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Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal
Bioproduction of optical pure (R)‐citronellal from (E/Z)‐citral at high substrate loading remains challenging. Low catalytic efficiency of (R)‐stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure‐based engineering strategy was adopted to en...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9049607/ https://www.ncbi.nlm.nih.gov/pubmed/34729922 http://dx.doi.org/10.1111/1751-7915.13958 |
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author | Zhang, Baoqi Du, Han Zheng, Yanqiu Sun, Jiale Shen, Yu Lin, Jinping Wei, Dongzhi |
author_facet | Zhang, Baoqi Du, Han Zheng, Yanqiu Sun, Jiale Shen, Yu Lin, Jinping Wei, Dongzhi |
author_sort | Zhang, Baoqi |
collection | PubMed |
description | Bioproduction of optical pure (R)‐citronellal from (E/Z)‐citral at high substrate loading remains challenging. Low catalytic efficiency of (R)‐stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure‐based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards (E/Z)‐citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site‐saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the (E/Z)‐citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency (k (cat)/K (m)) for (Z)‐citral and (E)‐citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in (E/Z)‐citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled (E)‐citral and (Z)‐citral to bind with a smaller distance to the key hydrogen donors at a modified (R)‐selective binding mode. The variant Y84V was then co‐expressed with glucose dehydrogenase from Bacillus megaterium in E. coli D4, in which competing prim‐alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l(−1) (E/Z)‐citral was completely converted into (R)‐citronellal with 95.4% ee value and a high space‐time yield of 121.6 g l(−1) day(−1). The work highlights the synthetic potential of Y84V, which enabled the highest productivity of (R)‐citronellal from (E/Z)‐citral in high enantiopurity so far. |
format | Online Article Text |
id | pubmed-9049607 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90496072022-05-02 Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal Zhang, Baoqi Du, Han Zheng, Yanqiu Sun, Jiale Shen, Yu Lin, Jinping Wei, Dongzhi Microb Biotechnol Engineering Biology and Synthetic Biology Bioproduction of optical pure (R)‐citronellal from (E/Z)‐citral at high substrate loading remains challenging. Low catalytic efficiency of (R)‐stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure‐based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards (E/Z)‐citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site‐saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the (E/Z)‐citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency (k (cat)/K (m)) for (Z)‐citral and (E)‐citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in (E/Z)‐citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled (E)‐citral and (Z)‐citral to bind with a smaller distance to the key hydrogen donors at a modified (R)‐selective binding mode. The variant Y84V was then co‐expressed with glucose dehydrogenase from Bacillus megaterium in E. coli D4, in which competing prim‐alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l(−1) (E/Z)‐citral was completely converted into (R)‐citronellal with 95.4% ee value and a high space‐time yield of 121.6 g l(−1) day(−1). The work highlights the synthetic potential of Y84V, which enabled the highest productivity of (R)‐citronellal from (E/Z)‐citral in high enantiopurity so far. John Wiley and Sons Inc. 2021-11-02 /pmc/articles/PMC9049607/ /pubmed/34729922 http://dx.doi.org/10.1111/1751-7915.13958 Text en © 2021 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. |
spellingShingle | Engineering Biology and Synthetic Biology Zhang, Baoqi Du, Han Zheng, Yanqiu Sun, Jiale Shen, Yu Lin, Jinping Wei, Dongzhi Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title | Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title_full | Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title_fullStr | Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title_full_unstemmed | Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title_short | Design and engineering of whole‐cell biocatalyst for efficient synthesis of (R)‐citronellal |
title_sort | design and engineering of whole‐cell biocatalyst for efficient synthesis of (r)‐citronellal |
topic | Engineering Biology and Synthetic Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9049607/ https://www.ncbi.nlm.nih.gov/pubmed/34729922 http://dx.doi.org/10.1111/1751-7915.13958 |
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