PNNL

Abstract

Recently, several transformation methods have been established for the oleaginous yeast Lipomyces starkeyi, a model strain being explored for use in the production of renewable fuels and chemicals in Lipomyces species. However, targeted gene disruption and/or integration in L. starkeyi have been impeded by its preference for the non-homologous end-joining (NHEJ) pathway. To augment homologous recombination (HR) by repressing the NHEJ function, the Lsku70 gene was identified and disrupted in L. starkeyi strain NRRL Y-11558. Targeted disruption efficiency of four selected genes (gsy1, mfe1, pex10, and tgl4) was increased to 50 to 100% in the Lsku70 mutant strain with 0.8 to 1.3 kb homologous flanking fragments. In contrast, HR frequency was 0 to 11% in the parent strain even with longer (1.34 kb) homologous flanking fragments. Furthermore, the minimum length of flanking homologous DNA fragments was about 0.6 kb for high-efficiency tgl4 gene disruption in the Lsku70 mutant strain, Site-specific gene insertion at intergenic regions near gsy1, ku70, mfe1, and pex10 genes were examined in the Lsku70 mutant and found to be 100% in all cases. Finally, the deletion of Lsku70 did not perturb the growth, sugar consumption, and total lipid production in the Lsku70 mutant. Therefore, the Lsku70 mutant can serve as a useful platform strain for targeted gene manipulation and improvement of fuel and value-added chemical production.