CRISPR-Cas9-Mediated Genome-editing And Reconstruction Of Biosynthetic Gene Cluster In Saccharopolyspora Erythraea

Erythromycin A（ErA）is a kind of macrolide antibiotic synthesized by Saccharopolyspora erythraea in secondary metabolism.As an important pharmaceutical antibiotic,it has a wide range of clinical applications,and improvement of its yield is very important.High-yield strains are required for overexpression of enzymes in biosynthesis.For this reason the application of genome editing strategies is an efficient way to enhance gene expression and increase yield of target products.With the development of genome editing technology,its application to further improve gene expression and increase the yield of target products has received increasing attention from researchers.S.erythraea is a high GC content Actinomycete,which is difficult to perform genetic editing.However,the traditional homologous double exchange is inefficient.Thus,a more efficient gene editing tool is needed for the increased capacity of ErA.The clustered regularly interspaced short palindromic repeats（CRISPR）-Cas9 system is widely used in many species for gene editing.The Streptomyces coelicolor has been reported in genetic manipulation studies by Cas9 system while it has not been applied to S.erythraea.Our research uses CRISPR-Cas9 efficient gene editing system to genetically engineer S.erythraea,including knock-in constitutive strong promoter,site-specific integration to increase the synthesis of precursors,CRISPRi optimizes metabolic flux,builds and screens an ErA high-yield industrial strain;in addition,the erythromycin biosynthesis gene cluster was knocked out by the CRISPR-Cas9 system,and gene cluster studies for other secondary metabolite biosynthesis were reconstructed.The main research contents are as follows:The S.erythraea genome was edited using the CRISPR-Cas9 system.A temperature-sensitive plasmid containing the PermE promoter,to drive codon-optimized expression.Pj23119 and PkasO promoters to drive sgRNAs.Results showed that with the CRISPR-Cas9 gene editing system,dual sgRNA targeting was highly efficient（65%）,and knockout indA（SACE₁229）with marker-free.Knock-out of SACE₁765 increased erythromycin production by 12.7%.Knock-in PermE-egfp at the SACE₀712 locus activated part of the gene in the erythromycin biosynthesis gene cluster（ery BGC）in wild type,increasing erythromycin production by 80.3%.The bidirectional promoter Pj23119-PkasO knock-in increases erythromycin production.The strength of promoter of the erythromycin biosynthesis gene cluster was detected by the mCherry reporter gene system,and the SACE 0720（eryBIV）-SACE 0721（eryAI）spacer was selected as the target regulatory region,and the combined bidirectional promoter Pj23119-PkasO was knock-in by the CRISPR-Cas9 dual sgRNAs method.Results showed that Pj23119-PkasO knock-in significantly enhanced the transcription levels of erythromycin biosynthesis genes eryBIV and eryAI（The gene transcription levels were increased by 32 and 79 times,respectively）,activating the entire gene cluster.High performance liquid chromatography（HPLC）results showed that,compared with the parent strain,the yield of erythromycin was increased（58.3%）in bidirectional promoter knock-in recombinant strains.Further studies found that the use of temperature-controlled CRISPRi technology to inhibit the expression of the sdhA gene further increased erythromycin production by 15.1%.These results fully demonstrate that the CRISPR-Cas9-based genome editing system and CRISPRi can be successfully applied to the gene editing and optimization of S.erythraea to increase the yield of ErA.Biosynthetic gene clusters were reconstituted in S.erythraea using CRISPR-Cas9.The erythromycin synthesis gene cluster（ery BGC,49491 bp）in the erythromycin industrial high-yield strain Ab（HP）was knocked out by double sgRNA to obtain the AbAery strain.This strain has the potential to provide methylmalonyl-CoA and propionyl-CoA,helping to transform S.erythraea into other sub-precursors with methylmalonyl-CoA and propionyl-CoA as precursors to heterologous synthesis of metabolites.This study constructed an effective CRISPR-Cas9 gene editing technology for S.erythraea.The gene-editing framework based on temperature-sensitive plasmid facilitates multiple genetic operations of multiple genes.We using dual sgRNAs targeting to knock-in bidirctional strong promoters in S.erythraea and increased the yield of erythromycin.The CRISPR-Cas9 system was used to knock-out the erythromycin biosynthesis gene cluster,and the constructed chassis strain has the potential to be applied to heterologous synthetic important secondary metabolites with methylmalonyl-CoA and propionyl-CoA.