NADPH Regeneration Research Articles

Copper oxide-based cathode for direct NADPH regeneration

Nearly a fourth of all enzymatic activities is attributable to oxidoreductases, and the redox reactions supported by this vast catalytic repertoire sustain cellular metabolism. In many biological processes, reduction depends on hydride transfer from either reduced nicotinamide adenine dinucleotide (NADH) or its phosphorylated derivative (NADPH). Despite longstanding efforts to regenerate NADPH by various methods and harness it to support chemoenzymatic synthesis strategies, the lack of product purity has been a major deterrent. Here, we demonstrate that a nanostructured heterolayer Ni–Cu2O–Cu cathode formed by a photoelectrochemical process has unexpected efficiency in direct electrochemical regeneration of NADPH from NADP+. Remarkably, two-thirds of NADP+ was converted to NADPH with no measurable production of the inactive (NADP)2 dimer and at the lowest reported overpotential [− 0.75 V versus Ag/AgCl (3 M NaCl) reference]. Sputtering of nickel on the copper-oxide electrode nucleated an unexpected surface morphology that was critical for high product selectivity. Our results should motivate design of integrated electrolyzer platforms that deploy this heterogeneous catalyst for direct electrochemical regeneration of NADH/NADPH, which is central to design of next-generation biofuel fermentation strategies, biological solar converters, energy-storage devices, and artificial photosynthesis.

Targeting NRF2 to suppress ferroptosis in brain injury.

Brain injury is accompanied by serious iron metabolism disorder and oxidative stress. As a novel form of regulated cell death (RCD) depending on lipid peroxidation caused by iron overload, ferroptosis (FPT) further aggravates brain injury, which is different from apoptosis, autophagy and other traditional cell death in terms of biochemistry, morphology and genetics. Noteworthy, transcriptional regulator NRF2 plays a key role in the cell antioxidant system, and many genes related to FPT are under the control of NRF2, including genes for iron regulation, thiol-dependent antioxidant system, enzymatic detoxification of RCS and carbonyls, NADPH regeneration and ROS sources from mitochondria or extra-mitochondria, which place NRF2 in the key position of regulating the ferroptotic death. Importantly, NRF2 can reduce iron load and resist FPT. In the future, it is expected to open up a new way to treat brain injury by targeting NRF2 to alleviate FPT in brain.

Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the “best” variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose Km, kcat/Km towards (5R)-1 was 0.66 mM and 210.77 s−1 mM−1, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L-1Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L-1Variant VI completely reduced (5R)-1 of up to 450 g L−1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.p) with a space–time yield (STY) of 1.24 kg L−1 day−1, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1Variant VI completely reduced 400 g L−1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.p) with a STY of 1.34 kg L−1 day−1. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery.

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

In Vivo Selection for Formate Dehydrogenases with High Efficiency and Specificity toward NADP.

The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usually specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity toward NADP+, and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

FoxM1-dependent and fatty acid oxidation-mediated ROS modulation is a cell-intrinsic drug resistance mechanism in cancer stem-like cells

Increased oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) levels are inherently linked. ROS are essential signaling molecules, with detrimental effects when produced in excess during chemotherapy, leading to cell death. Cancer stem-like cells (CSCs) are a subpopulation of tumor cells resistant to chemotherapy, highly invasive and metastagenic, driving malignant cancer behavior. In this study, we demonstrated that CSCs exhibit increased OXPHOS but paradoxically low ROS levels. Considering the detrimental effects of large amounts of ROS, CSCs have developed potential mechanisms for quenching excess ROS to maintain redox homeostasis. We aimed to investigate the distinct metabolic features and mechanisms of ROS regulation in gastric CSCs and explore potential therapeutic strategies targeting CSCs. Human gastric cancer cell lines, AGS and MKN1, were subjected to liquid chromatography/mass spectrometry-based metabolomic and microarray analyses. Mitochondrial properties such as mitochondrial mass, membrane potential, and ROS were assessed by flow cytometric analysis. CSCs with increased OXPHOS levels maintained low ROS levels by coupling FoxM1-dependent Prx3 expression and fatty acid oxidation-mediated NADPH regeneration. Thus, interventions targeting ROS homeostasis in CSCs may be a useful strategy for targeting this drug-resistant tumor cell subpopulation.

Deciphering the crucial roles of AraC-type transcriptional regulator Cgl2680 on NADPH metabolism and L-lysine production in Corynebacterium glutamicum.

Lysine is widely used in food, medical and feed industries. The biosynthesis of L-lysine is closely related to NADPH level, but the regulation mechanism between the biosynthesis of L-lysine in C. glutamicum and the cofactor NADPH is still not clear. Here, a high intracellular NADPH level strain C. glutamicum XQ-5Δpgi::(zwf-gnd) was constructed by blocking the glycolytic pathway and overexpressing the pentose phosphate pathway in the lysine-producing strain C. glutamicum XQ-5, and the intracellular NADPH level in strain XQ-5Δpgi::(zwf-gnd) was increased from 3.57 × 10-5nmol/(104 cells) to 1.8 × 10-4nmol/(104 cell). Transcriptome analyses pointed to Cgl2680 as an important regulator of NADPH levels and L-lysine biosynthesis in C. glutamicum. By knocking out the gene Cgl2680, the intracellular NADPH level of the recombinant C. glutamicum lysCfbr ΔCgl2680 was raised from 7.95 × 10-5nmol/(104 cells) to 2.04 × 10-4nmol/(104 cells), consequently leading to a 2.3-fold increase in the NADPH/NADP+ ratio. These results indicated that the regulator Cgl2680 showed the negative regulation for NADPH regeneration. In addition, Cgl2680-deficient strain C. glutamicum lysCfbr ΔCgl2680 showed the increase of yield of both L-lysine and L-leucine as well as the increase of H2O2 tolerance. Collectively, our data demonstrated that Cgl2680 plays an important role in negatively regulating NADPH regeneration, and these results provides new insights for breeding L-lysine or L-leucine high-yielding strain.

Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration.

Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (kcat/Km) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP+-dependent formate dehydrogenase reported and has the highest kcat/Km toward NADP+ (kcat/KmNADP+) compared with other FDHs that can utilize NADP+ as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO- and NADP+ affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO- and NADP+. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced KmNADP+ to 0.09mM, with a concomitant decrease in KmHCOO-, and gave 1.6-fold higher kcat/KmNADP+ than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest KmHCOO- of 8.51mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased KmNADP+ of 0.11mM. These results are beneficial for improving the performance of NADP+-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.

Tandem mass tag labeling to characterize muscle-specific proteome changes in beef during early postmortem period

Previous research has indicated that variation in the proteome profile of longissimus lumborum (LL) and psoas major (PM) post-rigor influences meat quality attributes such as tenderness and color stability during retail display. Tandem mass tag (TMT) labeling is a chemical labeling approach using isobaric mass tags for accurate mass spectrometry-based quantification and identification of biological macromolecules. The objective of this study was to use TMT labeling to examine proteome profile variation between beef LL and PM during the early postmortem period (45 min, 12 h, and 36 h). We identified a total of 629 proteins, of which 71 were differentially abundant (fold change > 1.5, P < .05) from three comparisons between the muscles (PM vs. LL at 45 min, 12 h and 36 h). These proteins were mainly involved in oxidative phosphorylation and ATP-related transport, tricarboxylic acid cycle, NADPH regeneration, fatty acid degradation, muscle contraction, calcium signaling, chaperone activity, oxygen transport, as well as degradation of the extracellular matrix. At early postmortem, more abundant antiapoptotic proteins in LL could cause high metabolic stability, enhanced autophagy, and delayed apoptosis, while overabundant metabolic enzymes and pro-apoptotic proteins in PM could accelerate the generation of reactive oxygen species and initiation of cell death. SignificanceLongissimus lumborum and psoas major are two highly valuable cuts in beef carcass exhibiting different quality attributes such as color and tenderness, partly due to their distinct muscle fiber proportion and metabolic property. We characterized the proteomic change of these two muscles at early postmortem using tandem mass tag labeling for the first time, which highlighted the potential relationships between metabolism, cell death, and color stability. Our work provides a new perspective on muscle-to-meat conversion, especially on the potential effect of muscle-specific cell death on meat color stability.

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl-CoA supply.

Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

Production of drug metabolites by human FMO3 in Escherichia coli

BackgroundIn the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites.ResultsThe effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated.ConclusionsThe study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.

Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination

Biosynthesizing unnatural chiral amino acids is challenging due to the limited reductive amination activity of amino acid dehydrogenase (AADH). Here, for the asymmetric synthesis of l-phosphinothricin from 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), a glutamate dehydrogenase gene (named GluDH3) from Pseudomonas monteilii was selected, cloned and expressed in Escherichia coli (E. coli). To boost its activity, a “two-step”-based computational approach was developed and applied to select the potential beneficial amino acid positions on GluDH3. l-phosphinothricin was synthesized by GluDH-catalyzed asymmetric amination using the d-glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration. Using lyophilized E. coli cells that co-expressed GluDH3_V375S and EsGDH, up to 89.04 g L−1 PPO loading was completely converted to l-phosphinothricin within 30 min at 35 °C with a space-time yield of up to 4.752 kg·L−1·d−1. The beneficial substitution V375S with increased polar interactions between K90, T193, and substrate PPO exhibited 168.2-fold improved catalytic efficiency (kcat/KM) and 344.8-fold enhanced specific activity. After the introduction of serine residues into other GluDHs at specific positions, forty engineered GluDHs exhibited the catalytic functions of “glufosinate dehydrogenase” towards PPO.

Improving the System Performance of the Asymmetric Biosynthesis of d-Pantoic Acid by Using Artificially Self-Assembled Enzymes in Escherichia coli

d-Pantoic acid (d-PA) is an important chiral precursor of a broad range of biologically active compounds. The asymmetric synthesis of d-PA through reductase coupling with NADPH regeneration systems is highly promising, but the process is restricted by expensive cofactor consumption and low cofactor recycling frequency. Here, an effective construction of self-assembled ketopantoic acid reductase and glucose dehydrogenase via protein-peptide interaction of PDZ domain and PDZ ligand was established. The self-assembled enzymes exhibited highly ordered two-dimensional threadlike macromolecular structures with improved cofactor regeneration. Furthermore, the bioconversion with whole-cell catalysis showed that the robustness and efficiency of the system with self-assembled enzymes were significantly higher than those of the unassembled enzymes. This study provides a strategy for the effective asymmetric biosynthesis of d-PA with a trace amount of cofactor and shows potential for industrial applications.

ATP citrate lyase: A central metabolic enzyme in cancer.

ACLY links energy metabolism provided by catabolic pathways to biosynthesis. ACLY, which has been found to be overexpressed in many cancers, converts citrate into acetyl-CoA and OAA. The first of these molecules supports protein acetylation, in particular that of histone, and de novo lipid synthesis, and the last one sustains the production of aspartate (required for nucleotide and polyamine synthesis) and the regeneration of NADPH,H+(consumed in redox reaction and biosynthesis). ACLY transcription is promoted by SREBP1, its activity is stabilized by acetylation and promoted by AKT phosphorylation (stimulated by growth factors and glucose abundance). ACLY plays a pivotal role in cancer metabolism through the potential deprivation of cytosolic citrate, a process promoting glycolysis through the enhancement of the activities of PFK 1 and 2 with concomitant activation of oncogenic drivers such as PI3K/AKT which activate ACLY and the Warburg effect in a feed-back loop. Pending the development of specific inhibitors and tailored methods for identifying which specific metabolism is involved in the development of each tumor, ACLY could be targeted by inhibitors such as hydroxycitrate and bempedoic acid. The administration of citrate at high level mimics a strong inhibition of ACLY and could be tested to strengthen the effects of current therapies.

Tandem Mass Tag Labeling to Identify Proteome Changes in Beef Longissimus Lumborum and Psoas Major Muscles During Early Postmortem Period

ObjectivesLongissimus lumborum (LL) and psoas major (PM) are important muscles in beef hindquarters that exhibit variation in meat quality attributes. Postmortem metabolism (muscle-to-meat conversion) affects biochemical properties of muscles and in turn influence the meat quality. Although previous research has indicated that variation in the proteome profile of LL and PM post-rigor influences meat quality attributes such as tenderness and color stability during retail display, limited research has examined the influence of early postmortem metabolism on meat quality. Tandem mass tag (TMT) labeling is a chemical labeling approach used for accurate mass spectrometry-based quantification and identification of biological macromolecules. Therefore, the objective of this study was to use TMT labeling to examine proteome profile variation between beef LL and PM during the early postmortem period.Materials and MethodsMuscle biopsy samples were collected from carcasses (n = 4) at 45 min, 12 h, and 36 h postmortem from a commercial beef processing facility. Samples were frozen immediately in liquid nitrogen and stored at –80°C until proteomic analysis. Proteome was analyzed using TMT label containing ten different isobaric compounds with the same mass and chemical structure composed of an amine-reactive NHS-ester group, a spacer arm, and a mass reporter. After labeling and peptide fractionation, all the samples were multiplexed and ran through the Orbitrap Velos mass spectrometer equipped with a Nanospray Flex ion source to identify differentially abundant proteins. The proteins exhibiting 1.5-fold or more intensity difference and a statistical difference (P &lt; 0.05) between LL and PM or within the muscles during the postmortem were reported as differentially abundant.ResultsSeventy differentially abundant proteins (P &lt; 0.05) were identified from three comparisons between the muscles (31 proteins in PM 45 min vs. LL 45 min, 41 proteins in PM 12 h vs. LL 12 h, 49 proteins in PM 36 h vs. LL 36 h). However, no difference (P &gt; 0.05) in protein expression within a muscle was observed during these time points. The differentially abundant proteins were mainly involved in oxidative phosphorylation and ATP-related transport, tricarboxylic acid cycle, NADPH regeneration, fatty acid degradation, muscle contraction, calcium signaling, chaperone activity, oxygen transport, as well as degradation of the extracellular matrix. At early postmortem, overabundant anti-apoptotic proteins in LL could cause high metabolic stability, enhanced autophagy, and delayed apoptosis, while overabundant metabolic enzymes and pro-apoptotic proteins in PM could accelerate the reactive oxygen species generation and programmed cell death.ConclusionDifferentially abundant proteins between LL and PM during the early postmortem were primarily associated with cellular metabolism and programmed cell death. The greater oxidative and color stability in LL compared to PM could be related to the increased expression of anti-apoptotic proteins and the decreased expression of metabolic enzymes and proapoptotic proteins in LL.

A new carotenoid, 6′-hydroxy-3′-didehydrolutein, produced by recombinant Escherichia coli that expresses the violaxanthin biosynthesis and chaperone AtCYO1 genes — Its structure and antioxidant activities

Recombinant Escherichia coli cells, which express the higher-plant CaZEP (zeaxanthin epoxidase) and AtCYO1 (molecular chaperone) genes and an NADPH regeneration system in addition to the IDI, crtE, crtB, crtI, crtY, and crtZ genes for zeaxanthin biosynthesis, were cultured in 2 × YT medium for the efficient production of antheraxanthin and violaxanthin. The carotenoids accumulated in the cells were purified by two-phase partition, silica gel column chromatography, and preparative silica gel HPLC. Mass spectrometry and nuclear magnetic resonance analyses of these carotenoids revealed that a novel carotenoid, determined to be (3R,6′S)-3,6′-dihydroxy-β,ε-caroten-3′-one and named as 6′-hydroxy-3′-didehydrolutein, was produced in addition to zeaxanthin, antheraxanthin and violaxanthin. It is likely that the new carotenoid was generated from an unstable epoxy-carotenoid antheraxanthin that was synthesized from zeaxanthin with CaZEP. Its antioxidant activities were also examined.

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues.

Engineering flavin-free NAD(P)+-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD+s) is of importance for eliminating the need for costly NAD(P)+ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD+ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD+-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD+-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD+-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN+). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (k cat/K M) and a specific activity of 17.7 U/mg on NMN+, which is comparable to the wild-type enzyme on its natural coenzyme, NADP+. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD+-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD+s similar to the levels of natural enzyme/coenzyme pairs.

Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1β was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1β expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H2O2) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

Characterization of a robust glucose 1-dehydrogenase, SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

Abstract To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The Km and catalytic efficiency (kcat/Km) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM−1 s−1, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM−1 s−1, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% eep and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% eep and 98.3% yield.

Engineering an Alcohol Dehydrogenase for Balancing Kinetics in NADPH Regeneration with 1,4-Butanediol as a Cosubstrate

Cofactor regeneration using diols as “smart cosubstrates” is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient ...