摘要： 旨在通过代谢工程手段提高谷氨酸非依赖性菌株的γ-聚谷氨酸（γ-PGA）发酵产率，最大程度挖掘出此类芽孢杆菌生物合成系统的潜力。以实验室保藏的一株能产生γ-PGA的谷氨酸非依赖性菌株解淀粉芽孢杆菌C10为研究对象，选择upp基因作为反向筛选标记构建解淀粉芽孢杆菌C10的无痕基因敲除体系，对解淀粉芽孢杆菌C10基因组中的oxdC基因快速无痕敲除，并发酵验证突变菌株γ-PGA的产率。结果显示，C10ΔuppΔoxdC和对照C10Δupp在发酵36 h后OD₆₀₀值分别为13.96，15.56，突变菌株生长能力下降；γ-PGA产量分别为12.33，22.22 g/L，发酵产物γ-PGA产量也低于初始菌株，说明如果草酸不被降解，反而不利于菌体生长和γ-PGA的合成。

关键词: 解淀粉芽孢杆菌, upp基因, 基因敲除, γ-PGA, 发酵

Abstract: In order to improve the γ-PGA fermentation yield of glutamate-independent strains through metabolic engineering methods, and to maximize the potential of this type of Bacillus biosynthetic system. Aglutamate-independent strain of Bacillus amyloliquefaciens C10 preserved in the laboratory that can produce γ-PGA was used as the research object, and the upp gene was selected as the reverse selection marker to construct a traceless gene knockout of Bacillus amyloliquefaciens C10 in addition to the system, the oxdC gene in the C10 genome of Bacillus amyloliquefaciens was quickly and seamlessly knocked out, and the yield of the mutant strain γ-PGA was verified by fermentation. The results showed that the OD₆₀₀ values of C10ΔuppΔoxdC and control C10Δupp were 13.96, 15.56 after 36 h of fermentation, respectively, and the mutant strain's growth ability decreased;the yield of γ-PGA was 12.33, 22.22 g/L, and the yield of fermentation product γ-PGA was also lower. The initial strains indicate that if oxalic acid was not degraded, it was not conducive to the growth of the bacteria and the synthesis of γ-PGA.

Key words: B.amyloliquefaciens, upp gene, Gene knockout, γ-PGA, Fermentation

中图分类号: 

• S432.4