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Background  Red yeast species in the  Rhodotorula/Rhodosporidium  genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts.    Results  The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in  Rhodotorula toruloides  were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene ( LDP1 ) displayed much stronger activity (4–11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene ( GPD1 ), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene ( ACC1 ) and fatty acid synthase β subunit gene ( FAS1 ) exhibited intermediate strength, displaying 50–160 and 20–90% levels of  GPD1  promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of  LDP1  ( LDP1in  promoter) enhanced its promoter activity by 1.6–3.0 folds. Similarly, the strength of  ACC1  promoter was enhanced by 1.5–3.2 folds if containing intron 1. The intron 1 sequences of  ACL1  and  FAS1  also played significant regulatory roles. When driven by the intronic promoters of  ACC1  and  LDP1  ( ACC1in  and  LDP1in  promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene ( DGA1 ) by  LDP1in  promoter was significantly more efficient than  GPD1  promoter in enhancing lipid accumulation.    Conclusion  Intronic sequences play an important role in regulating gene expression in  R. toruloides.  Three intronic promoters,  LDP1in ,  ACC1in  and  FAS1in , are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast,  R. toruloides.Electronic supplementary material  The online version of this article (doi:10.1186/s12934-016-0600-x) contains supplementary material, which is available to authorized users.

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species