Genetic Mapping of Mutations Affecting Phosphoglucose Isomerase and Fructose Diphosphatase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting phosphoglucose isomerase (pgi) and fructose diphosphatase (fdp) have been determined by conjugation and transduction experiments, chiefly three-factor crosses. The loci for these two constitutive enzymes of central intermediary metabolism lie in the same region of the chromosome, but they are not cotransducible. The order of some markers in this region of the chromosome is arg(ACFH) metA pgi malB uvrA fdp pyrB thr.     

Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K(m) values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.     

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.     

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase ( pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain ( E. coli JM109), growth of the E. coli mutant lacking pgi ( E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

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.     

Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli.

Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt. The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo. Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts.     

Bifunctional phosphoglucose/phosphomannose isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum constitute a novel enzyme family within the phosphoglucose isomerase superfamily

The hyperthermophilic crenarchaeon Aeropyrum pernix contains phosphoglucose isomerase (PGI) activity. However, obvious homologs with significant identity to known PGIs could not be identified in the sequenced genome of this organism. The PGI activity from A. pernix was purified and characterized. Kinetic analysis revealed that, unlike all known PGIs, the enzyme catalyzed reversible isomerization not only of glucose 6-phosphate but also of epimeric mannose 6-phosphate at similar catalytic efficiency, thus defining the protein as bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). The gene pgi/pmi encoding PGI/PMI (open reading frame APE0768) was identified by matrix-assisted laser desorption ionization time-of-flight analyses; the gene was overexpressed in Escherichia coli as functional PGI/PMI. Putative PGI/PMI homologs were identified in several (hyper)thermophilic archaea and two bacteria. The homolog from Thermoplasma acidophilum (Ta1419) was overexpressed in E. coli, and the recombinant enzyme was characterized as bifunctional PGI/PMI. PGI/PMIs showed low sequence identity to the PGI superfamily and formed a distinct phylogenetic cluster. However, secondary structure predictions and the presence of several conserved amino acids potentially involved in catalysis indicate some structural and functional similarity to the PGI superfamily. Thus, we propose that bifunctional PGI/PMI constitutes a novel protein family within the PGI superfamily.     

Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli.

Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt. The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo. Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts.     

Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a major metabolic gene

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

6-phosphogluconolactonase mutants of Escherichia coli and a maltose blue gene

Mutants lacking an enzyme of the oxidative branch of the hexose monophosphate shunt, 6-phosphogluconolactonase (pgl), have been selected as a new class of glucose-negative derivatives of a phosphoglucose isomerase (pgi) mutant. Glucose negativity is not as complete as in mutants lacking phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Pgi(+), pgl(-) strains have been constructed by transduction and grow almost normally on glucose. Genetic mapping shows that pgl lies between chlD and att-lambda, in the same position as and identical with a blu gene described by Adhya and Schwartz. These blu mutants grown on maltose were recognized by their property to turn blue after treatment with iodine. It is not known how phosphogluconolactonase deficiency causes this reaction; it might be related to accumulation of 6-phosphogluconolactone.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli.

The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself.     

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).     

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.     

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.     

Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains

Saccharomyces cerevisiae strains lacking phosphoglucose isomerase (pgi1) cannot use the pentose phosphate (PP) pathway to oxidize glucose, which has been explained by the lack of mechanism for reoxidation of the NADPH surplus. Consistent with this, the defective growth on glucose of a ENYpgi1 strain can be partially restored by expressing the Escherichia coli transhydrogenase udhA. In this work it was found that growth of V5 (wine yeast-derived) and FY1679 (isogenic to S288C) pgi1 mutants is not rescued by expression of udhA. Moreover, the flux through the PP pathway of 11 S. cerevisiae strains from various origins was estimated, by calculating the ratio between the enzymatic activity of the G6PDH and HXK, placed at the glycolysis-PP pathway branch point. The results show that ENY.WA-1A exhibited the highest ratio (1.5-3-fold) and the highest G6PDH activity. Overexpression of ZWF1 encoding the G6PDH in V5pgi1udhA did not rescue growth on glucose, suggesting that steps downstream the G6PDH might limit the PP pathway in this strain. As a whole, these data highlight a great intraspecies diversity in the PP pathway capacity among S. cerevisiae strains and suggest that a low capacity may be the prime limiting factor in glucose oxidation through this pathway.