The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

Background

Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P.

Results

We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1 ⁺ Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1 ⁺ Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose.

Conclusions

The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

NADPH-dependent pgi-gene knockout Escherichia coli metabolism producing shikimate on different carbon sources

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+) strain and its mixed glucose/fructose fermentation grew well. Interestingly, when fructose was used as a carbon source, the pgi(-) mutant showed the enhanced SA production compared with the pgi(+) strain. In silico analysis of a genome-scale E. coli model was then conducted to characterize the cellular metabolism and quantify NAPDH regeneration, which allowed us to understand such experimentally observed attenuated cell growth and enhanced SA production in glucose- and fructose-consuming pgi(-) mutant, respectively with respect to cofactor regeneration.     

Physiological Effects of Seven Different Blocks in Glycolysis in Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow and ferment glucose have been isolated. Of 45 clones isolated, 25 had single enzyme defects of one of the following activities: phosphoglucose isomerase (pgi), phosphofructokinase (pfk), triosephosphate isomerase (tpi), phosphoglycerate kinase (pgk), phosphoglyceromutase (pgm), and pyruvate kinase (pyk). Phosphofructokinase activities in crude extracts of the pfk mutant were only 2% of the wild-type level. However, normal growth on glucose medium and normal fermentation of glucose suggested either that the mutant enzyme was considerably more active in vivo or, alternatively, that 2% residual activity was sufficient for normal glycolysis. All other mutants were moderately to strongly inhibited by glucose. Unusually high concentrations of glycolytic metabolites were observed before the reaction catalyzed by the enzyme which was absent in a given mutant strain when incubated on glucose. This confirmed at the cellular level the location of the defect as determined by enzyme assays. With adh (lacks all three alcohol dehydrogenase isozymes) and pgk mutants, accumulation of the typical levels of hexosephosphates was prevented when respiration was blocked with antimycin A. A typical feature of all glycolytic mutants described here was the rapid depletion of the intracellular adenosine 5'-triphosphate pool after transfer to glucose medium. No correlation of low or high levels of fructose-1,6-bisphosphate with the degree of catabolite repression and inactivation could be found. This observation does not support the concept that hexose metabolites are directly involved in these regulatory mechanisms in yeast.     

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.     

Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli.

The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.     

Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae

Summary The structural gene PG11 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PG11 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PG11 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 strains did not grow in glucose as sole carbon source. On the other hand pgi1/pgi1 diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that (a) the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, (b) glucose-6-P is essential in yeasts, and (c) the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.     

Saccharomyces cerevisiae aldolase mutants.

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.     

Relationship between deficiency of phosphoglucose isomerase in Coprinus macrorhizus and fruiting body formation.

A mutant (pgi) of Coprinus macrorhizus deficient in phosphoglucose isomerase did not grow on fructose and grew poorly on glucose. The pgi mutation inhibited the formation of monokaryotic and dikaryotic fruiting bodies.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.     

Glucose and fructose consumption in a phosphoglucoseisomeraseless mutant in Saccharomyces cerevisiae

Saccharomyces cerevisiae with a practically complete absence of phosphoglucoseisomerase activity when grown in fructose or glucose minimal medium showed different consumption of fructose and glucose during different periods of the culture. At the beginning of growth, cells had a great quantity of glucose available relative to their requirements and a large quantity of trehalose accumulated from ¹⁴C-glucose in comparison with the wild type strain. A second phase arises when the concentration of glucose in the medium was practically absent and the cells obtain glucose by mobilisation of stored glucose containing compounds. It is very likely that at this moment a balance rate between glucose 6-phosphate formation and consumption occurs. Finally cells reach conditions of glucose starvation and fructose consumption increases in this last stage. The different consumption of fructose throughout different periods of cell growth most probably indicates a strict regulation at the level of sugar uptake.Non Standard Abbreviation pgi phosphoglucoseisomerase     

Glucose and Gluconate Metabolism in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.     

Constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Constitutive mutants for dextransucrase were isolated from cells of Leuconostoc mesenteroides NRRL B-512F by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, growing on an agar plate containing glucose as a carbon source and overlaying a soft agar with sucrose and tetracycline. These mutants were able to produce the enzyme in a liquid media containing sugars other than sucrose, such as glucose, fructose and maltose, without simultaneous synthesis of dextran. The enzyme activity of one mutant strain, SH 3002, was 2- to 3-fold higher than that of the wild strain grown on sucrose. When the concentration of glucose in the medium was increased from 2 to 4%, a 1.7-fold increase of enzyme activity was obtained for the mutant, whereas only a slight increase of the activity was observed on sucrose for both the wild strain and the mutant.     

New phosphoglucose isomerase mutants of Escherichia coli.

The mutants used to show that phosphoglucose isomerase, and glucose itself, are not essential components of Escherichia coli had not been characterized genetically, other than by mapping. We now describe two new pgi mutants, one amber and the other a Mu-phage insertion, presumably both complete inactivation mutations. The new mutations do not give a phenotype markedly different from those described earlier. However, they might be preferred for certain physiological studies, and we have prepared for this a new double mutant, strain DF214, with a Mu insertion in pgi and a deletion in zwf (flucose 6-phosphate dehydrogenase).     

Phosphoenolpyruvate: Sugar Phosphotransferase Systems and Sugar Metabolism in Brevibacterium flavum

Brevibacterium flavum mutants defective in the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) were selected with high frequency by 2-deoxyglucose-resistance. Most of them (DOG^r) still had the fructose-PTS and grew not only on fructose but also on glucose like the wild-type strain. A mutant having 1 /8th the fructose-PTS activity of the wild strain but normal glucose-PTS activity was isolated as a xylitol-resistant mutant. It grew on glucose but not on fructose. The glucose-PTS was active on glucose, glucosamine, 2-deoxyglucose and mannose, and slightly on methyl-a-glucoside and N-acetylglucosamine, but not on fructose or xylitol. The fructose-PTS acted on fructose and xylitol, and to some extent on glucose but not on glucosamine or 2-deoxyglucose. Mutants unable to grow on glucose (DOG^rGlc^-) derived from a DOG^r mutant were all defective in the fructose-PTS. All revertants able to grow on glucose derived from a DOG^rGlc“ mutant had the fructose-PTS. The glucokinase activity was about 2/3rds the glucose activity of the fructose-PTS. All the DOG^rGlc^- mutants had normal levels of glucokinase. One of these mutants grew on maltose and sucrose, which were hydrolyzed to glucose. Thus, glucokinase seems to contribute to the phosphorylation of glucose liberated inside the cell. The fructose-PTS was induced by fructose and repressed by glucose. The glucose repression was not observed in a mutant defective in the glucose-PTS.     

Differential effects of vanadium,tungsten and molybdenum on inhibition of glucose formation in renal tubules and hepatocytes of control and diabetic rabbits: Beneficial action of melatonin and N-acetylcysteine

Effect of vanadyl acetylacetonate (VAc), tungstate and molybdate on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules. In renal tubules of control and alloxan-diabetic animals, the rank order of the metal-compounds-induced (i) inhibition of glucose formation from alanine + glycerol + octanoate or aspartate + glycerol + octanoate, (ii) decrease in the mitochondrial membrane potential (_m), (iii) increase in the hydroxyl free radicals (HFR) generation and (iv) decline in glucose-6-phosphatase activity was the following: VAc > tungstate > molybdate. Moreover, in contrast to VAc, both tungstate and molybdate at 100 M concentration did not practically decrease glucose production in hepatocytes isolated from diabetic rabbits, and significantly increased the rate of lactate formation in renal tubules. N-acetylcysteine at 2 mM concentration partially attenuated vanadium-induced alterations in glucose formation, _m and the cellular glutathione redox state, whereas 0.1 mM melatonin did not abolish vanadium-induced changes in gluconeogenesis despite attenuation of vanadium effects on HFR formation and _m decline. However, similarly to control rabbits, following 6 days of intraperitoneal administration of both VAc (1.275 mg V/kg body weight daily) and melatonin (1 mg/kg body weight daily) to alloxan-diabetic animals, vanadium-induced elevated serum creatinine and urea levels were decreased, indicating the beneficial effect of melatonin on diabetes- and vanadium-induced nephrotoxicity in rabbits. As serum glucose levels were also significantly diminished by vanadium + melatonin treatment of diabetic animals, the combination therapy of vanadium compounds and melatonin needs a careful evaluation. (Mol Cell Biochem 261: 9–21, 2004)     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.