Abstract
BackgroundRed 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.ResultsThe 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.ConclusionIntronic 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 materialThe online version of this article (doi:10.1186/s12934-016-0600-x) contains supplementary material, which is available to authorized users.
Highlights
Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass
Characterization of genes involved in lipid accumulation Genomic sequences for acetyl-CoA carboxylase gene (ACC1), ATP:citrate lyase gene (ACL1), β subunit of fatty acid synthetase gene (FAS1), fatty acid transporter gene (FAT1) and urea amidolyase gene (DUR1) were identified by basic local alignment search tool (BLAST) search against the public database as well as in-house EST and genome database of R. toruloides strains [27, 28]
The putative homolog of ACC1, ACL1, FAS1, FAT1, DUR1 and lipid droplet protein 1 gene (LDP1) was found located in the genome sequencing scaffold No.18, 9, 18, 9, 25 and 10 of R. glutinis American type culture collection (ATCC) 204091, respectively (Table 1)
Summary
Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. Red yeast species in the Rhodosporidium (teleomorph) genus, which was recently revised as genus Rhodotorula (anamorphic) regarding to the implementation of “One Fungus = One Name” nomenclatural principle [1], are outstanding producers of lipids and carotenoids [2, 3]. Strong and robust promoters that function during lipid accumulation stage will be useful. The production of long chain acyl-CoA, triacylglycerol (TAG) and lipid bodies are the 3 major lipogenesis steps, and genes involved in these processes are the likely source of strong promoters in oleaginous yeasts. Other known abundant targets are the perilipin, adipophilin and tail-interacting (PAT) family proteins, which serve as dynamic scaffolds regulating the formation, growth and degradation of lipid bodies [15,16,17,18,19]
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