Glycerol and dihydroxyacetone metabolizing enzymes in fission yeasts of the genus Schizosaccharomyces

The only species of fission yeasts capable of growing on glycerol or dihydroxyacetone were Schizosaccharomyces pombe and S. malidevorans. When growing on glycerol or grown on glucose until it was exhausted, these species contained glycerol:NAD⁺ 2-oxidoreductase and dihydroxyacetone kinase but no glycerol kinase, consistent with utilization of glycerol via dihydroxyacetone. When grown to exhaustion of glucose, S. octosporus, S. slooffiae and S. japonicus contained dihydroxyacetone kinase but no glycerol:NAD⁺ 2-oxidoreductase or glycerol kinase. Prior to exhaustion of glucose in the medium, all species contained dihydroxyacetone kinase, all species except S. japonicus contained glycerol:NADP⁺ 2-oxidoreductase, and only S. pombe and S. malidevorans contained glycerol:NAD⁺ 2-oxidoreductase. Possible roles for the glycerol:NAD⁺ 2-oxidoreductase, glycerol:NADP⁺ 2-oxidoreductase and dihydroxyacetone kinase in metabolism of glycerol and dihydroxyacetone are discussed.Non-standard abbreviations DHA dihydroxyacetone - DHAK dihydroxyacetone kinase - DHAP dihydroxyacetone phosphate - GK glycerol kinase - G2DH-NAD glycerol - NAD⁺ 2-oxidoreductase - G2DH-NADP glycerol - NADP⁺ 2-oxidoreductase - MEA malt extract agar - YEP yeast extract phosphate medium     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Dihydroxyacetone production from methanol by a dihydroxyacetone kinase deficient mutant of Hansenula polymorpha

Summary A mutant, No. 65, of Hansenula polymorpha CBS 4732 was isolated which was impaired in its ability to grow on methanol and dihydroxyacetone. Mutant No. 65 produced dihydroxyacetone and glycerol from methanol with a 18.8% yield in a resting-cells reaction. The absence of dihydroxyacetone kinase activity in the mutant is believed to be the reason for its inability to grow on methanol and for the accumulation of trioses. This mutant, however, was able to grow on glycerol, and dihydroxyacetone kinase was found in the cells. The growth on glycerol was almost completely inhibited by the addition of methanol (0.1% v/v). As far as tested with partially purified enzymes, no property was found that could be used to distinguish between the kinases from methanol- and glycerol-grown cells. The evidence suggests that the phenotype of No. 65 is a lesion not in the structural gene but in its regulatory gene.     

Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva

An NADP⁺-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

The relation between the assimilation of methanol and glycerol in yeasts

Fifteen yeast strains of the genera Candida, Lodderomyces, Endomycopsis, Saccharomyces, Hansenula, Pichia and Torulopsis were investigated with respect to their ability to grow on methanol, glycerol and glucose as sole carbon and energy source. Eight of them can grow on both methanol and glycerol.Methanol is assimilated via triosephosphate (dihydroxyacetone) pathway. The dihydroxyacetone kinase is a key enzyme in methanol metabolism.The assimilation of glycerol can take place in bacteria via a phosphorylative or/and oxidative pathways. In general, the phosphorylative pathway is found in eucaryotes. In the present paper it is shown that in yeasts, which can utilize methanol and glycerol, too, glycerol is assimilated via an oxidative pathway, Dihydroxyacetone is a central intermediate in the assimilation of methanol as well as glycerol. It is metabolized by means of the dihydroxyacetone kinase.The enzyme formed during growth of Candida methylica on methanol does not differ from that of Candida valida H 122 after growing on glycerol as far as the regulatory properties are concerned.     

Isolation and characterization of mutants from Schyzosaccharomyces pombe defective in glycerol catabolism

Mutants unable to grow on glycerol were isolated from the fission yeast Schyzosaccharomyces pombe. Two types of mutants were obtained: one type was able to grow on dihydroxyacetone while the other one did not grow on this compound. The first type of mutants was defective in glycerol dehydrogenase while the second one was affected both in the glycerol dehydrogenase and in dihydroxyacetone kinase. It was found that the second type was defective in the derepression of several enzymes. The mutations were nuclear and monogenic and defined two complementation groups. Spontaneous revertants, able to grow on glycerol, were obtained from the first type of mutants. They have regained the glycerol dehydrogenase activity. The results presented provide genetic evidence for a pathway of glycerol catabolism in Sch. pombe involving dehydrogenation of glycerol as the first step followed by phosphorylation of the dihydroxyacetone formed.     

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h^?1 where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. ¹³C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO₂ production and oxygen consumption rates which were in good agreement.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

Alternation of the Dissimilation Pathway for Glycerol and Amplification of Glycerol Dehydrogenase in Mutant Strains of Cellulomonas sp. NT3060

Mutant strain ME544, which is able to grow on glycerol slowly, was derived from glycerol-negative mutant strain G011, which is a derivative strain of Cellulomonas sp. NT3060 and is defective in both the enzyme activities of glycerol kinase and glycerol 3-phosphate dehydrogenase. The mutant strain still lacked both the enzyme activities involved in the dissimilation of glycerol and had the same level of glycerol dehydrogenase activity as the parent strain. Dihydroxyacetone kinase activity in mutant strain ME544 was inducibly formed, reaching 4-fold the level in mutant strain G011 in glycerol medium. Thus, the mutant strain seemed to dissimilate glycerol by means of glycerol dehydrogenase followed by an increase in dihydroxyacetone kinase. Subsequently, a mutant strain, GP1807, which was resistant to the inhibition of growth on glycerol by 1,2-propanediol, was derived from mutant strain ME544. Glycerol dehydrogenase activity of the mutant strain was amplified about 6-fold compared to that of the wild type strain.     

l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD⁺ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD₆₀₀]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (Δpfl mutant) expressing the ldhL1_LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.     

Precursors of ricinine in the castor bean plant

Isotopic tracer experiments confirmed that glycerol and succinic acid are good precursors of the pyridine ring of ricinine in castor bean plants. Tritium from C-2 was lost from tritiated glycerol while tritium from C-1 was retained. Thus a derivative of dihydroxyacetone is likely to be intermediate. By simultaneous feeding of glycerol-1-(3)-[³H] and succinic acid-2(3)-[¹⁴C], it was hoped to find precursors of ricinine containing both labels, but none could be found. There was no evidence for the appearance of labeled quinolinic acid, which is presumed to be a precursor of ricinine.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

Crystal structure of a nucleotide-binding domain of fatty acid kinase FakA from Thermus thermophilus HB8

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg²⁺, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.     

Biosynthetic Pathways of Glycerol Accumulation under Salt Stress in Aspergillus nidulans

Redkar, R. J., Locy. R. D., and Singh, N. K. 1995. Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans. Experimental Mycology 19, 241-246. A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP⁺-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP⁺-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.     

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1⁺ gene encoding a putative A-type Fe-S scaffold, in addition to mas5⁺ and hsc1⁺ genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2⁺, but not isu1⁺ and ssc1⁺, complemented the growth phenotype of Δgrx5 mutant as isa1⁺ did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.     

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.Abbreviations XuMP xylulose monophosphate - DHA dihydroxyacetone - EMS ethyl methanesulphonate     

Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH₂ flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD⁺ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique