Study On The Mechanism Of Glycosyltransferase Med-ORF8 And Regulator Med-ORF10 Of Medermycin Biosynthesis

Medermycin,an aromatic polyketide antibiotic from Streptomyces,possesses potential bioactivity against many types of tumor cells,besides its inhibitory activity against Gram positive bacteria including Staphylococcus aureus.In the medermycin biosynthesis gene cluster,med-ORF8 encoding a C-glycosyitransferase is responsible for the formation of C-C glycosidic bond between glycosyl donor(Angola glycosamine)and sugar ligand(polyketide nucleus),and its catalytic mechanism has to be elucidated.Med-ORF10 is a positive regulator in medermycin biosynthesis and its regulatory mechanism also still keeps obsure.In this study,we performed investigation on the mechanism of these two genes by Red/ET seamless mutagenesis,MST(Microscale Thermophoresis),Yeast two-hybrid,Pull-Down and so on.(1)Study on the catalytic mechanism of C-glycosyltransferase(C-GT)Med-ORF8 using point mutation.Sequence alignment between O-GT and C-GT revealed that two regions may represent critical catalytic centers,which include the 8th and 51st?62nd amino acid residues along the sequence of Med-ORF8.Using Red/ET recombineering and ccdB screening system,we made three mutations respectively on med-ORF8 of the co-expression plasmid pAYT55 which has six possible glycosyl-synthase genes(med-ORF20,-18,-17,-16,-14,-15)and a glycosyltransferase gene(med-ORF8).All seven genes were proved previously to be sufficient for glycosyl biosynthesis and transfer.And two mutanted plasmids(pAYT55-mutA and pAYT55-mutB)were transferred by conjugation into Streptomyces coelicolor B135 which could offer a glycosyl acceptor.We found that the mutation on the 8th amino acid lead to decline in production of medermycin,implying its importance for the catalytic activity for Med-ORF8 Meanwhile two new compounds were isolated from B135/pAYT55-mutA and B135/pAYT55-mutB and identified as medermycin derivatives.Additionally,the comparable accumulaton of medermycin in B135/pAYT55-mutB suggested 51st-62nd amino acid residues seem not locaed in the catalytic center of Med-ORF8.(2)Study on the regulation mechanism of a new regulatory protein Med-ORF10Previous studies showed that Med-ORF10 can positively regulate the biosynthesis of medermycin,but not via a direct-protein-DNA-interaction mode.So we speculated that Med-ORF 10 may play a regulatory role via a protein-protein interaction mode.By the yeast two-hybrid assay,we obtained three positive pray plasmids named pGBKT7-1,pGBKT7-16 and pGBKT7-24 containing three genomic DNA fragments respectively from the wild-type medermycin-producing strain Streptomyces AM-7161.Here we focused on the No.1 fragment(named P1-S harboured by pGBKT7-1)and verified its encoding product posesseses direct interaction with Med-ORF10.Firstly,RT-PCR assay showed that No.1 sequence from pGBKT7-1 could express normally in Streptomyces sp AM-7161.Then,the pull-down assay and MST assay confirmed that there had an obvious binding effect between P1-S protein and Med-ORF10 in vitro.The bioinformatics analysis about P1-S protein showed that it has a conserved Ter-D domain and ANK which may regulate the biosynthesis of medermycin indirectly through phosphorylation-dependent signal transduction pathway after binding with Med-ORF10.