1.Reporter-guided Selection Of Clavulanic Acid Over-producing Mutants 2.Evaluation Of The Activities Of Two Promoters In Streptomycetes By Reporter Gene Method

Reporter-guided selection of clavulanic acid over-producing mutantsClavulanic acid(CA) is a secondary metabolite produced by Streptomyces clavuligerus.It is a bicyclicβ-lactam,and contains a typical four-memberβ-lactam ring and a five-member oxazolidine ring.Its characteristic 3R,5R stereoconfiguration is essential for itsβ-lactamase inhibitory activity.Clavulanic acid binds irreversibly to the serine hydroxyl group at the active center ofβ-lactamases,producing a stable acylated intermediate and resulting in the inactivation of the enzyme.Although CA possesses little antibacterial activity,it is used clinically with otherβ-lactam antibiotics to combat drug resistance caused byβ-lactamase.A reporter-guided mutant selection method was used to isolate CA over-producing strains.To construct a reporter vector,we cloned the promoter of ccaR,which encodes the key regulatory protein of CA biosynthesis,and placed the promoter in front of two reporter genes,i.e. kanamycin resistance gene(neo) and catechol 2,3-dioxygenase gene(xylE).This double reporter system could avoid some drawbacks of single reporter gene.We mutagenized the reporter vector containing strains by NTG and selection for mutants that grow on elevated kanamycin level and show yellow color.Because some promoters are present in both the vector and on the genome, the changes of reporter expression levels should reflect the expression level of native ccaR. Therefore,by selecting reporter over-expression mutant,we can obtain CA over-producing mutants;the CA titers of these mutants were further analysed by HPLC.Eventually,a high yield strain was obtained,its CA production level is four times that of the wild strain.We also developed a color based method to quickly detect CA production level variations. The method employs on plate filter paper colony detecting,96-well plates reconfirmation, followed by final HPLC confirming;it greatly simplified mutant selection practice. Evaluation of the activities of two promoters in Streptomycetes by reporter gene methodStreptomycetes are gram-positive soil bacteria with high GC-content and characterized by a complex morphology and secondary metabolism.Their metabolism is regulated by a complex,interrelated and hierarchical regulatory network,among it promoters and other cis-regulating elements constitute the basic parts.Promoters in Streptomycetes have diverse structure and transcription modes.The mutated promoter of the erythromycin resistance gene(PermE*) is a strong constitutive promoter often used in Streptomycetes.We evaluated the activities PermE* and a Psf,first reported by Gabriele Labes.A reporter based method was used,in which kanamycin resistance gene(neo) and catechol 2,3-dioxygenase gene(xy/E) were chosen as reporters.Both promoters exhibited high level of promoter activities in Streptomyces clavuligerus NRRL3585, Streptomyces coelicolor M145,Streptomyces venezuelae ISP5230 and Streptomyces lividans TK54.The activities of Psf were higher than those of PermE* in S.clavuligerus and S. coelicolor.Our results indicate that both Psf and PermE* are strong promoters suitable for gene over-expression in Streptomycetes and Psf offers an alternative for high-level gene expression.