CRISPR/Cas9-mediated genome editing of the <i>Komagataella phaffii</i> to obtain a phytase-producer markerless strain

Abstract

Using the CRISPR/Cas9 system, the recipient strains K. phaffii VKPM Y-5013 (His- phenotype) and K. phaffii VKPM Y-5014 (Leu- phenotype) were derived from the K. phaffii VKPM Y-4287 strain, which has a high expression potential. Based on developed recipients, markerless producers can be obtained. The gene inactivation efficiency with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for HIS4 and LEU2, respectively. The recipient strains retained the growth characteristics of the parent strain and have a high expression potential, as estimated by the production of heterologous phytase from Citrobacter gillenii. The average productivity of the transformants based on K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014 strains was 2.1 and 2.0 times higher than the productivity of the transformants of the commercial K. phaffii GS115 strain. Sequential integration of genetic material into the genome of the K. phaffii VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producing C. gillenii phytase was obtained.