Cofactor Regeneration Research Articles

A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex.

NAD+ -dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3±1.9 s-1 at 25°C compared to 7.7±1.0 s-1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2 O2 inactivation, showing a 200-fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2 O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H2 O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2 O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.

Cyano-modified poly(triazine imide) with extended π-conjugation for photocatalytic biological cofactor regeneration

A unique extended π-conjugated system of cyano-modified PTI is strategically developed for biological cofactor regeneration.

Enhanced Expression of Alcohol Dehydrogenase I in Pichia pastoris Reduces the Content of Acetaldehyde in Wines.

Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

Efficient and Recyclable Photobiocatalytic System via Cofactor Regeneration for Chiral Aryl Alcohol Synthesis

Efficient and Recyclable Photobiocatalytic System via Cofactor Regeneration for Chiral Aryl Alcohol Synthesis

Photoenzymatic CO2 Reduction Dominated by Collaborative Matching of Linkage and Linker in Covalent Organic Frameworks.

Artificial photoenzymatic systems based on covalent organic frameworks (COFs) provide an interesting platform for converting CO2 to value-added fuels. However, the dual roles of COFs as photocatalysts and enzyme hosts showcase contradictory preferences for structures, which poses a great challenge for their rational design. Herein, we report the collaborative matching of linkages and linkers in COFs on their ability to exert both photocatalytic activity and enzyme loading, which has been neglected until now. The linkage-dependent linker regulation pattern was elucidated, and the optimal match showed a record-breaking apparent quantum efficiency at 420 nm for photocatalytic cofactor regeneration of 13.95% with a high turnover frequency of 5.3 mmol g-1 h-1, outperforming other reported crystalline framework photocatalysts. Moreover, theoretical calculations and experiments revealed the mechanism underlying the effects of matching the linkage and linker on exciton dissociation and charge migration in photocatalysis. This newfound understanding enabled the construction of COFs with both high photoactivity and large pores closer in size to the formate dehydrogenase, achieving high loading capacity and a suitable confinement effect. Remarkably, the artificial photoenzymatic system constructed according to optimal linkage-linker matching exhibited highly efficient CO2 reduction, yielding formic acid with a specific activity as high as 1.46 mmol g-1 catalyst h-1 and good reusability, paving the way for sustainable CO2 conversion driven by visible light.

Expression of the synthetic CYP102A1-LG23 gene and functional analysis of recombinant P450 BM3-LG23 cytochrome in actinobacteria <i>Mycolicibacterium smegmatis</i>

Cytochrome CYP102A1 (P450 BM3) from Priestia megaterium (bas. Bacillus megaterium) has a number of specific features making it an ideal target for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme was obtained providing 7β-hydroxylation of steroid substrates of the androstane series with the formation of products possessing anti-inflammatory and neuroprotective activity. In this study, the synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant variant was expressed in Mycolicibacterium smegmatis cells as part of mono- and bicistronic operons together with the synthetic gdh or zwf2 genes encoding glucose dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PD), respectively. The functional activity of the recombinant enzymes was shown in vivo by the example of hydroxylation of androst-4-ene-3,17-dione (AD) to 7β-OH-AD in growing cultures of mycolicibacteria. Biocatalytic activity was doubled by increasing the CYP102A1-LG23 protein solubility in the cell and organizing the cofactor regeneration additional system by introducing GDH and G6PD. The maximum level of 7β-OH-AD amounting 37,68 mol % was achieved by co-expression the cyp102A1-LG23 and gdh genes in M. smegmatis. The results evidence to the perspective of using synthetic genes to obtain recombinant enzymes, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes and can be demand for the methods of microbiological production of 7β-hydroxylated steroids by genetically modified mycolicibacteria.

Lactic Acid from CO2: A Carbon Capture and Utilization Strategy Based on a Biocatalytic Approach.

The EU low-carbon economy aims to reduce the level of CO2 emission in the EU to 80% by 2050. High efforts are required to achieve this goal, where successful CCU (Carbon Capture and Utilization) technologies will have a high impact. Biocatalysts offer a greener alternative to chemical catalysts for the development of CCU strategies since biocatalysis conforms 10 of the 12 principles of green chemistry. In this study, a multienzymatic system, based on alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lactate dehydrogenase (LDH), that converts CO2 and ethanol into lactic acid leading to a 100% atom economy was studied. The system allows cofactor regeneration, thus reducing the process cost. Through reaction media engineering and enzyme ratio study, the performance of the system was able to produce up to 250 μM of lactic acid under the best conditions using 100% CO2, corresponding to the highest concentration of lactic acid obtained up to date using this multienzymatic approach. For the first time, the feasibility of the system to be applied under a real industrial environment has been tested using synthetic gas mimicking real blast furnace off-gases composition from the iron and steel industry. Under these conditions, the system was also capable of producing lactic acid, reaching 62 μM.

Light-Driven Metabolic Pathways in Non-Photosynthetic Biohybrid Bacteria.

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. This proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

Vigna radiata (mung) as superior natural biobased catalyst for asymmetric reduction of prochiral acetophenone

Asymmetric reduction of prochiral ketone via biocatalysis is one of the most promising and straight forward routes to prepare optically active alcohols. In this work, whole cells of Vigna radiata (also called mung) catalyzed enantioselective reduction of prochiral aromatic ketones was investigated. Effects of various parameters such as co-substrate, temperature, cell loading, substrate concentration on conversion and enantioselectivity were studied in a systematic manner by using acetophenone as the model compound. Glucose was found to be the most effective co-substrate for co-factor regeneration among different co-substrates employed. Whole cells of mung have shown excellent thermostability and high substrate tolerance (up to 200 mM). Under optimum conditions, maximum conversion of 93.44% and enantioselectivity of 98.92% were obtained in 9 h. Good conversion and enantioselectivity were observed with various substituted aromatic ketones. This is a cheap, clean, and eco-friendly process for preparation of chiral secondary alcohols compared to chemical processes.

An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.

Multidimensional optimization for accelerating light-powered biocatalysis in Rhodopseudomonas palustris

BackgroundWhole-cell biocatalysis has been exploited to convert a variety of substrates into high-value bulk or chiral fine chemicals. However, the traditional whole-cell biocatalysis typically utilizes the heterotrophic microbes as the biocatalyst, which requires carbohydrates to power the cofactor (ATP, NAD (P)H) regeneration.ResultsIn this study, we sought to harness purple non-sulfur photosynthetic bacterium (PNSB) as the biocatalyst to achieve light-driven cofactor regeneration for cascade biocatalysis. We substantially improved the performance of Rhodopseudomonas palustris-based biocatalysis using a highly active and conditional expression system, blocking the side-reactions, controlling the feeding strategy, and attenuating the light shading effect. Under light-anaerobic conditions, we found that 50 mM ferulic acid could be completely converted to vanillyl alcohol using the recombinant strain with 100% efficiency, and > 99.9% conversion of 50 mM p-coumaric acid to p-hydroxybenzyl alcohol was similarly achieved. Moreover, we examined the isoprenol utilization pathway for pinene synthesis and 92% conversion of 30 mM isoprenol to pinene was obtained.ConclusionsTaken together, these results suggested that R. palustris could be a promising host for light-powered biotransformation, which offers an efficient approach for synthesizing value-added chemicals in a green and sustainable manner.Graphical

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Artificial photosynthetic cells with biotic–abiotic hybrid energy modules for customized CO2 conversion

Programmable artificial photosynthetic cell is the ultimate goal for mimicking natural photosynthesis, offering tunable product selectivity via reductase selection toward device integration. However, this concept is limited by the capacity of regenerating the multiple cofactors that hold the key to various reductases. Here, we report the design of artificial photosynthetic cells using biotic–abiotic thylakoid–CdTe as hybrid energy modules. The rational integration of thylakoid with CdTe quantum dots substantially enhances the regeneration of bioactive NADPH, NADH and ATP cofactors without external supplements by promoting proton-coupled electron transfer. Particularly, this approach turns thylakoid highly active for NADH regeneration, providing a more versatile platform for programming artificial photosynthetic cells. Such artificial photosynthetic cells can be programmed by coupling with diverse reductases, such as formate dehydrogenase and remodeled nitrogenase for highly selective production of formate or methane, respectively. This work opens an avenue for customizing artificial photosynthetic cells toward multifarious demands for CO2 conversion.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Efficient Production of Chlorogenic Acid in Escherichia coli Via Modular Pathway and Cofactor Engineering.

Chlorogenic acid is a natural phenolic compound widely used in the food and daily chemical industries. Compared to plant extraction, microbial cell factories provide a green and sustainable production method for the production of chlorogenic acid. However, complex metabolic flux distribution and potential byproducts limited the biosynthesis of chlorogenic acid in microorganisms. A de novo biosynthesis pathway for chlorogenic acid was constructed in Escherichia coli via modular engineering. Increasing the shikimate pathway flux greatly promoted chlorogenic acid production, and the influence of pyruvate metabolism on chlorogenic acid synthesis was also explored. The supply of cofactors for the key enzymes quinate/shikimate 5-dehydrogenase (YdiB) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) was enhanced by a cofactor regeneration system. Furthermore, mutants of YdiB were verified for chlorogenic acid production in vivo. Chlorogenic acid browning occurred when the buffer pH of the buffer exceeded 6.0, but two-stage pH control achieved a chlorogenic acid titer of 2789.2 mg/L in a 5 L fermenter, the highest reported to date. This study provided a strategy for the efficient production of chlorogenic acid from simple carbon sources.

In the Lab: Heterogeneous Catalysis Mediated Interconversion between NAD(P)+ and NAD(P)H Accompanied by Consumption and Generation of Hydrogen

Dr. Wang is a Senior Lecturer in Chemical Engineering at Lancaster University. Prior to this, he was a Lecturer in Chemical Engineering at the University of Aberdeen, Postdoctoral Research Associate at Heriot-Watt University, where he also obtained his PhD (2014). He completed both MSc and BEng studies at Tianjin University. Wang has been the author of over 50 peer-reviewed publications, an editorial board member of Chinese Chemical Letters and his research has mainly been funded by the Engineering and Physical Sciences Research Council (EPSRC), Royal Society, UK Catalysis Hub and industry. Wang’s research interest ranges from reaction engineering, green energy and materials to chemicals, where heterogeneous catalysis is the core discipline. His recent work has focused on the innovative use of heterogeneous catalysts (e.g., supported metals) in enzymatic transformations via cofactor regeneration,1,2 paving the way to a potential new regeneration technology.

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes—isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7β with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7β-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7β-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7β-hydroxylated steroids in genetically modified Mycolicibacterium species.