Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides

<title>Abstract</title> Fungal natural products, including polyketides, are a rich source of bioactive molecules. Their biosynthetic enzymes are encoded within biosynthetic gene clusters, which are often activated by specific environmental conditions. As a result, many natural products are not produced under standard laboratory conditions. Heterologous expression bypasses native regulation, enabling a systematic approach for polyketide discovery. The most widely used fungal hosts for natural product production are Saccharomycetales yeasts, and filamentous Eurotiomycetes. Yeasts are highly tractable but have a narrow product scope due to their limited secondary metabolism, while filamentous Eurotiomycetes have a richer secondary metabolism but are more difficult to engineer. In this work, we established two yeasts of the genera <italic>Exophiala </italic>and <italic>Knufia </italic>as novel heterologous hosts for a broad range of polyketides. These hosts combine the genetic tractability of yeast with the metabolic robustness of filamentous fungi. We developed genetic engineering tools for precise gene integration and genome editing, allowing us to heterologously express five fungal polyketide synthases with different domain architectures, including one involved in the biosynthesis of a previously undescribed 2-pyridone. Our findings demonstrate that these novel yeast hosts can efficiently produce complex polyketides, paving the way for systematic polyketide synthase expression and engineering.