Establishment of a genome editing system and expression of heterologous proteins in the nonconventional yeast Pichia kudriavzevii

Impact statement Nonconventional yeast Pichia kudriavzevii possesses attractive phenotypic traits for industrial biomanufacturing; however, it remains genetically intractable compared with established yeast chassis. Here, we present an integrated synthetic biology framework that renders P. kudriavzevii a programmable production host. We established a highly efficient CRISPR/Cas9 genome editing system in this diploid yeast, enabling marker‐free editing with efficiencies of up to 88% and routine generation of homozygous mutants. By integrating efficient marker‐free CRISPR/Cas9 genome editing and locus‐aware expression strategies, we successfully expressed secretory mCherry and human IgG1 Fc fragment at different loci. Our results demonstrate that P. kudriavzevii is a promising expression system for protein production.