核黄素又名维生素B2,是人体必不可少的营养物质之一,而鸟苷三磷酸则是核黄素合成的重要前体。为在高产菌株的基础上进一步提高核黄素产量,该研究以高产核黄素的枯草芽孢杆菌S1为出发菌株,利用CRISPER/Cas9介导的基因组碱基编辑技术对嘌呤合成途径阻遏蛋白基因以及鸟嘌呤核苷酸(guanylate,GMP)还原酶基因进行失活,并利用质粒过表达的方法进行了一系列的代谢工程改造。结果显示,guaC基因的失活阻断了GMP到肌苷酸的回补途径,使GMP得到积累,使核黄素产量达到3.04 g/L,而purR基因的失活没有效果;使用PvegI启动子过表达希瓦氏菌来源的ribA后,核黄素产量提高到3.33 g/L。将失活guaC基因与PvegI启动子过表达希瓦氏菌ribA结合,最终使核黄素产量达到3.43 g/L,较出发菌株核黄素产量提升22%,转化率提升25.8%。
Riboflavin is one of the essential nutrients for human, and guanosine triphosphate (GTP) is an important precursor of riboflavin biosynthesis. In order to further increase riboflavin production on the basis of industrial producers, a riboflavin-producing strain Bacillus subtilis S1 was investigated. The purine synthesis pathway transcription repressor gene and GMP reductase gene were inactivated by CRISPR/Cas9-mediated genomic base editing technology, and a gene ribA from Shewanella oneidensis was overexpressed with pvegI promoter. The results showed that the production of riboflavin increased to 3.04 g/L with the inactivation of guaC gene in B. subtilis S1, which blocked the complementary pathway from GMP to IMP and accumulated GMP. The inactivation of purR gene had no effect. The production of riboflavin increased to 3.33g/L with the overexpressed ribA in B. subtilis S1. The production of riboflavin reached 3.43g/L with the inactivated guaC gene and the overexpressed ribA in B. subtilis S1, which increased 22% compared with B. subtilis S1.
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