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Enzymatic synthesis of an orlistat intermediate using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans
Institution:1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China;2. Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan, 316022, China;3. Zhoushan Institute for Food and Drug Control, Zhoushan, 316000, China;1. Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi 214122, PR China;2. National Key Laboratory for Food Science, Jiangnan University, Wuxi 214122, PR China;3. Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA;1. Key Laboratory of Textile Fibers and Products, Ministry of Education, P. R. C, College of Materials Science and Engineering, Wuhan Textile University, Wuhan 430073, China;2. Third Institute of Oceanography, Ministry of Natural Resources, P. R. C, Xiamen 361005, China;3. High-Tech Organic Fibers Key Laboratory of Sichuan Province, Sichuan Textile Research Institute, Chengdu 610072, China;1. College of Environmental Science and Engineering, Taiyuan University of Technology, Shanxi 030024, China;2. Key Laboratory of Coal Science and Technology of Shanxi Province and Ministry of Education, Taiyuan University of Technology, Shanxi 030024, China
Abstract:Methyl (R)-3-hydroxytetradeconoate ((R)-MHOT) is a crucial chiral intermediate for the chemical synthesis of the anti-obesity drug, orlistat. Here, (R)-MHOT was prepared from methyl 3-oxotetradecanoate (MOT) using a mutant of the short-chain dehydrogenase/reductase (SDR) from Novosphingobium aromaticivorans (NaSDR). Mutant NaSDR-G145A/I199L had a 3.23 times greater kcat value than that of wild type toward MOT. The conditions for the expression of recombinant NaSDR-G145A/I199L were further investigated and obtained cells were used for gram-scale preparation of (R)-MHOT with 50 g/L of MOT. The target product was extracted and confirmed by gas chromatography; the enantiomeric excess value of (R)-MHOT was 99.0 %. Molecular docking analysis was used to reveal the molecular basis of the enhanced catalytic activity of NaSDR-G145A/I199L; NaSDR-G145A/I199L presented a more effective docking posture than NaSDR. This is the first reported use of SDR for preparing (R)-MHOT via the reduction of MOT. Our study provides a foundation for greener preparation of (R)-MHOT.
Keywords:Short-chain dehydrogenase/reductase  Methyl (R)-3-hydroxytetradeconoate  Methyl 3-oxotetradecanoate  Biocatalysis  Orlistat
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