Exploring Natural Diversity of Limonene Synthases and Molecular Determinants Involved in Substrate Specificity in Escherichia coli

• Published in: Journal of agricultural and food chemistry Vol. 73; no. 22; pp. 13722 - 13735
• Main Authors: Scipion, Clement P. M., Esque, Jérémy, Borkar, Shreyash, Seah, Cristalle, Bozonnet, Sophie, Remaud-Siméon, Magali, Xue, Bo, Yew, Wen Shan, André, Isabelle, Chen, Xixian
• Format: Journal Article
• Language: English
• Published: United States 21.05.2025
• Subjects: Agastache rugosa; biocompatible materials; biofuels; biological production; biosynthesis; carbon; computer simulation; cosmetics; enantiomers; enzymes; Escherichia coli; food chemistry; limonene; mutagenesis; mutants; solvents; substrate specificity; therapeutics; enantioselectivity; limonene; enzyme engineering; monoterpene; neryl pyrophosphate; terpenoid; Agastache rugosa; geranyl pyrophosphate; limonene synthase
• ISSN: 0021-8561; 1520-5118; 1520-5118
• DOI: 10.1021/acs.jafc.5c01640

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 that displays a clear substrate preference for NPP over GPP . This enzyme was selected as a template for engineering. Using 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.