Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as foodadditives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic alpha-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene beta-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and beta-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms