The Development Of New Methods For Engineering Streptomyces

Streptomyces are known for their complex development and morphological differentiation,which suggest their rich control system and regulatory elements.Constructing the precise research method of regulation mechanism of Streptomyces,developing new regulatory compoments and transforming control elements into standardized components library,will have important application value.The present work contains three parts.First,the modified bacterial two-hybrid system:In our studies of the interaction of regulatory proteins in Streptomyces,it was found that the bacterial two-hybrid system was not sensitive enough by the blue-and-white selection on X-gal plate.The disturbance was diluted by introducing multicopy lacZ promoter,which drove another reporter gene gfp.By such design,the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also decreased the rate of the false positive clones.The interaction between BldM and WhiI protein from Streptomyces coelicolor was verified by the improved two-hybrid system,which indicated that the sensitivity was significantly improved.This improved technique can be used to study the interaction of peoteins,which will have an important meaning in studying biological processes of regulation and metabolism.Second,the library of constitutive promoters for Streptomyces was established:A total of 941 genes with constant gene expression profiles were selected based on systematic analysis of five sets of time-series transcriptome microarray of Streptomyces coelicolor M145 cultivated in different conditions.Then,166 putative constitutive promoters were picked out by following a rational selection workflow,containing disturbance analysis,function analysis,genetic loci analysis and transcript abundance analysis.Further,eight promoters with different transcript abundances were chosen at random and subjected to experimental validations by green fluorescent protein(GFP)reporter and real-time quantitative PCR in S.coelicolor,Streptomyces venezuelae and Streptomyces albus,respectively.The eight promoters drove the stable expression profiles of downstream genes in different conditions,implying the 166 promoters we identified might be constitutive in the genus Streptomyces.Four promoters were used in a plug-and-play platform to trigger the cryptic cluster of jadomycin B in S.venezuelae ISP5230,and resulted in different productions corresponding to promoter strengths.Third,a new method for screening of physiological promoter was developed:In S.coelicolor M1146,cumate-inducible expression system controlled Act productionby driving Act?-orf4 regulatory protein,and the best induction time and dose were determined through single factor experiment,orthogonal experiment and response surface analysis.The transcription temporal of cumate induced promoter and physiological promoter of S.coelicolor M145 was analyzed by RT-PCR and RNA-seq under the best induction conditions,and gene microarray of S.coelicolor M145.The physiological promoters of S.coelicolor M145 which had the same transcription temporal with induced promoter were selected out and verified.