Abstract
To elucidate the biotransformation from 5-oxomilbemycins A3 and A4 to milbemycins A3 and A4 in Streptomyces bingchengensis, the C5-ketoreductase gene (milF) was cloned using PCR with the specific primer designed from homologous nucleotide sequences. The C5-ketoreductase (MilF) was heterologously expressed in E. coli BL21 (DE3) as a His-tagged fusion protein. The characterization and biotransformation function of purified MilF was verified by in vitro enzyme assay. MilF is an NADPH-dependent reductase. The biotransformation products, analyzed by LC-APCI/MS, were identified as milbemycin A3 and milbemycin A4. MilF is thus present in Streptomyces bingchengensis and can transform 5-oxomilbemycins A3 and A4 to milbemycins A3 and A4. These findings are significant for understanding the biosynthetic pathway of milbemycins in Streptomyces bingchengensis and pave the way to obtain a producer strain of 5-oxomilbemycins directly by targeted milF disruption.
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Acknowledgements
This study was supported by the National Key Technology R&D Program (No. 2006BAD31B02), the Program for New Century Excellent Talents in University (No. NCET-08-0668, 1154-NCET-002), the Outstanding Youth Foundation of Heilongjiang Province (No. JC200706), the National Natural Science Foundation of China (No. 30971937), the National Key Project for Basic Research (No. 2010CB126102), the Program for New Teachers in University (No. 20092325120007) and the Innovative Program for Postgraduates in Heilongjiang Province (No. YJSCX2009-149HLJ).
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Wang, XJ., Wang, CQ., Sun, XL. et al. 5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization. Biotechnol Lett 32, 1497–1502 (2010). https://doi.org/10.1007/s10529-010-0320-y
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DOI: https://doi.org/10.1007/s10529-010-0320-y