Non-oxidative Pentose Phosphate Pathway Research Articles

A preliminary X-ray study of transketolase from Burkholderia pseudomallei.

TktA is the most critical enzyme in the nonoxidative pentose phosphate pathway. It catalyzes the conversion of xylulose 5-phosphate and ribose 5-phosphate into sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate, and its products are used in the biosynthesis of acetyl-CoA, aromatic amino acids, nucleic acids and ADP-L-glycero-β-D-manno-heptose. TktA also has an unexpected role in chromosome structure that is independent of its metabolic responsibilities. Therefore, it is a new potent antibiotic target. In this study, TktA from Burkholderia pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.0 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=146.2, b=74.6, c=61.6 Å, β=113.0°. A full structural determination is under way in order to provide insight into the structure-function relationship of this protein.

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-ALCS displayed significantly increased anaerobic growth rate (0.203±0.006h−1) and xylose consumption rate (1.866gg−1h−1) along with high ethanol conversion yield (0.41g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.

Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1.

Oncogenic Kras Maintains Pancreatic Tumors through Regulation of Anabolic Glucose Metabolism

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides invivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

Metabolic and Regulatory Rearrangements Underlying Efficient d-Xylose Utilization in Engineered Pseudomonas putida S12

Previously, an efficient D-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the D-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor D-xylose utilization was the redistribution of 6-phospho-D-gluconate (6-PG) between the Entner-Doudoroff pathway and the oxidative pentose phosphate (PP) pathway. This redistribution increased the availability of 6-PG for oxidative decarboxylation to D-ribose-5-phosphate, which is essential for the utilization of D-xylose via the nonoxidative PP pathway. The metabolic redistribution of 6-PG was procured by modified HexR regulation, which in addition appeared to control periplasmic sugar oxidation. Because the absence of periplasmic D-xylonate formation was previously demonstrated to be essential for achieving a high biomass yield on D-xylose, the aberrant HexR control appeared to underlie both the improved growth rate and biomass yield of the evolved D-xylose utilizing P. putida strain. The increased oxidative PP pathway activity furthermore resulted in an elevated NADH/NAD(+) ratio that caused the metabolic flux to be redirected from the TCA cycle to the glyoxylate shunt, which was also activated transcriptionally. Clearly, these findings may serve as an important case in point to engineer and improve the utilization of non-natural carbon sources in a wide range of industrial microorganisms.

Relevance of the MEK/ERK Signaling Pathway in the Metabolism of Activated Macrophages: A Metabolomic Approach

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.

Conservation of structure and mechanism within the transaldolase enzyme family

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (β/α)(8) -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes. Database The crystal structures have been deposited in the Protein Data Bank with accession code 3R8R for BsTal and 3R5E for CgTal.

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase–xylitol dehydrogenase (XR–XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP +-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06 h −1, 0.39 g g −1, and 0.13 g g −1 consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.

Riboneogenesis in Yeast

Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathway's committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.

Novel insights into the diversity of catabolic metabolism from ten haloarchaeal genomes.

BackgroundThe extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis.Methodology/Principal FindingsWe report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses.ConclusionsThese new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

BackgroundThe development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance.ResultsIn this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid.ConclusionsOur metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

Metabolic network adaptations in cancer as targets for novel therapies

Metabolite concentrations and fluxes are the system variables that characterize metabolism. The systematic study of metabolite profiles is known as metabolomics; however, knowledge of the complete set of metabolites may not be enough to predict distinct phenotypes. A complete understanding of metabolic processes requires detailed knowledge of enzyme-controlled intracellular fluxes. These can be estimated through quantitative measurements of metabolites at different times or by analysing the stable isotope patterns obtained after incubation with labelled substrates. We have identified distinct intracellular fluxes associated with metabolic adaptations accompanying cancer. The maintenance of an imbalance between fluxes for the oxidative and non-oxidative PPP (pentose phosphate pathway) has been shown to be critical for angiogenesis and cancer cell survival. Mouse NIH 3T3 cells transformed by different mutated K-ras oncogenes have differential routing of glucose to anaerobic glycolysis, the PPP and the Krebs cycle. These results indicate that knowledge of metabolic fingerprints associated with an altered genetic profile could be exploited in the rational design of new therapies. We conclude that the understanding of the multifactorial nature of metabolic adaptations in cancer may open new ways to develop novel multi-hit antitumoral therapies.

Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae

BackgroundOverexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway.ResultsIn the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium.ConclusionsPGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

The deletion of YLR042c improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

In a recent study combining transcriptome analyses of a number of recombinant laboratory and industrial S. cerevisiae strains with improved xylose utilization and their respective control strains, the ORF YLR042c was identified as a downregulated gene and it was shown that the gene deletion improved aerobic growth on xylose in the tested strain background. In the present study, the influence of deleting YLR042c on xylose fermentation was investigated in two different xylose-fermenting strains: TMB3001, which expresses genes from the initial xylose catabolizing pathway, including heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) and endogenous xylulokinase (XK); and TMB3057, which, in addition to the initial xylose catabolizing pathway, overexpresses the endogenous genes encoding the non-oxidative pentose phosphate pathway enzymes. The deletion of YLR042c led to improved aerobic growth on xylose in both strain backgrounds. However, the effect was more significant in the strain with the poorer growth rate on xylose (TMB3001). Under anaerobic conditions, the deletion of YLR042c increased the specific xylose consumption rate and the ethanol and xylitol yields. In strain TMB3057, xylose consumption was also improved at low concentrations and during co-fermentation of xylose and glucose. The effect of the gene deletion and overexpression was also tested for different carbon sources. Altogether, these results suggest that YLR042c influences xylose and the assimilation of carbon sources other than glucose, and that the effect could be at the level of sugar transport or sugar signalling.

Fructose Induces Transketolase Flux to Promote Pancreatic Cancer Growth

Carbohydrate metabolism via glycolysis and the tricarboxylic acid cycle is pivotal for cancer growth, and increased refined carbohydrate consumption adversely affects cancer survival. Traditionally, glucose and fructose have been considered as interchangeable monosaccharide substrates that are similarly metabolized, and little attention has been given to sugars other than glucose. However, fructose intake has increased dramatically in recent decades and cellular uptake of glucose and fructose uses distinct transporters. Here, we report that fructose provides an alternative substrate to induce pancreatic cancer cell proliferation. Importantly, fructose and glucose metabolism are quite different; in comparison with glucose, fructose induces thiamine-dependent transketolase flux and is preferentially metabolized via the nonoxidative pentose phosphate pathway to synthesize nucleic acids and increase uric acid production. These findings show that cancer cells can readily metabolize fructose to increase proliferation. They have major significance for cancer patients given dietary refined fructose consumption, and indicate that efforts to reduce refined fructose intake or inhibit fructose-mediated actions may disrupt cancer growth.

Imatinib resistance associated with BCR-ABL upregulation is dependent on HIF-1α-induced metabolic reprograming

As chronic myeloid leukemia (CML) progresses from the chronic phase to blast crisis, the levels of BCR-ABL increase. In addition, blast transformed leukemic cells display enhanced resistance to imatinib in the absence of BCR-ABL resistance mutations. Here we show that when BCR-ABL transformed cell lines were selected for imatinib resistance in vitro, the cells that grew out displayed higher BCR-ABL expression comparable to increase seen in accelerated forms of the disease. This enhanced expression of BCR-ABL was associated with an increased rate of glycolysis but a decreased rate of proliferation. The higher level of BCR-ABL expression in the selected cells correlated with a non-hypoxic induction of HIF-1α that was required for cells to tolerate enhanced BCR-ABL signaling. HIF-1α induction resulted in an enhanced rate of glycolysis but reduced glucose flux through both the TCA cycle and the oxidative arm of the pentose phosphate pathway (PPP). The reduction in oxidative PPP mediated ribose synthesis was compensated by the HIF-1α-dependent activation of the non-oxidative PPP enzyme, transketolase, in imatinib-resistant CML cells. In both primary cultures of cells from patients exhibiting blast transformation and in vivo xenograft tumors, use of oxythiamine which can inhibit both the pyruvate dehydrogenase complex and transketolase resulted in enhanced imatinib sensitivity of tumor cells. Together, these results suggest that oxythiamine can enhance imatinib efficacy in patients that present in the accelerated form of the disease.

A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC–IDMS

A gas chromatography isotope dilution mass spectrometry (GC–IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10 m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC–MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of 13C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of (13)C-labeling experiments in chemostat cultures of a wild-type strain, DeltacreB and DeltaarcA single mutants, and a DeltacreB DeltaarcA double mutant. Continuous cultures were conducted at D = 0.1 h(-1) under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its DeltaarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the DeltaarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

Proteome analysis of the xylose‐fermenting mutant yeast strain TMB 3400

Xylose fermentation in yeast has been a target of research for years, yet not all the factors that may affect xylose fermentation performance of yeast strains are known. In this study, the mutant S. cerevisiae strain TMB 3400, which has good xylose fermentation properties, was compared with its parental strain to examine the factors behind the improved xylose utilization at protein level. The proteome of the parental and the mutant strains were characterized by difference in gel electrophoresis (DiGE) to quantitatively identify proteins that are expressed at altered levels in the mutant. The most significant changes detected by proteome analysis were the 6-10-fold increased levels of xylose reductase, xylitol dehydrogenase and transketolase (Tkl1) in the mutant, which is in accordance with previous knowledge about xylose metabolism in yeast. The level of acetaldehyde dehydrogenase (Ald6) was also significantly increased. In addition, several proteins homologous to proteins from yeast species other than S. cerevisiae were identified in both strains, demonstrating the genetic heterogeneity of industrial yeast strains. The results were also compared with a previously reported transcription analysis performed with identical experimental set-up; however, very little correlation between the two datasets was observed. The results of the proteome analysis were in good agreement with a parallel study in which rationally designed overexpression of XR, XDH and the non-oxidative pentose phosphate pathway resulted in similar improvement in xylose utilization, which demonstrates the usefulness of proteome analysis for the identification of target genes for further metabolic engineering strategies in industrial yeast strains.