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 LbCR, 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 LbCRM6 (M154I/A155D/A201D/A202L) and LbCRM8 (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 intera...