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

Medermycin is an aromatic polyketide antibiotic from Streptomyces and it has the potential antibacterial and antitumor activity. wed-ORF8 and med-ORF10 are two functional genes located in the biosynthetic gene cluster of medermycin. In this study, we performed investigation on mechanism of these two genes by RED/ET seamless mutagenesis, EMSA, yeast two-hybrid and so on.?1? Study on the catalyzation of C-glycosyltransferase ?C-GT? Med-ORF8 using point mutation.Previous studies have proved med-ORF8 to encode a C-GT acted to the rare C-glycosylation in the biosynthesis of medermycin., Sequence alignment between O-GT and C-GT including Med-ORF8 revealed that several regions may represent critical catalytic centers. Among of them, (I³⁰⁷) for Med-ORF8 was selected for further analysis. pAYT55 was previously constructed for expression of Med-ORF8 and 6 glycosy-synthetases ?med-ORF20,18,17,16,14,15? and was functional for angolosamine biosynthesis and transfer. The wild type Med-ORF8 on pAYT55 was mutated firstly to generate Med-ORF8* containing a site mutation (I³⁰⁷-D³⁰⁷)via seamless point mutation based on Red/ET DNA recombineering and ccdB screening system. The genetic engineering pAYT55* was then transfered into streptomyces coelicolor B135 by conjugation. The host HPLC/MS measurement proved that B135 could accumulate an intermediate product kalafungin, which is identical to the core skeletal of medermycin. HPLC/MS measured that kalafungin was glycosylated into medermycin in the strain B135/pAYT55*, indicating that the mutant point selected was not located in the catalytic center of Med-ORF8.?2? Primary investigation of the mechanism of the positive regulator Med-ORF10 involved the medermycin biosynthesisPrevious studies have shown that med-ORF10 located in medermycin cluster encodes a positive regulator and could control the yield of medermycin. It has no any homology to the known-mechanism regulators and has no any DNA-binding domain.It could not bind with the promoter of its target gene med-ORF12.Firstly, since med-ORF29 is another target gene of Med-ORF10 in medermycin cluster, we intended to know if Med-ORF10 could bind to the promoter region of Med-ORF29 by EMSA. Pmed-ORF29 was obtained by PCR and labeled by biotin.Then incubated Pmerf-ORF29 with purified protein Med-ORF10 and detected by chemiluminescence measurement. The result of EMSA assay shown that there was no obvious binding between Med-ORF10 and Pmed-ORF29 under the current conditions.Secondly, we performed screening proteins interacted with Med-ORF10 by yeast two-hybrid assay. Firstly, we constructed genomic library of Streptomyces AM-7161 with vector pGBKT7. Then med-OKF10 was connected with pGADT7 as bait which was tested for toxicity and autoactivation in the yeast cell Y2Hgold. After transferring the "bait" and "prey" plasmids into yeast cell Y2Hgold the positive clones were screened by detecting the reported gene. By far we have obtained three positive plasmids and the BLAST results shown that they belong to SPRY_PRY_TRIM76 family, PGRP family and Amidinotransferases superfamily respectively.