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Abstract <p>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. <i>Yarrowia lipolytica</i> 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 <i>TdTomato</i>-2A-<i>hrGFP</i> reporters to compare the efficiency of various viral 2A peptides in <i>Y. lipolytica</i> and identify the most effective 2A sequences. Using the PNPV2A1 peptide in a bicistronic setup, we reconstructed and validated the partial <i>Aspergillus hancockii</i> putative patulin pathway, producing m-cresol in <i>Y. lipolytica</i> and achieving a titer of 306&#xa0;mg/L in comparison to 418&#xa0;mg/L in the monocistronic setup. Our work expands the polycistronic toolkit for <i>Y. lipolytica</i>, beyond the four canonical 2A peptides, by highlighting the distinct performance of several non-canonical 2As and streamlining pathway expression and fungal metabolite biosynthesis.</p> Key points <p>• <i>2 A peptides allow compact fungal BGC expression in Y. lipolytica.</i></p> <p>• <i>Polycistronic expression can facilitate fungal natural product discovery and biosynthesis.</i></p> <p>• <i>Noncanonical 2 A peptides expand the arsenal of polycistronic tools in Y. lipolytica.</i></p> Graphical Abstract <p></p>

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2A peptides enable simplified discovery and heterologous production of fungal bioactive metabolites in Yarrowia lipolytica

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月22日

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  • Mihaela Bejenari,
  • Mikkel Rank Nielsen,
  • Jens Laurids Sørensen

摘要

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.

Graphical Abstract