Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115 |
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| Institution: | 1. School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Republic of Korea;2. Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80309, USA;1. Otorhinolaryngology Hospital, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China;2. Department of Otolaryngology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China;3. Department of Otolaryngology, Head and Neck Surgery, Affiliated Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China;4. Department of Otolaryngology, Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China;1. A.M. Butlerov Institute of Chemistry, Kazan Federal University, Kremlevskaya str. 18, Kazan 420008, Russia;2. A.E. Arbuzov Institute of Organic and Physical Chemistry, Russian Academy of Sciences, Arbuzov str. 8, Kazan 420088, Russia;1. Department of Pathophysiology, Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, 227 South Chongqing Road, Shanghai 200025, PR China;2. Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, PR China |
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| Abstract: | Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%). |
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