钝齿棒杆菌的代谢改造:L-鸟氨酸与L-瓜氨酸合成菌株的构建  

Engineering Corynebacterium crenatum for construction of L-Ornithine and L-Citrulline producers

作　　者：赵芹芹[1] 罗玉常[1] 窦文芳[1] 张晓梅[1] 耿燕[1] 许正宏[1,2] 

机构地区：[1]江南大学药学院制药工程研究室,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学通报》2014年第10期1941-1947,共7页Microbiology China

基　　金：国家863计划项目(No.2012AA022102)

摘　　要：【目的】对一株产L-精氨酸的钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5进行代谢工程改造,构建L-鸟氨酸和L-瓜氨酸合成菌株,并考察其发酵生产相应氨基酸的性能。【方法】分别敲除菌株SYPA5-5鸟氨酸氨甲酰转移酶(Ornithine carbamoyltransferase,OTC)的编码基因argF和精胺琥珀酸合成酶(Argininosuccinate synthase,ASS)的编码基因argG,构建能够合成L-鸟氨酸及L-瓜氨酸的重组菌株SYPA5-5△argF和SYPA5-5△argG;考察不同营养条件对上述重组菌株生长和相应氨基酸积累的影响。【结果】添加0.3 g/L L-精氨酸可满足SYPA5-5△argF的生长及L-鸟氨酸积累所需,L-鸟氨酸产量可达21.5 g/L;添加L-精氨酸有利于SYPA5-5△argG的生长,但不利于L-瓜氨酸的积累;不添加L-精氨酸时,L-瓜氨酸产量可达15.2 g/L,同时积累6.8 g/L的L-谷氨酸。【结论】分别敲除L-精氨酸生产菌株SYPA5-5的argF及argG基因,可实现L-精氨酸合成途径的中间代谢物L-瓜氨酸和L-鸟氨酸的积累,拓展了该菌株的工业应用范围。[Objective] L-Ornithine and L-Citrulline producers were constructed based on the L-Arginine producer Corynebacterium crenatum SYPA5-5 and their fermentative performances were evaluated. [Methods] argF encoding ornithine carbamoyltransferase（OTC） and argG encoding argininosuccinate synthase（ASS） were deleted respectively in SYPA5-5 to block the biosyntheticpathway that converting L-Ornithine and L-Citrulline to L-arginine. Two recombinant strains SYPA5-5△argF and SYPA5-5△argG were constructed. The influence of different L-arginine concentration on growth and amino acids accumulation of the recombinant strains were determined.[Results] SYPA5-5△argF kept well growth status with the fermentation media supplied with 0.3 g/L L-arginine and the growth rate was similar to SYPA5-5. SYPA5-5△argF had the capacity of producing 21.5 g/L L-Ornithine. On the contrary, L-arginine had no use for SYPA5-5△argG to enhance the productivity of L-Citrulline even though it contributed to improve cell growth.SYPA5-5△argG produced 15.2 g/L L-Citrulline with the original media without adding L-arginine,and it produced 6.8 g/L L-glutamate simultaneously. [Conclusion] Deleting argF and argG respectively in SYPA5-5 made it accumulate high concentration of L-Ornithine and L-Citrulline.These expanded the applied scope of SYPA5-5 in amino acids industry.

关 键 词：L-鸟氨酸 L-瓜氨酸 钝齿棒杆菌 鸟氨酸氨甲酰转移酶 argF 精胺琥珀酸合成酶 ARGG 

分 类 号：TQ922[轻工技术与工程—发酵工程]