(424b) Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream 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, but no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through spontaneous adaptive evolution. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains. In addition, the phytosterol-producing yeast strains were also used to reconstitute the de novo synthesis of campesterol-derived specialized phytochemicals. 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.