Exploring Natural Diversity of Limonene Synthases and Molecular Determinants Involved in Substrate Specificity in Escherichia coli
Uloženo v:
| Název: | Exploring Natural Diversity of Limonene Synthases and Molecular Determinants Involved in Substrate Specificity in Escherichia coli |
|---|---|
| Autoři: | Scipion, Clement, Esque, Jérémy, Borkar, Shreyash, Seah, Cristalle, Bozonnet, Sophie, Remaud-Siméon, Magali, Xue, Bo, Yew, Wen Shan, André, Isabelle, Chen, Xixian |
| Přispěvatelé: | SCD, INSA Toulouse |
| Zdroj: | Journal of Agricultural and Food Chemistry. 73:13722-13735 |
| Informace o vydavateli: | American Chemical Society (ACS), 2025. |
| Rok vydání: | 2025 |
| Témata: | enzyme engineering, limonene synthase, enantioselectivity, limonene, neryl pyrophosphate, geranyl pyrophosphate, Agastache rugosa, terpenoid, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, monoterpene, [SDV.BIO] Life Sciences [q-bio]/Biotechnology |
| Popis: | Limonene is a chiral, high-demand monoterpene that has wide applications in therapeutics, cosmetics, biofuels, agri-food, biomaterials, and solvent industries. However, its biosynthesis by microbial cell factories is often limited by the poor activity of limonene synthase (LS). Optimization of the rate-limiting enzyme is thus crucial for boosting limonene production. Here, we report the identification of ten LS homologues from sequence data mining and their testing in cells accumulating geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) for limonene production. The selectivity of these enzymes toward GPP or NPP was investigated, leading to the identification of a limonene synthase from Agastache rugosa that displays a clear substrate preference for NPP over GPP in vivo. This enzyme was selected as a template for engineering. Using in silico analyses and mutagenesis, several mutants were engineered that revealed differences in substrate specificity. Among them, a combination of mutations (S8K/I265V/E276P/P277R/A281K/N282T/I285Q/I286L) improved limonene production by 4.8- and 1.9-fold with the GPP and NPP pathways, respectively. The mutant predominantly produced (+)-limonene from GPP and a mixture of limonene from NPP, with ∼85-90% of (+)-limonene. This decreased the selectivity for NPP by 2.4-fold. This supports the improved biological production of limonene enantiomers from renewable carbon sources. |
| Druh dokumentu: | Article |
| Jazyk: | English |
| ISSN: | 1520-5118 0021-8561 |
| DOI: | 10.1021/acs.jafc.5c01640 |
| Přístupová URL adresa: | https://pubmed.ncbi.nlm.nih.gov/40396278 https://hal.science/hal-05240689v1 https://doi.org/10.1021/acs.jafc.5c01640 |
| Rights: | STM Policy #29 |
| Přístupové číslo: | edsair.doi.dedup.....b051fc7eb14ebbb5a0bac118fd16b9c6 |
| Databáze: | OpenAIRE |
Nájsť tento článok vo Web of Science | Abstrakt: | Limonene is a chiral, high-demand monoterpene that has wide applications in therapeutics, cosmetics, biofuels, agri-food, biomaterials, and solvent industries. However, its biosynthesis by microbial cell factories is often limited by the poor activity of limonene synthase (LS). Optimization of the rate-limiting enzyme is thus crucial for boosting limonene production. Here, we report the identification of ten LS homologues from sequence data mining and their testing in cells accumulating geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) for limonene production. The selectivity of these enzymes toward GPP or NPP was investigated, leading to the identification of a limonene synthase from Agastache rugosa that displays a clear substrate preference for NPP over GPP in vivo. This enzyme was selected as a template for engineering. Using in silico analyses and mutagenesis, several mutants were engineered that revealed differences in substrate specificity. Among them, a combination of mutations (S8K/I265V/E276P/P277R/A281K/N282T/I285Q/I286L) improved limonene production by 4.8- and 1.9-fold with the GPP and NPP pathways, respectively. The mutant predominantly produced (+)-limonene from GPP and a mixture of limonene from NPP, with ∼85-90% of (+)-limonene. This decreased the selectivity for NPP by 2.4-fold. This supports the improved biological production of limonene enantiomers from renewable carbon sources. |
|---|---|
| ISSN: | 15205118 00218561 |
| DOI: | 10.1021/acs.jafc.5c01640 |