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

## TL;DR

Researchers used 2A peptides to simplify the production of fungal bioactive compounds in Yarrowia lipolytica, improving the efficiency of heterologous expression.

## Contribution

The study introduces non-canonical 2A peptides to enhance polycistronic expression in Y. lipolytica for fungal metabolite biosynthesis.

## Key findings

- The PNPV2A1 peptide enabled m-cresol production in Y. lipolytica at 306 mg/L.
- Non-canonical 2A peptides outperformed some canonical ones in bicistronic setups.
- The study expands the toolkit for polycistronic expression in Y. lipolytica.

## 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.

• 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.

The online version contains supplementary material available at 10.1007/s00253-026-13797-y.