Reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of Escherichia coli by using Zymomonas mobilis genes encoding the glucose facilitator protein and glucokinase.

Expression of the Zymomonas mobilis glf (glucose facilitator protein) and glk (glucokinase) genes in Escherichia coli ZSC113 (glucose negative) provided a new functional pathway for glucose uptake and phosphorylation. Both genes were essential for the restoration of growth in glucose minimal medium and for acid production on glucose-MacConkey agar plates.     

Control of glycolytic flux in Zymomonas mobilis by glucose 6-phosphate dehydrogenase activity

Glycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6-phosphate dehydrogenase (G6-P dehydrogenase), 6-phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO(2)) is highest. Almost half of flux control appears to reside with G6-P dehydrogenase (C(J) (G6-P dehydrogenase) = 0.4). Although Z. mobilis exhibits one of the highest rates of glycolysis known, recombinants with elevated G6-P dehydrogenase had a 10% to 13% higher glycolytic flux than the native organism. A small increase in flux was also observed for recombinants expressing glf. Results obtained did not allow a critical evaluation of glucokinase and this enzyme may also represent an important control point. 6-Phosphogluconate dehydratase appears to be saturating at native levels. With constructs containing the full operon, growth rate and flux were both reduced, complicating interpretations. However, results obtained were also consistent with G6-P dehydrogenase as a primary site of control. Flux was 17% higher in operon constructs which exhibited a 17% increase in G6-P dehydrogenase specific activity, relative to the average of other operon constructs which contain a frameshift mutation in zwf. It is unlikely that all flux control residues solely in G6-P dehydrogenase (calculated C(J) (G6-P dehydrogenase) = 1.0) although these results further support the importance of this enzyme. As reported in previous studies, changes in flux were not accompanied by changes in growth rate providing further evidence that ATP production does not limit biosynthesis in rich complex medium. (c) 1996 John Wiley & Sons, Inc.     

Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action.

The Zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a lacIq-Ptac system using Escherichia coli K-12 mutants deficient in uptake and phosphorylation of glucose and fructose. Growth on glucose or fructose was restored when the respective genes (glf-glk or glf-frk) were expressed. In E. coli glf+ strains, both glucose and fructose were taken up via facilitated diffusion (Km, 4.1 mM for glucose and 39 mM for fructose; Vmax at 15 degrees C, 75 and 93 nmol min-1 mg-1 [dry weight] for glucose and fructose, respectively). For both substrates, counterflow maxima were observed.     

Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.     

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf).     

大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida: genomic and flux analysis

In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase (encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g(-1) h(-1), which allowed a growth rate of 0.58 h(-1) and a biomass yield of 0.44 g/g carbon used. Flux analysis of (13)C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose.     

Genetic Mapping of Loci for Glucose-6-Phosphate Dehydrogenase, Gluconate-6-Phosphate Dehydrogenase, and Gluconate-6-Phosphate Dehydrase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting the constitutive enzymes glucose-6-phosphate dehydrogenase (zwf) and gluconate-6-phosphate dehydrogenase (gnd), and the inducible enzyme gluconate-6-phosphate dehydrase (edd), were determined by conjugation and transduction experiments, chiefly by three-factor crosses. They are in the same region of the chromosome, and their order is gnd-his-(edd, zwf)-aroD; gnd and his are cotransduceable, as are zwf and edd. The position of gnd in Salmonella typhimurium was shown to be similar to that in E. coli.     

Growth recovery on glucose under aerobic conditions of an Escherichia coli strain carrying a phosphoenolpyruvate:carbohydrate phosphotransferase system deletion by inactivating arcA and overexpressing the genes coding for glucokinase and galactose permease

In Escherichia coli the phosphotransferase system (PTS) consumes one molecule of phosphoenolpyruvate (PEP) to phosphorylate each molecule of internalized glucose. PEP bioavailability into the aromatic pathway can be increased by inactivating the PTS. However, the lack of the PTS results in decreased glucose transport and growth rates. To overcome such drawbacks in a PTS(-) strain and reconstitute rapid growth on glucose phenotype (Glc(+)), the glk and galP genes were cloned into a plasmid and the arcA gene was inactivated. Simultaneous overexpression of glk and galP increased the growth rate and regenerated a Glc(+) phenotype. However, the highest growth rate was obtained when glk and galP were overexpressed in the arcA(-) background. These results indicated that the arcA mutation enhanced glycolytic and respiratory capacities of the engineered strain.     

Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism.

Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli,gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.     

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.     

Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli

The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on (13)C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake. This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant. In general, the changes in enzyme activities more or less correlated with ratios of gene expression, while the changes in metabolic fluxes did not correlate with enzyme activities. For example, high flux ratios were obtained through the oxidative pentose phosphate pathway due to an increased concentration of glucose-6-phosphate rather than to favorable enzyme activity ratios. In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways.