Carbonyl reductase (CR)-catalyzed asymmetric reduction offers an approach for producing t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b), which serves as a key building block in atorvastatin (Liptor). However, controlling the stereopreference of CR with the desired selectivity remains challenging because natural CRs usually exhibit Prelog preference. Moreover, transferring knowledge from engineered anti-Prelog CRs to other CRs is difficult. Herein, the key residues that regulate the stereopreference of a CR from Kluyveromyces marxianus (KmCR) were identified by a semirational engineering toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1a). A structural switch that consists of the key residues was discovered, and related structural features were summarized to predict the stereopreference by analyzing the structural information and multiple-sequence alignment of structure-available CRs carefully. According to the obtained knowledge, the simultaneous mutation of four key residues enabled the conversion of Prelog-selectivity of KmCR into a complete anti-Prelog selectivity (de p > 99% (R) for (3R,5R)-1b). Moreover, the stereopreferences of 11 CRs that share 20–40% sequence identities with KmCR were predicted successfully and engineered experimentally. The knowledge gained from this protein engineering study on KmCR has universal significance for CRs toward (5R)-1a.

No abstract

Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCR M3 , coevolved thermostability, and activity. Compared with LsCR M3 , the catalytic efficiency k cat /K M of LsCR M3 -E145A (LsCR M4 ) was increased from 6.6 to 21.9 s −1 mM −1 . Moreover, E145A prolonged the half-life t 1/2 at 40°C from 4.1 to 117 h, T m was increased by 5°C, T 50 30 was increased by 14.6°C, and T opt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCR M4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% ee P , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g.Compared with LsCR M3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCR WT , the substrate loading was increased by 6.5 times. In contrast, LsCR M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

No abstract

Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (k cat /K M = 47.3 s −1 •mM −1 ), improved stereoselectivity (from moderate selectivity (de P = 66.7%) to strict (S)-selectivity (de P > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.