Systematic metabolic engineering enables highly efficient production of vitamin A in Saccharomyces cerevisiae.

Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism Saccharomyces cerevisiae (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining ß-carotene 15,15'-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from ß-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in S. cerevisiae was achieved in a 1-L bioreactor. This study unlocked the potential of S. cerevisiae for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A.

Indole-N-Linked Hydroperoxyl Adduct of Protein-Derived Cofactor Modulating Catalase-Peroxidase Functions.

Bifunctional catalase-peroxidase (KatG) features a posttranslational methionine-tyrosine-tryptophan (MYW) crosslinked cofactor crucial for its catalase function, enabling pathogens to neutralize hydrogen peroxide during infection. We discovered the presence of indole nitrogen-linked hydroperoxyl adduct (MYW-OOH) in Mycobacterium tuberculosis KatG in the solution state under ambient conditions, suggesting its natural occurrence. By isolating predominantly MYW-OOH-containing KatG protein, we investigated the chemical stability and functional impact of MYW-OOH. We discovered that MYW-OOH inhibits catalase activity, presenting a unique temporary lock. Exposure to peroxide or increased temperature removes the hydroperoxyl adduct from the protein cofactor, converting MYW-OOH to MYW and restoring the detoxifying ability of the enzyme against hydrogen peroxide. Thus, the N-linked hydroperoxyl group is releasable. KatG with MYW-OOH represents a catalase dormant, but primed, state of the enzyme. These findings provide insight into chemical strategies targeting the bifunctional enzyme KatG in pathogens, highlighting the role of N-linked hydroperoxyl modifications in enzymatic function.

Virtual screening and molecular dynamics simulations of phytochemicals targeting cofactor-independent phosphoglycerate mutase in antimicrobial-resistant Mycoplasma genitalium.

Mycoplasma genitalium (M. genitalium) poses a significant challenge in clinical treatment due to its increasing antimicrobial resistance. This study investigates alternative therapeutic approaches by targeting the cofactor-independent phosphoglycerate mutase (iPGM) enzyme with phytochemicals derived from ethnobotanical plants. In silico screening identified several promising inhibitors, with 2-carboxy-D-arabinitol demonstrating the highest binding affinity (- 9.77 kcal/mol), followed by gluconic acid (- 9.03 kcal/mol) and citric acid (- 8.68 kcal/mol). Further analysis through molecular dynamics (MD) simulations revealed insights into the binding mechanisms and stability of these phytochemicals within the iPGM active site. The MD simulations indicated initial fluctuations followed by stability, with intermittent spikes in RMSD values. The lowest RMSF values confirmed the stability of the ligand-protein complexes. Key residues, including Ser-61, Arg-188, Glu-62, Asp-397, and Arg-260, were found to play crucial roles in the binding and retention of inhibitors within the active pocket. These findings suggest that the identified phytochemicals could serve as novel antimicrobial agents against M. genitalium by effectively inhibiting iPGM activity.

An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.

Getting in Shape: Updates in Exercise Anaphylaxis.

PURPOSE OF REVIEW: Exercise induced anaphylaxis (EIA) can be difficult to diagnose due to the interplay of co-factors on clinical presentation and the lack of standardized, confirmatory testing. RECENT FINDINGS: EIA has been historically categorized as either food-independent or food-dependent. However, recent literature has suggested that perhaps EIA is more complex given the relationship between not only food on EIA but other various co-factors such as medications and alcohol ingestion that are either required to elicit symptoms in EIA or make symptoms worse. For the practicing clinician, understanding how these co-factors can be implicated in EIA can enable one to take a more personalized approach in treating patients with EIA and thus improve quality of life for patients.

[Recent advances in pyridoxal phosphate-dependent enzymes and their applications].

Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, ß-addition, ß-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

Synthesis of Substituted Acyclic and Cyclic N-Alkylhydrazines by Enzymatic Reductive Hydrazinations.

Imine reductases (IREDs) provide promising opportunities for the synthesis of various chiral amines. Initially, asymmetric imine reduction was reported, followed by reductive aminations of aldehydes and ketones via imines. Herein we present the reductive amination of structurally diverse carbonyls and dicarbonyls with hydrazines (reductive hydrazination), catalyzed by the IRED from Myxococcus stipitatus. In analogy to IRED-catalyzed reductive aminations, various carbonyls and dicarbonyls could react with simple hydrazines to produce substituted acyclic and cyclic N-alkylhydrazines. By incorporating and scaling up hydrogenase cofactor regeneration system, we demonstrated the scalability and atom-efficiency of an H2-driven double reductive hydrazination, highlightling the potential of IREDs in biocatalysis.

Constructing an artificial in vitro multi-enzyme cascade pathway to convert glycerol and CO2 into L-aspartic acid.

Developing utilization technologies for biomass resources, exploring their applications in the fields of energy and chemical engineering, holds significant importance for promoting sustainable development and constructing a green, low-carbon society. In this study, we designed a non-natural in vitro multi-enzyme system for converting glycerol and CO2 into L-aspartic acid (L-Asp). The coupled system utilized eight enzymes, including alditol oxidase (ALDO), catalase-peroxidase (CAT), lactaldehyde dehydrogenase (ALDH), glycerate 2-kinase (GK), phosphopyruvate hydratase (PPH), phosphoenolpyruvate carboxylase (PPC), L-aspartate dehydrogenase (ASPD), and polyphosphate kinase (PPK), to convert the raw materials into L-Asp in one-pot coupled with NADH and ATP regeneration. Under optimal reaction conditions, 18.6 mM of L-Asp could be produced within 2.0 h at a total enzyme addition of 4.85 mg/mL, demonstrating the high efficiency and productivity characteristics of the designed system. Our technological application provides new insights and methods for the development of biomass resource utilization technologies.

Identification and characterization of archaeal-type FAD synthase as a novel tractable drug target from the parasitic protozoa Entamoeba histolytica.

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica, the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica. Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting KM and kcat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica, the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections.

Pharmacodynamic profiling in three patients with molybdenum cofactor deficiency type A reveals prolonged biological effects after withdrawal of cyclic pyranopterin monophosphate.

Molybdenum cofactor deficiency type A has successfully been treated in a small number of children with daily intravenous administration of cyclic pyranopterin monophosphate. Pharmacodynamic data for this novel treatment have not been published and alternative dosing intervals have not been explored. We monitored pharmacodynamic biomarkers of sulfite oxidase and xanthine oxidoreductase activity in three patients with MoCD-A for a period of 2 to 9 months after discontinuation of cPMP substitution. We found that the clinical and metabolic effects were sustained for longer than expected, over 7 days at least. Our data implicate a biological half-life of the molybdenum cofactor dependent enzyme activities of approximately 3 days and suggest the possibility that less frequent than once daily dosing intervals could be a safe alternative to current practice.

Biotransformation of selected secondary metabolites by Alternaria species and the pharmaceutical, food and agricultural application of biotransformation products.

Biotransformation is a process in which molecules are modified in the presence of a biocatalyst or enzymes, as well as the metabolic alterations that occur in organisms from exposure to the molecules. Microbial biotransformation is an important process in natural product drug discovery as novel compounds are biosynthesised. Additionally, biotransformation products offer compounds with improved efficacy, solubility, reduced cytotoxic and allows for the understanding of structure activity relationships. One of the driving forces for these impeccable findings are associated with the presence of cytochrome P450 monooxygenases that is present in all organisms such as mammals, bacteria, and fungi. Numerous fungal strains have been used and reported for their ability to biotransform different compounds. This review focused on studies using Alternaria species as biocatalysts in the biotransformation of natural product compounds. Alternaria species facilitates reactions that favour stereoselectivity, regioselectivity under mild conditions. Additionally, microbial biotransformation products, their application in food, pharmaceutical and agricultural sector is discussed in this review.

[Recent advances in the bioproduction of mannitol].

D-mannitol is a six-carbon sugar alcohol and one of the most abundant polyols in the nature. With antioxidant and osmotic pressure-regulating effects and non-metabolism by the human body, D-mannitol has been widely used in functional food and pharmaceutical industries. At present, a major way for industrial production of D-mannitol is chemical hydrogenation. In addition, D-Mannitol can be produced by microbial metabolism or catalysis. Compared with the chemical hydrogenation, the microbial methods for synthesizing mannitol do not produce sorbitol as a by-product and have the advantages of mild reaction conditions, strong specificity, and high conversion rate. Microbial fermentation is praised for easy access of strains and raw materials and simple separation of the product. Microbial catalysis usually adopts a multi-enzyme coupling strategy, which uses enzymes produced by engineered bacteria for whole-cell catalysis, and the cofactor recycling pathway is introduced to replenish expensive cofactor. This method can achieve high yields with cheap substrates under mild conditions without the formation of by-products. However, the application of microbial methods in the industrial production of D-mannitol is limited by the high costs of fermentation media and substrates and the long reaction time. This article reviews the reported microbial methods for producing D-mannitol, including the use of high-yielding strains and their fermentation processes, the utilization of low-cost substrates, whole-cell catalytic strategies, and the process control for high productivity. The biosynthesis of mannitol is not only of great significance for promoting industrial upgrading and realizing green manufacturing, but also provides strong support for the development of new bio-based products to meet the growing market demand. With the continuous improvement of technological innovation and industrial chain, it is expected to become one of the main ways of mannitol production in the future.

One-Pot Biocatalytic Conversion of Chemically Inert Hydrocarbons into Chiral Amino Acids through Internal Cofactor and H2O2 Recycling.

Chemically inert hydrocarbons are the primary feedstocks used in the petrochemical industry and can be converted into more intricate and valuable chemicals. However, two major challenges impede this conversion process: selective activation of C-H bonds in hydrocarbons and systematic functionalization required to synthesize complex structures. To address these issues, we developed a multi-enzyme cascade conversion system based on internal cofactor and H2O2 recycling to achieve the one-pot deep conversion from heptane to chiral (S)-2-aminoheptanoic acid under mild conditions. First, a hydrogen-borrowing-cycle-based NADH regeneration method and H2O2in situ generation and consumption strategy were applied to realize selective C-H bond oxyfunctionalization, converting heptane into 2-hydroxyheptanoic acid. Integrating subsequent reductive amination driven by the second hydrogen-borrowing cycle, (S)-2-aminoheptanoic acid was finally accumulated at 4.57 mM with eep > 99%. Hexane, octane, 2-methylheptane, and butylbenzene were also successfully converted into the corresponding chiral amino acids with eep > 99%. Overall, the conversion system employed internal cofactor and H2O2 recycling, with O2 as the oxidant and ammonium as the amination reagent to fulfill the enzymatic conversion from chemically inert hydrocarbons into chiral amino acids under environmentally friendly conditions, which is a highly challenging transformation in traditional organic synthesis.

Nicotinamide Riboside: What It Takes to Incorporate It into RNA.

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR.

Nanoflower Microreactor Based Versatile Enhancer for Recognition Cofactor-Dependent Enzyme Biocatalysis toward Saxitoxin Detection.

Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.

The Toxoplasma gondii mitochondrial transporter ABCB7L is essential for the biogenesis of cytosolic and nuclear iron-sulfur cluster proteins and cytosolic translation.

Iron-sulfur (Fe-S) clusters are ubiquitous inorganic cofactors required for numerous essential cellular pathways. Since they cannot be scavenged from the environment, Fe-S clusters are synthesized de novo in cellular compartments such as the apicoplast, mitochondrion, and cytosol. The cytosolic Fe-S cluster biosynthesis pathway relies on the transport of an intermediate from the mitochondrial pathway. An ATP-binding cassette (ABC) transporter called ABCB7 is responsible for this role in numerous commonly studied organisms, but its role in the medically important apicomplexan parasites has not yet been studied. Here we identify and characterize a Toxoplasma gondii ABCB7 homolog, which we name ABCB7-like (ABCB7L). Genetic depletion shows that it is essential for parasite growth and that its disruption triggers partial stage conversion. Characterization of the knock-down line highlights a defect in the biogenesis of cytosolic and nuclear Fe-S proteins leading to defects in protein translation and other pathways including DNA and RNA replication and metabolism. Our work provides support for a broad conservation of the connection between mitochondrial and cytosolic pathways in Fe-S cluster biosynthesis and reveals its importance for parasite survival. IMPORTANCE: Iron-sulfur (Fe-S) clusters are inorganic cofactors of proteins that play key roles in numerous essential biological processes, for example, respiration and DNA replication. Cells possess dedicated biosynthetic pathways to assemble Fe-S clusters, including a pathway in the mitochondrion and cytosol. A single transporter, called ABCB7, connects these two pathways, allowing an essential intermediate generated by the mitochondrial pathway to be used in the cytosolic pathway. Cytosolic and nuclear Fe-S proteins are dependent on the mitochondrial pathway, mediated by ABCB7, in numerous organisms studied to date. Here, we study the role of a homolog of ABCB7, which we name ABCB7-like (ABCB7L), in the ubiquitous unicellular apicomplexan parasite Toxoplasma gondii. We generated a depletion mutant of Toxoplasma ABCB7L and showed its importance for parasite fitness. Using comparative quantitative proteomic analysis and experimental validation of the mutants, we show that ABCB7L is required for cytosolic and nuclear, but not mitochondrial, Fe-S protein biogenesis. Our study supports the conservation of a protein homologous to ABCB7 and which has a similar function in apicomplexan parasites and provides insight into an understudied aspect of parasite metabolism.

Glucocorticoid Receptor-Dependent Binding Analysis Using Chromatin Immunoprecipitation and Quantitative Polymerase Chain Reaction.

ChIP-qPCR offers the opportunity to identify interactions of DNA-binding proteins such as transcription factors and their respective DNA binding sites. Thereby, transcription factors can interfere with gene expression, resulting in up- or downregulation of their target genes. Utilizing ChIP, it is possible to identify specific DNA binding sites that are bound by the DNA-binding proteins in dependence on treatment or prevailing conditions. During ChIP, DNA-binding proteins are reversibly cross-linked to their DNA binding sites and the DNA itself is fragmented. Using bead-captured antibodies, the target proteins are isolated while still binding their respective DNA response element. Using quantitative PCR, these DNA fragments are amplified and quantified. In this protocol, DNA binding sites of the glucocorticoid receptor are identified by treatment with the synthetic glucocorticoid Dexamethasone in murine bone marrow-derived macrophages.

The INSIGHT platform: Enhancing NAD(P)-dependent specificity prediction for co-factor specificity engineering.

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Construction of a bioelectrocatalytic system with bacterial surface displayed enzyme-nanomaterial hybrids.

To take advantage of the high specificity of enzymatic catalysis along with the high efficiency of electrochemical cofactor regeneration, a bacterial surface displayed enzyme-nanomaterial hybrid bioelectrocatalytic system is herein developed. A cofactor-dependent xylose reductase, capable of reducing xylose to xylitol, is displayed on the surface of Bacillus subtilis, followed by the attachment of copper nanomaterials via the binding of His-tagged enzyme with the nickel ion. This hybrid system can regenerate NADPH with a highest efficiency of 71.6% in 4 h without the usage of extra electron mediators, and 2.35 mM of xylitol can be synthesized after a series of optimization processes. This work opens up new possibilities for the construction and application of bioelectrocatalytic systems with enzyme-nanomaterial hybrids.

AtDREB2G is involved in the regulation of riboflavin biosynthesis in response to low-temperature stress and abscisic acid treatment in Arabidopsis thaliana.

Riboflavin (RF) serves as a precursor to flavin mononucleotide and flavin adenine dinucleotide, which are crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the dehydration-responsive element binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G is involved in the positive regulation of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.