Regeneration Of Nicotinamide Adenine Dinucleotide Phosphate Research Articles

Engineering a Formate Dehydrogenase for NADPH Regeneration.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Simulation Photosynthesis Improves the Photoresponse Performance of Cr2Ge2Te6 Nanosheets for High-Performance Self-Powered Photoelectrochemical Photodetectors

Two-dimensional (2D) semiconductor materials have been extensively studied as excellent candidates for future optoelectronic devices. Cr2Ge2Te6, as a two-dimensional semiconductor material, has attracted extensive interest in the field of photodetectors due to its excellent light absorption properties and suitable band gap. Inspired by biological photosynthesis, this study constructed a high-performance biomimetic self-powered photoelectrochemical-type flexible photodetector using liquid-stripped Cr2Ge2Te6 nanosheets as the photoanode. The device exhibits obvious self-powered photoresponse behavior and excellent photoresponse performance with a responsivity of 54.4 μA/W. Furthermore, the addition of nicotinamide adenine dinucleotide phosphate (NADPH) effectively consumes the photo-excited holes on the Cr2Ge2Te6 nanosheets to inhibit the recombination of photogenerated electron–hole pairs, and the cyclic regeneration of NADPH is realized by adding adenosine triphosphate. In addition, the flexible photodetector also exhibits excellent stability and durability, and it still maintains good photoresponse characteristics after 1000 times of bending and folding. Our work fully confirms that it is an effective means to improve the photoresponse performance of 2D materials by simulating biological photosynthesis, and it also provides theoretical and experimental guidance for the research and development of high-performance biomimetic devices.

Hollow Rh-COF@COF S-Scheme Heterojunction for Photocatalytic Nicotinamide Cofactor Regeneration

Photocatalytic regeneration of a nicotinamide cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), has emerged as an ideal cascade partner for combination with enzymatic transformations, but it still suffers from low efficiency due to the recombination of photogenerated charge carriers. Herein, a hierarchical Rh-covalent organic framework (COF)@COF core–shell hollow sphere (CSHS) with S-scheme heterojunction is proposed to enhance the charge carrier transfer and utilization in photocatalysis to regenerate the expensive NADPH. The hierarchical Rh-COFBpy@ hollow spherical COF (HCOF) CSHS was prepared by simple sequential in situ growth. The turnover frequency of Rh-COFBpy@HCOF25 for NADPH regeneration reached 2.6 mmol·gRh-COF–1·h–1, which was 3.7 times that of pure Rh-COFBpy under visible light. Density functional theory calculation and X-ray photoelectron spectroscopy analysis showed that an internal electric field directed from Rh-COFBpy to HCOF was formed in Rh-COFBpy@HCOF CSHS, which accelerated the photogenerated electron transfer from HCOF to Rh-COFBpy. In situ irradiated XPS analyses and photoirradiated Kelvin probe measurement revealed the S-scheme charge-transfer mechanism within Rh-COFBpy@HCOF. The Rh-COFBpy@HCOF photocatalytic NADPH regeneration system was further coupled with enzymatic reduction of C═C, achieving a photoenzyme cascade reaction. This work provides a protocol for the design and utilization of COF-based artificial photosynthetic systems.

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

Computational design of highly stable and soluble alcohol dehydrogenase for NADPH regeneration

Nicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP+ to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP+-specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M (S24P/G182A/G196A/H222D/S250E/S254R) exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: Δ {T}_{1/2}^{60mathrm{min}}= + 3.6 °C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy using recombinant Escherichia coli, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH).

Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data.

The thermotolerant Kluyveromyces marxianus is a potential candidate for high-temperature ethanol fermentation. Although K. marxianus exhibited high ethanol productivity at 45°C during the early fermentation stage, we observed a fermentation arrest due to the accumulated inhibitors. The stress responses of K. marxianus during high-temperature fermentation were revealed based on integration of RNA sequencing (RNA-Seq) and metabolite data. High temperature stimulated mitochondrial respiration but repressed the tricarboxylic acid (TCA) cycle, leading to increased generation of reactive oxygen species (ROS) and a lowered ratio of reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+). Glycerol production was enhanced during the early fermentation stage, which might contribute to NADH reoxidation and ROS generation. Excess ROS could be neutralized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) that might be reserved in the following ways: (1) decreased biosynthesis of branched-chain amino acids (BCAAs) reduced NADPH consumption; (2) enhanced acetic acid production increased NADPH regeneration. The degree of fatty acid unsaturation was also reduced to adapt to high temperature. In addition, stress responses were also observed after the fermentation arrest at 45°C. Genes related to peroxidase activity, iron-sulfur cluster assembly, and flavin mononucleotide (FMN) binding were downregulated, while genes associated with DNA repair and lipid composition of the plasma were upregulated. The yeast also produced more ergosterol to deal with ethanol stress. This study gains comprehensive insights into the K. marxianus transcriptome under various stresses during high-temperature ethanol fermentation, providing rich information for further metabolic engineering towards improved stress tolerance and ethanol production.

Light-powered cell factories

Biohybrid Microbes Bacteria and fungi are used industrially to produce commodity fine chemicals at vast scale. Sugars are an economical feedstock, but many of the desired products require enzymatic reduction, meaning that some of the sugar must be diverted to regenerate the cellular reductant NADPH (reduced form of nicotinamide adenine dinucleotide phosphate). Guo et al. show that electrons from light-sensitive nanoparticles can drive reduction of cellular NADPH in yeast, which can then be used for reductive biosynthetic reactions. This system can reduce diversion of carbon to NADPH regeneration and should be compatible with many existing engineered strains of yeast. Science , this issue p. [813][1] [1]: /lookup/doi/10.1126/science.aat9777

Strengthening NADPH Regeneration for Improving Photo-biocatalytic Ketones Asymmetric Reduction Reaction by Synechocystis Through Overexpression of FNR

Microalgae are excellent biocatalyst candidates for photo-biocatalytic-asymmetric reduction of prochiral ketones to produce enantiomer alcohols. In the biocatalysis asymmetric reduction of carbonyl group process, the cofactor, NADPH, plays a key role. So the current study focused on the effect of NADPH availability on the reduction. Photosynthesis is a main pathway for NADPH regeneration in microalgae, and the ferredoxin-NADP+ oxidoreductase (FNR) is the key enzyme in this process. In this work, we constructed an engineered cyanobacterium Synechocystis sp. PCC6803::Ω-PpetE-petH to overexpress FNR gene petH to strengthen NADPH regeneration. The results show that the intracellular NADPH content was increased 80.8% compared to the wild type. Applying the Synechocystis sp. PCC6803::Ω-PpetE-petH to catalyze the model asymmetric reduction reaction (reduction of acetophenone to S-phenylethanol), the yield was improved 61.2% compared to the wild-type. This work makes the microalgal biocatalysis asymmetric reduction process become more preponderant via providing a new route to enhance the regeneration of cofactor NADPH. Putative metabolic process of nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and the photo-biocatalytic-asymmetric reduction reaction of prochiral ketones in microalgal cells.

An NADPH-dependent Lactobacillus composti short-chain dehydrogenase/reductase: characterization and application to (R)-1-phenylethanol synthesis.

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30kDa. The optimal pH and temperature were 6.5 and 30 °C, respectively. The maximal reaction rate Vmax was 133.9 Umg-1; the Michaelis-Menten constant K m of LcSDR were 0.345mM for acetophenone (1a), and 0.085mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time-space yield. Under the optimized conditions, 50 gl-1 1a was converted to (R)-1b in 2h with a yield of 93.8%, enantiomeric excess of product (e.e.p) above 99% and space-time yield of 562.8 gl-1d-1.

Promotion of the microalgal photo‐biocatalytic asymmetric reduction of prochiral ketone by NADPH metabolic regulation

AbstractA metabolic regulation method was proposed to increase the microalgal intracellular nicotinamide adenine dinucleotide phosphate (NADPH) by lowering NADPH consumption in the Calvin cycle or accelerating NADPH regeneration in photosynthesis, thereby to promote the asymmetric reduction of prochiral ketone to chiral alcohol catalyzed by microalgae cell. The NADPH consumption was efficiently decreased by inhibiting glyceraldehyde 3‐phosphate dehydrogenase with inhibitors, such as iodoacetic acid and Angeli's Salt, which increased the microalgal intracellular NADPH by 80% and 96%, respectively. Correspondingly, the yields of the asymmetric reduction reaction were enhanced by 12.9% and 63.3%, respectively. On the other hand, NADPH regeneration was accelerated by promoting electron transfer and ferredoxin‐NADP+ reductase activity with addition of sodium cholate, sodium acetate and sodium thiosulfate; the intracellular NADPH was increased 73.1%, 60.2% and 70.4%, respectively. The yields of the asymmetric reduction reaction were correspondingly enhanced by 118.4%, 36.7% and 26.2%, respectively. This provides a practical technique to promote microalgae intracellular NADPH amount to enhance asymmetric reduction reaction by photo‐biocatalytic processes. © 2016 Curtin University of Technology and John Wiley & Sons, Ltd.

NADPH-generating systems in bacteria and archaea.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

Response of Saccharomyces cerevisiae to D-limonene-induced oxidative stress

In the present study, we investigated the mode of cell response induced by D-limonene in Saccharomyces cerevisiae. D-limonene treatment was found to be accompanied by intracellular accumulation of reactive oxygen species (ROS). Since ROS impair cell membranes, an engineered strain with enhanced membrane biosynthesis exhibited a higher tolerance to D-limonene. Subsequent addition of an ROS scavenger significantly reduced the ROS level and alleviated cell growth inhibition. Thus, D-limonene-induced ROS accumulation plays an important role in cell death in S. cerevisiae. In D-limonene-treated S. cerevisiae strains, higher levels of antioxidants, antioxidant enzymes, and nicotinamide adenine dinucleotide phosphate (NADPH) were synthesized. Quantitative real-time PCR results also verified that D-limonene treatment triggered upregulation of genes involved in the antioxidant system and the regeneration of NADPH at the transcription level in S. cerevisiae. These data indicate that D-limonene treatment results in intracellular ROS accumulation, an important factor in cell death, and several antioxidant mechanisms in S. cerevisiae were enhanced in response to D-limonene treatment.

Efficient Regeneration of NADPH in a 3‐Enzyme Cascade Reaction by in situ Generation of Glucose 6‐Phosphate from Glucose and Pyrophosphate

AbstractWe report here a promising method to regenerate NADPH (nicotinamide adenine dinucleotide phosphate) using the intermediate formation of glucose 6‐phosphate (G6P) from glucose and pyrophosphate (PPi) catalyzed by the acid phosphatase from Shigella flexneri (PhoN‐Sf). The G6P formed is used in turn by glucose 6‐phosphate dehydrogenase (G6P DH) to mediate the reduction of NADP+ to NADPH. The method was tested in a one‐pot system with three enzymes in which the NADPH generated was subsequently used by an alcohol dehydrogenase (ADH) from Lactobacillus kefir or Thermoanaerobium brockii to catalyze the enantioselective reduction of acetophenone to R‐(+)‐1‐phenylethyl alcohol or S‐(−)‐1‐phenylethyl alcohol, respectively with NADP+ as starting cofactor. We were able to synthesize 50 mL of 50 mM R‐(+)‐1‐phenylethyl alcohol in the presence of 5 mM PPi and 0.4 mM NADP+. The substoichiometric amount of PPi needed demonstrates that phosphate cycling occurs. Under optimal conditions a total turnover number for NADPH higher than 3000 was reached.

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae

Industrial bakers' yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and D: -arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.

Transcription Analysis of Recombinant Saccharomyces cerevisiae Reveals Novel Responses to Xylose

Lignocellulosic biomass, rich in hexose and pentose sugars, is an attractive resource for commercially viable bioethanol production. Saccharomyces cerevisiae efficiently ferments hexoses but is naturally unable to utilize pentoses. Metabolic engineering of this yeast has resulted in strains capable of xylose utilization. However, even the best recombinant S. cerevisiae strains of today metabolize xylose with a low rate compared to glucose. This study compares the transcript profiles of an S. cerevisiae strain engineered to utilize xylose via the xylose reductase-xylitol dehydrogenase pathway in aerobic chemostat cultures with glucose or xylose as the main carbon source. Compared to the glucose culture, 125 genes were upregulated, whereas 100 genes were downregulated in the xylose culture. A number of genes encoding enzymes capable of nicotinamide adenine dinucleotide phosphate regeneration were upregulated in the xylose culture. Furthermore, xylose provoked increased activities of the pathways of acetyl-CoA synthesis and sterol biosynthesis. Notably, our results suggest that cells metabolizing xylose are not in a completely repressed or in a derepressed state either, indicating that xylose was recognized neither as a fermentable nor as a respirative carbon source. In addition, a considerable number of the changes observed in the gene expression between glucose and xylose samples were closely related to the starvation response.

Glycogen is mobilized during the disposal of peroxides by cultured astroglial cells from rat brain

Regeneration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for the activity of glutathione redox cycling during cellular peroxide detoxification. In order to test for a function of astroglial glycogen to serve as endogenous precursor for glucose-6-phosphate, the substrate for the regeneration of NADPH by the pentose phosphate pathway, the content of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats was determined after application of peroxides. In the presence of hydrogen peroxide or cumene hydroperoxide in concentrations of 200 μM glycogen was mobilized with a half-life of 16 min in incubation medium containing 20 mM glucose, whereas in the absence of peroxides the glycogen content decreased more slowly with a half-life of 42 min. After 30 min of incubation with or without peroxides 30 and 73%, respectively, of the initial glycogen content was found. The degree of glycogen mobilization was reduced by lowering the initial concentration of the peroxides. These results demonstrate that in astroglial cells (i) glucosyl residues of glycogen are mobilized after application of peroxides despite the presence of exogenous glucose, and (ii) that the demand for glucose-6-phosphate as substrate for NADPH regeneration via the pentose phosphate pathway can, at least partially, be met by mobilization of glycogen.

Oxidation and reduction of D-xylose by cell-free extract of Pichia quercuum.

The fermentation mechanism of the simultaneous production of D-xylonic acid and xylitol from D-xylose by Pichia quercuum was studied by using a cell-free enzyme preparation. Nicotinamide adenine dinucleotide phosphate (NADP)-dependent D-xylose dehydrogenase activity and NADP-dependent D-xylose reductase activity were detected, and the oxido-reduction reaction of D-xylose was able to couple through regeneration of NADP and NADPH to produce D-xylonic acid and xylitol.

Oxidation and Reduction of D-Xylose by Cell-Free Extract of Pichia quercuum

The fermentation mechanism of the simultaneous production of D-xylonic acid and xylitol from D-xylose by Pichia quercuum was studied by using a cell-free enzyme preparation. Nicotinamide adenine dinucleotide phosphate (NADP)-dependent D-xylose dehydrogenase activity and NADP-dependent D-xylose reductase activity were detected, and the oxido-reduction reaction of D-xylose was able to couple through regeneration of NADP and NADPH to produce D-xylonic acid and xylitol.