FPP-Producing S. cerevisiae Strains

We combined the previously reported HI-CRISPR system (Bao et al., 2015) and three plant-derived ATF/BSs, namely NLS-GAL4AD-JUB1-2X (Naseri et al., 2019), NLS-GAL4AD-ANAC102-2X (Naseri et al., 2019), and NLS-GAL4AD-ATAF1-4X (Naseri et al., 2019), to overexpress yeast genes, GDH2, tHMG1, and ERG20, respectively. To construct a strain overexpressing GDH2, tHMG1, and ERG20, previously characterized integration sites, shown to exhibit a high integration efficiency, were targeted (Bao et al., 2015). The GDH2, tHMG1, and ERG20 donors were integrated, respectively, at genomic ADE2.a, his3D1, and ura3-52 loci of yeast Gen 0.2, where each donor is integrated into a single locus (Figure 5A). Each donor contains an IPTG-inducible ATF/BS upstream of a CDS and ends with a yeast terminator. The 50-bp overhang sequences up- and downstream of each donor allow its integration into pre-designed genomic loci.

Production of β-carotene in yeast strains. (A) Scheme showing the β-carotene production from FPP precursor. BTS1, geranylgeranyl diphosphate synthase; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; McrtYB, optimized phytoene synthase/lycopene cyclase; McrtI, phytoene desaturase. (B) Scheme showing the β-carotene-encoding plasmid. The pCAROTENE plasmid was constructed as described by Naseri et al. (2019). McrtI, McrtYB, and BTS1 genes are under the control of ATF/BSs NLS-GAL4AD-GRF9-4X, NLS-GAL4AD-GRF7-4X, and NLS-GAL4AD-ANAC102-4X. The ATF/BS-McrtI, -McrtYB-, and -BTS1 modules are flanked by the IPTG inducible modified GAL1 promoter. Additionally, the ATF/BS-McrtI module encodes the LacI repressor. Selection on SC-URA media allows screening for successful plasmid integration. To simplify the figure, the CYC1 terminator located downstream of ATF is not shown. AD, activation domain; ATF, artificial transcription factor; BS_{plant TF}, binding site of the plant TF; DBD, DNA binding domain; IPTG, isopropyl-β-D-thiogalactopyranoside; NLS, nucleus localization signal; Pro_minCYC1, CYC1 minimal promoter; Ter, terminator. (C) Representative plate of the constructed β-carotene producing strains. The pCAROTENE plasmid was transformed to P. pastoris and S. cerevisiae to generate the yPPGN022 and ySCGN01, respectively. Upon adding IPTG, plant-derived ATFs were expressed and β-carotene was produced. Colors of carotenoid-producing yeast strains in YPDA media for the yPPGN022 and ySCGN01 strains. (D) Scheme illustration of FPP precursor production in yeast. To redirect yeast metabolisms toward the FPP production, GDH2, ERG20, and tHMG1 overexpression (blue), deleted or inactive DPP1, LPP1 and GDH1 (red) are needed. ERG20, FPP synthase; DPP1 and LPP1, lipid phosphate phosphatases; tHMG1, truncated HMG-CoA reductase, GDH1, NADP + -glutamate dehydrogenase; GDH2, NAD + -dependent glutamate dehydrogenase; HMG-CoA, 3-hydroxy-3methylglutaryl-CoA; IPP, isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate. (E) Scheme representing overexpressing donors for the enhanced production of FPP. ATFs NLS-GAL4AD-ANAC102-2X, NLS- GAL4AD-JUB1-2X, and NLS-GAL4AD-ATAF1-4X are fused to tHMG1-T_HMG1, GDH2-T_GDH2, and ERG20-T_ERG20. The tHMG1, GDH2, and ERG20 donors are flanked by 50-bp homology arms to integrate into the his3D1, ADE2.a, and ura3-52 loci, respectively. In each donor, a modified GAL1 promoter is located upstream of the plant-derived ATF. Additionally, the tHMG1 donor encodes LacI. The BS of ATFs is placed upstream of the CYC1 minimal promoter to drive gene expression. NLS, nuclear localization signal; JUB1, plant JUNGBRUNNEN1 transcription factor; GAL4AD, yeast GAL4 activation domain; ANAC102, NAC domain containing protein 102; ATAF1, Arabidopsis thaliana Activating Factor 1; “2X”, and “4X” indicate two, or four copies of the plant TF binding site, respectively. (F) Colors of the carotenoid-producing ySCGN02 S. cerevisiae strain (optimized for FPP production) in YPDA media. (G) SFC analysis of the carotenoid content of yeast strains. Values represent the mean ± SD of three independent colonies after 3 days of cultivation. P. pastoris strains yPPGN023 containing pCAROTENE-PGAP plasmid and J308 were used as a positive and negative controls, while Gen 0.2 was used as a negative S. cerevisiae control. Asterisks indicate a statistically significant difference (Student’s t-test; *p < 0.05; **p < 0.01). Cdw, cell dry weight. Full data are shown in Supplementary Data 4.

For inactivation of the GDH1, DPP1, and LPP1 genes, three donors, together with pCRCT-GDH1-DPP1-LPP1 (expressing gRNAs, tracer RNAs, and iCas9 protein), were used to transform yeast cells, where each donor is integrated into a single locus. Each donor contains the 50-bp overhang sequences up- and downstream of its integration into target genomic sites, and incorporates an 8-bp deletion included within the target gene, thus introducing a frameshift mutation. The ySCGN0.1 or ySCGN0.2 strains were obtained by integrating overexpression or inactivation donors into the yeast genome, respectively. The ySCGN1.2 strain harbors inactivated genes, lpp1, dpp1, and gdh1, in addition to overexpressed GDH2, tHMG1, and ERG20.