Cofactor Concentrations Research Articles

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid.

The coffee industry produces over 10 billion kg beans per year and generates high amounts of different waste products. Spent coffee grounds (SCG) are an industrially underutilized waste resource, which is rich in the polysaccharide galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups. Here, we present a cell-free reaction cascade for the conversion of mannose, the most abundant sugar in SCG, into L-lactic acid. The enzymatic conversion is based on a so far unknown oxidative mannose metabolism from Thermoplasma acidophilum and uses a previously characterized mannonate dehydratase to convert mannose into lactic acid via 4 enzymatic reactions. In comparison to known in vivo metabolisms the bioconversion is free of phosphorylated intermediates and cofactors. Assessment of enzymes, adjustment of enzyme loadings, substrate and cofactor concentrations, and buffer ionic strength allowed the identification of crucial reaction parameters and bottlenecks. Moreover, reactions with isotope labeled mannose enabled the monitoring of pathway intermediates and revealed a reverse flux in the conversion process. Finally, 4.4 ± 0.1 mM lactic acid was produced from 14.57 ± 0.7 mM SCG-derived mannose. While the conversion efficiency of the process can be further improved by enzyme engineering, the reaction demonstrates the first multi-enzyme cascade for the bioconversion of SCG.

Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations

Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form.

Co-factor demand and regeneration in the enzymatic one-step reduction of carboxylates to aldehydes in cell-free systems

Addressing the challenges associated with the development of in vitro biocatalytic carboxylate reductions for potential applications, important aspects of the co-factor regeneration systems and strategies for minimizing over-reduction were investigated. The ATP recycling can be performed with similarly high efficiency exploiting the polyphosphate source by combining Meiothermus ruber polyphosphate kinase and adenylate kinase or with Sinorhizobium meliloti polyphosphate kinase instead of the latter. Carboxylate reductions with the enzyme candidates used in this work allow operating at co-factor concentrations of adenosine 5′-triphosphate and β-nicotinamide adenine dinucleotide 2′-phosphate of 100 μM and, thereby, reducing the amounts of alcohols formed by side activities in the enzyme preparations. This study confirmed the expected benefits of carboxylic acid reductases in chemoselectively reducing the carboxylates to the corresponding aldehydes while leaving reductively-sensitive nitro, ester and cyano groups intact.

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

In vitro production of cysteine from glucose.

Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.

Reversible photocontrol of oxidase activity by inserting a photosensitive domain into the oxidase

BackgroundPhotocontrol of protein activity has become a helpful strategy for regulating biological pathways. Herein, a method for the precise and reversible photocontrol of oxidase activity was developed by using the conformational change of the AsLOV2 domain.ResultsThe AsLOV2 domain was inserted into the nonconserved sites exposed on the surface of the AdhP protein, and the alov9 fusion was successfully screened for subsequent optical experiments under the assumption that neither of these actions affected the original activity of AdhP protein. The activity of alov9 was noticeably inhibited when the fusion was exposed to 470 nm blue light and recovered within 30 min. As a result, we could precisely and reversibly photocontrol alov9 activity through the optimization of several parameters, including cofactor concentration, light intensity, and illumination time.ConclusionsAn efficient method was developed for the photoinhibition of enzymatic activity based on the insertion of the light-sensitive AsLOV2 domain, providing new ideas for photocontrolling metabolic pathways without carriers in the future.

Interlinked GTPase cascades provide a motif for both robust switches and oscillators.

GTPases regulate a wide range of cellular processes, such as intracellular vesicular transport, signal transduction and protein translation. These hydrolase enzymes operate as biochemical switches by toggling between an active guanosine triphosphate (GTP)-bound state and an inactive guanosine diphosphate (GDP)-bound state. We compare two network motifs, a single-species switch and an interlinked cascade that consists of two species coupled through positive and negative feedback loops. We find that interlinked cascades are closer to the ideal all-or-none switch and are more robust against fluctuating signals. While the single-species switch can only achieve bistability, interlinked cascades can be converted into oscillators by tuning the cofactor concentrations, which catalyse the activity of the cascade. These regimes can only be achieved with sufficient chemical driving provided by GTP hydrolysis. In this study, we present a thermodynamically consistent model that can achieve bistability and oscillations with the same feedback motif.

In-Vitro and In-Silico Characterization of Xylose Reductase from Emericella nidulans

Background: Lignocellulosic residues generated by various anthropogenic activities can be a potential raw material for many commercial products such as biofuels, organic acids and nutraceuticals including xylitol. Xylitol is a low-calorie nutritive sweetener for diabetic patients. Microbial production of xylitol can be helpful in overcoming the drawbacks of traditional chemical production process and lowring cost of production. Objective: Designing efficient production process needs the characterization of required enzyme/s. Hence current work was focused on in-vitro and in-silico characterization of xylose reductase from Emericella nidulans. Methods: Xylose reductase from one of the hyper-producer isolates, Emericella nidulans Xlt-11 was used for in-vitro characterization. For in-silico characterization, XR sequence (Accession No: Q5BGA7) was used. Results: Xylose reductase from various microorganisms has been studied but the quest for better enzymes, their stability at higher temperature and pH still continues. Xylose reductase from Emericella nidulans Xlt-11 was found NADH dependent and utilizes xylose as its sole substrate for xylitol production. In comparison to whole cells, enzyme exhibited higher enzyme activity at lower cofactor concentration and could tolerate higher substrate concentration. Thermal deactivation profile showed that whole cell catalysts were more stable than enzyme at higher temperature. In-silico analysis of XR sequence from Emericella nidulans (Accession No: Q5BGA7) suggested that the structure was dominated by random coiling. Enzyme sequences have conserved active site with net negative charge and PI value in acidic pH range. Conclusion: Current investigation supported the enzyme’s specific application i.e. bioconversion of xylose to xylitol due to its higher selectivity. In-silico analysis may provide significant structural and physiological information for modifications and improved stability.

Investigating the enzymatic CO2 reduction to formate with electrochemical NADH regeneration in batch and semi-continuous operations

The enzymatic production of formate from CO2 with the immobilized NADH-dependent formate dehydrogenase (FDH) on the activated electrospun polystyrene nanofibers (EPSNF) was investigated to develop a sustainable process for CO2 reduction. Direct electrochemical regeneration on a Cu foam electrode was employed to supply the reaction with the reduced cofactor (NADH). The formate production was studied in two modes of batch and semi-continuous operations with the cofactor recycle. Results indicated that the regenerated cofactor concentrations in both systems were nearly identical (0.5 mM) which ensured the desirable activity of the immobilized enzyme. This showed that the electrochemical regeneration system was effective even in the semi-continuous operation. Although the cumulative formate concentration in the batch operation was higher, the total amounts of the produced formate were higher for the semi-continuous mode for more than 42% which was justified by the fact that the lower formate concentration in the semi-continuous mode would be favorable to the progress of the enzymatic CO2 to formate conversion. Finally, it was concluded that the proposed semi-continuous process in this work could be considered as a promising process for the enzymatic CO2 conversion.

Biochemical Characterization of Heat-Tolerant Recombinant L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in D-Tagatose Production.

D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50°C, pH 5.5, and 0.3mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45°C and 50°C were 1427h and 11h, respectively, and 21.5h and 39.5h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50°C after 48h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.

Pareto Optimality Explanation of the Glycolytic Alternatives in Nature

The Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) glycolytic pathways are largely conserved across glycolytic species in nature. Is this a coincidence, convergent evolution or there exists a driving force towards either of the two pathway designs? We addressed this question by first employing a variant of the optStoic algorithm to exhaustively identify over 11,916 possible routes between glucose and pyruvate at different pre-determined stoichiometric yields of ATP. Subsequently, we analyzed the thermodynamic feasibility of all the pathways at physiological metabolite concentrations and quantified the protein cost of the feasible solutions. Pareto optimality analysis between energy efficiency and protein cost reveals that the naturally evolved ED and EMP pathways are indeed among the most protein cost-efficient pathways in their respective ATP yield categories and remain thermodynamically feasible across a wide range of ATP/ADP ratios and pathway intermediate metabolite concentration ranges. In contrast, pathways with higher ATP yield (>2) while feasible, are bound within stringent and often extreme operability ranges of cofactor and intermediate metabolite concentrations. The preponderance of EMP and ED is thus consistent with not only optimally balancing energy yield vs. enzyme cost but also with ensuring operability for wide metabolite concentration ranges and ATP/ADP ratios.

Improving the production of isoprene and 1,3-propanediol by metabolically engineered Escherichia coli through recycling redox cofactor between the dual pathways.

The biosynthesis of isoprene by microorganisms is a promising green route. However, the yield of isoprene is limited due to the generation of excess NAD(P)H via the mevalonate (MVA) pathway, which converts more glucose into CO2 or undesired reduced by-products. The production of 1,3-propanediol (1,3-PDO) from glycerol is a typical NAD(P)H-consuming process, which restricts 1,3-PDO yield to ~ 0.7mol/mol. In this study, we propose a strategy of redox cofactor balance by coupling the production of isoprene with 1,3-PDO fermentation. With the introduction and optimization of the dual pathways in an engineered Escherichia coli, ~ 85.2% of the excess NADPH from isoprene pathway was recycled for 1,3-PDO production. The best strain G05 simultaneously produced 665.2mg/L isoprene and 2532.1mg/L 1,3-PDO under flask fermentation conditions. The yields were 0.3mol/mol glucose and 1.0mol/mol glycerol, respectively, showing 3.3- and 4.3-fold improvements relative to either pathway independently. Since isoprene is a volatile organic compound (VOC) whereas 1,3-PDO is separated from the fermentation broth, their coproduction process does not increase the complexity or cost for the separation from each other. Hence, the presented strategy will be especially useful for developing efficient biocatalysts for other biofuels and biochemicals, which are driven by cofactor concentrations.

Highly efficient production of chiral amines in batch and continuous flow by immobilized ω-transaminases on controlled porosity glass metal-ion affinity carrier

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100 mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG3-AsR-ωTA could be recycled for at least 16 consecutive cycles (15 min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG3-AsR-ωTA was tested in a continuous flow packed-bed reactor (157 μL reactor volume), which produced more than 5 g of (S)-α-MBA (>49% conversion, >99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L−1 h−1.

Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element

Riboswitches modulate gene expression in response to small-molecule ligands. Switching is generally thought to occur via the stabilization of a specific RNA structure conferred by binding the cognate ligand. However, it is unclear whether any such stabilization occurs for riboswitches whose ligands also play functional roles, such as the glmS ribozyme riboswitch, which undergoes self-cleavage using its regulatory ligand, glucosamine 6-phosphate, as a catalytic cofactor. To address this question, it is necessary to determine both the conformational ensemble and its ligand dependence. We used optical tweezers to measure folding dynamics and cleavage rates for the core glmS ribozyme over a range of forces and ligand conditions. We found that the folding of a specific structural element, the P2.2 duplex, controls active-site formation and catalysis. However, the folded state is only weakly stable, regardless of cofactor concentration, supplying a clear exception to the ligand-based stabilization model of riboswitch function.

Noncanonical RNA-capping: Discovery, mechanism, and physiological role debate.

Recently a new type of 5'-RNA cap was discovered. In contrast to the specialized eukaryotic m7 G cap, the novel caps are abundant cellular cofactors like NAD+ . RNAs capped with cofactors are found in prokaryotes and eukaryotes. Unlike m7 G cap, installed by specialized enzymes, cofactors are attached by main enzyme of transcription, RNA polymerase (RNAP). Cofactors act as noncanonical initiating substrates, provided cofactor's nucleoside base-pairs with template DNA at the transcription start site. Adenosine-containing NAD(H), flavin adenine dinucleotide (FAD), and CoA modify transcripts on promoters starting with +1A. Similarly, uridine-containing cell wall precursors, for example, uridine diphosphate-N-acetylglucosamine were shown to cap RNA in vitro on +1U promoters. Noncanonical capping is a universal feature of evolutionary unrelated RNAPs-multisubunit bacterial and eukaryotic RNAPs, and single-subunit mitochondrial RNAP. Cellular concentrations of cofactors, for example, NAD(H) are significantly higher than their Km in transcription. Yet, only a small proportion of a given cellular RNA is noncanonically capped (if at all). This proportion is a net balance between capping, seemingly stochastic, and decapping, possibly determined by RNA folding, protein binding and transcription rate. NUDIX hydrolases in bacteria and eukaryotes, and DXO family proteins eukaryotes act as decapping enzymes for noncanonical caps. The physiological role of noncanonical RNA capping is only starting to emerge. It was demonstrated to affect RNA stability in vivo in bacteria and eukaryotes and to stimulate RNAP promoter escape in vitro in Escherichia coli. NAD+ /NADH capping ratio may connect transcription to cellular redox state. Potentially, noncanonical capping affects mRNA translation, RNA-protein binding and RNA localization. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry.

Enzymatic CO2 reduction to formate by formate dehydrogenase from Candida boidinii coupling with direct electrochemical regeneration of NADH

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Identification of steroidal derivatives inhibiting the transformations of allopregnanolone and estradiol by 17β-hydroxysteroid dehydrogenase type 10

17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a mitochondrial enzyme known for its potential role in Alzheimer’s Disease (AD). 17β-HSD10, by its oxidative activity, could decrease the concentration of two important neurosteroids, allopregnanolone (ALLOP) and 17β-estradiol (E2), respectively preventing their neurogenesis and neuroprotective effects. Since the inhibition of 17β-HSD10 could lead to a new treatment for AD, we developed two biological assays using labeled ALLOP or E2 as substrates to measure the inhibitory activity of compounds against pure 17β-HSD10 protein. After the optimization of different parameters (time, concentration of enzyme, substrate and cofactor), analogs of the first reported steroidal inhibitor of 17β-HSD10 in intact cells were screened to determine their inhibitory potency for the ALLOP or the E2 oxidation. One compound, androstane derivative 5, possesses the best dual inhibition against both transformations (ALLOP, IC50 = 235 μM and E2, IC50 = 610 μM). Some compounds are dual inhibitors to a lesser extent, and others seem selective for one of the transformations in particular. By developing two reliable assays and by identifying a first generation of steroidal inhibitors of pure 17β-HSD10, this preliminary study opens the door to new and more potent inhibitors.

Sinorhizobium meliloti RNase III: Catalytic Features and Impact on Symbiosis.

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its α–proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg2+ as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants.

Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine

In spite of huge endeavors in cell line engineering to produce glycoproteins with desired and uniform glycoforms, it is still not possible in vivo. Alternatively, in vitro glycoengineering can be used for the modification of glycans. However, in vitro glycoengineering relies on expensive nucleotide sugars, such as uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) which serves as GlcNAc donor for the synthesis of various glycans. In this work, we present a systematic study for the cell-free de novo synthesis and regeneration of UDP-GlcNAc from polyphosphate, UMP and GlcNAc by a cascade of five enzymes (N–acetylhexosamine kinase (NahK), Glc–1P uridyltransferase (GalU), uridine monophosphate kinase (URA6), polyphosphate kinase (PPK3), and inorganic diphosphatase (PmPpA). All enzymes were expressed in E. coli BL21 Gold (DE3) and purified using immobilized metal affinity chromatography (IMAC). Results from one-pot experiments demonstrate the successful production of UDP-GlcNAc with a yield approaching 100%. The highest volumetric productivity of the cascade was about 0.81 g L−1 h−1 of UDP-GlcNAc. A simple model based on mass action kinetics was sufficient to capture the dynamic behavior of the multienzyme pathway. Moreover, a design equation based on metabolic control analysis was established to investigate the effect of enzyme concentration on the UDP-GlcNAc flux and to demonstrate that the flux of UDP-GlcNAc can be controlled by means of the enzyme concentrations. The effect of temperature on the UDP-GlcNAc flux followed an Arrhenius equation and the optimal co-factor concentration (Mg2+) for high UDP-GlcNAc synthesis rates depended on the working temperature. In conclusion, the study covers the entire engineering process of a multienzyme cascade, i.e. pathway design, enzyme expression, enzyme purification, reaction kinetics and investigation of the influence of basic parameters (temperature, co-factor concentration, enzyme concentration) on the synthesis rate. Thus, the study lays the foundation for future cascade optimization, preparative scale UDP-GlcNAc synthesis and for in situ coupling of the network with UDP-GlcNAc transferases to efficiently regenerate UDP-GlcNAc. Hence, this study provides a further step towards cost-effective in vitro glycoengineering of antibodies and other glycosylated proteins.

Production of glutaric acid from 5-aminovaleric acid using Escherichia coli whole cell bio-catalyst overexpressing GabTD from Bacillus subtilis

Glutaric acid is one of the promising C5 platform compounds in the biochemical industry. It can be produced chemically, through the ring-opening of butyrolactone followed by hydrolysis. Alternatively, glutaric acid can be produced via lysine degradation pathways by microorganisms. In microorganisms, the overexpression of enzymes involved in this pathway from E. coli and C. glutamicum has resulted in high accumulation of 5-aminovaleric acid. However, the conversion from 5-aminovaleric acid to glutaric acid has resulted in a relatively low conversion yield for unknown reasons. In this study, as a solution to improve the production of glutaric acid, we introduced gabTD genes from B. subtilis to E. coli for a whole cell biocatalytic approach. This approach enabled us to determine the effect of co-factors on reaction and to achieve a high conversion yield from 5-aminovaleric acid at the optimized reaction condition. Optimization of whole cell reaction by different plasmids, pH, temperature, substrate concentration, and cofactor concentration achieved full conversion with 100 mM of 5-aminovaleric acid to glutaric acid. Nicotinamide adenine dinucleotide phosphate (NAD(P)+) and α-ketoglutaric acid were found to be critical factors in the enhancement of conversion in selected conditions. Whole cell reaction with a higher concentration of substrates gave 141 mM of glutaric acid from 300 mM 5-aminovaleric acid, 150 mM α-ketoglutaric acid, and 60 mM NAD+ at 30 °C, with a pH of 8.5 within 24 h (47.1% and 94.2% of conversion based on 5-aminovaleric acid and α-ketoglutaric acid, respectively). The whole cell biocatalyst was recycled 5 times with the addition of substrates; this enabled the accumulation of extra glutaric acid.