Molecular Cloning, Expression And In Vitro Functional Identification Of C5-O-methyltransferase From Streptomyces Bingchengensis

In order to demonstrate the biosynthetic pathway of milbemycins in Streptomyces bingchengensis, our reserch focused on amplification of full-length binD, expression and in vitro functional identification, the present investigation provided that binD codes for a C5-O-methylating enzyme able to convert the milbemycins A3 and A4 into C5-O-methlmilbemycins A3 and A4. And the results were as follows:(1) Amplification of complete binD. To amplify the complete sequence of binD, the degenerate primers derived from conserved regions of C5-O-methltransferase gene sequences from three Streptomyces strains (S. griseochromogenes, S. avermilitis, and S. cyaneogriseus) were employed to amplify a 795-bp portion of the binD gene. The partial sequence of binD was the highest identical to the nucleotide sequence of milD (about 91%). Hence, full-length primers were designed based on the sequences of milD to clone the entire binD gene. A full-length DNA encoding binD gene was cloned from the genomic DNA of S. bingchenggensis and deposited in the GenBank database under Accession No. Sequence FJ531497.2. Basic local alignment search tool (BLAST) analysis of BinD showed 87% similarity with MilD from S. griseochromogenes, 56% and 53% similarity with NemD and AveD from S. cyaneogriseus and S. avermitilis, respectively.(2) Expression of BinD. According to multiple cloning sites of prokaryotic expression plasmid pET-30a(+) and restriction map of the cloned binD gene, another pair of primers were designed and synthesized, then the gene including restriction sites of BamHI and HindIII was amplified and ligased with plasmid pET-30a to generate pET-30a-binD, then transformed into E.coli BL21(DE3). The result of SDS-PAGE analysis of expressed production show that BinD had been expressed, but informed including body which is about 31 KDa.(3) Methyltransferase reaction by whole cell of E. coli expressing BinD. The E. coli transformant containing the BinD were incubated with milbemycin A3 and milbemycin A4, respectively. LC analysis showed the appearance of a new peak at a different time (8.95 min), when compared to the parent compound. The corresponding mass spectrum of the new peak demonstrated a molecular ion at m/z = 543 [M+H]+ for C5-O-methylmilbemycin A3. When milbemycin A4 was used as a substrate, C5-O-methylmilbemycin A4 had a retention time of 10.24 min and the corresponding mass spectrum indicated a molecular ion at m/z = 557 [M+H]+.(4) In vitro functional identification. To further confirm the function of BinD, our in vitro analysis was initiated by testing directly the ability of BinD to convert the substrates milbemycin A3 and milbemycin A4, respectively. The C5-O-methylation of milbemycins A3 and A4 was revealed by HPLC analysis and comparison with the negative control. The reaction products from milbemycins A3 and A4 had the same retention time (8.95 min, 10.24 min, respectively) as those of the methylation in E. coli. The results suggested that BinD could catalyze C5-methyl-transfer reaction in S. bingchengensis.(5) The effects of pH and temperature on purified BinD activity were quantitatively assayed. The results showed that BinD was optimally active approximately at pH 7.4 and 28 oC.