Development of metabolic engineering toolbox for lipid production in oleaginous red yeast, Sporidiobolus pararoseus

A number of economically viable technologies for converting feedstocks into biofuel, through large-scale fermentation of oleaginous microorganisms, have been developed. One of the major goals in biofuel production is improvement of fermentation characteristics, such as yield and productivity, via metabolic engineering in order to develop a low-cost biodiesel product. Here, we describe the development of a molecular toolbox for metabolic engineering of a Basidiomycetous red yeast, Sporidiobolus pararoseus.

Basidiomycetes are a unique group of fungi that possess a genetic architecture which can make them exceedingly difficult to genetically manipulate. S. pararoseus has a genome of 21.8 MB, and it is common for a 3 kb gene to have upwards of 10 introns, complicating gene predictions and metabolic engineering. The molecular tool box developed for this oleaginous yeast includes a polyethylene glycol (PEG)-mediated transformation system preceded by protoplasting with a cocktail of enzymes; auxotrophic and dominant selectable markers; working combinations of promoters and introns; and a preliminary genome-scale metabolic model for target identification.

Using these molecular biology tools, it was possible to successfully perform over-expression of native genes, integrate functions from other organisms, and perform targeted gene knock-outs and other genetic modifications. These tools enabled the metabolic engineering of S. pararoseus for optimal yields and production of middle distillate lipids, and resulted in improvements of 10% in fermentation yield over parental strain. These findings may help in the development of other novel yeasts and strategies for yield improvement of other oleaginous microorganisms.