钝齿棒杆菌异源表达乙酰辅酶A合成酶对L-精氨酸合成的影响  

Enhancement of L-arginine yield by heterologously expressing acetyl-CoA synthetase in Corynebacterium crenatum

作　　者：吴洁琴 石电 徐华 张显[1] 杨套伟[1] 徐美娟[1] 饶志明[1] WU Jieqin;SHI Dian;XU Hua;ZHANG Xian;YANG Taowei;XU Meijuan;RAO Zhiming(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2022年第21期9-16,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2021YFC2100900);国家自然科学基金项目(32070035);宁夏回族自治区重点研究开发计划项目(2019BCH01002);111工程资助项目(111-2-06)。

摘　　要：L-精氨酸是一种人体必需氨基酸,在医疗保健、食品添加等制造行业中商业价值显著。针对1株产L-精氨酸的钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5进行代谢改造,通过比较异源乙酰辅酶A合成酶(acetyl-CoA synthetases,ACS)的酶学性质,构建了过表达ACS的重组钝齿棒杆菌SYPA-ACS,能有效利用乙酸提高菌株产L-精氨酸能力。首先在大肠杆菌中分别克隆表达及纯化4种不同细菌来源的ACS,发现巴氏醋酸杆菌(Acetobacter pasteurianus)ATCC 33445来源的ApACS1酶活力最高,为784.59 U/mL,最适反应的pH为7.0,最适反应温度37℃,与其他3种ACS来源相比,ApACS1具有更好的pH和温度稳定性。然后通过pXMJ19载体,将A.pasteurianus来源的编码基因acs_(1)在C.crenatum中表达,有效提高了乙酰辅酶A含量。利用5 L发酵罐,将SYPA-ACS菌株培养96 h后,L-精氨酸的产量达53.43 g/L,产率为0.577 g/(L·h),相较于出发菌株SYPA5-5,产量提高了27.48%。该策略既可以利用乙酸,降低乙酸对菌株的危害作用,又能提高乙酰辅酶A的含量,促进L-精氨酸的积累。研究结果为L-精氨酸的制备提供了绿色、高效的合成策略,具有工业化应用潜力。L-Arginine is essential amino acid that is widely used in medicine,food addition,and other industries.The metabolic pathway engineering of Corynebacterium crenatum SYPA5-5 was performed to further enhance the flux of L-arginine biosynthesis.Comparing the enzymatic properties of acetyl-CoA synthase(ACS) from different bacterial sources,and heterologous expression in C.crenatum SYPA-ACS to effectively utilize acetic acid and increase L-arginine yield.Firstly,the four genes encoding ACS from different bacterial sources were cloned and expressed in Escherichia coli,and the highest activity of ApACS1 from Acetobacter pasteurianus ATCC 33445 was 784.59 U/mL,the optimum pH was 7.0 and temperature was 37 ℃.Compared with the other ACSs,ApACS1 has better pH and temperature stability.The acs_(1) from A.pasteurianus was then cloned into plasmid pXMJ19 and expressed in C.crenatum,which was effectively improving the acetyl-CoA content.The recombinant strain SYPA-ACS was cultured in 5 L fermenter for 96 h,L-arginine accumulation increased to 53.43 g/L with the productivity of 0.577 g/(L·h) in the recombinant SYPA-ACS,which was approximately 27.48% higher than that of SYPA5-5.The ACS from A.pasteurianus has been screened and overexpressed in C.crenatum,which could use acetic acid to improve the content of acetyl-CoA and achieve L-arginine accumulation.Thus,this strategy provides a safe and efficient synthesis of L-arginine and has important industrial application potential.

关 键 词：L-精氨酸 钝齿棒杆菌 乙酰辅酶A合成酶 ApACS1 乙酸 

分 类 号：TQ922[轻工技术与工程—发酵工程]