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Fig. 1 | Microbial Cell Factories

Fig. 1

From: A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

Fig. 1

Experimental workflow. a A KU70-deficient strain of haploid R. toruloides IFO0880 was used for all experiments. Each promoter-reporter cassette was constructed in two orientations (1 and 2, above) in order to construct strains that report expression of both EGFP and mRuby2 from every promoter. 13 of the 29 promoters were predicted to be bidirectional (not shown). b Constructs were integrated into the CAR2 locus of the R. toruloides genome using a lithium-acetate-based transformation method. Strains harboring a correctly-targeted construct were determined by a white Δcar2 phenotype and fluorescence. c The Δku70 parent strain, a control ∆car2 strain, and three transformants for each of the 58 promoter strains were cultivated in 24-deep-well plates in four different media: YPD, SD supplemented with 1% (w/v) glucose, SD supplemented with 1% (w/v) xylose, and SD supplemented with 1% (w/v) each of glucose and xylose. Cultures were grown for 7 days, with sample collection at 8, 24, 48, 96 and 168 h. d Reporter gene expression was analyzed by flow cytometry. The upper and lower histograms show fluorescence from EGFP (upper, green histogram) and mRuby2 (lower, red histogram) driven by promoter P9, compared to the control ∆car2 strain (blue histograms); these populations were sampled at 24 h in SD supplemented with 1% (w/v) each of glucose and xylose

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