代谢工程改造谷氨酸棒杆菌生成丙酮酸

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.033

摘要/Abstract

摘要： 旨在增加谷氨酸棒杆菌代谢过程中丙酮酸的积累、减少副产物的生成,利用同源重组的方法,敲除了丙酮酸代谢过程中支流代谢途径的关键基因：丙酮酸醌氧化还原酶pqo基因、丙酮酸脱氢酶pdh基因和乳酸脱氢酶lldh基因,获得了基因缺失工程菌株。利用4.5%的葡萄糖复合培养基,工程菌经摇瓶发酵48 h,丙酮酸的浓度达到14.6 g/L,而野生菌株仅为0.45 g/L;工程菌的糖酸转化率为33.18%,比野生菌提高了32.5倍。

关键词: 丙酮酸, 谷氨酸棒状杆菌, 丙酮酸醌氧化还原酶, 丙酮酸脱氢酶, 乳酸脱氢酶

Abstract: In order to increase the accumulation of pyruvate while decrease the production of by-products during the metabolic process of Corynebacterium glutamicum,the homologous recombination was employed to knock out the key genes of pyruvate：quinone oxidoreductase （pqo）,pyruvate dehydrogenase（pdh）and L-lactate dehydrogenase（lldh）in the tributary metabolic pathways,thus the gene deletion mutation strains were constructed. Using compound medium with 4.5% glucose as carbon sources,the concentration of pyruvate with mutation strain at shake flask fermentation for 48 hours reached 14.6 g/L,while that with the wild type only was 0.45 g/L. The conversion rate of sugar to acid with the mutation strain was 33.18% and 32.5 times higher than the wild type.

Key words: pyruvate, Corynebacterium glutamicum, pyruvate：quinone oxidoreductase, pyruvate dehydrogenase, L-lactate dehydrogenase

于莹,许湄雪,刘金雷,范荣,冯惠勇,李天明. 代谢工程改造谷氨酸棒杆菌生成丙酮酸[J]. 生物技术通报, 2016, 32(8): 226-232.

YU Ying, XU Mei-xue ,LIU Jin-lei, FAN Rong ,FENG Hui-yong, LI Tian-ming. Metabolic Engineering for Modifying Corynebacterium glutamicum to Produce More Pyruvate[J]. Biotechnology Bulletin, 2016, 32(8): 226-232.

参考文献

[1] 郭英, 付涛. 发酵法生产丙酮酸的工艺条件优化研究[J]. 石家庄职业技术学院学报, 2010, 22（2）：26-28.
[2] 刘立明, 李寅, 堵国成, 等. 生物技术法生产丙酮酸的研究进展[J]. 生物工程学报, 2002, 18（6）：651-655.
[3] Wieschalka S, Blombach B, EikmannsI BJ. Engineering Corynebacterium glutamicum for the production of pyruvate[J]. Applied Microbiology and Biotechnology, 2012, 94：449-459.
[4] 吴新阳, 裴广胜, 等. 不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响[J]. 生物技术通报, 2013（7）：172-178.
[5] 张君胜, 王扬, 杨晓志, 张力. 谷氨酸棒杆菌关键酶活性与苏氨酸高产的关系[J]. 湖南农业科学, 2011（9）：23-25.
[6] Wieschalka S, Blombach B, Bott M, et al. Bio-based production of organic acids with Corynebacterium glutamicum[J]. Microbial Biotechnology, 2012, 6：87-102.
[7] Lin LH, Hu XQ, Xu DQ, et al. Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum[J]. Metabolic Engineering, 2012, 14：542-550.
[8] Radoš D, Turner DL, Fonseca LL, et al. Carbon flux analysis by 13 C nuclear magnetic resonance to determine the effect of CO 2 on anaerobic succinate production by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2014, 80（10）：3015-3024.
[9] Bommareddy RR, Chen Z, Rappert S, et al. A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase[J]. Metabolic Engineering, 2014, 25：30-37.
[10] Vogt M, Haas S, Klaffl S, et al. Pushing product formation to its limit：metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction[J]. Metabolic Engineering, 2014, 22：40-52.
[11] Baumgart M, Unthan S, Rückert C, et al. Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology[J]. Applied and Environmental Microbiology, 2013, 79（19）：6006-6015.
[12] Zhang Y, Shang XL, Lai SHJ, et al. Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2012, 78（16）：5831-5838.
[13] Yamamoto S, Gunji W, Suzuki H, et al. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions[J]. Applied and Environmental Microbiology, 2012, 78（12）：4447-4457.
[14] Blombach B, Schreiner ME, Holátko J, et al. L-valine production with pyruvate dehyrogenase complex-deficient Corynebacterium glutamicum[J]. Appl Environ Microbiol, 2007, 73（7）：2079-2084.
[15] Blombach B, Schreiner ME, Bartek T, et al. Corynebacterium glutamicum tailored for high-yield L-valine production[J]. Appl Microbiol Biotechnol, 2008, 79：471-479.
[16] Litsanov B, Brocker M, Bott M. Toward homosuccinate fermentat-ion：metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate[J]. Applied and Environmental Microbiology, 2012, 78（9）：3325-3337.
[17] Yamamoto S, Gunji W, Suzuki H, et al. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions[J]. Applied and Environmental Microbiology, 2012, 78（12）：4447-4457.
[18] Zahoor A, Lindner SN, Wendisch VF. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products[J]. Computational and Structural Biotechnology Journal, 2012, 3：1-11.
[19] Litsanov B, Kabus A, Brocker M, et al. Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum[J]. Microbial Biotechnology, 2012, 5（1）：116-128.
[20] Li Y, Chen J, Lun SY. Biotechnological production of pyruvic acid[J]. Applied Microbiology and Biotechnology, 2001, 57：451-459.
[21] Zelić B, Gerharz T, Bott M, et al. Fed-batch process for pyruvate production by recombinant Escherichia coli YYC202 strain[J]. Eng in life Sci, 2003, 3：299-305.
[22] Zhu Y, Eiteman MA, Altman R, Altman E. High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain[J]. Appl Environ Microbiol, 2008, 74（21）：6649-6655.