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

Snoep, Jacky L.; Arfman, N; Yomano, Lorraine P.; Fliege, Ronda; Conway, Tyrrell; Ingram, L. O.

doi:10.1128/jb.176.7.2133-2135.1994

Cited by 50 publications

(33 citation statements)

References 21 publications

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“…Expression of glf on high-copy-number plasmids such as pZY600 and pUCBM20glf or on pZY507glf invariably led to glucose-positive derivatives of strain LR2-177, whereas ZSC112L and its ⌬pts derivative stayed negative. LR2-177/pZY507glf grew on glucose with growth rates up to 0.41 h Ϫ1 (Table 1); coexpression of glk led to enhanced growth ( of 0.53) similar to rates shown recently by another group (23).…”

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confidence: 52%

“…The sequence of a presumptive glf gene from Z. mobilis was reported (6), and on the basis of sequence comparison, this open reading frame was found to be a member of a superfamily of sugar transporters from both prokaryotes and eukaryotes (for a recent review, see reference 3). Recently, it was shown that the glf plus glk genes of Z. mobilis complemented an Escherichia coli mutant strain, which was deficient in glucokinase and in glucose uptake, to growth on glucose (23). However, detailed glucose uptake studies using this heterologous system have not yet been reported, nor was the substrate range of this carrier studied further.…”

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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

et al. 1995

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The Zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a lacI q -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 (K m , 4.1 mM for glucose and 39 mM for fructose; V max at 15؇C, 75 and 93 nmol min ؊1 mg ؊1 [dry weight] for glucose and fructose, respectively). For both substrates, counterflow maxima were observed.Glucose can be transported in bacteria by carrier systems which belong either to the phosphoenolpyruvate-dependent phosphotransferase system (PTS) type, to proton-or cationlinked permeases, or to ATP-binding cassette (ABC)-type carriers (4, 17). A rare case is the facilitator-type transport (uniport) of glucose in bacteria, exemplified by the system of the gram-negative, strictly fermentative bacterium Zymomonas mobilis. This type of sugar uptake does not use metabolic energy in the form of a proton potential or phosphoenolpyruvate (10, 17). Biochemical evidence for facilitated diffusion of glucose in this organism came from earlier studies (10, 26) and from in vivo nuclear magnetic resonance measurements using 13 C-labeled glucose and xylose as substrates (22). Mainly on the basis of competition studies, it was stated that the glucose facilitator (GLF) of Z. mobilis is a low-affinity, high-velocity carrier which in addition to glucose also transports fructose, xylose, or nonmetabolizable glucose analogs, such as 2-deoxyglucose (10,22,26). The sequence of a presumptive glf gene from Z. mobilis was reported (6), and on the basis of sequence comparison, this open reading frame was found to be a member of a superfamily of sugar transporters from both prokaryotes and eukaryotes (for a recent review, see reference 3). Recently, it was shown that the glf plus glk genes of Z. mobilis complemented an Escherichia coli mutant strain, which was deficient in glucokinase and in glucose uptake, to growth on glucose (23). However, detailed glucose uptake studies using this heterologous system have not yet been reported, nor was the substrate range of this carrier studied further.Plasmid constructions. DNA preparation, cloning, restriction analysis, and transformation were done according to established methods (20). Chromosomal DNA from Z. mobilis ZM6 (27) was prepared according to the lysozyme freeze-thaw method (12). The Z. mobilis gene glf from strain CP4 (15) had been fortuitously cloned in our laboratory as a 2.3-kb HindIII DNA fragment (pZY600) and shown to confer Glf activity to E. coli Glc Ϫ strains (18). DNA sequencing established the identity with the glf sequence reported earlier (6). The glf gene was amplified by PCR (16) using plasmid pZY600 as a template, and glk (6) and frk (31) genes were amplified from chromosomal DNA of Z. mobilis ZM6. The PCR primers for glf wer...

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confidence: 52%

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confidence: 99%

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

et al. 1995

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“…The transport mechanism was not determined. It is known that in the absence of the glucose PTS, E-coli can transport glucose by the mannose-PTS system (29), but other possibilities may also exist.…”

Section: Elimination Of the Glucose Pts System Onlymentioning

confidence: 99%

“…Expression of this plasmid has been shown to allow simultaneous utilization of glucose and arabinose in PTS-negative E-coli mutants (24). Plasmid pLOI740 contains the full Z. mobilis glf-zwf-edd-glk operon and has been shown to restore glucose utilization in glucose-negative mutants of E-coli (29).…”

Section: Cloning and Expressing Transportersmentioning

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Biological research survey for the efficient conversion of biomass to biofuels.

Kent¹,

Andrews

2007

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

Snoep

Arfman

Yomano

et al. 1996

Biotechnol. Bioeng.

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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 (qCO2) is highest. Almost half of flux control appears to reside with G6‐P dehydrogenase (CJG6‐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 CJG6‐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. © 1996 John Wiley & Sons, Inc.

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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

Cited by 50 publications

References 21 publications

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

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

Biological research survey for the efficient conversion of biomass to biofuels.

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

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