Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream Brassinosteroid Biosynthetic Pathways

As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. In this study, we engineered campesterol production in yeast using plant enzymes and enhance the titer of campesterol by upregulating the mevalonate pathway. Further engineer to inactivate yeast acyltransferases enhanced the production of free campesterol. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains and reconstitute and characterize the early brassinosteroids biosynthetic pathways on this phytosterol platform. In addition, the phytosterol-producing yeast platform were also used to reconstitute the plant membrane-anchored enzymes with enhanced enzymatic activities. This work resolves the technical bottlenecks in phytosterol-derived pathway reconstitution in the backer’s yeast and opens up opportunities for efficient bioproduction and pathway elucidation of this group of phytochemicals.