Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae

The anaerobic performance of gpd1Δ and gpd2Δ mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2Δ mutant compared to the wild-type. Also the gpd1Δ mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2Δ mutant was elevated by 13%. In terms of growth, the gpd1Δ mutant and the wild-type were indistinguishable. The gpd2Δ mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2Δ mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2Δ mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2Δ mutant did not show any decrease in the protein content of the biomass. Received: 16 February 1998 / Received revision: 16 March 1998 / Accepted: 1 June 1998     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae

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.     

Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter.

The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment. Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters. Chromosomal mapping indicates that this genomic fragment is located on chromosome XII. The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.     

Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance of Saccharomyces cerevisiae.

In a cell culture of Saccharomyces cerevisiae exponentially growing in basal medium, only 0.02% of the cells were osmotolerant, i.e., survived transfer to medium containing 1.4 M NaCl. Short-time conditioning in 0.7 M NaCl medium transformed the whole population into an osmotolerance phenotype. During this conditioning, the rate of formation of glycerol, the main compatible solute in S. cerevisiae, increased threefold and the specific activity of glycerol-3-phosphate dehydrogenase (NAD+) (GPDH) (EC 1.1.1.8) was enhanced sixfold. The apparent flux control coefficient for GPDH in the formation of glycerol was estimated to be 0.6. Glycerol production was also favored by regulated activities of alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase [NAD(P)]+ (EC 1.2.1.5). About 50% of the total glycerol produced during conditioning in 0.7 M NaCl was retained intracellularly, and the increased glycerol accumulation was shown to be not merely a result of enhanced production rate but also of increased retention of glycerol. Washing the cells with solutions of lower salinities resulted in loss of glycerol, with retained levels proportional to the concentration of NaCl in the washing solution. Cycloheximide addition inhibited the development of acquired osmotolerance and conditioned cells washed free of glycerol retained a high degree of osmotolerance, which indicate that protein synthesis was required to establish the osmotolerance state.     

Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.     

Phosphatidylcholine is essential for efficient functioning of the mitochondrial glycerol-3-phosphate dehydrogenase Gut2 in Saccharomyces cerevisiae

Gut2, the mitochondrial glycerol-3-phosphate dehydrogenase, was previously shown to become preferentially labelled with photoactivatable phosphatidylcholine (PC), pointing to a functional relation between these molecules. In the present study we analyzed whether Gut2 functioning depends on the PC content of yeast cells, using PC biosynthetic mutants in which the PC content was lowered. PC depletion was found to reduce growth on glycerol and to increase glycerol excretion, both indicating that PC is needed for optimal Gut2 functioning in vivo. Using several in vitro approaches the nature of the dependence of Gut2 functioning on cellular PC contents was investigated. The results of these experiments suggest that it is unlikely that the effects observed in vivo are due to changes in cellular Gut2 content, in specific activity of Gut2 in isolated mitochondria, or in the membrane association of Gut2, upon lowering the PC level. The in vivo effects are more likely an indirect result of PC depletion-induced changes in the cellular context in which Gut2 functions, that are not manifested in the in vitro systems used.     

Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ?GPD1?GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.     

Osmoregulation in Saccharomyces cerevisiae. Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+)

Production of glycerol and a key enzyme in glycerol production, glycerol 3-phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiae cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source. At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumulated to high intracellular concentration in cells grown on glucose and raffinose. Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity. However, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content. Immunoblot analysis demonstrated an increased production of GPD at high salinity. This increase was osmotically mediated but was lower when glycerol was substituted for NaCl or sorbitol as the stress-solute. The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was significantly lower in cells grown on glucose, than on raffinose or ethanol.     

A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene.

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.     

Divergence of glyceraldehyde-3-phosphate dehydrogenase isozymes in Saccharomyces cerevisiae complex

Although sake yeasts are placed in Saccharomyces cerevisiae, we have been interested in their difference from the other subgroups of the species, and examined their proteins. When SDS-PAGE patterns of their soluble proteins were compared, specific differences between subgroups were found in their 36,000 Da regions. Proteins isolated therefrom were found to be subunits of three isomers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from their N-terminal amino acid sequences and identified with anti-GAPDH serum. Therefore, comparison of zymogram was carried out by a modified method: denatured monomers were observed and the enzyme activity of their oligomers was not considered. SDS-PAGE patterns of all the sake yeasts differed from those of the other strains of S. cerevisiae. Strains of Saccharomyces bayanus showed uniform patterns which are different from the above two groups. Saccharomyces pastorianus strains resembled S. bayanus and were partly similar to S. cerevisiae in their patterns, in agreement with the hypothesis that S. pastorianus is a hybrid between these two species. Patterns of S. paradoxus appeared to be rather similar to those of sake yeasts. Results on the other species of the genus and on the preliminary experiments on PAGE of native isozymes are also described.     

The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae.

The complete nucleotide sequence of the coding, as well as the flanking noncoding regions, of a yeast glyceraldehyde-3-phosphate dehydrogenase gene was determined. Both the 5' and 3' noncoding sequences are extremely AT-rich and regions of partial dyad symmetry are present immediately adjacent to the 5' and 3' ends of the translated portion of the gene. The sequence AAUAAA is present in the 3' noncoding region of this gene and is a part of an extensive region of dyad symmetry which is structurally related to the 3'-terminal portion of both procaryotic mRNAs, as well as some eukaryotic mRNAs. The coding region of this gene does not contain intervening sequences. Establishment of the primary structure of this glyceraldehyde-3-phosphate dehydrogenase gene provides a basis for further studies involving in vitro mutation of the gene and subsequent analysis of gene expression in vivo.     

Developmental regulation of glycerol-3-phosphate dehydrogenase synthesis inDrosophila

Methods have been developed to measure the synthesis of glycerol-3-phosphate dehydrogenase (GPDH) during the development of Drosophila melanogaster. In emerged adult flies, GPDH is a principal component of protein synthesis, comprising between 1 and 2% of the protein synthetic effort. This high relative rate of protein synthesis continues throughout adult life during a period of stable enzyme concentration. Therefore, it is evident that GPDH undergoes continual turnover. Analysis of GPDH synthesis in the adult segments reveals that this enzyme is synthesized in head, thorax, and abdomen. In 5-day-old flies, the relative rates of GPDH synthesis in the thorax and abdomen are similar. However, the concentration of GPDH in the thorax greatly exceeds that found in the abdomen. Therefore, it appears that the turnover rate of GPDH in the abdomen must be greater than the turnover rate of GPDH in the GPDH-containing cells (flight muscle) of the thorax. GPDH represents between 0.5 and 0.9% of the protein synthetic effort of larvae. The principle GPDH-containing tissue of larvae is fat body. The turnover of GPDH in larvae is similar to that in adult abdomen. This may be related to the concurrent presence of GPDH isozyme-3 in both tissues. Our studies indicate that the cell type-specific control of GPDH occurs at several levels.     

Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

The localization of glycerol-3-phosphate dehydrogenase inEscherichia coli

Summary Starved cells ofEscherichia coli are dependent on an exogenous source of energy. It was of interest to ask whether compounds that are commonly used to supply energy must themselves be transported or whether they can be utilized on the outer portion of the cytoplasmic membrane. The utilization of glycerol-3-phosphate an energy source is totally dependent on the membrane-bound glycerol-3-phosphate dehydrogenase. In the present report glycerol-3-phosphate was used as the energy source for uptake of amino acids. A mutant was constructed which is unable to transport this ester and the starved mutant could not drive the uptake of glutamine with glycerol-3-phosphate. It is concluded that the enzyme is located on the internal surface of the membrane in intactE. coli cells. Further evidence was obtained by showing that no glycerol-3-phosphate dehydrogenase activity could be measured in either intact cells or spheroplasts using ferricyanide as electron acceptor, due to its impermeability. The activity could be measured after destruction of the membrane permeability barrier by toluenization. With membrane vesicles prepared according to Kaback's procedure nearly half of the dehydrogenase activity was accessible to ferricyanide as well as to impermeable competitive inhibitors of the enzyme. Partial inversion during preparation of vesicles is the most probable explanation for the results.A protion of this work was presented at the Miami Winter Symposia on the Molecular Basis of Biological Transprot, 1972.     

Purification and some properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae

An NAD-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD⁺ oxidoreductase, EC 1.1.1.8) has been isolated and purified from Saccharomyces cerevisiae by affinity and exclusion chromatography. The enzyme was purified 5100-fold to a specific activity of 158. It has a molecular weight of approximately 31,000, a pH optimum between 6.8 and 7.2, and is sensitive to high-ionic-strength salt solutions. The enzyme is most strongly inhibited by phosphate and chloride ions.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Isoenzymic forms of NAD-linked glycerol-3-phosphate dehydrogenase from rabbit brain.

Two major enzyme forms of cytosolic NAD-linked glycerol-3-phosphate dehydrogenase in rabbit brain have been purified to apparent homogeneity. One major enzyme form designated I6.5 exhibits an iso-electric point at pH 6.5, and is indistinguishable from the major form I6.5 found in other tissues. The other major form, designated I5.9, has an isolectric point at pH 5.9, and by amino acid analysis is shown to be a true isoenzyme distinct from form I6.5. Form I5.9 appears to be closely related to or identical with the major enzyme characteristic of heart. Neither the brain enzyme form I5.9 nor the major heart isoenzyme are inhibited by antiserum to the muscle enzyme. Because of the high apparent Km for NADH, it is postulated that the brain isoenzyme I5.9 serves to maintain glycolysis when NADH levels rise under relatively anaerobic conditions especially during fetal and neonatal development.