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Molecules (Basel, Switzerland) Jun 2024The strain LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone...
The strain LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone was determined, and the influence of the pH of the culture medium and the initial concentration of glycerol on maximizing the concentration of dihydroxyacetone and on the yield and speed of obtaining dihydroxyacetone by bioconversion was examined. The feeding strategy of the substrate (crude glycerol) during the process was based on measuring the dissolved oxygen tension of the culture medium. The highest concentration of dihydroxyacetone P = 175.8 g·L and the highest yield Y = 94.3% were obtained when the initial concentration of crude glycerol was S = 70.0 g·L and the pH of the substrate was maintained during the process at level 5.0.
Topics: Gluconobacter oxydans; Dihydroxyacetone; Glycerol; Batch Cell Culture Techniques; Culture Media; Hydrogen-Ion Concentration; Fermentation
PubMed: 38930996
DOI: 10.3390/molecules29122932 -
Microbial Cell Factories May 2024Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of...
BACKGROUND
Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative.
RESULTS
This study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the -35 region and into the spacer region between the -10 and -35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 μM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 μM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants.
CONCLUSIONS
The methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.
Topics: Pyruvaldehyde; Biosensing Techniques; Dihydroxyacetone; Escherichia coli; Promoter Regions, Genetic; Metabolic Engineering; Escherichia coli Proteins
PubMed: 38796416
DOI: 10.1186/s12934-024-02393-2 -
ELife May 2024Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight , and the...
Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight , and the human intestinal protozoan . In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.
Topics: Blastocystis; Glycolysis; Humans; Mitochondria; Cytosol; Biological Transport; Protozoan Proteins
PubMed: 38780415
DOI: 10.7554/eLife.94187 -
Bioresources and Bioprocessing May 2024Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3...
BACKGROUND
Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity.
FINDINGS
FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation.
CONCLUSIONS
This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.
PubMed: 38735884
DOI: 10.1186/s40643-024-00767-3 -
Frontiers in Veterinary Science 2024Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus . While most , , and biovars grow slowly in complex media, they multiply intensely in...
Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus . While most , , and biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in and , GlpX is disabled in biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in . We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.
PubMed: 38601913
DOI: 10.3389/fvets.2024.1328293 -
PloS One 2024Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol...
Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible β-hairpin and its significance. Another commonly reported structural feature is the enzyme's octameric oligomerization, though its structural details and functional significance remained unclear. Here, with a newly reported GDH structure, complexed with both NAD+ and glycerol, we analyzed the octamerization of GDH. Structural analyses revealed that octamerization reduces the structural dynamics of the N-domain, which contributes to more consistently maintaining a distance required for catalysis between the cofactor and substrate. This suggests that octamerization may play a key role in increasing the likelihood of the enzyme reaction by maintaining the ligands in an appropriate configuration for catalysis. These findings expand our understanding of the structure of GDH and its relation to the enzyme's activity.
Topics: NAD; Glycerol; Sugar Alcohol Dehydrogenases; Oxidation-Reduction; Glutamate Dehydrogenase
PubMed: 38483875
DOI: 10.1371/journal.pone.0300541 -
Journal of the Dermatology Nurses'... 2023The use of indoor UV tanning devices (also known as "indoor tanning") has declined in recent years. Less is known about use of dihydroxyacetone-containing products used...
The use of indoor UV tanning devices (also known as "indoor tanning") has declined in recent years. Less is known about use of dihydroxyacetone-containing products used for tanning (also known as "sunless tanning"). We analyzed data from the 2015 National Health Interview Survey. Analysis was limited to non-Hispanic White women ages 18-49 years. We estimated the proportion of women reporting spray tanning, self-applied lotion tanning, and indoor tanning and used weighted multivariable logistic regression models to examine the relationships between socio-demographic characteristics, skin cancer risk factors, and other cancer risk factors with sunless and indoor tanning. Overall, 17.7% of women reported sunless tanning. Lotion tanning was more common (15.3%) than spray tanning (6.8%), while 12.0% of women engaged in indoor tanning. Among sunless tanners, 23.7% also engaged in indoor tanning. Younger age, ever having a skin exam, skin reactions to the sun, binge drinking, and being at a healthy weight were associated with sunless tanning. While sunless tanning may be less harmful for skin cancer risk than indoor tanning, the frequency with which the two behaviors co-occur suggests that efforts to address societal pressures for women to alter their skin color may have important public health benefits.
PubMed: 38463180
DOI: 10.1097/jdn.0000000000000736 -
Microbiology Spectrum Apr 2024All organisms utilize -adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM...
UNLABELLED
All organisms utilize -adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as -adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in ATCC 25922, when introduced into K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt.
IMPORTANCE
The acquisition and utilization of organic compounds that serve as growth substrates are essential for to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic , but its function in possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.
Topics: S-Adenosylmethionine; Escherichia coli; Dihydroxyacetone Phosphate; Methionine; Bacteria; Pentoses; Carbon; Sugars; Deoxyadenosines; Methylamines; Thionucleosides
PubMed: 38441472
DOI: 10.1128/spectrum.03086-23 -
PLoS Pathogens Feb 2024The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher...
The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulate Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage-defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2, label preferentially accumulates in dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accrued lactate toxicity via the deleterious buildup of sugar phosphates. The evolved lldD2 variants increase lactate incorporation to pyruvate while altering triose phosphate flux, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX- 1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provides context for the selective advantage of lldD2 mutations in adapting to host stress.
Topics: Humans; Mycobacterium tuberculosis; L-Lactate Dehydrogenase; Lactic Acid; Pyruvates; Quinones; Phosphates
PubMed: 38422159
DOI: 10.1371/journal.ppat.1012050 -
ACS Sustainable Chemistry & Engineering Feb 2024Potassium exchanged Sn-β and Sn-USY zeolites have been tested for the transformation of various aldoses (hexoses and pentoses), exhibiting outstanding catalytic...
Potassium exchanged Sn-β and Sn-USY zeolites have been tested for the transformation of various aldoses (hexoses and pentoses), exhibiting outstanding catalytic activity and selectivity toward methyl lactate. Insights into the transformation pathways using reaction intermediates-dihydroxyacetone and glycolaldehyde-as substrates revealed a very high catalytic proficiency of both zeolites in aldol and retro-aldol reactions, showcasing their ability to convert small sugars into large sugars, and vice versa. This feature makes the studied Sn-zeolites outstanding catalysts for the transformation of a wide variety of sugars into a limited range of commercially valuable alkyl lactates and derivatives. [K]Sn-β proved to be superior to [K]Sn-USY in terms of shape selectivity, exerting tight control on the distribution of produced α-hydroxy methyl esters. This shape selectivity was evident in the transformation of several complex sugar mixtures emulating different hemicelluloses-sugar cane bagasse, Scots pine, and white birch-that, despite showing very different sugar compositions, were almost exclusively converted into methyl lactate and methyl vinyl glycolate in very similar proportions. Moreover, the conversion of a real hemicellulose hydrolysate obtained from Scots pine through a simple GVL-based organosolv process confirmed the high activity and selectivity of [K]Sn-β in the studied transformation, opening new pathways for the chemical valorization of this plentiful, but underutilized, sugar feedstock.
PubMed: 38389903
DOI: 10.1021/acssuschemeng.3c07356