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 ␤-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 ␤-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of ␤-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 ␤-carotene by S. cerevisiae.

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

“…Intracellular accumulation of different types of carotenoids was determined by HPLC as described previously (Verwaal et al, 2007). Intracellular accumulation of total carotenoids was analysed by resuspension of the cells in 1 ml sterile water, 1 g 0.50-0.75 mm glass beads was added and the cells were broken by vortexing for 3 min.…”

“…Expression of all genes was controlled by the TDH3 promoter and CYC1 terminator. Detailed construction of the expression plasmids and strains was described previously (Verwaal et al, 2007). The Xanthophyllomyces dendrorhous reference strain CBS6938 and the β-carotene accumulation mutant β-Carotene accumulation mutant of X. dendrorhous (Girard et al, 1994) PR-1-104 (Girard et al, 1994) were also used in this study.…”

“…The Xanthophyllomyces dendrorhous reference strain CBS6938 and the β-carotene accumulation mutant β-Carotene accumulation mutant of X. dendrorhous (Girard et al, 1994) PR-1-104 (Girard et al, 1994) were also used in this study. Single S. cerevisiae deletion mutants were obtained from the BY4741 MATa deletion collection from EUROSCARF (http://web.unifrankfurt.de/fb15/mikro/euroscarf/) (Table 1) and were transformed with YEPlac195 YB/I/E (Verwaal et al, 2007) and YEPlac195 as control. Transformation plates were incubated for 3 days at 30 • C.…”

“…However, Saccharomyces cerevisiae may be an attractive alternative, due to its well-known genetic and fermentation characteristics. We previously transformed S. cerevisiae with carotenogenic genes from X. dendrorhous (Verwaal et al, 2007; Figure 1). Introduction of crtYB and crtI into S. cerevisiae resulted in a low level of carotenoid production.…”

Biofuel alternatives to ethanol: pumping the microbial well

“…phytoene [C40]) serve as inter-mediates in the production of large isoprenoid compounds, and they might be suitable as biocrude. Both E. coli and S. cerevisiae have been successfully engineered for the pro-duction of the phytoene-based carotenoids [51,[57][58][59]; unfortunately, the current yields of these compounds fall far short of what is required if they are to produce viable fuel alternatives [47,50,60]. However, if sufficient titers of phytoene are reached, it could be refined into motor and jet fuel through the infrastructure currently used to process petroleum.…”