5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization.
To elucidate the biotransformation from 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4) 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 A(3) and milbemycin A(4). MilF is thus present in Streptomyces bingchengensis and can transform 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4). 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.
Wang
Xiang-Jing
XJ
School of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China.
Wang
Cheng-Qin
CQ
Sun
Xiao-Lin
XL
Xiang
Wen-Sheng
WS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018942:Macrolides / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D018942:Macrolides; D009249:NADP; D010088:Oxidoreductases; C027837:moxidectin