Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the (beta-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.