Presentation Information
[P03-390]A Promoter-Replacement Library Platform for Genome-Scale Overexpression Screening in Komagataella phaffii
○Yoichiro Ito1, Masahiro Tominaga1, Takahiro Sawada1, Yuri Kyo1, Akihiko Kondo1, Jun Ishii1 (1. Kobe university (Japan))
Keywords:
Overexpression library,Genome-scale screening,Komagataella phaffii,recombinant protein production
Gene overexpression has long been used to associate genotype with phenotype. Conventional cDNA and genomic DNA libraries have supported large-scale overexpression studies; however, these approaches often suffer from substantial bias and incomplete representation of target genes. To address these limitations, we developed a novel promoter-replacement library construction platform by combining genomic sequence information with single-stranded DNA pool synthesis technology. This platform enables generation of a genome-scale overexpression library through promoter replacement and phenotype-based screening in yeast.
Promoter-replacement libraries were constructed in Komagataella phaffii. In this platform, promoter-replacement constructs were designed based on genomic sequence information and introduced as a pooled DNA library, enabling replacement of native promoters with the constitutive GAPDH promoter in a population of clones. To verify that this strategy functioned as intended, two model genes were selected and overexpression strains were generated using the same method. RT-qPCR analysis showed that mRNA levels of both model genes were increased in the corresponding promoter-replaced strains, demonstrating that the method successfully induced gene overexpression at the transcriptional level.
As an application example, we applied this platform to antibody production in K. phaffii. Promoter-replacement libraries were constructed for each of the strains producing one of three antibodies, including blinatumomab, a difficult-to-secrete bispecific antibody. The resulting libraries were screened in a 96-well plate-based high-throughput evaluation system, with antibody productivity as the screening index. Screening of approximately 19,000 clones for each target antibody identified around 30 genes whose overexpression improved antibody production relative to the parental strain. The effect of single-gene overexpression ranged from 1.1- to 2.6-fold, depending on the target protein and factor identified. In addition, multiplex overexpression of selected factors further enhanced productivity, and optimization of factor combinations resulted in more than a 15-fold improvement in blinatumomab production.
These results demonstrate that the promoter-replacement library functions as designed and provides a useful platform for yeast strain engineering.
Promoter-replacement libraries were constructed in Komagataella phaffii. In this platform, promoter-replacement constructs were designed based on genomic sequence information and introduced as a pooled DNA library, enabling replacement of native promoters with the constitutive GAPDH promoter in a population of clones. To verify that this strategy functioned as intended, two model genes were selected and overexpression strains were generated using the same method. RT-qPCR analysis showed that mRNA levels of both model genes were increased in the corresponding promoter-replaced strains, demonstrating that the method successfully induced gene overexpression at the transcriptional level.
As an application example, we applied this platform to antibody production in K. phaffii. Promoter-replacement libraries were constructed for each of the strains producing one of three antibodies, including blinatumomab, a difficult-to-secrete bispecific antibody. The resulting libraries were screened in a 96-well plate-based high-throughput evaluation system, with antibody productivity as the screening index. Screening of approximately 19,000 clones for each target antibody identified around 30 genes whose overexpression improved antibody production relative to the parental strain. The effect of single-gene overexpression ranged from 1.1- to 2.6-fold, depending on the target protein and factor identified. In addition, multiplex overexpression of selected factors further enhanced productivity, and optimization of factor combinations resulted in more than a 15-fold improvement in blinatumomab production.
These results demonstrate that the promoter-replacement library functions as designed and provides a useful platform for yeast strain engineering.
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