title: 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.
authors: Biotechnology Laboratory, Toyota Central R&D Laboratories, Inc., Nagakute, Aichi 480-1192, Japan. tokuhiro@mosk.tytlabs.co.jp
authors: Kenro
authors: K
authors: Tokuhiro
authors: Masayoshi
authors: M
authors: Muramatsu
authors: Chikara
authors: C
authors: Ohto
authors: Toshiya
authors: T
authors: Kawaguchi
authors: Shusei
authors: S
authors: Obata
authors: Nobuhiko
authors: N
authors: Muramoto
authors: Masana
authors: M
authors: Hirai
authors: Haruo
authors: H
authors: Takahashi
authors: Akihiko
authors: A
authors: Kondo
authors: Eiji
authors: E
authors: Sakuradani
authors: Sakayu
authors: S
authors: Shimizu
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics*; D004224:Diterpenes / Q000378:metabolism*; D051231:Farnesyltranstransferase / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D051228:Geranyltranstransferase / Q000235:genetics / Q000378:metabolism; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013185:Squalene / Q000378:metabolism
chemical_list: D004224:Diterpenes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013185:Squalene; C017338:geranylgeraniol; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D051228:Geranyltranstransferase; D051231:Farnesyltranstransferase; C519642:DPP1 protein, S cerevisiae; D011755:Pyrophosphatases