Analysis of Carbon Metabolism in Escherichia coli Strains with an Inactive Phosphotransferase System by 13C Labeling and NMR Spectroscopy

Flores, Sergio; Gosset, Guillermo; Flores, Noemí; Graaf, Albert A. de; Bolívar, Francisco

doi:10.1006/mben.2001.0209

Cited by 136 publications

(187 citation statements)

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“…In E. coli, the PTS is the main glucose transport system and is also responsible for carbon catabolite repression (CCR) and chemotaxis (Hernandez-Montalvo et al 2003). Thus, deletion of the PTS is also pleiotropic and its mutant has several distinct characteristics over the wild-type strain, such as enhancement of intracellular PEP concentration, coexistence of glycolytic and gluconeogenic pathways, and the simultaneous utilization of different carbon sources (Flores et al 2002). The PTS mutant is able to co-utilize xylose and glucose (Nichols et al 2001) and produces higher titers of various compounds such as recombinant proteins (Chou et al 1994), aromatics (Flores et al 1996), lysine (Lindner et al 2011), L-phenylalanine (Baez-Viveros et al 2007, and succinate (Zhang et al 2009b).…”

Section: Discussionmentioning

confidence: 99%

“…The PTS mutant is able to co-utilize xylose and glucose (Nichols et al 2001) and produces higher titers of various compounds such as recombinant proteins (Chou et al 1994), aromatics (Flores et al 1996), lysine (Lindner et al 2011), L-phenylalanine (Baez-Viveros et al 2007, and succinate (Zhang et al 2009b). However, the uptake rate of glucose in PTS mutant is known to be low Flores et al 2002) and consequently the growth rate is exceptionally low. Therefore, in this study, the GGS was overexpressed in the PTS-deficient mutant in an attempt to increase the biomass when grown on glucose.…”

Section: Discussionmentioning

confidence: 99%

“…To address this issue, adaptive evolution was used to select fast-growing strains (Flores et al 1996). Transcriptome analysis in these fast-growing strains indicated that the expressions of gene galP (coding for galactose permease), glk (coding for glucose kinase), and pgi (coding for phosphoglucose isomerase) were increased as compared to the wild-type strain, suggesting that these genes were important in enhancing glucose uptake Flores et al 2002). Hence, it was reported that the overexpression of galP and glk enhanced the growth rate of microbes in glucose medium Lu et al 2012) and improved the yields of valuable products such as aromatics (Yi et al 2003), recombinant protein (De Anda et al 2006), and ethanol (Hernandez-Montalvo et al 2003).…”

Section: Introductionmentioning

confidence: 99%

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Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Zhang

Zou

Chen

et al. 2015

Appl Microbiol Biotechnol

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Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways.These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Zhang

Zou

Chen

et al. 2015

Appl Microbiol Biotechnol

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“…Moreover, 13 C-labeling and nuclear magnetic resonance studies have shown that when cells are growing aerobically in minimal medium with glucose as the carbon source, the main biosynthetic pathway in E. coli for ribose-5-phosphate and erythrose-4-phosphate, both of which are precursors for nucleotide and aromatic amino acid biosynthesis, is the oxidative branch of the PP pathway (22). It has been reported that 22% of the glucose transported into E. coli is driven by the glucose-6-phosphate dehydrogenase and that part is diverted to nucleotide, amino acid, and vitamin biosynthesis (7). Another important product of the PP oxidative branch is NADPH, which participates mostly in anabolic pathways (e.g., protein biosynthesis).…”

Section: Figmentioning

confidence: 99%

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Aon¹,

Caimi²,

Taylor³

et al. 2008

Appl Environ Microbiol

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Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

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“…In Escherichia coli, overexpression of the soluble transhydrogenase UdhA was shown to partially restore the growth phenotype of a phosphoglucose isomerase mutant with impaired NADPH metabolism (26), but its phys-iological role in wild-type E. coli remains obscure. Whether or not the membrane-bound isoform actually contributes to the formation of NADPH in E. coli is unclear at present, but isotopic tracer experiments indicated that it is not an important source of NADPH during growth on glucose (8,(27)(28)(29).…”

mentioning

confidence: 99%

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

Sauer

Canonaco

Heri

et al. 2004

Journal of Biological Chemistry

523

526

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Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiological function that could potentially transfer electrons directly from NADH to NADP ؉ and vice versa. Using defined mutants and metabolic flux analysis, we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During standard aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20 -25%, respectively. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiological functions: energy-dependent reduction of NADP ؉ with NADH by PntAB and reoxidation of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metabolism, because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, respectively. The two transhydrogenase isoforms provide E. coli primary metabolism with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metabolism?About 1,000 anabolic reactions synthesize the macromolecular components that make up functional cells (1, 2), but only 11 intermediates of central carbon metabolism and the cofactors ATP, NADH, and NADPH constitute the core of this intricate biochemical network (3, 4). These intermediates and cofactors must be supplied through the catabolism of different substrates at appropriate rates and stoichiometries for balanced growth; hence, anabolism and catabolism are delicately balanced and regulated to enable growth under fluctuating environmental conditions. Although chemically very similar, the redox cofactors NADH and NADPH serve distinct biochemical functions and participate in more than 100 enzymatic reactions (5). The electrons of the main respiratory cofactor NADH are transferred primarily to oxygen, thereby driving oxidative phosphorylation of ADP to ATP (3,4,6). NADPH, in contrast, exclusively drives anabolic reduction reactions. Despite the important role in linking the fundamental processes of catabolism and anabolism, however, even a qualitative understanding of NADPH metabolism is still missing for most organisms.The primary NADPH-generating reactions are considered to be the oxidative pentose phosphate (PP) 1 pathway and the NADPH-dependent isocitrate dehydrogenase in the TCA cycle (Fig.

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Analysis of Carbon Metabolism in Escherichia coli Strains with an Inactive Phosphotransferase System by 13C Labeling and NMR Spectroscopy

Cited by 136 publications

References 31 publications

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

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