵条件的研究

以谷氨酸棒杆菌为出发菌株 ,经硫酸二乙酯、紫外线诱变 ,定向选育出一株鸟氨酸生产菌A1157,其遗传标记为Arg- +D Argr+SGr,并能耐受高浓度葡萄糖。对菌株A1157的发酵条件进行了研究 ,在最佳培养条件下 ,该菌株L 鸟氨酸产量可达 9.85g/L。     

Modelling of the Corynebacterium glutamicum biosynthesis under aerobic fermentation conditions

This work concerns the modelling of the Corynebacterium glutamicum biosynthesis. At the beginning, the impulsive growth model of C. glutamicum under glutamate inhibition is established. According to this model, the analysis of the bioprocess stability is presented. Then, new objective functions are proposed and optimisation of C. glutamicum growth is presented. The results of the optimisation indicate that, although the growth of C. glutamicum is an aerobic process, the maximum of biomass productivity occurs under controlled penetration into the area of aerobic fermentation. Analytical results presented in this work are validated by numerical simulations.     

L－脯氨酸产生菌的选育

以嗜乙酰乙酸棒杆菌ＡＴＣＣｌ３８７０为出发菌株，经硫酸二乙酯（ＤＥＳ）和亚硝基胍（ＮＴＧ）逐级诱变处理，结构类似物定向选育，获得一株Ｌ－脯氨酸高产菌ＺＱ－３（ＳＧｒ、Ｓｕｃｇ、ＤＨＰｒ）。在含１６％葡萄糖的培养基中，摇瓶发酵７２ｈ，产酸率为５．３％～５．５％。     

Mechanistic understanding of the fermentative L‐glutamic acid overproduction by Corynebacterium glutamicum through combined metabolic flux profiling and transmembrane transport characteristics

Since the 1950s when Micrococcus glutamicus later renamed Corynebacterium glutamicum was discovered, the production of amino acids by fermentative methods has become an important aspect of industrial microbiology. Numerous studies to understand and improve the metabolic conditions leading to amino acid overproduction have been carried out. Most amino acids are currently produced by use of mutants that contain combinations of auxotrophic and regulatory mutations. L ‐Glutamic acid is the amino acid produced in the greatest quantities (10⁶ tonnes per year) and Corynebacteria are central to its industrial production. However, further improvements to strain performance are difficult to obtain by empirical optimization and a more rational approach is required. The use of metabolic flux analysis provides valuable information regarding bottlenecks in the formation of desired metabolites. Such techniques have found application in elucidating flux control, provided insight into metabolic network function and developed methods to amplify or redirect fluxes in engineered bioprocesses. Hence, branch points in biosynthesis, precursor supply in fuelling reactions and export of metabolites can be manipulated, resulting in high glutamic acid overproduction by Corynebacterium glutamicum fermentations. In this review, in addition to reviewing the state of play in metabolic flux analysis for glutamate overproduction, the metabolic pathways involved in the production of L ‐glutamic acid, the mechanisms mediating its efflux and secretion as well as their manipulation to achieve higher glutamate production, are discussed. The link between metabolic flux and transmembrane transport of glutamic acid are also considered. Copyright © 2004 Society of Chemical Industry     

生物素对L-缬氨酸发酵的影响

为研究生物素添加量对谷氨酸棒状杆菌（Corynebacterium glutamate）发酵生产L-缬氨酸的影响,以谷氨酸棒状杆菌XV0505（Leu－＋Ile－＋2-TAr＋α-ABr＋SGr）为供试菌株,考察不同生物素添加量条件下菌体量、耗糖、产酸以及副产物L-丙氨酸的情况,确定了生物素最适添加量为50 μg/L;利用膜偶联透析发酵方式有效解除了发酵生产过程中产生的反馈抑制现象,降低了副产物的产量,提高了L-缬氨酸的转化率及产量。与原单批次发酵的工艺相比,新工艺的最终L-缬氨酸总量达到106.1 g/L,产量提高了47.4%,糖酸转化率提高到34.5%。     

常温常压等离子体诱变选育高产L-异亮氨酸谷氨酸棒杆菌

以谷氨酸棒杆菌（Corynebacterium glutamicum）23798为原始菌株,对其进行常温常压等离子体（ARTP）诱变,以磺胺胍抗性和氨基酸与茚三酮特异显色为筛选标记,以期得到高产L-异亮氨酸的诱变谷氨酸棒杆菌,并对其遗传稳定性进行研究。结果表明,原始菌株23798经过ARTP诱变处理180 s后,经0.4 mg/mL磺胺胍抗性筛选、多孔板高通量筛选、发酵培养复筛,选育出一株高产L-异亮氨酸诱变谷氨酸棒杆菌（Corynebacterium glutamicum）B1。该菌株在摇瓶中发酵培养48 h,L-异亮氨酸产量达18.5 g/L,比原始菌株提高62.03%,且遗传性状稳定。     

改造乙酰羟酸合成酶提高L-亮氨酸产量

乙酰羟酸合成酶（acetohydroxy acid synthase,AHAS,编码基因ilvBN）是L-亮氨酸合成途径的第一个限速酶。以谷氨酸棒杆菌XL-3（Corynebacterium glutamicum XL-3）为底盘细胞,通过分析并改造AHAS增加其对底物丙酮酸的偏好性,从而提高L--亮氨酸产量。首先利用AHAS的氨基酸序列进行同源建模,根据蛋白质结构进行丙氨酸扫描,找到突变的潜在位点,通过测定突变体酶活力和重组菌株的L--亮氨酸产量寻找最适突变体。测定结果发现将157位Gln突变成Arg能够有效提高AHAS催化丙酮酸的能力,最终重组菌株的L--亮氨酸产量达到（23.5±1.8）g/L,比出发菌株谷氨酸棒杆菌XL-3增加了51%,同时副产物L--异亮氨酸产量有所下降。因此,通过对AHAS的理性改造促进了L--亮氨酸的合成,该研究结果对后续利用蛋白质工程强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

新型表面活性剂在谷氨酸发酵中的应用

通过对预先选择的8种表面活性剂和3种溶剂作为谷氨酸发酵促进剂效果的初步筛选,得知聚氧乙烯基失水山梨醇单脂肪酸酯三季铵盐(CTTE)和二甲基亚砜(DMSO)对产酸率提高有较为明显的效果;进而从加入时间,加入剂量两个方面研究了筛选出的两种表面活性剂对谷氨酸棒状杆菌(Corynebacterium glutamicum)T-613菌体生长和谷氨酸发酵产酸的影响,同时研究了CITE和DMSO同时加入的协同效果,从而得到最佳的发酵工艺参数. 研究结果表明:菌种生长对数期(10h)加入DMSO为0. 1%(V/V),发酵中期(18h)加入(CTTE)为0. 08%(V/V),在不影响菌种生长的前提下,促使谷氨酸发酵产酸率由4. 86%提高到6. 55%,较原产量提高了36. 46%,同时发酵的周期由44h下降为36h,缩短了8h,有效的提高了生产效率;最终结合染色显微照片,分析了表面活性剂促进谷氨酸发酵的机理.