Effect of OPI1 Gene Inactivation on the Production of Heterologous Phytase and β-Amylase in Komagataella phaffii

A new recipient strain, Komagataella phaffii VKPM Y-5229 (Δhis4Δopi1), has been developed. Its use allows constructing strains with increased production of recombinant proteins. The OPI1 gene encoding the Opi1 transcription regulator, a functional analogue of the negative regulator of lipid biosynthesis Opi1p from Saccharomyces cerevisiae, was identified. The OPI1 gene was knocked out in the K. phaffii VKPM Y-5013 (Δhis4) strain using the highly efficient CRISPR/Cas9 genome editing system. The effect of OPI1 gene inactivation on the production of recombinant heterologous enzymes, Citrobacter gillenii phytase and Priestia flexa β-amylase, was studied. The average productivity of transformants based on the VKPM Y-5229 (Δhis4Δopi1) strain secreting phytase or β-amylase increased by 18 and 25%, respectively, compared to the productivity of transformants derived from the parent strain. Furthermore, it was demonstrated that inactivation of the OPI1 gene in a multicopy phytase-producing strain led to an increase in productivity by more than 26%. The results obtained can be employed for developing industrial strains producing heterologous proteins based on K. phaffii, as well as for increasing the production of targeted proteins in existing producing strains.