Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli

Abstract Background: R-(-)-linalool is a versatile acyclic monoterpene alcohol with applications in the flavor and fragrance, pharmaceutical, and agrochemical industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method of R-(-)-linalool synthesis is urgently needed. Results: To induce the R-(-)-linalool biosynthesis pathway in E. coli , we expressed heterologous (3R)-linalool synthase (LIS) from Lavandula angustifolia (laLIS). We then enhanced R-(-)-linalool production in the cells using a suitable LIS from Streptomyces clavuligerus (bLIS). The bLIS expression was markedly elevated by using optimized ribosomal binding sites (RBSs) and protein fusion tags. R-(-)-linalool output rose from 4.8 mg L -1 to 33.4 mg L -1 . To increase the geranyl diphosphate (GPP) content in E. coli , we tested various alterations in geranyl diphosphate synthases (GPPSs) and their mutants. The final E. coli strain harboring GPPS from Abies grandis ( Ag GPPS) accumulated ≤ 100.1 mg L -1 R-(-)-linalool after 72 h shake-flask fermentation. This yield gain constitutes a 60.7-fold improvement in R-(-)-linalool biosynthesis over the parent strain. Fed-batch cultivation of the engineered strain in a 1.3-L fermenter yielded 1,027.3 mg L -1 R-(-)-linalool. Conclusions: In this study, an efficient R-(-)-linalool production pathway was induced in E. coli via the heterologous MVA pathway, AgGPPS, and (3R)-linalool synthase (bLIS). By overexpressing the key enzyme in the engineered E. coli strain, R-(-)-linalool production reached 100.1 mg L -1 and 1,027.3 mg L -1 under shake flask- and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date using an engineered E. coli strain. The strategies of key enzyme overexpression and mutation could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.