Journal
JOURNAL OF BIOTECHNOLOGY
Volume 224, Issue -, Pages 20-26Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2016.03.008
Keywords
Aldo-keto reductase; Atorvastatin calcium; Rational engineering; Site-saturation mutagenesis; T-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate
Categories
Funding
- National Basic Research Program of China (973 Program) [2011CB710800]
- Major Program for Key Science and Technology of Zhejiang Province of China [2014C03010]
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Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, de(p) >99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (k(cat)/K-m) toward 1a up to 12.37s(-1) mM(-1), which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7mM with de(p) value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis. (C) 2016 Elsevier B.V. All rights reserved
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