Rational Enzyme Modification and Expression Enhancement Strategies for 1,8-cineole Production in Serratia marcescens Cell Factories.

1,8-cineole, a bicyclic monoterpenoid compound with significant application value, is widely used in the fields of flavoring, pharmaceuticals, and biofuels. Although recent advances have enabled its biosynthesis in model microorganisms (Escherichia coli and Saccharomyces cerevisiae), the inherent cytotoxicity of terpenoids and the low catalytic efficiency of terpene synthases remain major bottlenecks limiting further improvement in biosynthetic efficiency. To address these limitations, we employed Serratia marcescens HBQA7-a non-model microorganism with high terpene tolerance, previously isolated in our lab-as a chassis for constructing an efficient 1,8-cineole biosynthetic system. Heterologous expression and functional screening of six 1,8-cineole synthase (CinS) genes from different sources were performed, among which SoCinS from Salvia officinalis exhibited the highest catalytic activity. Rational enzyme engineering through site-directed mutagenesis further improved SoCinS activity. In addition, fusion with high-expression tag proteins significantly improved enzyme expression levels, leading to increased 1,8-cineole titers. Under 72-hour shake flask fermentation conditions, the 1,8-cineole concentration reached 2.1 g/L. Finally, fed-batch fermentation in a 5-L bioreactor yielded a final 1,8-cineole titer of 10.2 g/L, demonstrating a higher productivity and greater industrial application potential than current model microbial systems. This work highlights the promising potential of non-model microorganisms in the efficient biosynthesis of terpenoid natural products.