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

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.