One-Step Assembly of Multiple DNA Fragments and Genomic Integration in .

The methylotrophic yeast species (synonym: ) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on , advanced methods such as DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step assembly of multiple DNA fragments and genomic integration in . To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus , a 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 strains.