Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor

Protein engineering is a powerful strategy for enhancing the properties of enzymes for industrial applications. However, thermostabilizing an enzyme via this strategy while simultaneously improving its activity is challenging due to the well-known stability–activity trade-off. Herein, using native ketoreductase Lb CR, thermostability and activity were evolved separately by directed evolution, generating mutations V198I and M154I/A155D with increased thermostability and mutations A201D/A202L with increased enzymatic activity. On the basis of additivity and cooperative mutational effects, variants Lb CR M6 (M154I/A155D/A201D/A202L) and Lb CR M8 (M154I/A155D/V198I/A201D/A202L) with simultaneously improved thermostability and activity were subsequently constructed by combining mutations. Analysis of variant structures demonstrated that increased thermostability was largely attributed to rigidification of flexible loops around the active site through the formation of additional hydrogen bonds and hydrophobic interactions. The best variant Lb CR M8 displayed a 1944-fold increase in half-life at 40 °C and a 3.2-fold improvement in catalytic efficiency compared with the wide-type enzyme. Using only 1 g L –1 of lyophilized E. coli cells coexpressing this Lb CR M8 and glucose dehydrogenase Bm GDH as a catalyst, t -butyl 6-cyano-(5 R )-hydroxy-3-oxo-hexanoate up to 300 g L –1 loading was completely reduced within 6 h at 40 °C, yielding the corresponding t -butyl 6-cyano-(3 R,5 R )-dihydroxyhexanoate (ATS-7) with >99.5% de and a space-time yield of up to 1.05 kg L –1 day –1 . These results demonstrated that Lb CR M8 is an attractive biocatalyst for the synthesis of ATS-7, an advanced chiral intermediate for the production of the cholesterol-lowering drug atorvastatin.