缬氨酸生产菌株的定向改造及发酵优化
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摘要
缬氨酸是人体必需氨基酸之一,具有重要的生理生化作用。为进一步提高缬氨酸产量及优化发酵过程的温度控制,从谷氨酸棒杆菌细胞壁肽聚糖合成途径的2个关键基因murA(cgl0352)和murB(cgl0353)入手,从1株缬氨酸生产菌中敲除murA和murB基因构建出重组工程菌株C.glutamicum A301。摇瓶发酵结果显示,该菌株具有温度敏感性,在变温发酵的条件下,能从生长型状态转化为产酸型状态,促进缬氨酸的积累。在5 L发酵罐中采用分段控温策略发酵,即前15 h控温33℃,15 h之后提温至39℃,重组工程菌株的缬氨酸产量达到35.5 g/L,与全程33℃发酵相比提高了55.7%。研究为缬氨酸发酵过程温度控制提供了经验,同时对于谷氨酸棒杆菌的定向改造提供了一种思路。
Valine is one of the essential amino acids in human body and has important physiological and biochemical functions. In order to further increase the production of valine and optimize the temperature control of valine fermentation process, two key genes murA(cgl0352) and murB(cgl0353) of Corynebacterium glutamicum cell wall peptidoglycan biosynthesis pathway were studied. A recombinant engineering strain C. glutamicum A301 was constructed by knocking out murA and murB of a valine producing strain. The shake-flask fermentation results showed that the strain had temperature-sensitive trait. Under the condition of temperature shift fermentation, the state of the strain could be transformed from growth form to amino acid producing form, and the accumulation of valine was improved. Using a two-step temperature control strategy(temperature of 33 ℃ for the first 15 h and then increased the temperature to 39 ℃), the yield of valine from the recombinant strain reached 35.5 g/L, which was 55.7% higher than that using a one-step temperature control strategy(temperature of 33 ℃) in a 5 L fermentor. This study provides experience for controlling temperature for valine fermentation process, and provides an idea for directional transformation of C. glutamicum.
Valine is one of the essential amino acids in human body and has important physiological and biochemical functions. In order to further increase the production of valine and optimize the temperature control of valine fermentation process, two key genes murA(cgl0352) and murB(cgl0353) of Corynebacterium glutamicum cell wall peptidoglycan biosynthesis pathway were studied. A recombinant engineering strain C. glutamicum A301 was constructed by knocking out murA and murB of a valine producing strain. The shake-flask fermentation results showed that the strain had temperature-sensitive trait. Under the condition of temperature shift fermentation, the state of the strain could be transformed from growth form to amino acid producing form, and the accumulation of valine was improved. Using a two-step temperature control strategy(temperature of 33 ℃ for the first 15 h and then increased the temperature to 39 ℃), the yield of valine from the recombinant strain reached 35.5 g/L, which was 55.7% higher than that using a one-step temperature control strategy(temperature of 33 ℃) in a 5 L fermentor. This study provides experience for controlling temperature for valine fermentation process, and provides an idea for directional transformation of C. glutamicum.
引文
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[15] WEI ZH,WU H,BAI LQ,et al.Temperature shift-induced reactive oxygen species enhanced validamycin A production in fermentation of Streptomyces hygroscopicus 5008[J]. Bioprocess and Biosystems Engineering, 2012, 35(8): 1 309-1 316
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[17] ASAKURA Y, KIMURA E, USUDA Y,et al. Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2007, 73(4): 1 308-1 319
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[21] DU W,BROWN J R,SYLVESTER D R,et al.Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria[J]. Journal of Bacteriology, 2000, 182(15): 4 146-4 152.
[22] LOVERING A L, SAFADI S S, STRYNADKA N C J. Structural perspective of peptidoglycan biosynthesis and assembly[J]. Annual Review of Biochemistry, 2012, 81: 451-478.
[23] SCH?FER A,TAUCH A,J?GEr W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]. Gene, 1994, 145(1): 69-73.
[24] 乐军,胡宏.双歧双歧杆菌细胞壁完整肽聚糖的分离纯化[J].中国微生态学杂志, 1997(5):10-13.
[25] 杨媛,潘道东,曾小群,等.嗜酸乳杆菌胞壁肽聚糖的提取及结构分析[J].中国食品学报, 2014, 14(5):202-208.
[26] 余秉琦,沈微,诸葛健.温度和溶菌酶敏感菌株的L-谷氨酸合成[J].应用与环境生物学报, 2009, 15(6):840-845.
[2] BLOMSTRAND E, ELIASSON J, KARLSSON H K R, et al. Branched-chain amino acids activate key enzymes in protein synthesis after physical exercise[J]. The Journal of Nutrition, 2006, 136(1): 269S-273S.
[3] SHIMOMURA Y, YAMAMOTO Y, BAJOTTO G, et al. Nutraceutical effects of branched-chain amino acids on skeletal muscle[J]. The Journal of Nutrition, 2006, 136(2): 529S-532S.
[4] 张伟国,郭燕风.支链氨基酸生物合成及其代谢工程育种研究进展[J].食品与生物技术学报, 2014, 33(2):14-20.
[5] 苏跃稳,张昕,王健. L-缬氨酸代谢工程研究进展[J]. 发酵科技通讯, 2016, 45(2): 118-122.
[6] OLDIGES M,EIKMANNS B J,BLOMBACH B.Application of metabolic engineering for the biotechnological production of L-valine[J]. Applied Microbiology and Biotechnology, 2014, 98(13): 5 859-5 870.
[7] ZHANG H,LI Y,WANG C,et al.Understanding the high l-valine production in Corynebacterium glutamicum VWB-1 using transcriptomics and proteomics[J]. Scientific Reports, 2018, 8(1): 3 632.
[8] Schwentner A, Feith A, Muench E, et al. Metabolic engineering to guide evolution–Creating a novel mode for L-valine production with Corynebacterium glutamicum[J]. Metabolic engineering, 2018, 47: 31-41.
[9] MA Y, CUI Y, DU L, et al. Identification and application of a growth-regulated promoter for improving L-valine production in Corynebacterium glutamicum[J]. Microbial Cell Factories, 2018, 17(1): 185.
[10] HASEGAWA S,SUDA M,UEMATSU K,et al. Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions[J]. Applied and Environmental Microbiology, 2013, 79(4): 1 250-1 257.
[11] HASEGAWA S,UEMATSU K,NATSUMA Y,et al.Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions[J]. Applied and Environmental Microbiology, 2012, 78(3): 865-875.
[12] 张海灵. 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[J]. 生物工程学报, 2018, 34(10):1 606-1 619.
[13] HOU X, CHEN X, ZHANG Y, et al. L-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum[J]. Amino Acids, 2012, 43(6): 2 301-2 311.
[14] DELAUNAY S, GOURDON P, LAPUJADE P, et al. An improved temperature-triggered process for glutamate production with Corynebacterium glutamicum[J]. Enzyme and Microbial Technology, 1999, 25(8-9): 762-768.
[15] WEI ZH,WU H,BAI LQ,et al.Temperature shift-induced reactive oxygen species enhanced validamycin A production in fermentation of Streptomyces hygroscopicus 5008[J]. Bioprocess and Biosystems Engineering, 2012, 35(8): 1 309-1 316
[16] HASAN CM, SHIMIZU K. Effect of temperature up-shift on fermentation and metabolic characteristics in view of gene expressions in Escherichia coli[J]. Microbial Cell Factories, 2008, 7(1): 35.
[17] ASAKURA Y, KIMURA E, USUDA Y,et al. Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2007, 73(4): 1 308-1 319
[18] HIRASAWA T, WACHI M, NAGAI K. A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production[J]. Journal of Bacteriology, 2000, 182(10): 2 696-2 701.
[19] HIRASAWA T, WACHI M, NAGAI K. L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains[J]. BMC Biotechnology, 2001, 1(1): 9.
[20] 张成林,李志华,梁静波,等. ldhA基因敲除对Corynebacterium glutamicum TCCC11822发酵生产L-谷氨酸的影响[J]. 中国酿造, 2014, 33(4):106-109.
[21] DU W,BROWN J R,SYLVESTER D R,et al.Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria[J]. Journal of Bacteriology, 2000, 182(15): 4 146-4 152.
[22] LOVERING A L, SAFADI S S, STRYNADKA N C J. Structural perspective of peptidoglycan biosynthesis and assembly[J]. Annual Review of Biochemistry, 2012, 81: 451-478.
[23] SCH?FER A,TAUCH A,J?GEr W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]. Gene, 1994, 145(1): 69-73.
[24] 乐军,胡宏.双歧双歧杆菌细胞壁完整肽聚糖的分离纯化[J].中国微生态学杂志, 1997(5):10-13.
[25] 杨媛,潘道东,曾小群,等.嗜酸乳杆菌胞壁肽聚糖的提取及结构分析[J].中国食品学报, 2014, 14(5):202-208.
[26] 余秉琦,沈微,诸葛健.温度和溶菌酶敏感菌株的L-谷氨酸合成[J].应用与环境生物学报, 2009, 15(6):840-845.