Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus

Abstract

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34mgL−1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sga15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.