CRISPR/Cas9-Mediated Genome Editing of the Komagataella phaffii to Obtain a Phytase-Producer Markerless Strain

AbstractUsing CRISPR/Cas9 system, the recipient strainsK.  phaffii VKPM Y-5013 (His– phenotype) andK.  phaffii VKPM Y-5014 (Leu– phenotype) were derived from theK.  phaffii VKPM Y-4287 strain, which has a high expression potential. Based on the developed recipient strains, markerless producers of heterologous proteins could be obtained. Efficiency of the gene inactivation with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for theHIS4 andLEU2 genes, respectively. The recipient strains retained growth characteristics of the parent strain and exhibited high expression potential, as estimated by the production of heterologous phytase fromCitrobacter gillenii. Average productivity of the transformants based on theK.  phaffii VKPM Y-5013 andK.  phaffii VKPM Y-5014 strains was 2.1 and 2.0 times higher than productivity of the transformants of the commercialK.  phaffii GS115 strain. Method for sequential integration of genetic material into genome of theK.  phaffii VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producingC.  gillenii phytase was obtained.

Source: Biochemistry (Moscow) - September 1, 2023 Category: Biochemistry Source Type: research