CRISPR/Cas9-mediated Genome Editing In Amycolatopsis Keratiniphila And Expression Of Gene CobA

This paper intends to construct a CRISPR/Cas9 genome editing system in the vancomycin-producing strain Amycolatopsis keratiniphila HCCB10007 and to verify the range of editing length in this industrial strain.In this paper,the key enzyme cobA in the VB₁₂ synthesis pathway was studied not only by traditional overexpression method but also CRISPR/Cas9 to detect the influence in secondary metabolism.It provides a new and efficient method for the genetic manipulation of this strain.1.CRISPR/Cas9 genome editing system in A.keratiniphila HCCB10007A sg RNA,targeting glycosyltransferase gene gtfD in the vancomycin biosynthesis pathway was designed and coupled with the plasmid p KCCas9d O framework from Streptomyces.The plasmids of three different promoter combinations were constructed named as p KCCas9dgtfD-NA,p KCCas9EgdgtfD-NA and p KCp GCas9EgdgtfD-NA,respectively.On this basis,the homology arm of the gene gtfD flanking e GFP was constructed and then three editing plasmids was constructed named p LYNY02,p LYNY03,p LYNY04,respectively.It presents that p LYNY04,in which the promoter of Cas9 was P_GADPH from A.keratiniphila HCCB1007 and the promoter of sg RNA was P_{erm E*},could be successfully applied to A.keratiniphila.Results showed that the GFP can detected clearly in the mycelia of genetically engineered strain A.keratiniphila LYNY04 by confocal laser microscopy;p LYNY04,knocking out gtfD(1.2kb)and p LYNY05,knocking out four gene fragments gtfD,gtfE,vas C,Lmb E(5.5kb)were successfully electrotransformed into the strain A.keratiniphila HCCB10007;Results showed that CRISPR/Cas9 editing plasmid p LYNY04 successfully blocked the vancomycin biosynthesis.2.Study of endogenous cobA and exogenous cobA geneIn this paper,endogenous cobA overexpression plasmid p LYNY01 was constructed and then transformed into competent cells of A.keratiniphila HCCB10393 by electroporation.The positive strain A.keratiniphila LYNY01 was obtained.The results of UPLC-MS showed that coproporphyrin III(the product of VB₁₂branch metabolic pathway)was significantly reduced;The cobA of P.denitrificans SC510 was selected and then the exogenous cobA knock-in plasmid p LYNY06 was constructed using CRISPR/Cas9 method.Exogenous cobA knock-in strain A.keratiniphila LYNY06-10393 was obtained by transforming into the original strain A.keratiniphila HCCB10393.The metabolism of the engineered strains changed significantly compared with the original strains,revealing that the CRISPR/Cas9 plasmids worked in this strain.