Optimization Of Streptomyces Lividans Host And Its Application In The High-throughput Genomic Library Expression Analysis System

Streptomyces secondary metabolites provide important lead compounds for the development of clinical anti-infective and anti-tumor drugs.Though Streptomyces genome-wide sequence information has been reported increasingly,the active compounds that have been discovered account for only small part of all secondary metabolites.Meanwhile,most wild-type strains have many defects such as unclear genetic background,difficult molecular biology operations,and low yields of compounds.In order to effectively exploit antibiotic resources in Streptomyces,functional genomic mining is generally used to screen active compounds and activate cryptic antibiotic biosynthetic gene clusters in wild-type strains.High-throughput genomic library expression analysis system(LEXAS)is a high-throughput gene cluster resource mining method based on heterologous expression and functional screening for the discovery of active secondary metabolites from Streptomyces genome libraries.The excellent heterologous expression host is the basis for improving the screening efficiency of LEXAS system.In order to fully exploit the active compound resources in actinomycetes,we firstly optimized the Streptomyces lividans as library-expression hosts.To improve the heterologous expression of biosynthetic gene clusters(BGCs),herein we optimized S.lividans SBT5 via the stepwise integration of three global regulatory genes and two codon optimized multi-drug efflux pump genes and deletion of a negative regulatory gene,yielding four engineered strains(GX25,GX27,GX28,LJ1018).In order to verify the function of the optimized Streptomyces lividans,we applied it in the heterologous expression of polyketides,non-ribosomal peptides and other antibiotics.As a result,the heterologous production of the above antibiotics was improved.For example,the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the optimized strain S.lividans LJ1018 was used as the heterologous expression host.The heterologous production of these antibiotics in S.lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated.Therefore,these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs.In this paper,we successfully conducted high-throughput library expression and functional screening of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S.lividans GX28 as the library-expression host.We screened an actinomycin D biosynthesis gene cluster from the cosmid library of S.parvulus 10#,and subsequently increased the production of actinomycin D by increasing the copy number of the gene cluster and replacing the heterologous expression host strains.The dehydrorabelomycin biosynthetic gene cluster from the cosmid library of S.galtieri 48 was successfully expressed.The BAC library of S.griseus Sgr29 was screened by GX28 as the LEXAS expression host to detect a complete piericidin A1 BGC(pie).These results demonstrate the feasibility and superiority of S.lividans GX28 as a host strain for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.Based on this study,the optimization of Streptomyces lividans can improve the screening efficiency of the functional genomic mining system by markedly promoted the heterologous production of secondary metabolites,without requiring manipulation of gene expression components such as promoters on individual gene clusters.This study demonstrated that the superiority of GX28 and LJ1018 as heterologous expression hosts,and also demonstrated the GX28 strain is an excellent expression host for the LEXAS procedure to screen for functional BGCs in arrayed cosmid libraries and BAC libraries.