scholarly journals Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

2018 ◽  
Author(s):  
Pavel Dvořák ◽  
Víctor de Lorenzo
Keyword(s):  
Glucose Dehydrogenase ◽  
Sugar Metabolism ◽  
Xylose Isomerase ◽  
Pentose Phosphate ◽  
Soil Bacterium ◽  
Biochemical Network ◽  
Xylose Metabolism ◽  
Native Proteins ◽  
Dead End ◽  
A Genome

AbstractGiven its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strainPseudomonas putidaEM42, a genome-edited derivative of the soil bacteriumP. putidaKT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire ofP. putidaEM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network ofP. putidaEM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC fromThermobifida fuscacatalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by by deleting thegcdgene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing theEscherichia coliproton-coupled symporter XylE. The sugar was further metabolized by the products ofE. coli xylA(xylose isomerase) andxylB(xylulokinase) towards the pentose phosphate pathway. The resultingP. putidastrain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promoteP. putidaEM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85

1992 ◽  
Vol 38 (5) ◽  
pp. 370-376 ◽  
Author(s):  
Allan Matte ◽  
Cecil W. Forsberg ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Xylose Isomerase ◽  
Sole Source ◽  
Pentose Phosphate ◽  
Binding Potential ◽  
Fibrobacter Succinogenes ◽  
Xylose Metabolism ◽  
Ruminal Bacteria ◽  
Selenomonas Ruminantium ◽  
Pentose Phosphate Cycle ◽  
Prevotella Ruminicola

Prevotella (Bacteroides) ruminicola strains B14 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohyrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B14 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B14 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase,xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B14 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle. Key words: Fibrobacter succinogenes, Prevotella, Selenomonas, xylose metabolism, rumen bacteria, pentose phosphate cycle.

scholarly journals Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

2011 ◽  
Vol 77 (22) ◽  
pp. 7886-7895 ◽  
Author(s):  
Han Xiao ◽  
Yang Gu ◽  
Yuanyuan Ning ◽  
Yunliu Yang ◽  
Wilfrid J. Mitchell ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Xylose Isomerase ◽  
Cost Effective ◽  
Pentose Phosphate ◽  
Xylose Metabolism ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Content Type ◽  
Sugar Mixtures ◽  
Residual Glucose

ABSTRACTEfficient cofermentation ofd-glucose,d-xylose, andl-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacteriumClostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predictedglcGgene, encoding enzyme II of thed-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strainClostridium acetobutylicumATCC 824, resulting in greatly improvedd-xylose andl-arabinose consumption in the presence ofd-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermentedd-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result ofglcGdisruption. Furthermore, the inherent rate-limiting steps of thed-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of thed-xylose proton-symporter (cac1345),d-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integratingglcGdisruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures ofd-glucose,d-xylose, andl-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

scholarly journals Engineering Pseudomonas putida S12 for Efficient Utilization of d-Xylose and l-Arabinose

2008 ◽  
Vol 74 (16) ◽  
pp. 5031-5037 ◽  
Author(s):  
Jean-Paul Meijnen ◽  
Johannes H. de Winde ◽  
Harald J. Ruijssenaars
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Glucose Dehydrogenase ◽  
Xylose Isomerase ◽  
Dry Weight ◽  
High Yield ◽  
Efficient Production ◽  
Primary Metabolism ◽  
Dead End ◽  
Solvent Tolerant

ABSTRACT The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate−1], where CDW is cell dry weight), and the growth rate was poor (0.01 h−1). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose−1]) at a considerably improved growth rate (0.35 h−1). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved d-xylose-utilizing strain metabolized l-arabinose as efficiently as d-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

scholarly journals Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Minhye Shin ◽  
Heeyoung Park ◽  
Sooah Kim ◽  
Eun Joong Oh ◽  
Deokyeol Jeong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Sugar Metabolism ◽  
Pentose Phosphate ◽  
Biofuel Production ◽  
Rna Seq ◽  
Loss Of Function ◽  
Lignocellulosic Biofuel ◽  
Pentose Sugar ◽  
Transcriptomic Changes

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

scholarly journals The Bacillus subtilis yqjI Gene Encodes the NADP+-Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway

2004 ◽  
Vol 186 (14) ◽  
pp. 4528-4534 ◽  
Author(s):  
Nicola Zamboni ◽  
Eliane Fischer ◽  
Dietmar Laudert ◽  
Stéphane Aymerich ◽  
Hans-Peter Hohmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Glucose Dehydrogenase ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Metabolic Intermediates ◽  
The Third ◽  
Key Enzyme ◽  
Sequence Similarities

ABSTRACT Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.

scholarly journals Concomitant Regulation by a LacI-Type Transcriptional Repressor XylR on Genes Involved in Xylan and Xylose Metabolism and the Type III Secretion System in Rice Pathogen Xanthomonas oryzae pv. oryzae

2018 ◽  
Vol 31 (6) ◽  
pp. 605-613 ◽  
Author(s):  
Yumi Ikawa ◽  
Sayaka Ohnishi ◽  
Akiko Shoji ◽  
Ayako Furutani ◽  
Seiji Tsuge
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Xylose Isomerase ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Upstream Region ◽  
Xylose Metabolism ◽  
Type Iii ◽  
Hrp Genes

The hypersensitive response and pathogenicity (hrp) genes of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, encode components of the type III secretion system and are essential for virulence. Expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX; HrpG regulates hrpX and hrpA, and HrpX regulates the other hrp genes on hrpB-hrpF operons. We previously reported the sugar-dependent quantitative regulation of HrpX; the regulator highly accumulates in the presence of xylose, followed by high hrp gene expression. Here, we found that, in a mutant lacking the LacI-type transcriptional regulator XylR, HrpX accumulation and hrp gene expression were high even in the medium without xylose, reaching the similar levels present in the wild type incubated in the xylose-containing medium. XylR also negatively regulated one of two xylose isomerase genes (xylA2 but not xylA1) by binding to the motif sequence in the upstream region of the gene. Xylose isomerase is an essential enzyme in xylose metabolism and interconverts between xylose and xylulose. Our results suggest that, in the presence of xylose, inactivation of XylR leads to greater xylan and xylose utilization and, simultaneously, to higher accumulation of HrpX, followed by higher hrp gene expression in the bacterium.

scholarly journals Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase

2020 ◽  
Vol 69 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Brenna M. Zimmer ◽  
Joseph J. Barycki ◽  
Melanie A. Simpson
Keyword(s):  
Glucose Dehydrogenase ◽  
Sugar Metabolism ◽  
Structural Features ◽  
Proteoglycan Synthesis ◽  
Allosteric Modulators ◽  
Uridine Diphosphate ◽  
Cellular Mechanisms ◽  
Glycosaminoglycan Synthesis ◽  
Post Translational Modifications ◽  
Multiple Levels

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:

scholarly journals Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

2013 ◽  
Vol 79 (7) ◽  
pp. 2336-2348 ◽  
Author(s):  
Tanja Hanke ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
Tino Polen ◽  
Stephanie Bringer ◽  
...  
Keyword(s):  
Phase Ii ◽  
Growth Phase ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Gluconobacter Oxydans ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Content Type ◽  
Activity Measurements

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

scholarly journals Lateral acquisition of genes is affected by the friendliness of their products

2010 ◽  
Vol 108 (1) ◽  
pp. 343-348 ◽  
Author(s):  
Uri Gophna ◽  
Yanay Ofran
Keyword(s):  
New Species ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Interaction Sites ◽  
Native Proteins ◽  
Multiple Protein ◽  
A Genome ◽  
Genes Encoding ◽  
Foreign Genes ◽  
New Interactions

A major factor in the evolution of microbial genomes is the lateral acquisition of genes that evolved under the functional constraints of other species. Integration of foreign genes into a genome that has different components and circuits poses an evolutionary challenge. Moreover, genes belonging to complex modules in the pretransfer species are unlikely to maintain their functionality when transferred alone to new species. Thus, it is widely accepted that lateral gene transfer favors proteins with only a few protein–protein interactions. The propensity of proteins to participate in protein–protein interactions can be assessed using computational methods that identify putative interaction sites on the protein. Here we report that laterally acquired proteins contain significantly more putative interaction sites than native proteins. Thus, genes encoding proteins with multiple protein–protein interactions may in fact be more prone to transfer than genes with fewer interactions. We suggest that these proteins have a greater chance of forming new interactions in new species, thus integrating into existing modules. These results reveal basic principles for the incorporation of novel genes into existing systems.

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