钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究  

Producing L-ornithine by Heterologous Expression of N-acetyl-L-ornithine Deacetylase in Corynebacterium crenatum

作　　者：舒群峰 徐美娟[1] 李静[1] 张显[1] 杨套伟[1] 许正宏[1] 饶志明[1] SHU Qun-feng;XU Mei-juan;LI Jing;ZHANG Xian;YANG Tao-wei;XU Zheng-hong;RAO Zhi-ming(The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室

出　　处：《中国生物工程杂志》2018年第7期29-39,共11页China Biotechnology

基　　金：国家自然科学基金(31770058);江苏省杰出青年科学基金(BK20150002);教育部重点研究项目(113033A);中央高校基本科研业务费专项资金(JUSRP51708A);江苏高校优势学科建设工程资助项目

摘　　要：目的：对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF（SYPO-1）进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法：通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果：来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg^（2＋）、Li^＋、Mn^（2＋）促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/（L·h）。结论：筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum（SYPO-1）中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。Objective： The metabolic pathway engineering of Corynebacterium crenatum SYPA5-5/△proB/△argF（ SYPO-1） has been performed to further enhance the pathway flux of L-ornithine biosynthesis. Firstly,the four genes encoding N-acetyl-L-ornithine deacetylase（ NAOD） from different bacterial sources were screened,cloned and expressed in Escherichia coli BL21（ DE3）. Then the recombinant NAODs were purified and characterized. The arg E gene from Serratia marcescens Y213 was overexpressed in the L-ornithine producing strain C. crenatum SYPO-1 to increase the L-ornithine production. Methods： The genes from different sources were sub-cloned into the pDXW10 plasmid and expressed under the tac M promoter in E. coli BL21（ DE3）. Then the recombinant N-acetyl-L-ornithine deacetylation were purified and their characterization were studied. The optimal N-acetyl-L-ornithine deacetylase was expressed in recombinant C. crenatum. The parameters of the recombinant strains during fermentation were also investigated. Results： The recombinant arg E coding NAOD enzyme from S. marcescens showed a very higher activity than the other NAOD enzymes from E. coli BL21（ DE3）,K. pneumoniae and B. subtilis,the activity was 798. 98 U/mg,the optimum pH and temperature were7℃ and 37℃ respectively. Sm NAOD was expressed in C. crenatum,and the activity was 128. 4 U/ml,which was significantly increasing intracellular acetyl cycle levels. At the end of fermentation, L-ornithine yield increased to 38. 5 g/L with the overall productivity of 0. 401 g/（ L·h） in the recombinant SYPO-2,which was approximately 21. 3% and 33. 2% higher than that of SYPO-1 and SYPO-3,respectively. Conclusion： The Nacetyl-L-ornithine deacetylase from S. marcescens Y213 has been screened and overexpressed in the L-ornithine producing strain C. crenatum SYPO-1,which could promote the rapid consumption of L-ornithine precursors and achieve L-ornithine accumulation. A huge potential of C. crenatum to overproduce not only L-ornithine but also L-citrul

关 键 词：L-鸟氨酸 钝齿棒杆菌 N-乙酰鸟氨酸脱乙酰基酶 ARGE 

分 类 号：Q819[生物学—生物工程]