5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization

• Published in: Biotechnology letters Vol. 32; no. 10; pp. 1497 - 1502
• Main Authors: Wang, Xiang-Jing, Wang, Cheng-Qin, Sun, Xiao-Lin, Xiang, Wen-Sheng
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.10.2010; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: 5-Oxomilbemycins A₃ and A; Applied Microbiology; Bacterial Proteins; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; biochemical pathways; Biochemistry; Bioconversions. Hemisynthesis; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Biotransformation; C5-ketoreductase (MilF); chemistry; Chromatography, Liquid; Cloning; Cloning, Molecular; Coenzymes; Coenzymes - metabolism; DNA, Bacterial; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; enzymology; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes; genetics; Life Sciences; Macrolides; Macrolides - metabolism; Mass Spectrometry; metabolism; Methods. Procedures. Technologies; Microbiology; Milbemycin; milbemycins; Milbemycins A₃ and A; Molecular Sequence Data; NADP; NADP - metabolism; nucleotide sequences; Nucleotides; Original Research Paper; Oxidation-Reduction; Oxidoreductases; Oxidoreductases - genetics; Oxidoreductases - metabolism; polymerase chain reaction; Proteins; Sequence Analysis, DNA; Strain; Streptomyces; Streptomyces - enzymology; Streptomyces - genetics; Streptomyces bingchengensis; Transforms; Milbemycins A; and A; Biotransformation; C5-ketoreductase (MilF); 5-Oxomilbemycins A; Streptomyces bingchengensis; Streptomycetaceae; Streptomyces; Gene overexpression; Actinomycetales; Characterization; Bacteria; Actinomycetes
• ISSN: 0141-5492; 1573-6776; 1573-6776
• DOI: 10.1007/s10529-010-0320-y

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.