Engineering an ATP-saving mevalonate pathway for high-efficiency S-(+)-linalool production in Serratia marcescens

(S)-(+)-linalool, a valuable acyclic monoterpenol secondary metabolite of plants, finds extensive applications in the food, flavor and fragrance, pharmaceutical, and daily chemical industries. Microbial synthesis offers two pathways for its production, among which the mevalonate (MVA) pathway derived from halophilic archaea is more widely employed. However, the traditional Haloarchaea-type MVA pathway relies on bifunctional enzyme catalysis, consuming 3 molecules of ATP. Moreover, the catalytic activity of natural linalool synthase (LIS) is generally low, failing to meet industrial requirements. Additionally, linalool exhibits significant toxicity to microbial hosts, thereby limiting the production capacity of conventional chassis microorganisms. To address these bottlenecks, this study implemented systematic optimizations: Firstly, the Archaeal mevalonate pathway was reconstructed by replacing the original bifunctional enzyme with two monofunctional enzymes, successfully reducing ATP consumption to 2 molecules. Secondly, through directed screening and rational design, a high-activity linalool synthase mutant, CsMLIS^I331V/I444L, derived from Coriandrum sativum, was obtained. Furthermore, an enzyme fusion strategy was adopted, involving the introduction of a long flexible linker between key genes, which significantly enhanced catalytic efficiency. Finally, S. marcescens HBQA7ΔsIaAB-pyc, a strain previously screened in our laboratory with broad-spectrum tolerance to terpenoids, was selected as the novel chassis cell. Collectively, these efforts resulted in the construction of a microbial cell factory for the efficient synthesis of (S)-(+)-linalool, laying a solid foundation for industrial-scale production.