Establishment Of The Genetic Manipulation System Of Am-7161 And Prokaryotic Expression Study On The C-glycosyltransferase Gene Med-orf8

Streptomyces sp AM-7161(AM-7161) is a producer of aromatic polyketide antibiotic medermycin showing strong antibacterial and anticancer activity.It is quite interesting to study biosynthetic problems concerning tailoring modifications of medermycin biosynthesis,including deoxyhexose biosynthesis,C-C glycosidic band formation.However,it has been hard to perform genetic manipulations in medermycin-producing Streptomyces sp AM-7161.Here,firstly,we established the genetic manipulation system of AM-7161,and then constructed the expression system of a C-glycosyltransferase gene wed-ORF8 from this strain:(1) Establishment of the genetic manipulation system of AM-7161:Firstly, systematic optimization of conditions for mycelium growth,protoplast preparation and protoplast regeneration were conducted.Based on these results,two types of foreign DNAs(integrative and auto-replicating) were successfully introduced into this strain with high efficiency.Then,we built the conjugation system between AM-7161 and Escherichia coli to make plasmid DNA successfully enter into this strain by conjugation. Our data also suggested that there is at least one integration site for an integrative plasmid pSET152 on the chromosome of streptomyces sp AM-7161 since this strain could accept pSET152.Additionally,streptomyces sp AM-7161 could not restrict methylated foreign DNAs because the DNAs used for protoplast transformation here were isolated from Escherichia coli DH5α.(2) Establishment of the prokaryotic expression system of med-ORF8:med-ORF8 located in the biosynthetic gene cluster of the medermycin is essential for medermycin biosynthesis and was speculated to be involved in the formation of a novel C-C glycosidic bond.In this study,med-ORF8 amplified by PCR was ligated into an expression vector pET23a(+) to gain an expression plasmid,designated as pHSL51. After introducing pHSL51 into E.coli BL21(DE3),the expression of target protein was detected based on optimizations of induction conditions,SDS-PAGE and western blotting.Further purification of this protein with Ni-NTR agarose chromatography was under the way.