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

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。

Abstract:

[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 biosynthetic pathway 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.