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1.
Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.  相似文献   

2.
In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.  相似文献   

3.
Neves L  Lages F  Lucas C 《FEBS letters》2004,565(1-3):160-162
Previous studies evidenced in Saccharomyces cerevisiae the activity of a H(+)/glycerol symport, derepressed by growth on non-fermentable carbon sources, later associated with GUP1 and GUP2 genes. It was also demonstrated that only the combined deletion of GUP1, GUP2 together with GUT1 (glycerol kinase) abolished active transport in ethanol-induced cells. In this work, we show that a glycerol H(+)/symport, with identical characteristics to the previously described, was found in gup1gup2gut1 grown under salt-stress, particularly high in cells collected during diauxic-shift. These results suggest different roles for Gup1/2p than glycerol transport. The gene encoding for glycerol active uptake is thus yet unknown.  相似文献   

4.
In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.  相似文献   

5.
6.
The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.  相似文献   

7.
In the yeast Saccharomyces cerevisiae, the most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are the external NADH dehydrogenases (Nde1p and Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p. Subsequently, glycerol 3-phosphate donates electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). At saturating concentrations of NADH, the activation of external NADH dehydrogenases completely inhibits glycerol 3-phosphate oxidation. Studies on the functionally isolated enzymes demonstrated that neither Nde1p nor Nde2p directly inhibits Gut2p. Thus, the inhibition of glycerol 3-phosphate oxidation may be caused by competition for the entrance of electrons into the respiratory chain. Using single deletion mutants of Nde1p or Nde2p, we have shown that glycerol 3-phosphate oxidation via Gut2p is inhibited fully when NADH is oxidized via Nde1p, whereas only 50% of glycerol 3-phosphate oxidation is inhibited when Nde2p is functioning. By comparing respiratory rates with different respiratory substrates, we show that electrons from Nde1p are favored over electrons coming from Ndip (internal NADH dehydrogenase) and that when electrons come from either Nde1p or Nde2p and succinodehydrogenase, their use by the respiratory chain is shared to a comparable extent. This suggests a very specific competition for electron entrance into the respiratory chain, which may be caused by the supramolecular organization of the respiratory chain. The physiological consequences of such regulation are discussed.  相似文献   

8.
Glycerol, one of the most important by-products of alcoholic fermentation, has positive effects on the sensory properties of fermented beverages. It was recently shown that the most direct approach for increasing glycerol formation is to overexpress GPD1, which encodes the glycerol-3-phosphate dehydrogenase (GPDH) isoform Gpd1p. We aimed to identify other steps in glycerol synthesis or transport that limit glycerol flux during glucose fermentation. We showed that the overexpression of GPD2, encoding the other isoform of glycerol-3-phosphate dehydrogenase (Gpd2p), is equally as effective as the overexpression of GPD1 in increasing glycerol production (3.3-fold increase compared to the wild-type strain) and has similar effects on yeast metabolism. In contrast, overexpression of GPP1, encoding glycerol 3-phosphatase (Gpp1p), did not enhance glycerol production. Strains that simultaneously overexpress GPD1 and GPP1 did not produce higher amounts of glycerol than a GPD1-overexpressing strain. These results demonstrate that GPDH, but not the glycerol 3-phosphatase, is rate-limiting for glycerol production. The channel protein Fps1p mediates glycerol export. It has recently been shown that mutants lacking a region in the N-terminal domain of Fps1p constitutively release glycerol. We showed that cells producing truncated Fps1p constructs during glucose fermentation compensate for glycerol loss by increasing glycerol production. Interestingly, the strain with a deregulated Fps1 glycerol channel had a different phenotype to the strain overexpressing GPD genes and showed poor growth during fermentation. Overexpression of GPD1 in this strain increased the amount of glycerol produced but led to a pronounced growth defect.  相似文献   

9.
10.
During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.  相似文献   

11.
12.
Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 106 cells ml−1 Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.  相似文献   

13.
Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.  相似文献   

14.
15.
16.
The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.  相似文献   

17.
BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.  相似文献   

18.
The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.  相似文献   

19.
Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of 13C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.  相似文献   

20.
Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).  相似文献   

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