Using gene editing to create a Komagataella phaffii strain that produces phytase without added markers

Gene inactivation efficiency ranged from 65 to 98% for the HIS4 gene across different sgRNA variants.

• Two recipient strains, K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014, were derived from a high-expression parent strain.
• Markerless producers of heterologous proteins were achieved using these recipient strains.
• The recipient strains maintained the growth characteristics of the parent strain while showing high expression potential.
• Average productivity of transformants from K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014 was 2.1 and 2.0 times higher than that of the commercial K. phaffii GS115 strain.
• A method for the sequential integration of genetic material into the genome of K. phaffii VKPM Y-5013 was proposed.
• A multicopy markerless strain producing phytase from Citrobacter gillenii was successfully developed.

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