Cofactor Regeneration Research Articles

Renewable hydrogen production and storage via enzymatic interconversion of CO2 and formate with electrochemical cofactor regeneration.

The urgent need to reduce CO2 emissions has motivated the development of CO2 capture and utilization technologies. An emerging application is CO2 transformation into storage chemicals for clean energy carriers. Formic acid (FA), a valuable product of CO2 reduction, is an excellent hydrogen carrier. CO2 conversion to FA, followed by H2 release from FA, are conventionally chemically catalyzed. Biocatalysts offer a highly specific and less energy intensive alternative. CO2 conversion to formate is catalyzed by formate dehydrogenase (FDH), which usually requires a cofactor to function. Several FDHs have been incorporated in bioelectrochemical systems where formate is produced by the biocathode and the cofactor is electrochemically regenerated. H2 production from formate is also catalyzed by several microorganisms possessing either formate hydrogenlyase or hydrogen-dependent CO2 reductase complexes. Combination of these two processes can lead to a CO2-recycling cycle for H2 production, storage, and release with potentially lower environmental impact than conventional methods.

Chemoenzymatic Oxidation of Diols Catalyzed by Co‐Immobilized Flavins and Dehydrogenases**

AbstractEnzyme driven oxidations catalyzed by alcohol dehydrogenases rely on the in situ NAD(P)+ regeneration. A wide variety of chemoenzymatic and fully enzymatic methods have been reported over the last 30 years to integrate the cofactor regeneration in biocatalytic oxidations. However, the majority of examples are limited to homogeneous systems where the reuse of both enzymes and chemical catalysts are challenging. In this work, we co‐immobilize an alcohol dehydrogenase from Bacillus stearothermophilus with a flavin derivative (FMN), which performs as an organocatalyst that oxidizes NADH back to NAD+. This latter oxidized cofactor is sequentially utilized by the dehydrogenase to oxidize 1,ω‐diols. Remarkably, the immobilization chemistry of FMN determines its efficiency to oxidize NADH and, unlike in its free state, the immobilized FMN can recycle NAD+ in dark. This is possible because the support where both enzyme and FMN are immobilized also captures NADH, making the electron transfer from the substrates to the cofactors more efficient. This work illustrates how the co‐immobilization and confinement of bio and chemical catalysts on solid materials (heterogeneous phase) enable chemoenzymatic cascades that are precluded in solution (homogeneous phase).

Easy to make highly efficient FG@ACC stable platform for sp3C-CF3 bond generation and NADH regeneration under sun light

The conversion of sun light energy into sustainble greener chemicals are a major obstacle due to the use of expensive and toxic materials. Sun light induced trifluoromethylation emerges as a highly efficient procedure to insert trifluoromethyl groups into the organic compounds. Yet, the expensive and toxic properties of the metal-based photocatalysts creates a major obstacle for the insertion of trifluoromethyl groups. Metal free activated carbon cloth (ACC) emerged as a highly efficient pillar in the area of material science. In this work, we have successfully synthesized self-assembled metal free fast green with activated carbon cloth (FG@ACC) photocatalyst for photocatalytic trifluoromethylation and reduced nicotinamide adenine dinucleotide (NADH) cofactor regeneration (85.89 %, 2 h) under sun light. The sun light induced organic transformation promotes the use of low-cost CF3SO2Na as the CF3 radical source to produce highly selective products with 97% yield.

Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids.

Efficient formate dehydrogenase (FDH)-based cofactor regeneration systems are widely used for biocatalytic processes due to their ready availability, low reduction potential, and production of only benign byproducts. However, FDHs are usually specific to NAD+, and NADPH regeneration with formate is challenging. Herein, an FDH with a preference for NAD+ from Azospirillum palustre (ApFDH) was selected owing to its high activity. By static and dynamic structural analyses, a beneficial substitution, D222Q, was identified for cofactor-preference switching. However, its total activity was substantially decreased by 90% owing to the activity-specificity trade-off. Subsequently, a semirational library was designed and screened, which yielded a variant ApFDHD222Q+A199G+H380S with satisfactory activity and NADP+ specificity. Our analysis of dynamical cross-correlations revealed a substitution combination that brought balance to the dynamical correlation network. This combination successfully overcame the activity-specificity-stability trade-off and resulted in a beneficial outcome. The substitution combination (D222Q-A199G/H380S-C256A/C146S) enabled the simultaneous improvement of activity, specificity, and stability and was successfully applied to other 17 FDHs. Finally, by employing engineered ApFDH, an NADPH regeneration system was developed, optimized, and utilized for the asymmetric biosynthesis of l-phosphinothricin.

Metabolism: How a eukaryote adapted to life without respiration

A new study finds that Schizosaccharomyces japonicus, a eukaryote that lost the ability to respire, modified its central carbon metabolism to maintain efficient ATP production, cofactor regeneration, and amino-acid production. This remarkable metabolic flexibility opens new avenues towards applications.

Efficient biosynthesis of high-value 5‑Hydroxytryptophan using a multienzyme cascade

5-Hydroxytryptophan (5-HTP) is a precursor of serotonin. The increasing demand for 5-HTP is driving the search for a more efficient and cost-effective production method. In this study, a new in vitro multi-enzyme cascade system (MECCS) was developed to convert L-tryptophan (L-trp) to 5-HTP in a single container while achieving the cycling and regeneration of cofactors and coenzymes. The rate-limiting enzyme tryptophan hydroxylase (TPH) was truncated to improve the overall efficiency of the cascade. After screening, the mutant M1 (NΔ143/CΔ26) showed excellent enzymatic performance, with a 2.3-fold increase in catalytic activity. Molecular dynamics simulations showed that the increase in active center binding force and the improvement of substrate tunnel in M1 improved the overall catalytic efficiency. In addition, the rigidity enhancement of the ligand binding pocket surrounding the region improved the thermal stability of the enzyme. Under optimized conditions, the MECCS converted 50 mM L-trp to 36.9 mM 5-HTP, with a conversion rate of 73.8%. This study provides an alternative pathway for the efficient production of 5-HTP.

Catalytic Promiscuity of Horseradish Peroxidase: Aerobic Oxidative Regeneration of Oxidized Nicotinamide Cofactors

AbstractNAD(P)+ regeneration is of great importance for dehydrogenase (DH)‐catalyzed oxidations. In this work, we report horseradish peroxidase (HRP)‐catalyzed aerobic oxidation of the reduced nicotinamide cofactors, including natural and non‐natural ones, into the oxidized form at neutral and alkaline pH enabled by external amino acids (AAs). HRP showed promiscuous yet extremely low oxidase activity toward NADH at pH 7 in the absence of both AAs and mediators, as evidenced by the observation of compound III. The mechanistic study revealed that AAs as decoy molecules (likely act as acid‐base catalysts) and mediators significantly promoted the formation of compound III as well as its decay back to the ground state. The applicability of the aerobic cofactor regeneration method was demonstrated by the HRP total turnover number up to 88 000 as well as high yields (91–99 %) in bi‐enzymatic synthesis of carboxylic acids. The present work offers an alternative method to recycle NAD(P)+. Also, it may open up possibilities for HRP‐catalyzed aerobic oxidations.

Constructing a continuous-flow bioreactor with co-immobilized KmAKR and BmGDH for synthesizing tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate

tert-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2), an important precursor of atorvastatin, can be synthesized through asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) by the aldo-keto reductases (AKRs), coupled with BmGDHs for cofactor regeneration. Herein, we employed glutaraldehyde (GLA) and polyethyleneimine (PEI) resin microsphere to co-immobilize KmAKR and BmGDH. The immobilizate activity of KmAKR&BmGDH@amino resin-PEI peaked at 73 U/g, and 80.7% activity remained after 10 cycles of reuse. Continuous flow bioreactor packed with the KmAKR&BmGDH@amino resin-PEI had ∼3.7-fold higher space-time yield (STY, 374.7 g/L/d) than the batch mode. Moreover, excellent operational stability was achieved, remaining over 95% conversion after 90 h consecutive usage. Hence, a self-sustaining closed-loop bienzymatic platform running in the continuous flow mode was developed.

Investigation of process stability of a whole-cell biocatalyst with Baeyer–Villiger monooxygenase activity in continuous bioreactors

The stability of a whole-cell biocatalyst based on genetically modified Escherichia coli cells harboring cyclohexanone monooxygenase was investigated in a free and immobilized form in continuous bioreactors. The biotransformation of a bicyclic ketone to a racemic mixture of the corresponding lactones by an enzymatic cascade was performed in a membrane bioreactor (MBR) for free cells and in a fluidized-bed bioreactor (FBBR) for immobilized cells. Both reactors were operated at the residence time of 6.25 h with the biocatalyst concentration of 1 % w/w. Different oxygen mass transfer rates were set via the impeller speed in MBR or air flow rate in FBBR, respectively. The potential of continuous bioreactors to achieve a steady state was utilized for the analysis of different factors affecting the biocatalyst stability. An apparent steady state could be maintained in these bioreactors up to 40 h. The operation at high conversions or high oxygenation rates allowed to exclude poisoning by substrate or oxygen as causes of biocatalyst activity loss. Further experiments showed that the cells did not lose viability or plasmid. The problem in biocatalyst stability was not in the stability of cyclohexanone monooxygenase but in insufficient regeneration of cofactors. The only partially effective measure was the addition of catalase with the feed concentration of 0.025 g/L that decomposed hydrogen peroxide formed in the enzymatic cascade. However, both free and immobilized biocatalysts lost their activity completely in about a week.

Modelling of a hybrid bioelectrocatalytic flow reactor with NADH cofactor regeneration by immobilized rhodium mediator for pyruvate bioconversion

Modelling of a hybrid bioelectrocatalytic flow reactor with NADH cofactor regeneration by immobilized rhodium mediator for pyruvate bioconversion

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.

Irreversible biosynthesis of D-Allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

As a rare hexose with low calories and various physiological functions, D-allulose has drawn increasing attention. The current industrial production of D-allulose from D-fructose or D-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of D-fructose to D-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 D-allulose from 30 g L-1 D-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and D-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in D-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the D-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a D-glucose conversion rate of 84.8%. This study presented a novel strategy for high-yield D-allulose production from low-cost substrate. This article is protected by copyright. All rights reserved.

Light-driven bioprocesses

Abstract Enzyme catalysis and photocatalysis are two research areas that have become of major interest in organic synthesis. This is mainly because both represent attractive strategies for making chemical synthesis more efficient and sustainable. Because enzyme catalysis offers several inherent advantages, such as high substrate specificity, regio-, and stereoselectivity, and activity under environmentally benign reaction conditions, biocatalysts are increasingly being adopted by the pharmaceutical and chemical industries. In addition, photocatalysis has proven to be a powerful approach for accessing unique reactivities upon light irradiation and performing reactions with an extended substrate range under milder conditions compared to light-independent alternatives. It is therefore not surprising that bio- and photocatalytic approaches are now often combined to exploit the exquisite selectivity of enzymes and the unique chemical transformations accessible to photocatalysis. In this chapter, we provide an overview of the wide variety of light-driven bioprocesses, ranging from photochemical delivery of reducing equivalents to redox enzymes, photochemical cofactor regeneration, to direct photoactivation of enzymes. We also highlight the possibility of catalyzing non-natural reactions via photoinduced enzyme promiscuity and the combination of photo- and biocatalytic reactions used to create new synthetic methodologies.

Application of cofactors in the regulation of microbial metabolism: A state of the art review.

Cofactors are crucial chemicals that maintain cellular redox balance and drive the cell to do synthetic and catabolic reactions. They are involved in practically all enzymatic activities that occur in live cells. It has been a hot research topic in recent years to manage their concentrations and forms in microbial cells by using appropriate techniques to obtain more high-quality target products. In this review, we first summarize the physiological functions of common cofactors, and give a brief overview of common cofactors acetyl coenzyme A, NAD(P)H/NAD(P)+, and ATP/ADP; then we provide a detailed introduction of intracellular cofactor regeneration pathways, review the regulation of cofactor forms and concentrations by molecular biological means, and review the existing regulatory strategies of microbial cellular cofactors and their application progress, to maximize and rapidly direct the metabolic flux to target metabolites. Finally, we speculate on the future of cofactor engineering applications in cell factories. Graphical Abstract.

Characterization and Application of a Novel Glucose Dehydrogenase with Excellent Organic Solvent Tolerance for Cofactor Regeneration in Carbonyl Reduction.

An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of βD-αE-αF-αG-βG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40°C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55°C for 5h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.

N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase.

Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD+ in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize cytosolic NADH to NAD+ without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD+ regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD+ regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD+ regeneration applications under a neutral environment.

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media.

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.

Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction.

The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy "Modular-Enzymes Assembly by Spatial Segregation" (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.

Enzyme Grafting with a Cofactor-Decorated Metal-Organic Capsule for Solar-to-Chemical Conversion.

Semi-artificial approaches to solar-to-chemical conversion can achieve chemical transformations that are beyond the capability of natural enzymes, but face marked challenges to facilitate in vivo cascades, due to their inevitable need for cofactor shuttling and regeneration. Here, we report on an enzyme grafting strategy to build a metal-organic capsule-docking artificial enzyme (metal-organic-enzyme, MOE) that comprised the self-assembly of a cofactor-decorated capsule and the supramolecular enzyme-recognition features between the enzyme scaffold and the capsule to bypass cofactor shuttling and regeneration. The incorporated NADH mimics within the metal-organic capsule interacted with the imine intermediate that formed from the condensation of the amines and the dehydrogenation of alcohol substrates in the microenvironment to form complexes within the capsule and subsequently served as an in situ-generated photoresponsive cofactor. Upon illumination, the photoresponsive cofactor facilitates efficient proton/electron transport between the inner space (supramolecular hydrogenation) and outer space (enzymatic dehydrogenation) of the capsule to dehydrogenize the alcohols and hydrogenize the imine intermediates, respectively, circumventing the conventionally complex multistep cofactor shuttling and regeneration. The semi-artificial enzyme endows the conversion of diverse types of alcohol to amine products in both aqueous/organic solutions and Escherichia coli with high efficiency, offering a wide range of opportunities for sustainable and environmentally friendly biomanufacturing of commodity and fine chemicals.

A novel NADH-dependent leucine dehydrogenase for multi-step cascade synthesis of L-phosphinothricin

L-Phosphinothricin (L-PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L-PPT has always been the focus in pesticide industry. In recent years, “one-pot, two-step” enzyme-mediated cascade strategy is a mainstream pathway to obtain L-PPT. Herein, RgDAAO and BsLeuDH were applied to expand “one-pot, two-step” process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis (BsLeuDH) was firstly characterized and attempted to generate L-PPT, achieving an excellent enantioselectivity (99.9% ee). Meanwhile, a formate dehydrogenase from Pichia pastoris (PpFDH) was utilized to implement NADH cofactor regeneration and only CO2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D-PPT relying on a D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) with content conversion (46.1%). L-PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by RgDAAO and reductive amination catalyzed by BsLeuDH, achieving 80.3% overall yield and > 99.9% ee value.