Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

Abstract

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter ؊1 ) rather than GGOH (0.2 mg liter ؊1 ) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter ؊1 GGOH and 6.5 mg liter ؊1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter ؊1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.(E,E,E)-Geranylgeraniol (GGOH) can be used as an important ingredient for perfumes and as a desirable raw material for synthesizing vitamins A and E (4, 13). It is also known to induce apoptosis in various cancer and tumor cell lines (24,36). GGOH is the dephosphorylated derivative of (E,E,E)-geranylgeranyl diphosphate (GGPP) (Fig. 1). GGPP is a significant intermediate of ubiquinone and carotenoid biosyntheses, especially in carotenoid-producing microorganisms and plant cells. It is also utilized as the lipid anchor of geranylgeranylated proteins. In the yeast Saccharomyces cerevisiae, GGPP is synthesized by GGPP synthase (GGPS), encoded by the BTS1 gene, which catalyzes the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) rather than the successive addition of IPP molecules to dimethylallyl diphosphate, geranyl diphosphate, and FPP that is detected in mammalian tissues (14). Biologically synthesized GGOH comprises only (E,E,E)-geometric isomers, and only the (E,E,E)-isomers have significant biological activities (23). The chemically synthesized form is usually obtained as mixtures of (E)-and (Z)-isomers and thus has lower potency. Therefore, there is a greater possibility of attaining efficient production of (E,E,E)-GGOH through fermentative production.Some yeast strains accumulate ergosterol up to 4.6% d...

Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli

“…DPP1 and LPP1 were proved to produce geranylgeraniol by removing the phosphatate of geranylgeranyl diphosphate. 13 However, they cannot use GPP as substrate to form geraniol in this study, which is consistent with the result no significant increase in geraniol formation by overexpressed DPP1 in vivo, as proved by Oswald. 11 NudF can remove the pyrophosphate from IPP and DMAPP to form prenol and isoprenol by introducing NudF into the strain that employed the whole MVA pathway, but not useful in this reaction.…”

“…The activity of alkaline phosphatase (PhoA) was measured as described by Kojima et al 18 with the following modification: the incubation mixture contained 1 M Tris-Cl, pH 8.0, 10 mM MgSO 4 , 50 mM ZnSO 4 , 5 mM GPP and the enzyme extract (about 0.5 mg protein), incubated for 30 min at 37 C. The activity of bifunctional diacylglycerol diphosphate phosphatase (DPP1) and lipid phosphate phosphatase (LPP1) were assayed using a modification of existing methods. 13,19 The assay mixture (100 ml) was comprised of citrate buffer (120 mM, pH 4.3), 5 mM MgCl 2 , the enzyme extract (about 0.5 mg protein), and 5 mM GPP and was incubated for 30 min at 37 C. The activity of ADP-ribose pyrophosphatase (NudF) was measured as described by Dunn et al 20 with the following modification: the incubation mixture contained 50 mM Tris-Cl (pH 8.0), 2 mM MgCl 2 , 5 mM GPP and the enzyme extract (about 0.5 mg protein) and was incubated for 15 min at 37 C. The products of the reactions were analyzed by GC-MS. Every reaction has 3 parallels and cell extract with empty pCOLADuet-1 was used as control.…”

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

“…The leucine and/or methionine auxotrophic strains were constructed as a previous report. 19 Diploid strain YJ2X was constructed by mating the methionine auxotrophic strain YJ27 and BY4742 as described previously. 25 Verification of the Assembled Pathways.…”

Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes

“…Second, experiments carried out with purified fusion proteins in polyethylene glycol solutions to mimic the crowded environment inside the cell indicate that the positive effects of enzyme fusion may be even greater in vivo (38,25,5). Last, the production of geranylgeraniol in S. cerevisiae was significantly increased by overexpressing a fusion of the yeast proteins Bts1 and Dpp1 (33). Encouraged by these results, we have explored the possibilities of expressing fusion enzymes, with components originating from different biological kingdoms, in the cell factory Saccharomyces cerevisiae to promote formation of a nonyeast product.…”