Scipion, C. P. M., Esque, J., Borkar, S., Seah, C., Bozonnet, S., Remaud-Siméon, M., Xue, B., Yew, W. S., André, I., & Chen, X. (2025). Exploring Natural Diversity of Limonene Synthases and Molecular Determinants Involved in Substrate Specificity in Escherichia coli. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.5c01640
Abstract:
Limonene is a chiral, high-demand monoterpene that has wide applications in therapeutics, cosmetics, biofuels, agrifood, 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
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Funding Info:
This work is funded by the EcoCTs project with the support of
the National Research Foundation, Prime Minister’s Office,
Singapore, under its Campus for Research Excellence and
Technological Enterprise (CREATE) program
This research is supported by core funding from: Singapore Institute of Food and Biotechnology Innovation (SIFBI)
Grant Reference no. : NA
This research / project is supported by the Agency for Science, Technology and Research - Central Research Fund (Applied/Translational Research)
Grant Reference no. : NA