High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous

Abstract

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

FIG. 1.

Overview of the ergosterol biosynthetic pathway in S. cerevisiae and the carotenogenic pathway in X. dendrorhous. The carotenogenic pathway in X. dendrorhous consists of GGPP synthase encoded by crtE, the bifunctional enzyme phytoene synthase and lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI. S. cerevisiae contains a GGPP synthase, encoded by BTS1, which is able to convert FPP into GGPP. HMG1 encodes HMG-CoA reductase, which is the main regulatory point in the ergosterol biosynthetic pathway in many organisms. IPP, isopentenyl diphosphate; DMAP, dimethylallyl diphosphate; GPP, geranyl diphosphate.

FIG. 2.

Construction of expression vectors used in this study. (a) Scheme representing the construction of the different vectors used in this study. GPDp is the 680-bp sequence of the TDH3 promoter, and CYC1t is the 250-bp sequence of the CYC1 terminator. Restriction enzyme abbreviations: B, BamHI; Sa, SalI; E, EcoRI; P, PstI; K, KpnI; S, SmaI. Sequences used for primers are indicated in Table 1. Integration into the ura3-52 locus requires linearization of the YIplac211 constructs with StuI. Because crtE contains a StuI restriction site, A at position 551 was changed into G by site-directed mutagenesis (indicated by an asterisk). See Materials and Methods for details concerning the cloning strategy used. (b) Orientation of the carotenogenic genes in the vectors used in this study, as determined by restriction analysis. All genes are present in tandem head-to-tail orientation on the expression vectors.

FIG. 3.

qPCR studies to determine overexpression of carotenogenic genes in carotenoid-producing yeast strains. Three cultures of each strain, the wild type, YB/I/E, and YB/I/BTS1, were inoculated in YNB with 2% glucose at the same optical density. Six hours after inoculation, samples were taken and further processed for cDNA synthesis. qPCR experiments were performed to determine the relative abundances of crtYB, crtI, crtE, and BTS1 in each strain with respect to that of ACT1, which encodes actin and served as an internal control. The data represent the average and standard deviation of three independently grown cultures.

FIG. 4.

Colors of different carotenoid-producing S. cerevisiae mutants. All strains were initially transformed with YIplac211 crtYB/crtI/crtE* to obtain carotenoid-producing cells and subsequently with the integrated vectors containing the indicated genes. (a) Strain YB/I/E, CEN.PK 113-5D; (b) strain YB/I/E+extra I, CEN.PK 113-6B transformed with YIplac204 crtI; (c) strain YB/I/E+tHMG1, CEN.PK 113-6B transformed with YIplac128 tHMG1; (d) strain YB/I/E+extra I+tHMG1, CEN.PK 113-6B transformed with YIplac128 crtI and YIplac204 tHMG1. Individual transformants were grown for 2 days on YNB-2% glucose medium supplemented with amino acids where required and streaked onto yeast extract-peptone-2% glucose plates. After 3 days of incubation at 30°C, the plates were photographed.