Construction And Preliminary Application Of CRISPR/Cas9 Genome Editing Systems In Two Rare Actinomycetes

Actinomycetes natural products contribute greatly in the history of human disease by providing many antibiotics,such as streptomycin,vancomycin,etc.Genetic manipulation plays an important role in the research of actinomycetes natural products.Traditional homologous recombination methods often increase the workload due to the long cycle,the dependence on screening markers,and the large amount of screening.All of these limit its scope of application.Therefore,we urgently need to improve the genetic system of actinomycetes to study the secondary metabolic potential of actinomycetes more efficiently.Our laboratory has been working on the mining of actinomycetes natural products,and constructed the "10,000 genomes of actinomycetes" library.In this study,we constructed the CRISPR/Cas9 genome editing technology in two rare actinomycetes Verrucosispora sp.MS100137 and A.methanolica,and applied it to the study of secondary metabolites.Mainly made a few results:1.Analysis the biosynthesis of proximicins by CRISPR/Cas9 genetic methods(1)A highly efficient CRISPR/Cas9 gene editing technique was successfully established in the genus Verrucosispora,and the efficiency of the gene deletion was as high as 75%.Through the fermentation of MS 100137 strain,many new proximicin compounds were found in the product,and a proximicin with relative molecular weight of 307 was detected.The structure of the proximicin was predicted to be a methyl group based on proximicin A,showing the structural diversity and active potency of proximicins.(2)Deletions of NRPS gene clusters in MS 100137 by CRISPR/Cas9 revealed that C12 and C17 were involved in the biosynthesis of proximicins.Both of these clusters contained a large number of unknown functional genes,suggesting new mechanisms..(3)Through the fermentation of MS 100137 strain,many new proximicin compounds were found in the product,and a proximicin with relative molecular weight of 307 was detected.The structure of this proximicin was predicted to be a methyl group based on proximicin A,showing the structural diversity and active potency of proximicins.(4)Based on the structures of proximicins and the annotation of genes in C12 and C17,we predict the biosynthesis pathway of proximicins.2.Isolation of new analogues of amychelin from A.methanolica and construction of chassis(1)The CRISPR/Cas9 gene editing technique was established in A.methanolica 239T,and the phiC31 integration site was introduced to the genome while the amychelin gene cluster was deleted with the efficiency of 100%.The powerful gene editing ability of CRISPR/Cas9 laid the foundation of the study of these strains.(2)Five new amychelin compounds were isolated from the new compound produced by the fed-batch amS gene knockout strain,and 4F-2 rescued P.aeruginosa infected C.elegans in pathogenesis models with EC50 value of 1.352 ?M.At the same time,a number of salicylic acid analogues were produced in fed-batch fermentations of some compounds,such as 5-F-salicylic acid,5-Br-salicylic acid,4-CH3-salicylic acid and so on.In this study,a genomic editing system based on CRISPR/Cas9 was successfully constructed for two strains of rare actinomycetes Verrucosispora sp.MS100137 and Amycolatopsis methanolica 239'.In this study,the proximicin biosynthetic gene cluster was identified by CRISPR/Cas9,and its synthetic pathway was deduced,which laid the foundation for further in-depth study.On the other hand,this study removed the amychelin gene cluster ofA.methanolica 239T and introduced a phiC31 integration site.In addition,based on the mutant strain constructed by amychelin synthesis pathway based on genetic means,we isolated high activity compounds,laid a solid foundation for the follow-up of antimicrobial drugs in-depth study.