1-07: Metabolic engineering of the oleaginous yeast Lipomyces starkeyi for isoprene production

Replacing fossil fuels with renewable alternatives will be a daunting challenge for human civilization in the 21^st century.  Isoprenoids are a diverse class of naturally occurring hydrocarbons, many of which have favorable characteristics and the potential to replace gasoline, diesel, or jet fuels.  Oleaginous yeasts, such as Lipomyces starkeyi, are well known for their capacity to accumulate high levels of intracellular lipids.  Metabolic engineering may be able to divert the flux of acetyl-CoA away from fatty acids biosynthesis and towards the production of isoprenes in these species.  Lipomyces is a particularly well suited host given its ability to thrive on mixtures of glucose and xylose at ratios similar to those obtained from renewable agricultural hydrolysate feedstocks.

Here we report the development of the first genetic transformation system for Lipomyces starkeyi.  Lipomyces URA- auxotrophic colonies were isolated by negative selection on 5’-FOA plates.  URA- strains were sequenced to identify those with mutations in the orotidine 5-phosphate decarboxylase (URA3) gene.  Lipomyces URA3 mutants were then transformed with a cloned wild-type URA3, using electroporation and the lithium acetate method.  These tools will be very useful for future genetic manipulations. Further, the Lipomyces starkeyi genome has been sequenced in collaboration with the Joint Genome Institute’s Fungal Genomics Program.  We have identified the members of the Lipomyces mevalonic acid pathway based on homology to previously sequenced species.  These genes will be of interest for designing constructs to maximize isoprene production in Lipomyces.