Presentation Information
[P03-324]Development of a high-efficiency genome editing platform in Komagataella phaffii for biopharmaceutical production
○Masaki Matsumoto1, Yoichiro Ito1, Kento Yamada1, Noriko Hashiba1, Keiji Nishida1, Masahiro Tominaga1, Rintaro Sato1, Jun Ishii1 (1. Kobe Univ. (Japan))
Keywords:
Komagataella phaffii,Multiplex genome editing,Heterologous protein production,Proteolytic degradation,Biopharmaceutical production
[Purpose]
With the increasing demand for protein therapeutics, including antibody-based drugs, there is a growing need for manufacturing platforms that enable both cost-effectiveness and high productivity. Komagataella phaffii is a promising microbial host due to its strong methanol-inducible expression system, high secretion capacity, and proven track record in producing FDA-approved biopharmaceuticals. However, proteolytic degradation by endogenous proteases remains a major challenge, leading to reduced yields and product heterogeneity. Because the mechanisms of proteolysis are not fully understood, rational host strain design is difficult. Therefore, this study aimed to establish a platform for high-quality protein production in K. phaffii.[Method]
We developed (1) a highly efficient multiplex genome editing system enabling multiple gene modifications in a single transformation and (2) a high-throughput, high-resolution platform for evaluating degradation of heterologous proteins. For genome editing, a Δdnl4 strain deficient in non-homologous end joining (NHEJ) was used to promote homologous recombination. Gene disruption was achieved by knocking in stop codons near the start codon region of target genes. For degradation analysis, human serum albumin (HSA), single-chain variable fragment (scFv), and bispecific T-cell engager (BiTE) were used as model proteins, and strains with individual gene disruptions were constructed. Degradation levels were quantitatively evaluated using an automated Western blotting system.[Results]
The developed genome editing system enabled efficient multi-gene modification, achieving double gene disruption at 87.5% and triple gene disruption at 85.7% in a single transformation. Disruption of YPS1 consistently suppressed degradation across all tested proteins. In contrast, other gene disruptions exhibited protein-dependent effects.[Consideration]
These results suggest that YPS1 plays a key role in the degradation of heterologous proteins in K. phaffii. The protein-dependent effects observed for other gene disruptions indicate that degradation mechanisms are influenced by the structural and physicochemical properties of target proteins. The combination of multiplex genome editing and systematic evaluation provides an effective approach for identifying factors governing protein stability.[Conclusion]
We established an integrated platform combining multiplex genome editing and degradation evaluation in K. phaffii. This platform enables systematic host optimization and provides a foundation for improving protein stability, yield, and product quality, contributing to the development of next-generation microbial cell factories for biopharmaceutical production.
With the increasing demand for protein therapeutics, including antibody-based drugs, there is a growing need for manufacturing platforms that enable both cost-effectiveness and high productivity. Komagataella phaffii is a promising microbial host due to its strong methanol-inducible expression system, high secretion capacity, and proven track record in producing FDA-approved biopharmaceuticals. However, proteolytic degradation by endogenous proteases remains a major challenge, leading to reduced yields and product heterogeneity. Because the mechanisms of proteolysis are not fully understood, rational host strain design is difficult. Therefore, this study aimed to establish a platform for high-quality protein production in K. phaffii.[Method]
We developed (1) a highly efficient multiplex genome editing system enabling multiple gene modifications in a single transformation and (2) a high-throughput, high-resolution platform for evaluating degradation of heterologous proteins. For genome editing, a Δdnl4 strain deficient in non-homologous end joining (NHEJ) was used to promote homologous recombination. Gene disruption was achieved by knocking in stop codons near the start codon region of target genes. For degradation analysis, human serum albumin (HSA), single-chain variable fragment (scFv), and bispecific T-cell engager (BiTE) were used as model proteins, and strains with individual gene disruptions were constructed. Degradation levels were quantitatively evaluated using an automated Western blotting system.[Results]
The developed genome editing system enabled efficient multi-gene modification, achieving double gene disruption at 87.5% and triple gene disruption at 85.7% in a single transformation. Disruption of YPS1 consistently suppressed degradation across all tested proteins. In contrast, other gene disruptions exhibited protein-dependent effects.[Consideration]
These results suggest that YPS1 plays a key role in the degradation of heterologous proteins in K. phaffii. The protein-dependent effects observed for other gene disruptions indicate that degradation mechanisms are influenced by the structural and physicochemical properties of target proteins. The combination of multiplex genome editing and systematic evaluation provides an effective approach for identifying factors governing protein stability.[Conclusion]
We established an integrated platform combining multiplex genome editing and degradation evaluation in K. phaffii. This platform enables systematic host optimization and provides a foundation for improving protein stability, yield, and product quality, contributing to the development of next-generation microbial cell factories for biopharmaceutical production.
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