Construction And Application Of "Superplasmid" For Enhanced Production Of Endogenous Antibiotics Of Actinomyces

Microorganisms produce abundant secondary metabolites which have rich diversity in chemical structure and biological activity.More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces.Unfortunately,the production rate of Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand.In this study,the production of antibiotics from Streptomyces was enhanced by a"superplasmid"which contained 4 positive regulation factors,including the global regulatory factor afs R,the cyclic adenosine receptor protein ecoding gene?crp?,the dot-mutated RNA polymerase beta subunit gene?rpo B?and the acetyl coenzyme A carboxylase gene?acc A2BE?,under the control of the constitutive strong promoter?P_{erm E*.}These positive regulator factors were overexpressed in different antibiotic producing strains by using the Streptomyces integrative vector p SET152 in order to improve the yield of antibiotics.Introduction of the"superplasmid"increased the production of polyketide actibiotic actinorhodin?2.019 times?and undecylindricin?2.816times?in Streptomyces coelicolor M145,ansamycin antibiotic hygrocin A?2.100 times?and rapamycin?2.634 times?in Streptomyces hygroscopicus ATCC29253,actinomycin D?2.178 times?in Streptomyces mutabilis TRM45540 isolated from alkaline land of Xinjiang.Even though introduction of the"superplasmid"decreased the yield of geldanamycin in Streptomyces hygroscopicus XM201,the production of new metabolites detected by HPLC and LC-MS analyses.The expression level of genes mostly depends on the activity of their promoter activity.For coexpression of more positive regulation factors in the"superplasmid",different reporting systems including kanamycin resistance gene,difluoroprotein reporting system and catechol dioxygenase gene were used to detect promoter activity in Streptomyces,and then P_{azi A4}from azinomycin B biosynthetic gene cluster was proved to be a constitutive strong promoter with significantly higher activity than P_{erm E*}.Truncation of P_{azi A4} significantly reduced its promoter activity.We speculated that P_{azi A4} might be composed of multiple promoters in tandem array.Where after,P_{azi A4}was chosed as the template to construct a promoter library by error prone PCR.High throughput screening of the promoter library was carried out by specific color reaction of catechol dioxygenase,and quantitative analysis of promoter activity was performed by enzyme activity detection.The activity of most mutants was significantly lower than that of wild type,while two mutants showed nearly the same activity as wild type.15 mutated promoters were introduced into the upstream region of aur D,the pathway specific regulatory gene of aureothin biosynthetic gene cluster,so that the aureothin production can be precisely adjusted by control of the aur D expression level with different promoters.