An advanced genome engineering platform for Komagataella phaffii enabling versatile in vivo DNA assembly and multiplex integration.

The transition toward a sustainable bioeconomy necessitates the development of robust microbial platforms capable of C1 chemical valorization, particularly the conversion of methanol into high-value chemicals. Komagataella phaffii is a premier methylotrophic host for such applications due to its natural methanol utilization pathways and high-density fermentation capabilities. However, its potential as a C1-based synthetic biology chassis has been hampered by inefficient homologous recombination and the technical complexity of multigene pathway integration. To accelerate the design-build-test-learn (DBTL) cycle for methanol-based biomanufacturing, a more sophisticated and high-throughput genome engineering toolkit is required. We report the development of an HR-enhanced K. phaffii chassis (dKR) achieved by synergistically deleting Ku70 and overexpressing RAD52 at the chromosomal level. This engineering markedly improved the robustness and reproducibility of CRISPR/Cas9-mediated genome editing, increasing integration efficiency from 15.3% in the wild-type to 68.1% in the dKR chassis when using short 50-bp homology arms. The platform demonstrated exceptional capacity for vector-free multi-fragment assembly, achieving an integration efficiency of 86.5% for nine linear DNA fragments. Furthermore, the implementation of a tRNA-ribozyme CRISPR system enabled simultaneous multi-locus integration at three distinct genomic sites with 44.6% efficiency. Notably, the chassis supported direct in vivo plasmid assembly of ten fragments (9 fragments + 1 backbone) with 44.8% efficiency, effectively bypassing E. coli-based cloning. Functional validation via a carotenoid biosynthetic pathway resulted in β-carotene production while maintaining normal cellular growth and metabolic productivity. This study established a versatile genome-engineering platform that advances K. phaffii from a protein-expression host to a broadly applicable biological engineering chassis. By enabling streamlined cellular design and reproducible multigene integration, the platform supports applications spanning synthetic biology, metabolic engineering, and bioproduction. More broadly, the approach illustrates how rational manipulation of DNA repair pathways can enhance genome engineering performance in non-conventional hosts, providing transferable design principles relevant to biological engineering across multiple domains.