High-Efficiency Production of (S)-Limonene in Monoterpene-Resistant <i>Serratia Marcescens</i> through Bicistronic Translation of Engineered (S)-Limonene Synthase

Limonene is a vital monoterpenoid with wide applications in food, medicine, cosmetics, and other fields. Microbial limonene production is a green and sustainable strategy, but its yield is limited by product toxicity and the low terpene synthase activity. In this study, using <i>Serratia. marcescens</i> HBQA7 Δ<i>slaAB-pyc</i> as the chassis strain, we first screened the most active (S)-limonene synthase from <i>Mentha spicata</i> among five plant-derived ones. Then, to further improve its activity, we rationally engineered its active site and enhanced its expression via a bicistronic structure. Finally, 4.5 g/L (S)-limonene was achieved in shake flasks with glycerol as the carbon source after 72 h. In a 5 L bioreactor, production reached 22.8 g/L within 100 h, representing the highest microbial (S)-limonene yield reported to date.