Expanding the Genetic Toolkit of Pichia kudriavzevii E1 for Enhanced Mycoprotein Production.

Pichia kudriavzevii is gaining attention as a non-conventional yeast with significant industrial potential, owing to its robust tolerance to low pH and diverse process-related stresses. However, the lack of a reliable episomal expression system and the scarcity of well-characterized promoters currently impede predictable gene expression and modular metabolic engineering in this workhorse. Here, we report development of the stable episomal expression vector for P. kudriavzevii E1. This was achieved through identification of a functional autonomously replicating sequence from the endogenous genome, guided by the established host-vector GC compatibility workflow. Leveraging this episomal platform, we then constructed an endogenous promoter library for P. kudriavzevii E1 via transcriptomic sequencing data analysis. Among the characterized promoters, the endogenous PkTDH3 promoter exhibited the highest activity. Notably, PkTDH3 demonstrated more stable and sustained expression compared to the strong promoter ScTDH3 from Saccharomyces cerevisiae, underscoring the value of establishing native promoter resources for P. kudriavzevii. Furthermore, we expanded the strength range of the promoter library by engineering the upstream activating sequence of PkTDH3 through random mutagenesis. As a proof of concept, the developed promoter library was employed to construct P. kudriavzevii mutants with elevated protein content, demonstrating the utility of this integrated genetic toolkit for strain engineering in P. kudriavzevii.