Study On Regulation Of Polymyxin Analogues Of Paenibacillus Polymyxa CJX518

As a "last line of defense" against superbugs,polymyxin B has limitation in clinical use for the uncertainty of components.Therefore,there is an urgent need to find a method for regulating the polymyxin components and an efficient gene editing system.In this paper,by sequencing the genome of P.polymyxa CJX518 and analyzing the genes involved in lipopeptide synthesis,we have studied methods to regulate the proportion of polymyxin components,and at the same time,in order to achieve molecular genetic transformation of P.polymyxa,an efficient gene knockout system was constructed.The main results are as follows:1)The fermentation supernatant of P.polymyxa CJX518 has inhibitory effects on Escherichia coli and Rhizoctonia solani,and its genome includes 14 secondary metabolite biosynthetic gene clusters,three of which are similar to known polymyxin,fusaricidin and tridcaptin with 100% similarity.LC-MS was used to detect fermentation supernatant,in which polymyxin B1,B2,B3,and B1-1,and fusaricidin with C15,C16,C17,and C19 carbon chain lengths were detected.2)In adenylation domain swapping,the proportion of polymyxin B1 increased from 41.36% to 52.90%,B1-1 decrease from 18.25% to 3.09%.The ratio of polymyxin B1 and B3 of Starter condensation domain swapping changed from41.36% and 16.99% to 55.03% and 6.39%,respectively.The two domain swapping strains produced 62.96% of polymyxin B1,6.70% of B3 and 3.32% of B1-1.The production of polymyxin is related to the metabolism of acetoin and 2,3-butanediol.The production of polymyxin is significantly reduced,but it promotes the production of acetoin and 2,3-butanediol.3)In the CJX518 genome,the genes alsD(acetolactate synthase),bdh(2,3-butanediol dehydrogenase),pmx(polymyxin synthetase),and fus(fusaricidin synthase)were selected as target genes to constructed four CRISPR / Cas9 vectors.Four single gene knockout strains(ΔalsD,Δbdh,Δpmx A,Δfus A)were obtained.The above studies can not only regulate the polymyxin homologue component ratio effectively,provide a new strategy for regulating lipopeptide homologues,but also provide a theoretical basis for molecular genetic modification of P.polymyxa.