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BMC Biology Sep 2015Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia...
BACKGROUND
Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date.
RESULTS
In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation.
CONCLUSIONS
Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.
Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Glucose; Glycerol; Methanol; Pichia
PubMed: 26400155
DOI: 10.1186/s12915-015-0186-5 -
MicrobiologyOpen Dec 2019In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a...
In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.
Topics: Biotransformation; Carbohydrate Metabolism; Chromatography, High Pressure Liquid; Dihydroxyacetone; Gluconobacter; Glyceric Acids; Glycerol; Kinetics; Magnetic Resonance Spectroscopy
PubMed: 31532065
DOI: 10.1002/mbo3.926 -
Microbial Cell Factories Jun 2021Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an...
BACKGROUND
Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway.
RESULTS
tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose.
CONCLUSIONS
This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.
Topics: Dihydroxyacetone; Dihydroxyacetone Phosphate; Diphosphoglyceric Acids; Fermentation; Genes, Bacterial; Glucose; Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycerol; Hydrogen-Ion Concentration; Klebsiella pneumoniae; Metabolic Engineering; Metabolic Networks and Pathways; Phosphotransferases (Alcohol Group Acceptor); Thermodynamics
PubMed: 34187467
DOI: 10.1186/s12934-021-01608-0 -
Journal of Oral Microbiology 2022The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the...
BACKGROUND
The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional signatures at the onset of dental caries.
METHODS
Since it is unethical to induce carious lesions clinically, we developed an innovative longitudinal model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics.
FINDINGS
For the first time, we induced longitudinally monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated with it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; , and , regardless of the abundance of other taxa.
INTERPRETATION
These findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.
PubMed: 36189437
DOI: 10.1080/20002297.2022.2123624 -
Biochemistry Jun 2023Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in...
Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in mutagenesis studies. The ND2 side chain atom of N11 is hydrogen bonded to the O-1 hydroxyl of enzyme-bound dihydroxyacetone phosphate (DHAP), and it sits in an extended chain of hydrogen-bonded side chains that includes T75' from the second subunit. The N11A variants of wild-type TIM from (TIM) and (TIM) undergo dissociation from the dimer to monomer under our assay conditions. Values of = 8 × 10 and 1 × 10 M, respectively, were determined for the conversion of monomeric N11A TIM and TIM into their homodimers. The N11A substitution at the variant of TIM previously stabilized by the E65Q substitution gives the N11A/E65Q variant that is stable to dissociation under our assay conditions. The X-ray crystal structure of N11A/E65Q TIM shows an active site that is essentially superimposable on that for wild-type TIM, which also has a glutamine at position 65. A comparison of the kinetic parameters for E65Q TIM and N11A/E65Q TIM-catalyzed reactions of ()-glyceraldehyde 3-phosphate (GAP) and (DHAP) shows that the N11A substitution results in a (13-14)-fold decrease in / for substrate isomerization and a similar decrease in for DHAP but only a 2-fold decrease in for GAP.
Topics: Triose-Phosphate Isomerase; Catalysis; Amino Acids; Hydrogen
PubMed: 37162263
DOI: 10.1021/acs.biochem.3c00133 -
The Journal of Biological Chemistry Nov 2023Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl...
Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high K values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
Topics: Aldehyde-Lyases; Carbon; Fructose-Bisphosphate Aldolase; Metals; Phosphates
PubMed: 37838169
DOI: 10.1016/j.jbc.2023.105338 -
Chemical Reviews Feb 2016The utility of metabolic synthons as the building blocks for new biomaterials is based on the early application and success of hydroxy acid based polyesters as... (Review)
Review
The utility of metabolic synthons as the building blocks for new biomaterials is based on the early application and success of hydroxy acid based polyesters as degradable sutures and controlled drug delivery matrices. The sheer number of potential monomers derived from the metabolome (e.g., lactic acid, dihydroxyacetone, glycerol, fumarate) gives rise to almost limitless biomaterial structural possibilities, functionality, and performance characteristics, as well as opportunities for the synthesis of new polymers. This review describes recent advances in new chemistries, as well as the inventive use of traditional chemistries, toward the design and synthesis of new polymers. Specific polymeric biomaterials can be prepared for use in varied medical applications (e.g., drug delivery, tissue engineering, wound repair, etc.) through judicious selection of the monomer and backbone linkage.
Topics: Amino Acids; Biocompatible Materials; Dihydroxyacetone; Glycerol; Hydroxy Acids; Molecular Structure; Polymers
PubMed: 26821863
DOI: 10.1021/acs.chemrev.5b00465 -
Cells Mar 2022Upregulation of glycolysis, induction of epithelial-mesenchymal transition (EMT) and macroautophagy (hereafter autophagy), are phenotypic changes that occur in tumor... (Review)
Review
Upregulation of glycolysis, induction of epithelial-mesenchymal transition (EMT) and macroautophagy (hereafter autophagy), are phenotypic changes that occur in tumor cells, in response to similar stimuli, either tumor cell-autonomous or from the tumor microenvironment. Available evidence, herein reviewed, suggests that glycolysis can play a causative role in the induction of EMT and autophagy in tumor cells. Thus, glycolysis has been shown to induce EMT and either induce or inhibit autophagy. Glycolysis-induced autophagy occurs both in the presence (glucose starvation) or absence (glucose sufficiency) of metabolic stress. In order to explain these, in part, contradictory experimental observations, we propose that in the presence of stimuli, tumor cells respond by upregulating glycolysis, which will then induce EMT and inhibit autophagy. In the presence of stimuli and glucose starvation, upregulated glycolysis leads to adenosine monophosphate-activated protein kinase (AMPK) activation and autophagy induction. In the presence of stimuli and glucose sufficiency, upregulated glycolytic enzymes (e.g., aldolase or glyceraldehyde 3-phosphate dehydrogenase) or decreased levels of glycolytic metabolites (e.g., dihydroxyacetone phosphate) may mimic a situation of metabolic stress (herein referred to as "pseudostarvation"), leading, directly or indirectly, to AMPK activation and autophagy induction. We also discuss possible mechanisms, whereby glycolysis can induce a mixed mesenchymal/autophagic phenotype in tumor cells. Subsequently, we address unresolved problems in this field and possible therapeutic consequences.
Topics: AMP-Activated Protein Kinases; Autophagy; Epithelial-Mesenchymal Transition; Glucose; Glycolysis
PubMed: 35326492
DOI: 10.3390/cells11061041 -
Science and Technology of Advanced... Jun 2015In recent decades, the substitution of non-renewable fossil resources by renewable biomass as a sustainable feedstock has been extensively investigated for the... (Review)
Review
In recent decades, the substitution of non-renewable fossil resources by renewable biomass as a sustainable feedstock has been extensively investigated for the manufacture of high value-added products such as biofuels, commodity chemicals, and new bio-based materials such as bioplastics. Numerous solid catalyst systems for the effective conversion of biomass feedstocks into value-added chemicals and fuels have been developed. Solid catalysts are classified into four main groups with respect to their structures and substrate activation properties: (a) micro- and mesoporous materials, (b) metal oxides, (c) supported metal catalysts, and (d) sulfonated polymers. This review article focuses on the activation of substrates and/or reagents on the basis of groups (a)-(d), and the corresponding reaction mechanisms. In addition, recent progress in chemocatalytic processes for the production of five industrially important products (5-hydroxymethylfurfural, lactic acid, glyceraldehyde, 1,3-dihydroxyacetone, and furan-2,5-dicarboxylic acid) as bio-based plastic monomers and their intermediates is comprehensively summarized.
PubMed: 27877800
DOI: 10.1088/1468-6996/16/3/034903 -
Foods (Basel, Switzerland) Jul 2021Soil properties in the foraging range of honeybees influence honey composition. We aimed to determine relationships between the antimicrobial properties of New Zealand...
Soil properties in the foraging range of honeybees influence honey composition. We aimed to determine relationships between the antimicrobial properties of New Zealand mānuka () honey and elemental concentrations in the honey, plants, and soils. We analyzed soils, plants, and fresh mānuka honey samples from the Wairarapa region of New Zealand for the chemical elements and the antimicrobial activity of the honey as indicated by methylglyoxal (MGO) and dihydroxyacetone (DHA). There were significant negative correlations between honey MGO and the concentrations of Mn, Cu, Mg, S, Na, Ba, K, Zn, and Al. These elements may provide a low-cost means of assessing mānuka honey quality. For individual elements, except for K, there were no correlations between the honeys, plants, and soils. Soil nitrate concentrations were negatively correlated with concentrations of MGO and DHA in the honey, which implies that soil fertility may be a determiner of mānuka honey quality.
PubMed: 34359540
DOI: 10.3390/foods10071670