Inducible Expression Vector Set For Engineering In The Oleaginous Yeast Rhodosporidium Toruloides

Rhodosporidium toruloides, which is an attractive candidate for microbial oil production, can accumulate oils to more than 60% of its biomass, under nutrient stress conditions such as nitrogen or phosphorus limitation, and has also been extensively used in a broad range of other industrial applications: carotenoids production, biotechnologically important enzymes(i.e. cephalosporin esterase and epoxide hydrolase) production, and bioremediation. Oleaginous R. toruloieds capable of accumulating oils in short fermentation period and metabolizing diverse monosaccharide, which from renewable lignocellulose biomass by hydrolysis. With respect to Saccharomyces cerevisiae, Escherichia coli and other conventional molecular biology operating mode strain, study of the molecular mechanisms concerning lipid accumulate and further rational engineering R. toruloides remains challenging because of the lack of a mature and efficient platform for genetic manipulation.[Objective] To construct a inducible expression vector set for functional integration and expression of exogenous genes in R. toruloides. [Methods] The aim of this study is to clone the R.toruloides adh2 promoter(Padh2), pho89 promoter(Ppho89), gal1 promoter(Pgal1), gpd terminator(Tgpd), and hsp70 terminator(Thsp), construct a set of inducible expression vectors for functional integration and expression of exogenous genes in R. toruloides. These three promoters and two terminators were predicted by bioinformatics assay, isolated from the oleaginous yeast R. toruloides NP11 by PCR, and replaced promoter Ppgk and terminator Tnos in pZPK-pPGK-hyg-tNOS vector by restriction free(RF) cloning method. Hygromycin phosphotransferase gene hyg was retained and as a report gene. The resulting six of vectors were transferred into haploid R. toruloides NP11 and diploid R. toruloides Y4 by the agrobacterium-mediated transformation(ATMT) method, and obtained the hygromycin resistant transformants, which can determine the promoters and terminators activity. Furthermore, three new inducible vectors which contain two expression cassettes driven by the constitutive Ppgk promoter and inducible promoter have been developed. [Results] Malic enzyme(ME) expression result shows that, Ppho89, Padh2 and Pgal1 were tightly regulated by phosphate or glucose, and were highly activated when the recombinant R. toruloides strains were grown in a phosphate-limited or glucose-deprived condition. [Conclusion] The Ppho89, Padh2 and Pgal1 are ideal promoters in terms of tight regulation by nutrition condition, comparative response strength, simplicity, and cost effectiveness. These ATMT vectors will able to provide useful manipulation tools for rational engineering of oleaginous yeast to produce fatty acid derived biofuels and biochemical.