Systematic engineering to enhance valencene production in Rhodobacter sphaeroides

Microbial synthesis of (+)-valencene from agricultural wastes such as cornstalk will serve as a sustainable alternative to the traditional plant extraction method. In this study, Rhodobacter sphaeroides strains were engineered for valencene production through a stepwise manner: (1) heterologous expression of Callitropsis nootkatensis valencene synthase (CnVS) in combination with phaB/gdhA/ladH knockouts enabled de novo biosynthesis of valencene from glucose at a titer of 34.21 ± 3.1 mg/L; (2) a quorum-sensing promoter P_cer to decouple growth and production phase further improved the valencene titer to 80.75 ± 3.0 mg/L; and (3) transposon-mediated genomic integration of the heterologous mevalonate pathway to enhance farnesyl pyrophosphate supply resulted in 120.53 ± 10.34 mg/L valencene. Subsequently, the alkali-pretreated cornstalk hydrolysate was used as the substrate, and 100.51 ± 14.15 mg/L valencene was achieved under the optimized carbon-to-nitrogen ratio. In summary, the engineered R. sphaeroides offers an alternative mean to valorize the cheap agricultural waste for high-value valencene production.