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91.
Kenro Tokuhiro Masayoshi Muramatsu Chikara Ohto Toshiya Kawaguchi Shusei Obata Nobuhiko Muramoto Masana Hirai Haruo Takahashi Akihiko Kondo Eiji Sakuradani Sakayu Shimizu 《Applied and environmental microbiology》2009,75(17):5536-5543
(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. (Fig.1).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.Open in a separate windowFIG. 1.Biosynthetic pathway for GGOH in S. cerevisiae. The solid arrows indicate the one-step conversions in the biosynthesis, and the dashed arrows indicate the several steps. Intermediates: HMG-CoA, 3-hydroxy-3-methylflutaryl coenzyme A; DMAPP, dimethylallyl diphosphate. Enzymes: HMG-R, HMG-coenzyme A reductase (encoded by the HMG1 gene); FPS, FPP synthase (ERG20).Some yeast strains accumulate ergosterol up to 4.6% dry mass (1). Thus, yeasts have the potential to produce large amounts of GGOH if it is possible to enhance and redirect the metabolic flux to GGOH synthesis. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), encoded by the HMG1 gene has been shown to be the major rate-limiting enzyme in the mevalonate pathway in S. cerevisiae (12). Overproduction of the catalytic domain of HMG-R in an S. cerevisiae strain resulted in squalene accumulation of up to 1% (27) and 2% (8) dry mass but did not cause any difference in the contents of isoprenoid alcohols such as farnesol (FOH) and geraniol (27). These results suggest that squalene is preferably accumulated rather than GGOH when the mevalonate pathway is enhanced by overexpression of the HMG1 gene. Squalene is synthesized through the condensation of two molecules of FPP catalyzed by squalene synthase (SQS) encoded by the ERG9 gene in S. cerevisiae (Fig. (Fig.1).1). The addition of an SQS inhibitor to cultures of S. cerevisiae strains resulted in the production of considerable amounts of FOH (∼77.5 mg liter−1) and relatively small amounts of GGOH (∼2.2 mg liter−1) (20). It has also been reported that SQS-deficient (Δerg9) S. cerevisiae strains, which are sterol auxotrophic, accumulated FPP in their cells (35) and excreted 1.3 mg liter−1 of FOH into the culture medium (5). Therefore, inactivation of SQS seems to enhance FOH rather than GGOH production. This is probably because of the low GGPS activity in S. cerevisiae. Indeed, a carotenoid-producing Rhodotorula yeast strain showed higher GGOH (24.4 mg liter−1) than FOH (4.4 mg liter−1) production on cultivation with an SQS inhibitor (20). Our group previously found that GGOH production could be enhanced by overexpression of the BTS1 gene in S. cerevisiae without SQS inhibition. In addition, coexpression of a fusion of the BTS1 and farnesyl diphosphate synthetase (ERG20) genes along with the HMG1 gene resulted in the production of a substantial amount of GGOH with only a small amount of FOH (C. Ohto, M. Muramatsu, E. Sakuradani, S. Shimizu, and S. Obata, submitted for publication).These results suggest that GGOH can be produced from GGPP through some endogenous phosphatase activities when GGPP synthesis is enhanced. We therefore hypothesized that enhancement of the phosphatase activity could increase the productivity of GGOH. However, it is not clear what kind of phosphatase enhances the GGOH production. It has been reported that the products of the diacylglycerol diphosphate phosphatase (DPP1) gene and lipid phosphate phosphatase (LPP1) gene account for most of the FPP and GGPP phosphatase activities in a particulate (membrane associated) fraction of S. cerevisiae (9). In this study, we found that GGOH production could be enhanced by overexpression of these phosphatase genes. We also demonstrated that overexpression of the BTS1-DPP1 and BTS1-ERG20 fusion genes along with the HMG1 gene further increased GGOH production. Finally, we constructed a high-level GGOH-producing yeast available for industrial processes involving multicopy integration vectors. The productivity of GGOH was evaluated in test tube cultures and 10-liter jar fermentors. 相似文献
92.
Hideyuki Matsuura Arata Aoi Chizuru Satou Mino Nakaya Chikara Masuta Kensuke Nabeta 《Plant Growth Regulation》2009,57(3):293-301
Jasmonic acid (JA) and salicylic acid (SA) are plant hormones involved in plant growth and development. Recent studies demonstrated
that presence of a complex interplay between JA and SA signaling pathways to response to pathogenesis attack and biotic stresses.
To our best knowledge, no method has existed for simultaneous analyses of JA, SA, and their related compounds. Especially,
the glucosides are thought to be the storages or the inactivated compounds, but their contribution should be considered for
elucidating the amount of the aglycons. It is also valuable for measuring the endogenous amount of phenylalanine, cinnamic
acid, and benzoic acid that are the biosynthetic intermediates of SA due to the existence of isochorismate pathway to synthesize
SA. We established this method using deuterium labeled compounds as internal standards. This is the first report of simultaneous
analysis of endogenous JA, SA, and their related compounds. Measuring the endogenous JA, SA, and their related compounds that
had been accumulated in tobacco plants proved the practicality of the newly developed method. It was demonstrated that accumulation
of JA, SA and their related compounds were induced in both case of TMV infection and abiotic stresses. 相似文献
93.
Takashi Hirasawa Kazuyuki Yamada Keisuke Nagahisa Thai Nho Dinh Chikara Furusawa Yoshio Katakura Suteaki Shioya Hiroshi Shimizu 《Process Biochemistry》2009,44(6):647-653
The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics. 相似文献
94.
Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
Chikara Ohto Masayoshi Muramatsu Shusei Obata Eiji Sakuradani Sakayu Shimizu 《Applied microbiology and biotechnology》2010,87(4):1327-1334
An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene
(SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes
the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any
supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The
GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions. 相似文献
95.
Sawada T Miyoshi H Shimada K Suzuki A Okamatsu-Ogura Y Perfield JW Kondo T Nagai S Shimizu C Yoshioka N Greenberg AS Kimura K Koike T 《PloS one》2010,5(11):e14006
Background
Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model.Methodology and Principal Findings
When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size.Conclusions
These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders. 相似文献96.
Bando T Sasaki S Minoshima M Dohno C Shinohara K Narita A Sugiyama H 《Bioconjugate chemistry》2006,17(3):715-720
Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5'-ACAAATCCA-3'. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides. 相似文献
97.
Fujita T Ashikaga A Shiba H Uchida Y Hirono C Iwata T Takeda K Kishimoto A Hirata R Kawaguchi H Shiba Y Kurihara H 《Cytokine》2006,34(5-6):271-277
Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different. 相似文献
98.
Ichihashi N Kurokawa K Matsuo M Kaito C Sekimizu K 《The Journal of biological chemistry》2003,278(31):28778-28786
DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s). 相似文献
99.
100.
When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?
Koide T Umesono K Hashimoto C 《The International journal of developmental biology》2002,46(6):777-783
During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements. 相似文献