Abstract
Filamentous fungi are a valuable source of bioactive secondary metabolites (SMs), the discovery of which is hindered by the challenges in cultivation and metabolic engineering. Yarrowia lipolytica emerges as a promising heterologous host for SMs, yet efficient expression of multigene biosynthetic clusters remains a bottleneck due to limited polycistronic expression strategies. In this study, we developed bicistronic TdTomato-2A-hrGFP reporters to compare the efficiency of various viral 2A peptides in Y. lipolytica and identify the most effective 2A sequences. Using the PNPV2A1 peptide in a bicistronic setup, we reconstructed and validated the partial Aspergillus hancockii putative patulin pathway, producing m-cresol in Y. lipolytica and achieving a titer of 306 mg/L in comparison to 418 mg/L in the monocistronic setup. Our work expands the polycistronic toolkit for Y. lipolytica, beyond the four canonical 2A peptides, by highlighting the distinct performance of several non-canonical 2As and streamlining pathway expression and fungal metabolite biosynthesis. KEY POINTS: • 2 A peptides allow compact fungal BGC expression in Y. lipolytica. • Polycistronic expression can facilitate fungal natural product discovery and biosynthesis. • Noncanonical 2 A peptides expand the arsenal of polycistronic tools in Y. lipolytica.