The High Shushan Research Group of the Institute of Microbiology of the Chinese Academy of Sciences established a gene editing system suitable for multinuclear industrial strains.

Recently, Gao Shushan research group has constructed a gene editing system suitable for multinucleated industrial bacteria, which can delete large DNA fragments without trace, and provide a new and efficient gene editing tool for genetic transformation of industrial fermentation strains in China "Fengi" has been published in ACS synthetic biology, an authoritative journal of synthetic biology.through this method, the team of Gao Shushan has successfully knocked out the gene cluster (15 KB) of citrinin biosynthesis in Monascus purpureus.the new strain has been strictly verified by Cologne's small tank fermentation, medium tank fermentation and large tank fermentation. The quality of fermentation products is significantly higher than that of similar products on the market. It has been applied to the industrial production of 1000 tons of Monascus red pigment and has achieved remarkable economic benefits for the enterprise. Red kojic red pigment is a valuable scientific heritage of the Chinese nation. It is widely used in the coloring of food industry, and can also be used as a health care product to alleviate cardiovascular and cerebrovascular diseases, hyperlipidemia and other related diseases.however, the industrial strain of Monascus red pigment can produce both nephrotoxic and carcinogenic citrinin at the same time; China, the European Union and Japan have made restrictions on the content of citrinin in the products, and the Chinese standard is 4 mg / kg.due to the lack of good genetic operation tools, at present, the relevant production enterprises meet the citrinin content standard by changing the fermentation process and random mutation, resulting in a huge waste of production capacity of enterprises; on the other hand, the export of Monascus red products of most enterprises is blocked due to the excessive citrinin content, which reduces China's international competitiveness in this field.therefore, it is urgent to select new Monascus strains with low or no citrinin production.compared with prokaryotes such as actinomycetes, the genome of multinucleated bacteria is larger and more complex, and there are different nuclei in a single cell. It is difficult to completely knock out the target gene by common gene knockout methods, so there is no efficient gene editing method applied to industrial multinuclear strains.in view of this dilemma, researchers have constructed a new double plasmid gene editing system: cas9 plasmid containing codon optimization and homologous recombinant plasmid containing homologous arms of upstream and downstream of the target gene.in order to ensure the knockout efficiency, the resistance gene and the polykaryote autonomous replicon were introduced into the cas9 plasmid.this method successfully realized the efficient and traceless deletion of & gt; 15 KB gene cluster of citrinin biosynthesis. The results of LC-MS showed that citrinin was not detected in the mutant after 10 rounds of culture, and the yield of Monascus red pigment was increased by about 5% compared with that before transformation.Liu Weiwei, an assistant researcher of the Institute of Microbiology, Chinese Academy of Sciences, and an Chunyan, a postdoctoral fellow, are the co authors of the paper, while Gao Shushan and Gao Xue, assistant professor of chemistry and molecular biological function department of Rice University, are co authors. Researcher Xiang Hua and Professor Chen Fusheng of Huazhong Agricultural University have provided valuable suggestions for revision of this paper.this project is supported by the national key R & D program, the strategic leading science and technology project of CAS, the National Natural Science Foundation of China and the cross innovation platform of CAS.the research group of Gao Shushan thanks researchers Chen Yihua, fan Keqiang, Wang Weishan and pan Guohui for their valuable experimental space.Article links: