Dihydroxyacetone Kinase Research Articles

Improving an Alternative Glycerol Catabolism Pathway in Yarrowia lipolytica to Enhance Erythritol Production.

Engineering the glycerol-3-phosphate pathway could enhance erythritol production by accelerating glycerol uptake. However, little work has been conducted on the alternative dihydroxyacetone (DHA) pathway in Yarrowia lipolytica. Herein, this route was identified and characterized in Y. lipolytica by metabolomic and transcriptomic analysis. Moreover, the reaction catalyzed by dihydroxyacetone kinase encoded by dak2 was identified as the rate-limiting step. By combining NHEJ-mediated insertion mutagenesis with a push-and-pull strategy, Y. lipolytica strains with high-yield erythritol synthesis from glycerol were obtained. Screening of a library of insertion mutants allows the identification of a mutant with fourfold increased erythritol production. Overexpression of DAK2 and glycerol dehydrogenase GCY3 together with gene encoding transketolase and transaldolase from the nonoxidative part of the pentose phosphate pathway led to a strain with further increased productivity with a titer of 53.1 g/L and a yield 0.56 g/g glycerol, which were 8.1- and 4.2-fold of starting strain.

Ethanologenesis from glycerol by the gut acetogen Blautia schinkii.

The human gut is an anoxic environment that harbours a multitude of microorganisms that not only contribute to food digestion. The microbiome is also involved in malfunctions such as diseases, inflammation processes or development of obesity, but it is also involved in processes that increase the human well-being. Both, the good and the bad, are mediated by fermentation end products of bacterial metabolism, among others. However, despite a steadily growing knowledge of 'who lives out there', little in known of 'what do they do out there'. The genus Blautia is commonly found in the gut and associated with human well-being, but the exploration of their metabolic potential has just started. We demonstrate that B. schinkii grows on glycerol by producing acetate and ethanol. Transcriptome studies and biochemical analyses revealed a glycerol dehydrogenase and dihydroxyacetone kinase that funnel the substrate into glycolysis. Consequently, cells also grew on dihydroxyacetone. Cells could be adapted to grow at high (up to 1.5 M) glycerol concentrations but then only ethanol was formed. Ethanol production from glycerol is not only of relevance for the human host but also for potential bioindustrial production of bioethanol from waste glycerol.

Cohort effects in mild to moderate AD trials: an empirical assessment utilizing data from the Alzheimer’s disease cooperative study

AbstractBackgroundHeterogeneity in patient populations can impact clinical trial conclusions and generalizability. Long recruitment windows may exacerbate this in Alzheimer’s disease (AD) trials, which often recruit over extended periods.MethodWe used data from 8 Alzheimer’s Disease Cooperative Study (ADCS) clinical trials (DHA, Homocysteine, Huperzine, Intravenous Immunoglobulin, Resveratrol, Valproate, NSAIDs, and FYN Kinase inhibitor) in patients with mild to moderate AD to investigate cohort effects by time‐of‐trial‐enrollment.For each participant, we assigned a study‐ and site‐specific enrollment time defined as the number of days between a patient’s screening and the first screening visit at the site. We used study‐ and site‐specific time origins to account for calendar time differences between studies and, within a study, differences in site times to activation.To investigate differences in baseline characteristics, we split our sample into tertiles by enrollment time and compared sex, race and ethnicity, age, years of education, and baseline scores on the Alzheimer’s Disease Assessment Scale ‐ Cognitive Subscale (ADAS‐Cog).To assess cohort effects on trial outcomes, we used the 12‐month within‐subject change in ADAS‐Cog. We regressed the 12‐month change in ADAS‐Cog on a patient’s enrollment time while controlling for their baseline score and demographic information and allowing for clustering between patients in the same study.ResultGroups were demographically similar at baseline across enrollment tertiles. We found a significant difference in 12‐month change in ADAS‐Cog for patients with different enrollment times: later enrolled patients had an expected 12‐month change in ADAS‐Cog 0.146 points higher than similar patients who enrolled one quarter (91.2 days) earlier (95% CI: 0.082‐0.210). This magnitude of difference was roughly on par with an additional year of education. We also found a negative relationship with age, with older patients tending to experience a smaller 12‐month decline.ConclusionIn these 8 trials, we found minimal evidence of cohort effects that would meaningfully change study generalizability or treatment effect. Future analyses will account for the overall pace of accrual in the trials and assess for potential differential effects in fast vs. slow‐accruing studies.

Crystal structure of a nucleotide-binding domain of fatty acid kinase FakA from Thermus thermophilus HB8

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg2+, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.

Glycerol Utilization as a Sole Carbon Source Disrupts the Membrane Architecture and Solventogenesis in Clostridium beijerinckii NCIMB 8052

Efficient bioconversion of abundant waste glycerol to value-added chemicals calls for a wider range of fermentative workhorses that can catabolize glycerol. In this study, we used quantitative gene expression and solvent profiling, qualitative metabolite analysis, and enzyme activity assays to investigate the factors that limit glycerol utilization as a sole carbon source by Clostridium beijerinckii NCIMB 8052. C. beijerinckii NCIMB 8052 did not produce acetate, acetone and butanol on glycerol. Congruently, the genes encoding the coenzyme A transferase subunits (ctfAB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE) were down-regulated up to 135- and 21-fold, respectively, at 12 h in glycerol-grown cells compared to glucose-grown cells. Conversely, NADH-dependent butanol dehydrogenase A (bdhA) was upregulated 2-fold. Glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (subunit dhaK) were upregulated up to 5- and 881-fold, respectively. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) showed mostly similar expression profiles at 12 h on glucose and glycerol. At 24 h, gapdh was downregulated 1.5-fold, while NADP+-dependent gapdh was upregulated up to 1.9-fold. Glycerol-grown cells showed higher or similar activity profiles for all solventogenic enzymes studied, compared to glucose-grown cells. Butyraldehyde (3 g/L) supplementation led to the production of ~0.1 g/L butanol, whilst butyrate (3.5 g/L) supplementation produced 0.7 and 0.5 g/L acetone and butanol, respectively, with glycerol. Further, the long chain saturated fatty acids cyclopentaneundecanoic acid, methyl ester and hexadecanoic acid, butyl ester were detected in glucose- but not in glycerol-grown cells. Collectively, growth on glycerol appears to disrupt synthesis of saturated long chain fatty acids, as well as solventogenesis in C. beijerinckii NCIMB 8052.

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.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350rpm found to be optimal. In the fed-batch fermentation, 9.3g/L of 1,2-propanediol was produced after 144h of cultivation, and the substrate conversion ratio was 0.2g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3g/L of 1,2-propanediol was produced in fed-batch fermentation.

In silico and invivo analyses reveal key metabolic pathways enabling the fermentative utilization of glycerol in Escherichia coli.

SummaryMost microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2, complementing the previously reported role of 1,2‐propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol‐3‐phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase‐dependent H2 evolution. Despite the importance of the GK‐G3PDH route under alkaline conditions, we found that the NADH‐generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)‐dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system‐wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA‐DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose‐1,6‐bisphosphatases and the fructose phosphate bypass.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

DAK inhibits MDA5-mediated signaling in the antiviral innate immunity of black carp

Dihydroxyacetone kinase (DAK) functions as a negative regulator of melanoma differentiation-associated gene 5 (MDA5)-mediated interferon (IFN) production in human. To explore its role in teleost fish, DAK homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this paper. The transcription of black carp DAK (bcDAK) variated in host cells in response to LPS, poly (I:C) and virus stimulation, and bcDAK was majorly distributed in the cytoplasm. Overexpressed bcDAK in EPC cells showed little IFN promoter-inducing ability in the reporter assay and no antiviral activity in plaque assay. When co-expressed with black carp MDA5 (bcMDA5) in EPC cells, bcDAK obviously inhibited bcMDA5-mediated IFN promoter transcription in reporter assay and the antiviral activity in plaque assay. The knockdown of bcDAK enhanced the antiviral activity of the host cells. The association between bcDAK and bcMDA5 has been identified through immunofluorescent staining and co-immunoprecipitation (co-IP) assay. Thus, the data generated in this study support the conclusion that black carp DAK interacts with MDA5 and negatively regulates MDA5-mediated antiviral signaling.

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.

Three ATP-dependent phosphorylating enzymes in the first committed step of dihydroxyacetone metabolism in Gluconobacter thailandicus NBRC3255.

Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose-glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. KEY POINTS: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties.

Elevated aldolase 1A, retrogene 1 expression induces cardiac apoptosis in rat experimental autoimmune myocarditis model.

Acute myocarditis is an unpredictable heart disease that is caused by inflammation-associated cell death. Although viral infection and drug exposure are known to induce acute myocarditis, the molecular basis for its development remains undefined. Using proteomics and molecular analyses in myosin-induced rat experimental autoimmune myocarditis (EAM), we identified that elevated expression of aldolase 1A, retrogene 1 (Aldoart1) is critical to induce mitochondrial dysfunction and acute myocarditis development. Here, we demonstrate that cardiac cell death is associated with increased expressions of proapoptotic genes in addition to high levels of glucose, lactate, and triglyceride in metabolite profiling. The functional protein association network analysis also suggests that Aldoart1 upregulation correlates with high levels of dihydroxyacetone kinase and triglyceride. In H9c2 cardiac cells, lipopolysaccharides (LPS) or high glucose exposure significantly increases the cytochrome c release and the conversion of pro-caspase 3 into the cleaved form of caspase 3. We also found that LPS- or glucose-induced toxicities are almost completely reversed by siRNA-mediated knockdown of Aldoartl, which consequently increases cell viability. Together, our study strongly suggests that Aldoart1 may be involved in inducing mitochondrial apoptotic processes and can be a novel therapeutic target to prevent the onset of acute myocarditis or cardiac apoptosis.

Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli

Metabolic engineering of microorganisms for production of succinate from glycerol remains challenging. It was thought that energy supply is a severe problem for anaerobic fermentation of glycerol to produce succinate. In this study, an energy-conserving glycerol utilization pathway was recruited to improve anaerobic succinate production in Escherichia coli. ATP dependent dihydroxyacetone kinase (DhaK) from Klebsiella was used to replace the native phosphoenolpyruvate (PEP) dependent dihydroxyacetone kinase (DhaKLM) of E. coli so that 2 NADH, instead of 1 NADH and 1 menaquinol, can be produced from glycerol to PEP. Besides consumption of 1 NADH and 1 menaquinol for converting PEP to succinate, NADH dehydrogenase I transfers electrons from 1 NADH to 1 menaquinone and pumps 4 protons. Proton-motive force are generated as the additional energy to support cell growth and succinate export. After using this energy-conserving glycerol utilization pathway, succinate titer, productivity and intercellular ATP content increased 282%, 63% and 338% respectively. The best strain YY-GS004 produced 483 mM succinate in 96 h with a yield of 0.92 mol mol-1 glycerol under anaerobic conditions. The specific succinate productivity was 0.47 g g-1 (DCW) h-1, which was the highest one up to date for succinate production from glycerol. This study demonstrated that the energy-conserving glycerol utilization pathway GldA-DhaK can solve the energy problem during anaerobic fermentation of glycerol to produce succinate.

A mutation in the glycosyltransferase gene lafB causes daptomycin hypersusceptibility in Enterococcus faecium.

To verify dissemination of daptomycin-non-susceptible Enterococcus faecium in a hospital where daptomycin was not in use and to understand the evolutionary pathways connecting daptomycin hypersusceptibility to non-susceptibility. Clonality of 26 E. faecium was assessed by PFGE and the STs of these isolates were determined. The most daptomycin-susceptible isolate was evolved in vitro by stepwise daptomycin selection, generating isolates for genome comparisons. The spread of a high-risk daptomycin-non-susceptible VRE clone was detected, as was the occurrence of an unusual daptomycin-hypersusceptible strain (HBSJRP18). To determine the basis for daptomycin hypersusceptibility, we evolved HBSJRP18 in vitro and identified candidate genetic alterations potentially related to daptomycin susceptibility. Both lafB, encoding glycosyltransferase, which is putatively involved in lipoteichoic acid (LTA) biosynthesis, and dak, encoding a dihydroxyacetone kinase likely involved in fatty acid metabolism, were mutated in multiple independent experiments. Trans-complementation showed that the lafB polymorphism naturally occurring in HBSJRP18 caused its daptomycin hypersusceptibility. Fourier-transform infrared spectroscopy identified differences between the extracted LTA spectra from the hypersusceptible isolate and its revertant, as well as other non-susceptible variants, supporting a role for LafB in E. faecium LTA biosynthesis. Zeta potential difference was detected in one evolved dak mutant derivative. While much more susceptible to daptomycin, HBSJRP18 showed enhanced growth in the presence of piperacillin, suggesting that this, or another cell wall-targeting antibiotic, may have selected for the daptomycin-hypersusceptible phenotype. Our findings provide new information on the basis for daptomycin susceptibility in E. faecium, with implications for limiting the development and spread of daptomycin resistance.

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA++/dhaD1K++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K++ consumed more glycerol than hydA++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Biosynthesis of dendroketose from different carbon sources using in vitro and in vivo metabolic engineering strategies

BackgroundAsymmetric aldol-type C–C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential.ResultsThe detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi–Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource.ConclusionsThe detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.

Front Cover: Phosphorylation Catalyzed by Dihydroxyacetone Kinase (Eur. J. Org. Chem. 23/2018)

Front Cover: Phosphorylation Catalyzed by Dihydroxyacetone Kinase (Eur. J. Org. Chem. 23/2018)

Phosphorylation Catalyzed by Dihydroxyacetone Kinase

Site‐ and enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites. Biocatalytic phosphorylations catalyzed by recombinant dihydroxyacetone kinase beyond the dihydroxyacetone substrate have been investigated with quantitative 31P NMR spectroscopy using pyruvate‐kinase‐catalyzed ATP regeneration. A nearly 100 % conversion of d‐glyceraldehyde to d‐glyceraldehyde 3‐phosphate has been found. Interestingly, with pure l‐glyceraldehyde as substrate, practically no formation of l‐glyceraldehyde 3‐phosphate was observed.

Computational study of the phosphoryl donor activity of dihydroxyacetone kinase from ATP to inorganic polyphosphate

AbstractAdenosine triphosphate (ATP) is the main biological phosphoryl donor required in many enzymes including dihydroxyacetone kinases (DHAKs) that convert dihydroxyacetone (Dha) into dihydroxyacetone phosphate (Dha‐P), a key species with potential applications in synthesis. Herein, we present a theoretical study of the molecular mechanism for the phosphoryl transfer reaction from an inorganic polyphosphate to Dha catalyzed by DHAK from C. freundii. This is part of a project devoted to modify the phosphoryl donor specificity of this enzyme avoiding the use of the problematic direct addition of ATP. Based on the use of hybrid QM/MM potentials, with the QM region described by semiempirical and DFT methods, the reaction mechanism of the wild‐type enzyme and the most active experimentally measured mutant (Glu526Lys) with poly‐P as phosphoryl donor has been explored to elucidate the origin of the activity of this mutant. The similar energy barriers obtained in both systems confirm our previous studies on the binding step (Sánchez‐Moreno et al., Int. J. Mol. Sci. 2015, 16, 27835) suggesting that this mutation favors a more adequate position of the poly‐P in the active site for the following step, the chemical reaction, to take place.