Wang, J.; Chen, G. G.

Producing high-value plant-derived unusual fatty acids in microorganisms via synthetic biology is attractive, but increasing their contents through rational step-by-step gene stacking is challenging. Using Saccharomyces cerevisiae and pomegranate-derived punicic acid (PuA) as representatives, an efficient, result-driven gene shuffling strategy was developed to facilitate the production of value-added plant lipids. By targeting yeast Ty retrotransposon regions, candidate genes related to PuA production were directly shuffled within the yeast genome to create recombinant libraries. Subsequent screening and bioprocess optimization led to a recombinant yeast strain with 26.7% of total fatty acids as PuA through neosynthesis. Further analyses revealed that the strain hosts multiple genes, contains over 22% PuA in the storage lipid triacylglycerol, and has substantial changes in its lipidome. Overall, this work provided an efficient strategy for improving PuA content in yeast, which could be adopted to engineer microorganisms for the production of other high-value plant-derived fatty acids and bioproducts.