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 b...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2023-06-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.14249 |
| _version_ | 1827940455271104512 |
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| author | Xiaoyan Guo Yunfang Gao Fangzheng Liu Yong Tao Haibo Jin Jianjun Wang Sheng Wu |
| author_facet | Xiaoyan Guo Yunfang Gao Fangzheng Liu Yong Tao Haibo Jin Jianjun Wang Sheng Wu |
| author_sort | Xiaoyan Guo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T09:10:07Z |
| format | Article |
| id | doaj.art-97a3556a8069497591b852534efc21e2 |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-03-13T09:10:07Z |
| publishDate | 2023-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-97a3556a8069497591b852534efc21e22023-05-27T09:33:44ZengWileyMicrobial Biotechnology1751-79152023-06-011661333134310.1111/1751-7915.14249A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediolXiaoyan Guo0Yunfang Gao1Fangzheng Liu2Yong Tao3Haibo Jin4Jianjun Wang5Sheng Wu6College of New Materials and Chemical Engineering Beijing Institute of Petrochemical Technology Beijing ChinaCollege of New Materials and Chemical Engineering Beijing Institute of Petrochemical Technology Beijing ChinaCollege of New Materials and Chemical Engineering Beijing Institute of Petrochemical Technology Beijing ChinaCAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology, Chinese Academy of Sciences Beijing ChinaCollege of New Materials and Chemical Engineering Beijing Institute of Petrochemical Technology Beijing ChinaCAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology, Chinese Academy of Sciences Beijing ChinaCAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology, Chinese Academy of Sciences Beijing ChinaAbstract 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.https://doi.org/10.1111/1751-7915.14249 |
| spellingShingle | Xiaoyan Guo Yunfang Gao Fangzheng Liu Yong Tao Haibo Jin Jianjun Wang Sheng Wu A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol Microbial Biotechnology |
| title | A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol |
| title_full | A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol |
| title_fullStr | A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol |
| title_full_unstemmed | A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol |
| title_short | A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol |
| title_sort | short chain carbonyl reductase mutant is an efficient catalyst in the production of r 1 3 butanediol |
| url | https://doi.org/10.1111/1751-7915.14249 |
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