PgmNr Y3107: Molecular genetic tools for manipulation of the oleaginous red yeast Rhodotorula toruloides.

Authors:
S. J. Aves; A. M. B. Johns; J. Love


Institutes
University of Exeter, Exeter, UK.


Keyword: Transcription

Abstract:

Rhodotorula toruloides (Rhodosporidium toruloides) is a red, basidiomycete yeast.  When grown under nitrogen limited conditions it can accumulate lipid to over 70 % of its dry biomass making it of interest for production of biofuels as well as having other potential uses in biotechnology.  In order to be able to fully investigate and exploit this organism, genetic tools for manipulation of this yeast are required. 

The R. toruloides genome has a high genomic GC content which can cause cloning problems, making in vitro genetic manipulations difficult.  To overcome this, plasmid vectors have been constructed for Agrobacterium tumefaciens-mediated transformation of R. toruloides, incorporating elements for yeast in vivo assembly in Saccharomyces cerevisiae.  These vectors have been developed to facilitate easy exchange of promoters and genes to be expressed, and can be used with either antibiotic or auxotrophic selection.  

To permit controlled expression of genes in R. toruloides, we have identified four homologous inducible promoters.  Their properties and basic structure have been characterized by fusion to a codon-optimized EGFP reporter gene, measuring fluorescence by flow cytometry.  Each promoter can give tight regulation with induction times of between 4 and 16 hours.  Because each promoter uses different induction and repression conditions, this provides a toolset of regulatable promoters such that for any given experiment there should be suitable promoter(s) for which conditions should not affect the results. 

We have developed a suite of molecular genetic tools for recombinant DNA technology and controlled gene expression which will facilitate molecular genetic investigation and biotechnological exploitation of oleaginous red yeast.