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

Abstract

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.

Keywords: Lipid; Metabolic engineering; Oleaginous yeast; Promoter; Rhodosporidium/Rhodotorula.

Fig. 1

Schematic diagrams of promoters. a DUR1 and DUR1in promoters. b FAT1 and FAT1in promoter. c FAS1 and FAS1in promoters. d ACL1 and ACL1in promoters. e ACC1 and ACC1in promoters. f LDP1 and LDP1in promoters. tss represents the transcription start site, blue bars represent exons. Translational starts (ATG) in intronic promoters are indicated. Nucleotides in red letters indicate modifications from the genome sequences. The scaffold number is based on the genome sequence of R. glutinis ATCC 204091 [28]

Fig. 2

Time-course studies of promoter strength by luciferase gene reporter assay. a DUR1 and DUR1in promoter. b FAT1 and FAT1in promoter. c FAS1 and FAS1in promoter. d ACL1 and ACL1in promoter. e ACC1 and ACC1in promoter. f LDP1 and LDP1in promoter. Cells were cultured in MinRL3 medium at 30 °C. Results were derived from three biological replicates and error bars indicate standard deviation. RPA relative promoter activity normalized against that of GPD1 promoter

Fig. 3

qRT-PCR analysis of mRNA levels. a Relative mRNA levels of ACC1, FAS1 and LDP1. Calculation of mRNA levels were done using 2^−ΔCt method by normalizing against actin gene ACT1 (reference). b Dynamic changes of mRNA levels of ACC1, FAS1 and LDP1. Fold change of mRNA level was normalized against its own mRNA level at day 0. WT strain was cultured in MinRL3 medium for 6 days at 30 °C. Results were derived from three biological replicates and error bars indicate standard deviation

Fig. 4

Promoter strength in lipid production-deficient mutant dlad. The dlad strain is mutant with targeted deletion of four diacylglycerol acyltransferase genes (our unpublished data). All reporter mutants were cultured in MinRL3 for 5 days and luciferase reporter assays were performed daily. Results were derived from three biological replicates and error bars indicate standard deviation

Fig. 5

Enhancement of lipid content by DGA1 overexpression. a Quantification of lipid content as gram lipids per gram dry cell weight (%). b qRT-PCR analysis of DGA1 expression. WT, P_GPD1 ::DGA1 and P_LDP1in ::DGA1 strains were cultured in GJ2013 medium for 3 days at 30 °C. Results were derived from three biological replicates and error bars indicate standard deviation