Optimizing enzyme properties to enhance dihydroxyacetone production via methylglyoxal biosensor development.

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.

Continuous catalytic production of 1,3-dihydroxyacetone: Sustainable approach combining perfusion cultures and immobilized cells.

Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L-1, it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L-1·h-1, representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses.

A tipping point in dihydroxyacetone exposure: mitochondrial stress and metabolic reprogramming alter survival in rat cardiomyocytes H9c2 cells.

Exogenous exposures to the triose sugar dihydroxyacetone (DHA) occur from sunless tanning products and electronic cigarette aerosol. Once inhaled or absorbed, DHA enters cells, is converted to dihydroxyacetone phosphate (DHAP), and incorporated into several metabolic pathways. Cytotoxic effects of DHA vary across the cell types depending on the metabolic needs of the cells, and differences in the generation of reactive oxygen species (ROS), cell cycle arrest, and mitochondrial dysfunction have been reported. We have shown that cytotoxic doses of DHA induced metabolic imbalances in glycolysis and oxidative phosphorylation in liver and kidney cell models. Here, we examine the dose-dependent effects of DHA on the rat cardiomyocyte cell line, H9c2. Cells begin to experience cytotoxic effects at low millimolar doses, but an increase in cell survival was observed at 2 mM DHA. We confirmed that 2 mM DHA increased cell survival compared to the low cytotoxic 1 mM dose and investigated the metabolic differences between these two low DHA doses. Exposure to 1 mM DHA showed changes in the cell's fuel utilization, mitochondrial reactive oxygen species (ROS), and transient changes in the glycolysis and mitochondrial energetics, which normalized 24 h after exposure. The 2 mM dose induced robust changes in mitochondrial flux through acetyl CoA and elevated expression of fatty acid synthase. Distinct from the 1 mM dose, the 2 mM exposure increased mitochondrial ROS and NAD(P)H levels, and sustained changes in LDHA/LDHB and acetyl CoA-associated enzymes were observed. Although the cells were exposed to low cytotoxic (1 mM) and non-cytotoxic (2 mM) acute doses of DHA, significant changes in mitochondrial metabolic pathways occurred. Further, the proliferation increase at the acute 2 mM DHA dose suggests a metabolic adaption occurred with sustained consequences in survival and proliferation. With increased exogenous exposure to DHA through e-cigarette aerosol, this work suggests cell metabolic changes induced by acute or potentially chronic exposures could impact cell function and survival.

A phosphatase gene is linked to nectar dihydroxyacetone accumulation in manuka (Leptospermum scoparium).

Floral nectar composition beyond common sugars shows great diversity but contributing genetic factors are generally unknown. Manuka (Leptospermum scoparium) is renowned for the antimicrobial compound methylglyoxal in its derived honey, which originates from the precursor, dihydroxyacetone (DHA), accumulating in the nectar. Although this nectar trait is highly variable, genetic contribution to the trait is unclear. Therefore, we investigated key gene(s) and genomic regions underpinning this trait. We used RNAseq analysis to identify nectary-associated genes differentially expressed between high and low nectar DHA genotypes. We also used a manuka high-density linkage map and quantitative trait loci (QTL) mapping population, supported by an improved genome assembly, to reveal genetic regions associated with nectar DHA content. Expression and QTL analyses both pointed to the involvement of a phosphatase gene, LsSgpp2. The expression pattern of LsSgpp2 correlated with nectar DHA accumulation, and it co-located with a QTL on chromosome 4. The identification of three QTLs, some of the first reported for a plant nectar trait, indicates polygenic control of DHA content. We have established plant genetics as a key influence on DHA accumulation. The data suggest the hypothesis of LsSGPP2 releasing DHA from DHA-phosphate and variability in LsSgpp2 gene expression contributing to the trait variability.

Metabolomic exhibits different profiles and potential biomarkers of Vitis spp. co-cultivated with Fusarium oxysporum for short, medium, and long times.

Differential rootstock tolerance to Fusarium spp. supports viticulture worldwide. However, how plants stand against the fungus still needs to be explored. We hypothesize it involves a differential metabolite modulation. Thus, we performed a gas chromatography coupled with mass spectrometry (GC-MS) analysis of Paulsen P1103 and BDMG573 rootstocks, co-cultured with Fusarium oxysporum (FUS) for short, medium, and long time (0, 4, and 8 days after treatment [DAT]). In shoots, principal component analysis (PCA) showed a complete overlap between BDMG573 non-co-cultivated and FUS at 0 DAT, and P1103 treatments showed a slight overlap at both 4 and 8 DAT. In roots, PCA exhibited overlapping between BDMG573 treatments at 0 DAT, while P1103 treatments showed overlapping at 0 and 4 DAT. Further, there is a complete overlapping between BDMG573 and P1103 FUS profiles at 8 DAT. In shoots, 1,3-dihydroxyacetone at 0 and 4 DAT and maltose at 4 and 8 DAT were biomarkers for BDMG573. For P1103, glyceric acid, proline, and sorbitol stood out at 0, 4, and 8 DAT, respectively. In BDMG573 roots, the biomarkers were ß-alanine at 0 DAT, cellobiose and sorbitol at both 4 and 8 DAT. While in P1103 roots, they were galactose at 0 and 4 DAT and 1,3-dihydroxyacetone at 8 DAT. Overall, there is an increase in amino acids, glycolysis, and tricarboxylic acid components in tolerant Paulsen P1103 shoots. Thus, it provides a new perspective on the primary metabolism of grapevine rootstocks to F. oxysporum that may contribute to strategies for genotype tolerance and early disease identification.

Selective Photoelectrocatalytic Glycerol Oxidation to Dihydroxyacetone via Enhanced Middle Hydroxyl Adsorption over a Bi2O3-Incorporated Catalyst.

Photoelectrocatalytic (PEC) glycerol oxidation offers a sustainable approach to produce dihydroxyacetone (DHA) as a valuable chemical, which can find use in cosmetic, pharmaceutical industries, etc. However, it still suffers from the low selectivity (≤60%) that substantially limits the application. Here, we report the PEC oxidation of glycerol to DHA with a selectivity of 75.4% over a heterogeneous photoanode of Bi2O3 nanoparticles on TiO2 nanorod arrays (Bi2O3/TiO2). The selectivity of DHA can be maintained at ∼65% under a relatively high conversion of glycerol (∼50%). The existing p-n junction between Bi2O3 and TiO2 promotes charge transfer and thus guarantees high photocurrent density. Experimental combined with theoretical studies reveal that Bi2O3 prefers to interact with the middle hydroxyl of glycerol that facilitates the selective oxidation of glycerol to DHA. Comprehensive reaction mechanism studies suggest that the reaction follows two parallel pathways, including electrophilic OH* (major) and lattice oxygen (minor) oxidations. Finally, we designed a self-powered PEC system, achieving a DHA productivity of 1.04 mg cm-2 h-1 with >70% selectivity and a H2 productivity of 0.32 mL cm-2 h-1. This work may shed light on the potential of PEC strategy for biomass valorization toward value-added products via PEC anode surface engineering.

The first in-depth exploration of the genome of the engineered bacterium, Gluconobacter thailandicus.

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.

Identification and biochemical characterization of an ATP-dependent dihydroxyacetone kinase from Trypanosoma cruzi.

Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production.

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.

Analysis of glycerol and dihydroxyacetone metabolism in Enterococcus faecium.

Glycerol (Gly) can be dissimilated by two pathways in bacteria. Either this sugar alcohol is first oxidized to dihydroxyacetone (DHA) and then phosphorylated or it is first phosphorylated to glycerol-3-phosphate (GlyP) followed by oxidation. Oxidation of GlyP can be achieved by NAD-dependent dehydrogenases or by a GlyP oxidase. In both cases, dihydroxyacetone phosphate is the product. Genomic analysis showed that Enterococcus faecium harbors numerous genes annotated to encode activities for the two pathways. However, our physiological analyses of growth on glycerol showed that dissimilation is limited to aerobic conditions and that despite the presence of genes encoding presumed GlyP dehydrogenases, the GlyP oxidase is essential in this process. Although E. faecium contains an operon encoding the phosphotransfer protein DhaM and DHA kinase, which are required for DHA phosphorylation, it is unable to grow on DHA. This operon is highly expressed in stationary phase but its physiological role remains unknown. Finally, data obtained from sequencing of a transposon mutant bank of E. faecium grown on BHI revealed that the GlyP dehydrogenases and a major intrinsic family protein have important but hitherto unknown physiological functions.

Exogenous exposure to dihydroxyacetone mimics high fructose induced oxidative stress and mitochondrial dysfunction.

Dihydroxyacetone (DHA) is a three-carbon sugar that is the active ingredient in sunless tanning products and a by-product of electronic cigarette (e-cigarette) combustion. Increased use of sunless tanning products and e-cigarettes has elevated exposures to DHA through inhalation and absorption. Studies have confirmed that DHA is rapidly absorbed into cells and can enter into metabolic pathways following phosphorylation to dihydroxyacetone phosphate (DHAP), a product of fructose metabolism. Recent reports have suggested metabolic imbalance and cellular stress results from DHA exposures. However, the impact of elevated exposure to DHA on human health is currently under-investigated. We propose that exogenous exposures to DHA increase DHAP levels in cells and mimic fructose exposures to produce oxidative stress, mitochondrial dysfunction, and gene and protein expression changes. Here, we review cell line and animal model exposures to fructose to highlight similarities in the effects produced by exogenous exposures to DHA. Given the long-term health consequences of fructose exposure, this review emphasizes the pressing need to further examine DHA exposures from sunless tanning products and e-cigarettes.

Dihydroxyacetone phosphate signals glucose availability to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.

Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration.

Cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD+ in enzyme-catalyzed reactions. Herein, glycerol dehydrogenase (GDH) and NADH oxidase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for effective regeneration of NAD+. After optimization, the activity of combi-CLEAs and separate CLEAs mixtures were 950 and 580 U/g, respectively. And the catalytic stability of combi-CLEAs against pH and temperature was superior to the free enzyme mixture. After ten cycles of reuse, the catalytic efficiency could still retain 63.3% of its initial activity, indicating that the constructed combi-CLEAs system had excellent reusability. Also, the conversion of glycerol to 1,3-dihydroxyacetone (DHA) was improved by the constructed NAD+ regeneration system, resulting in 4.6%, which was 2.5 times of the free enzyme system. Thus, wide applications of this co-immobilization method in the production of various chiral chemicals could be expected in the industry for its high efficiency at a low cost.

Efficient glycerol transformation by resting Gluconobacter cells.

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.

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli.

In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA.IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

Metformin inhibits gluconeogenesis via a redox-dependent mechanism in vivo.

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products.

Methane can be converted to triose dihydroxyacetone (DHA) by chemical processes with formaldehyde as an intermediate. Carbon dioxide, a by-product of various industries including ethanol/butanol biorefineries, can also be converted to formaldehyde and then to DHA. DHA, upon entry into a cell and phosphorylation to DHA-3-phosphate, enters the glycolytic pathway and can be fermented to any one of several products. However, DHA is inhibitory to microbes due to its chemical interaction with cellular components. Fermentation of DHA to d-lactate by Escherichia coli strain TG113 was inefficient, and growth was inhibited by 30 g⋅L-1 DHA. An ATP-dependent DHA kinase from Klebsiella oxytoca (pDC117d) permitted growth of strain TG113 in a medium with 30 g⋅L-1 DHA, and in a fed-batch fermentation the d-lactate titer of TG113(pDC117d) was 580 ± 21 mM at a yield of 0.92 g⋅g-1 DHA fermented. Klebsiella variicola strain LW225, with a higher glucose flux than E. coli, produced 811 ± 26 mM d-lactic acid at an average volumetric productivity of 2.0 g-1⋅L-1⋅h-1 Fermentation of DHA required a balance between transport of the triose and utilization by the microorganism. Using other engineered E. coli strains, we also fermented DHA to succinic acid and ethanol, demonstrating the potential of converting CH4 and CO2 to value-added chemicals and fuels by a combination of chemical/biological processes.

Low-cost biotransformation of glycerol to 1,3-dihydroxyacetone through Gluconobacter frateurii in medium with inorganic salts only.

Existing dihydroxyacetone (DHA) production practices require the use of yeast extracts, leading to relatively high production costs. This study explores the use of low-cost media comprising glycerol, inorganic salts and Gluconobacter frateurii BCC 36199 in the production of DHA. The medium components are also quantitatively optimized. Regression models describing the linear correlations between the nutrient concentrations and the generated DHA concentration (p), and between the nutrient concentrations and the yield (ysp ) are developed. Under the optimal conditions according to our regression models, the highest values for p and ysp are 29·36 g l-1 and 97·86% g g-1 respectively. Quantitatively, this study shows positive effects of inorganic salts and adverse effects of excessive amounts of glycerol on DHA production. In particular, the results suggest that low levels of biomass production lead to high levels of DHA production. Consequently, the media containing inorganic nitrogen source from (NH4 )2 SO4 lead to higher yields than organic media containing yeast extract. This study has identified an optimal, low-cost, minimal medium that can effectively enhance DHA production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates the advantages of inorganic nutrients supplementation over organic nutrient supplementation for a lower media cost and a higher dihydroxyacetone (DHA) production yield through Gluconobacter frateurii BCC 36199 cultivation. The study found that the use of media that contain only glycerol and inorganic salts enhanced DHA production (DHA-Prod) while keeping the production of bacterial biomass at a sufficient level. Most of the starting material, that is, glycerol, is converted into DHA, which is the target of the production process. The cost of the nitrogen supplement in the DHA-Prod process may be reduced by up to 80% through the use of the inorganic culture medium that has been developed in this study.

Sensitivity enhancement for detection of hyperpolarized 13 C MRI probes with 1 H spin coupling introduced by enzymatic transformation in vivo.

PURPOSE: Although 1 H spin coupling is generally avoided in probes for hyperpolarized (HP) 13 C MRI, enzymatic transformations of biological interest can introduce large 13 C-1 H couplings in vivo. The purpose of this study was to develop and investigate the application of 1 H decoupling for enhancing the sensitivity for detection of affected HP 13 C metabolic products. METHODS: A standalone 1 H decoupler system and custom concentric 13 C/1 H paddle coil setup were integrated with a clinical 3T MRI scanner for in vivo 13 C MR studies using HP [2-13 C]dihydroxyacetone, a novel sensor of hepatic energy status. Major 13 C-1 H coupling JCH = ∼150 Hz) is introduced after adenosine triphosphate-dependent enzymatic transformation of HP [2-13 C]dihydroxyacetone to [2-13 C]glycerol-3-phosphate in vivo. Application of WALTZ-16 1 H decoupling for elimination of large 13 C-1 H couplings was first tested in thermally polarized glycerol phantoms and then for in vivo HP MR studies in three rats, scanned both with and without decoupling. RESULTS: As configured, 1 H-decoupled 13 C MR of thermally polarized glycerol and the HP metabolic product [2-13 C]glycerol-3-phosphate was achieved at forward power of approximately 15 W. High-quality 3-s dynamic in vivo HP 13 C MR scans were acquired with decoupling duty cycle of 5%. Application of 1 H decoupling resulted in sensitivity enhancement of 1.7-fold for detection of metabolic conversion of [2-13 C]dihydroxyacetone to HP [2-13 C]glycerol-3-phosphate in vivo. CONCLUSIONS: Application of 1 H decoupling provides significant sensitivity enhancement for detection of HP 13 C metabolic products with large 1 H spin couplings, and is therefore expected to be useful for preclinical and potentially clinical HP 13 C MR studies. Magn Reson Med 80:36-41, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli.

Protein kinases, representing one of the largest protein families involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this article an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine, or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM, or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein, such as Gly78B, Thr79A, Ser80A, Arg178B, and one Mg2+ cation, would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rational design of new more specific inhibitors.