One-Step In Vivo Assembly of Multiple DNA Fragments and Genomic Integration in Komagataella phaffii

The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii. To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl4p-defective strain permits direct and easy engineering of K. phaffii strains.

Automated summary

In this study, a technique for in vivo DNA assembly and integration was established in the methylotrophic yeast species Komagataella phaffii. The use of a defective strain to improve gene targeting efficiency was demonstrated through the synthesis of green fluorescent protein and lycopene. This method allows for direct and easy engineering of K. phaffii strains.