Engineering an Extremely Monoterpene-Tolerant Serratia marcescens for High-Yield Geraniol Production via a Rationally Modified Insect Phosphatase

Geraniol is a monoterpenoid alcohol with diverse applications in medicine, agriculture, and food. The utilization of microorganisms for geraniol production represents a green and sustainable strategy. However, the yield is often constrained by product toxicity and the inefficiency of terpenoid synthases. To address these challenges, this study isolated Serratia marcescens HBQA7, a strain exhibiting broad-spectrum tolerance to monoterpenoid compounds. We constructed a Haloarchaea-type MVA pathway to supply geranyl diphosphate (GPP), the precursor of geraniol. The AaFPPase-2 phosphatase from Aedes aegypti was employed as a substitute for geraniol synthase, with directed modifications to enhance its hydrolytic efficiency, thereby increasing the flux of GPP toward geraniol production. The supply of GPP was further optimized by screening various geranyl diphosphate synthases (GPPS). Additionally, we minimized metabolic losses during geraniol biosynthesis by knocking out genes involved in competing pathways. In shake flask cultures with 40 g/L glycerol, a geraniol titer of 5.23 g/L was achieved, and in a 5 L bioreactor, 25.12 g/L of geraniol was produced in 96 h. To the best of our knowledge, this represents the highest reported yield of geraniol to date. This study provides a promising approach for large-scale microbial production of geraniol.