Hybrid Methylotrophic Pathway in Serratia marcescens for Sustainable Terpenoid Biosynthesis

Terpenoids are valuable chemicals used across industries. Methanol, a nonsugar-based feedstock, offers an eco-friendly approach to terpenoid production. In this study, a hybrid methylotrophic pathway was engineered in Serratia marcescens (S. marcescens) HBQA7, which is tolerant to both methanol and terpenoid compounds. The pathway utilizes bacterial methanol dehydrogenase (Mdh) and yeast dihydroxyacetone synthase (Das) to produce monoterpenes and sesquiterpenes from methanol and xylose. 13C labeling experiments identified the optimal enzyme pair: Mdh from Acinetobacter gerneri and Das from Pichia angusta, achieving 7.63% 13C enrichment of the central metabolic intermediate pyruvate. Deletion of pentose phosphate pathway genes (rpiAB) enhanced methanol utilization, achieving 22.99% 13C enrichment. Optimization of the mevalonate (MVA) biosynthetic pathway enabled the production of 5.12 g/L mevalonate in shake flask culture from methanol and xylose. Further construction of a haloarchaea-type MVA pathway enabled the production of geraniol (574.12 mg/L) and (-)-α-bisabolol (1256.41 mg/L) in shake flask cultures. This study demonstrates the first methanol conversion into valuable terpenoids in S. marcescens.