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

• Published in: Biochemistry (Moscow) Vol. 88; no. 9; pp. 1338 - 1346
• Main Authors: Tkachenko, Artur A., Borshchevskaya, Larisa N., Sineoky, Sergey P., Gordeeva, Tatiana L.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-09-2023; Springer Nature B.V
• Subjects: Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Citrobacter; CRISPR; Genome editing; Genomes; Inactivation; Life Sciences; Microbiology; Phenotypes; Phytase; Productivity; CRISPR/Cas9; genome editing; phytase
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297923090134

Using 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 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 the
HIS4
and
LEU2
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 from
Citrobacter gillenii
. Average productivity of the transformants based on the
K. phaffii
VKPM Y-5013 and
K. phaffii
VKPM Y-5014 strains was 2.1 and 2.0 times higher than productivity of the transformants of the commercial
K. phaffii
GS115 strain. Method for sequential integration of genetic material into genome of the
K. phaffii
VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producing
C. gillenii
phytase was obtained.