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A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

ABSTRACT: R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from -17.0 kJ/mol to -29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation-reduction cascade at larger scales.

SUBMITTER: Guo X

PROVIDER: S-EPMC10221522 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

Guo Xiaoyan X Gao Yunfang Y Liu Fangzheng F Tao Yong Y Jin Haibo H Wang Jianjun J Wu Sheng S

Microbial biotechnology 20230322 6

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and de ...[more]

PMID: 36946330

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Project description:Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter-1 day-1 These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Dataset Information

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

Publications

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

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