Enhanced Production of (+)-Limonene through Targeted Engineering of Citrus sinensis Limonene Synthase

Enhanced Production of (+)-Limonene through Targeted Engineering of Citrus sinensis Limonene Synthase

ACS Synthetic Biology

10.1021/acssynbio.5c00078

https://doi.org/10.1021/acssynbio.5c00078

Clement P. M. Scipion, Jérémy Esque, Shreyash Borkar, Jing Sen Ong, Cristalle Seah, Leonard Ong, Pnelope Ng, Yoganathan Kanagasundaram, Magali Remaud-Siméon, Sophie Bozonnet, Bo Xue, Wen Shan Yew, Isabelle André, Xixian Chen

monoterpene, limonene, Enzyme engineering, limonene synthase, citrus sinensis

28 May 2025

Scipion, C. P. M., Esque, J., Borkar, S., Ong, J. S., Seah, C., Ong, L., Ng, P., Kanagasundaram, Y., Remaud-Simeon, M., Bozonnet, S., Xue, B., Yew, W. S., André, I., & Chen, X. (2025). Enhanced Production of (+)-Limonene through Targeted Engineering of Citrus sinensis Limonene Synthase. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.5c00078

Limonene is a high-value monoterpene with numerous industrial applications including flavors, fragrances, and pharmaceuticals. Its biosynthesis in microbes is often limited by limonene synthase and can be enhanced through targeted enzyme engineering. This study aims to optimize the Citrus sinensis limonene synthase (CsLS) in order to boost (+)-limonene yield. Site-saturation mutagenesis was carried out to create a library of truncated CsLS variants, which were evaluated for their enzymatic activity, enantioselectivity, and kinetic properties. Through medium-throughput screening, a mutant, tCsLS-Q8K, was found to improve limonene production by 2.1-fold. Bioreactor experiments with this optimized mutant, in combination with the ispA S80F mutation to enhance monoterpene precursor availability, achieved a (+)-limonene titer of 4.9 g/L, representing the highest production level of limonene reported to date. This work not only deepens our understanding of CsLS function but also offers a scalable approach to enhanced (+)-limonene production. The findings underscore the potential of enzyme engineering and pathway optimization to transform biotechnological processes, significantly impacting the commercial viability of the flavor and fragrance industries.

Publisher Copyright

This research / project is supported by the National Research Foundation Singapore - Intra-CREATE Thematic Grant (Cities)
Grant Reference no. : NRF2019-THE001-0007

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

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see 10.1021/acssynbio.5c00078.

https://oar.a-star.edu.sg/communities-collections/articles/21867

2161-5063
2161-5063

Singapore Institute of Food and Biotechnology Innovation