Agrobacterium tumefaciens-mediated transformation of the oleaginous yeast genus Lipomyces

Due to interest in renewable bio-hydrocarbon fuel production, various organisms are being explored for their potential use in fuel and chemical production.  Numerous lipid-producing yeasts, e.g. Lipomyces starkeyi, are being actively investigated for their potential in biofuel production utilizing various carbon and nutrient sources.  Although L. starkeyi genome is available, there are few established genetic tools for engineering this organism.  Establishing effective transformation methods with suitable selectable markers and genetic elements for the selected gene expression will provide the tools needed to genetically modify these organisms.  Alteration of targeted gene expressions and metabolic pathways will help realize improvements in lipid and related chemical productivity. 

       Initially, we performed the transformation by following two recent published methods (Curr Genet 60:223; Biotechnol Lett 33:1993) with intact cells or protoplasts of L. starkeyi or L. konoenkoae and failed to generate transformed strains. Therefore, Agrobacterium-mediated transformation (ATMT) was investigated for transformation of Lipomyces starkeyi and other Lipomyces species.  We have demonstrated that DNA can be effectively integrated into the chromosomes of five different L. starkeyi strains and six other Lipomyces species by ATMT using a bacterial hph gene as an antibiotic selection marker.  As proof of principle, ATMT was used to produce transformants expressing a bacterial β-glucuronidase (GUS) and the L. starkeyi NADP-malic enzyme under the control of L. starkeyi translation elongation factor 1a (tef1) promoter. 

       The results from this study clearly demonstrate that Agrobacterium-mediated transformation is an effective and reliable genetic tool for integration of foreign genes into L. starkeyi and other Lipomyces species.