Alcohol Dehydrogenase Research Articles

Design of a Self-Sufficient Whole-Cell Cascade for the Production of (R)-Citronellal from Geraniol.

(R)-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (-)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of (R)-citronellal from geraniol using alcohol dehydrogenase from Escherichia coli K12 (AdhP) and ene-reductase from Saccharomyces cerevisiae YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP260T/284A/268P (M3) was obtained that demonstrated a 1.28-fold improvement in catalytic efficiency (kcat/Km) toward geraniol. After optimization of the reaction conditions, the hydrogen-borrowing cascade system achieved the conversion of 23.14 g/L geraniol into (R)-citronellal at a conversion rate of 98.23% with 96.7% ee. This work represents an alternative approach for the biosynthesis of (R)-citronellal without sacrificing a cosubstrate or additional enzymes.

Metabolic engineering of Escherichia coli for upcycling of polyethylene terephthalate waste to vanillin.

Polyethylene terephthalate (PET) waste presents a significant environmental challenge due to its durability and resistance to degradation. Innovative approaches for upcycling PET waste into high-value chemicals can mitigate these issues while contributing to a circular economy. In this study, we developed a multi-enzyme cascade system in E. coli to convert PET-derived monomer terephthalic acid (TPA) into vanillin (VAN). The metabolic engineering approach was then employed to increase VAN production, including 1) inhibition of VAN degradation by knocking out endogenous aldehyde reductases and alcohol dehydrogenases and 2) enhancement of TPA uptake by modifying membrane proteins to increase cell permeability. The engineered E. coli demonstrated a VAN production of 658.55 mg/L from 1992 mg/L of TPA with a molar conversion rate of 71.1 %, representing the highest production of VAN using TPA as the substrate. Additionally, the engineered E. coli effectively converted post-consumer PET waste into VAN under mild conditions, with the highest production of 259.2 mg/L in 20× diluted PET hydrolysates, highlighting its potential for application in PET waste upcycling. This approach not only provides an environmentally friendly alternative to traditional chemical synthesis but also offers substantial economic benefits by transforming low-value waste into high-value chemicals.

Ancestor-originated engineering of medium-chain alcohol dehydrogenase enhances its catalytic compatibility by balancing activity, stability, and selectivity trade-offs

The role of medium-chain alcohol dehydrogenases (MDR) in the preparation of chiral drug intermediates is indispensable; however, limited activity and stability towards unnatural substrates are crucial factors that restrict their application. Further, research on the ancestral MDRs remains unclear. Therefore, enhancing the activity, selectivity, and stability of MDRs is necessary. In this study, we resurrected ancestors of the MDR family using ancestral sequence reconstruction with descendant GcADH as a probe. Among these ancestors, ancestor 136 exhibited increased thermal stability (ΔTm = 19.5 °C) and had a comparable conversion and selectivity as GcADH to the selected substrates. After in-silico screening using the Funclib tool, we obtained the mutant anc136-R2, which exhibited strengthened selectivity and activity towards N-Benzyl-3,3-difluoropiperidin-4-one (1a). Moreover, the mutations of anc136-R2 were simultaneously mapped to GcADH, resulting GcADH-R2. Based on the results of substrate spectrum characterisation, anc136 was more receptive to mutations. Specifically, for the substrates N-Boc-3-piperidone (4a) and N-boc-3-oxoazepane (7a), the conversion rates of anc136-R2 were 29 and 57 times higher, respectively, than those of GcADH-R2, without compromising selectivity. In conclusion, this work provides guidance for exploring the evolutionary trajectory of the MDR family and further illustrates the superior balanced “trade-off” of activity–selectivity–stability on ancestral enzymes.

Switching the Stereopreference of TeSADH: Enabling Anti‐Prelog Asymmetric Reduction of Aryl‐Ring‐Containing Ketones

AbstractThe biocatalytic asymmetric reduction of prochiral ketones offers a promising approach for producing optically active secondary alcohols. Alcohol dehydrogenases (ADHs) are enzymes that facilitate the reversible conversion of prochiral ketones into their corresponding optically active secondary alcohols, and thus are pivotal for this process. Most ADHs adhere to Prelog's rule in determining the stereopreference for the asymmetric reduction of prochiral ketones. This study focuses on the ΔP84/A85G mutation of TeSADH, demonstrating its capability to reduce aryl‐ring‐containing ketones to their corresponding alcohols in anti‐Prelog mode with high stereoselectivities. The study also highlights the crucial role of P84 in switching the stereopreference of TeSADH in the asymmetric reduction of aryl‐ring‐containing ketones. Furthermore, this mutant exhibits a broad substrate scope, including substrates accepted by the previously reported W110 mutants of TeSADH with opposite stereopreference. The ability to create mutants of the same enzyme with opposite stereopreferences presents intriguing opportunities for fascinating transformations, such as bienzymatic racemization, which can be utilized in dynamic kinetic resolution, and stereoinversion using two enantiocomplementary mutants of the same enzyme.

Degradation of polystyrene plastics by alkane monooxygenase and alcohol dehydrogenase

<p style="text-indent:0.0000pt; text-align:justify"><span style="font-size:10.5pt"><span style="line-height:200%"><span style="font-family:"Times New Roman""><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">The consumption of plastic products has led to the generation of large amounts of plastic waste, which is persistent and difficult to degrade. Polystyrene (PS) is one of the six most important plastics in the world and is difficult to degrade in the environment owing to its high stability. To investigate PS degradation by biological enzymes, two oxidoreductases, alkane hydroxylase (AlkB) and alcohol dehydrogenase (Adh), were selected from the bacterial strain </span></span></span><i><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">Acinetobacter johnsonii</span></span></span></i><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%"> JUN01, which has been proven to be capable of degrading PS. Genetically engineered bacteria capable of expressing AlkB and Adh were constructed using genetic engineering technology, and the degradation activities of AlkB monoenzyme, Adh monoenzyme, and AlkB–Adh composite enzyme were investigated. Thermal field emission scanning electron microscopy </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">(SEM) </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">and water contact angle </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">(WCA) </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">measurements demonstrated that the investigated enzymes transformed PS from hydrophobic to hydrophilic. Fourier transform infrared </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">(FTIR)</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%"> results showed that after enzymatic hydrolysis, the number of hydroxyl groups (–OH) increased, the number of C=C and C=O bonds increased, and the structure of benzene ring was disrupted by degradation using AlkB monoenzyme and AlkB–Adh composite enzyme. X-ray photoelectron spectroscopy </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">(XPS) </span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">showed that the characteristic C–C bonds of PS decreased, and the number of C–O bonds and C=O bonds increased. The molecular weight of PS changed after digestion, as determined by high-temperature gel chromatography</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%"> (</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">GPC)</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">. Thermogravimetric analysis</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%"> (TGA)</span></span></span><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%"> was used to demonstrate a decrease in the thermal stability of PS after digestion. These results showed that the AlkB monoenzyme, Adh monoenzyme, and AlkB–Adh composite enzyme all had PS degradation activity, demonstrating that the idea of using a composite enzyme to degrade PS was feasible. In addition, Adh exhibited degradation activity in two different coenzyme reaction systems. Therefore, these results provide a theoretical basis and data support for the future degradation of PS by bioenzymes.</span></span></span></span></span></span></p> <p style="text-indent:0.0000pt; text-align:justify"><span style="font-size:10.5pt"><span style="line-height:200%"><span style="font-family:"Times New Roman""><span style="font-size:12.0000pt"><span style="font-family:'Times New Roman'"><span style="line-height:200%">Keywords: Plastic degrading enzymes; Microplastics; Biodegradation</span></span></span></span></span></span></p>

MYB168 and WRKY20 transcription factors synergistically regulate lignin monomer synthesis during potato tuber wound healing.

Lignin is a critical component of the closing layer of the potato (Solanum tuberosum L.) tuber during healing; however, the molecular mechanism of its formation remains poorly understood. To elucidate the molecular mechanism of tuber healing, we screened the genes encoding transcription factors that regulate lignin synthesis(StMYB24/49/105/144/168, StWRKY19/20/22/23/34) and the key genes involved in lignin monomer synthesis (PHENYLALANINE AMMONIA LYASE 5 (StPAL5) and CINNAMYL ALCOHOL DEHYDROGENASE 14 (StCAD14)) for induced expression after wounding using transcriptome data. DLR, Y1H, EMSA, and ChIP-qPCR assays revealed that StMYB168 could bind directly to the StPAL5 and StCAD14 promoters to activate their expression and that StWRKY20 enhanced this regulation with a synergistic effect. Y2H, BiFC, and Co-IP assays showed that StMYB168 interacted with StWRKY20 to form a MYB-WRKY complex. Furthermore, transient overexpression of StMYB168 and StWRKY20 in Nicotiana benthamiana leaves upregulated the expression of NbPAL and NbCAD10 and promoted lignin accumulation in the leaves. In addition, overexpression of StWRKY20 and StMYB168 together resulted in higher expression levels of NbPAL and NbCAD10 and higher levels of lignin monomer and total lignin. In contrast, silencing of StMYB168 and StWRKY20 in potato significantly reduced the lignin content of wounded tubers. In conclusion, StMYB168 and StWRKY20 are important regulators of lignin biosynthesis in potato tubers during healing and can positively regulate lignin biosynthesis by forming a complex. The elucidation of this regulatory module provides information on the regulatory mechanism of lignin monomer synthesis in wounded tubers at the transcriptional level.

Comparative Analysis of Two Soybean Cultivars Revealed Tolerance Mechanisms Underlying Soybean Adaptation to Flooding

Flooding stress poses a significant challenge to soybean cultivation, impacting plant growth, development, and ultimately yield. In this study, we investigated the responses of two distinct soybean cultivars: flooding-tolerant Nanxiadou 38 (ND38) and flooding-sensitive Nanxiadou 45 (ND45). To achieve this, healthy seedlings were cultivated with the water surface consistently maintained at 5 cm above the soil surface. Our objective was to elucidate the physiological and molecular adaptations of the two cultivars. Under flooding stress, seedlings of both cultivars exhibited significant dwarfing and a notable decrease in root length. While there were no significant differences in the dry weight of aboveground shoots, the dry weight of underground shoots in ND38 was strikingly decreased following flooding. Additionally, total chlorophyll content decreased significantly following flooding stress, indicating impaired photosynthetic performance of the cultivars. Moreover, malondialdehyde (MDA) levels increased significantly after flooding, particularly in the ND45 cultivar, suggesting heightened oxidative stress. Expression analysis of methylation and demethylation genes indicated that MET1 and DME play crucial roles in response to flooding stress in soybeans. Meanwhile, analysis of the hemoglobin family (GLBs), aquaporin family (AQPs), glycolytic pathway-related genes, and NAC transcription factor-related genes identified GLB1-1 and GLB1-2, GLB2-2, PIP2-6, PIP2-7, TIP2-2, TIP4-1, TIP5-1, Gm02G222400 (fructose-bisphosphate aldolase), Gm19G017200 (glucose-6-phosphate isomerase), and Gm04G213900 (alcohol dehydrogenase 1) as key contributors to flooding tolerance in both soybean cultivars. These findings provide crucial insights into the physiological and molecular mechanisms underlying flooding tolerance in soybeans, which could guide future molecular breeding strategies for the development of flooding-tolerant soybean cultivars.

Combining GWAS and RNA-Seq approaches identifies the FtADH1 gene for drought resistance in Tartary buckwheat

Drought is one of the major environmental constraints that significantly affects seedling emergence, yield, and quality of Tartary buckwheat, thereby hindering the development of its industry. However, the molecular mechanisms underlying drought tolerance genes in Tartary buckwheat remain largely unexplored. Alcohol dehydrogenase (ADH), one of the essential plant proteins, plays a crucial role in growth, development, and stress responses, but its specific role in drought resistance is still unclear. In this study, we identified an ADH gene FtADH1, using a membership function value of drought tolerance (MFVD) combined with a genome-wide association study (GWAS) and transcriptomic profiles that confers drought tolerance in Tartary buckwheat. Our findings demonstrated that the overexpression of FtADH1 in Arabidopsis and Tartary buckwheat hairy roots enhances drought tolerance by promoting root elongation and mitigating elevated levels of reactive oxygen species (ROS). Our findings demonstrated that FtADH1 can enhanced tolerance to drought stresses in both Tartary buckwheat and Arabidopsis. This study identifies the FtADH1 as a new player in affecting ROS level and the stress response of Tartary buckwheat by regulating protective enzyme activities at a high level to scavenge ROS and modulating root growth under drought stress. Further, we identified proteins interacting with FtADH1 through a prokaryotic expression pull-down assay combined with mass spectrometry, revealing that FtADH1 specifically interacts with the S-adenosyl-L-methionine (SAM) synthetase protein, FtSAMS1. Overexpression of FtSAMS1 was found to enhance ADH enzymatic activity, leading to increased SAM content in overexpressing Tartary buckwheat hairy roots under water-deficit conditions. Additionally, FtSAMS1 overexpression induced a drought-resistant phenotype in Arabidopsis and Tartary buckwheat hairy roots under drought stress, revealing the biological function of FtADH1. Evolutionary analysis indicates that ADH1 in Fagopyrum species has undergone significant evolutionary events, including duplication and purifying selection, which may contribute to functional diversification and adaptive advantages such as drought resistance in cultivated buckwheat. In summary, this study proposes that FtADH1 is a key contributor to drought tolerance, and its interaction with FtSAMS1 holds potential for the development of drought-resistant varieties in Tartary buckwheat and its relative species.

Vanadium bioreduction in an ethane-based membrane biofilm reactor: Performance and mechanism

Vanadium (V(V)) contamination in groundwater poses a serious threat to human health. V(V) bioreduction is an effective method for vanadium contamination remediation. This study confirmed that ethane (C2H6) as an electron donor could realize efficient V(V) bioreduction. V(V) reduction by C2H6-based membrane biofilm reactor was enhanced with the increase of V(V) loading in the influent. Vanadium in the biofilm was mainly present as V(IV) precipitation. The C2H6-oxidizing bacteria Mycobacterium and Methylocystis used alkane 1-monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase to generate metabolic intermediates, such as ethanol, acetaldehyde, and acetic acid, with the participation of O2, which were then utilized by V(V)-reducing bacteria Geothrix and Rudaea to reduce V(V) to V(IV) intracellularly and extracellularly under nicotinamide adenine dinucleotide (NADH) and cytochrome-c oxidase. This result could promote the application of C2H6 in the ex-situ remediation of V(V)-contamination groundwater.

Alcohol dehydrogenase (ADH)-activating peptides from Acaudina leucoprocta: Isolation, identification, molecular docking and peptide-enzyme interaction

Alcohol dehydrogenase (ADH)-activating peptides from Acaudina leucoprocta: Isolation, identification, molecular docking and peptide-enzyme interaction

Controllable synthesis of hydrogen-bonded organic framework encapsulated enzyme for continuous production of chiral hydroxybutyric acid in a two-stage cascade microreactor

Constructing a framework carrier to stabilize protein conformation, induce high embedding efficiency, and acquire low mass-transfer resistance is an urgent issue in the development of immobilized enzymes. Hydrogen-bonded organic frameworks (HOFs) have promising application potential for embedding enzymes. In fact, no metal involvement is required, and HOFs exhibit superior biocompatibility, and free access to substrates in mesoporous channels. Herein, a facile in situ growth approach was proposed for the self-assembly of alcohol dehydrogenase encapsulated in HOF. The micron-scale bio-catalytic composite was rapidly synthesized under mild conditions (aqueous phase and ambient temperature) with a controllable embedding rate. The high crystallinity and periodic arrangement channels of HOF were preserved at a high enzyme encapsulation efficiency of 59%. This bio-composite improved the tolerance of the enzyme to the acid-base environment and retained 81% of its initial activity after five cycles of batch hydrogenation involving NADH coenzyme. Based on this controllably synthesized bio-catalytic material and a common lipase, we further developed a two-stage cascade microchemical system and achieved the continuous production of chiral hydroxybutyric acid (R-3-HBA).

Dielectric Barrier Discharge Cold Plasma Improves Storage Stability in Paddy Rice by Activating the Phenylpropanoid Biosynthesis Pathway.

A nonthermal pretreatment using dielectric barrier discharge cold plasma (DBD-CP) was developed to improve the stress resistance of paddy rice during postharvest storage. The physicochemical properties, bioactive characteristics, and secondary metabolites of paddy rice were assessed after applying an optimized DBD-CP procedure, with enzyme activities and gene expression monitored over a 60 day storage period at 35 °C. A 17.06% reduction in the total color change index was noted in the DBD-CP group. Bioactive compounds, particularly gallic acid, were significantly increased, enhancing the defense mechanisms against high-temperature stress. Nontargeted metabolomics analysis indicated an upregulation of phenylpropanoid metabolism in DBD-CP-treated rice compared to controls, with notable increases in secondary metabolites such as coumaric acid, caffeic acid, and sinapic acid, suggesting potential biomarkers for stress resistance. Further verification showed significant enhancements in key enzymes of phenylpropanoid metabolism, including phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), plant coumaric acid-3-hydroxylase (C3H), and cinnamyl alcohol dehydrogenase (CAD), with increases ranging from 1.71 to 2.28 times. Gene expression levels of OsPAL7, OsC4H4, and OsCAD2 aligned with these enzymatic changes post-DBD-CP treatment. In conclusion, DBD-CP treatment can modulate phenylpropanoid metabolism in paddy rice, thereby enhancing bioactive compound levels to reduce stress damage during high-temperature storage.

Mature Tubers of Cyperus rotundus Confer Flooding Tolerance by Adopting A “Low-oxygen Quiescence Strategy”, which may Contribute to Its Emergence in Rice Fields

Abstract Purple nutsedge (Cyperus rotundus L.) is one of the world’s resilient upland weeds, primarily spreading through its tubers. Its emergence in rice fields has been increasing, likely due to changing paddy farming practices. This study aimed to investigate how C. rotundus, an upland weed, can withstand soil flooding and become a problematic weed in rice (Oryza sativa L.) fields. The first comparative analysis focused on the survival and recovery characteristics of growing and mature tubers of C. rotundus exposed to soil flooding conditions. Notably, mature tubers exhibited significant survival and recovery abilities in these environments. Based on this observation, further investigation was carried out to explore the morphological structure, non-structural carbohydrates, and respiratory mechanisms of mature tubers in response to prolonged soil flooding. Over time, the mature tubers did not form aerenchyma but instead gradually accumulated lignified sclerenchyma fibers, with lignin content also increasing. After 90 days, the lignified sclerenchyma fibers and lignin contents were 4.0 and 1.1 times higher than those in no soil flooding (CK). Concurrently, soluble sugar content decreased while starch content increased, providing energy storage, and alcohol dehydrogenase (ADH) activity rose to support anaerobic respiration via alcohol fermentation. These results indicated that mature tubers survived in soil flooding conditions by adopting a “low-oxygen quiescence strategy”, which involves morphological adaptations through the development of lignified sclerenchyma fibers, increased starch reserves for energy storage, and enhanced anaerobic respiration. This mechanism likely underpins the flooding tolerance of mature C. rotundus tubers, allowing them to endure unfavorable conditions and subsequently germinate and grow once flooding subsides. This study provides a preliminary explanation of the mechanism by which mature tubers of C. rotundus from the upland areas confer flooding tolerance, shedding light on the reasons behind this weed’s increasing presence in rice fields.

Systematic Analysis of Cinnamyl Alcohol Dehydrogenase Family in Cassava and Validation of MeCAD13 and MeCAD28 in Lignin Synthesis and Postharvest Physiological Deterioration.

Cassava (Manihot esculenta Crantz) is used as a biomass energy material and an effective supplement for food and feed. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of lignin biosynthesis and is responsible for various stresses. However, systematic investigations of the CAD gene family in cassava have been poorly understood. In this study, a genome-wide survey and bioinformatics analysis of CAD gene family was performed, transcriptomics, qRT-PCR, gene silencing and stress of yeast cell were used for excavate and validate the candidate MeCADs gene. 36 MeCADs genes unevenly distributed across 12 chromosomes were identified. Through phylogenetic analyses alongside their Arabidopsis counterparts, these MeCADs were divided into four groups, each containing a similar structure and conserved motifs. Interestingly, transcriptome data analysis revealed that 32 MeCAD genes were involved in the postharvest physiological deterioration (PPD) process, whereas 27 MeCAD genes showed significant changes. Additionally, the relative quantitative analysis of 6 MeCAD genes demonstrated that they were sensitive to PPD, suggesting that they may be involved in the regulation of PPD. Silencing MeCAD13 and MeCAD28 further showed that lignin content significantly decreased in the leaves. The wound-stress tolerance of transgenic yeast cells was enhanced after transformation with MeCAD13 and MeCAD28. MeCAD13 and MeCAD28 may play positive roles in lignin biosynthesis and PPD response, respectively. These results provided a systematic functional analysis of MeCADs in cassava and paved a new way to genetically modify lignin biosynthesis and PPD tolerance.

Characterization of human alcohol dehydrogenase 4 and aldehyde dehydrogenase 2 as enzymes involved in the formation of 5-carboxylpirfenidone, a major metabolite of pirfenidone.

Pirfenidone (PIR) is used to treatment of idiopathic pulmonary fibrosis. After oral administration, it is metabolized by cytochrome P450 1A2 to 5-hydroxylpirfenidone (5-OH PIR) and further oxidized to 5-carboxylpirfenidone (5-COOH PIR), a major metabolite excreted in the urine (90% of the dose). This study aimed to identify enzymes that catalyze the formation of 5-COOH PIR from 5-OH PIR in the human liver. 5-COOH PIR was formed from 5-OH PIR in the presence of NAD+ by human liver microsomes (HLM) more than by human liver cytosol (HLC), with the concomitant formation of the aldehyde form (5-CHO PIR) as an intermediate metabolite. By purifying enzymes from HLM, alcohol dehydrogenases (ADHs) were identified as candidate enzymes catalyzing 5-CHO PIR formation, although ADHs are localized in the cytoplasm. Among constructed recombinant ADH1-5 expressed in HEK293T cells, only ADH4 efficiently catalyzed 5-CHO PIR formation from 5-OH PIR with a K m value (29.0 {plus minus} 4.9 µM), which was close to that by HLM (59.1 {plus minus} 4.6 µM). In contrast to commercially available HLC, in-house prepared HLC clearly showed substantial 5-CHO PIR formation, and ADH4 protein levels were significantly (rs = 0.772, P < 0.0001) correlated with 5-CHO PIR formation in 25 in-house prepared HLC samples. Some components of the commercially available HLC may inhibit ADH4 activity. Disulfiram, an inhibitor of aldehyde dehydrogenases (ALDH), decreased 5-COOH PIR formation and increased 5-CHO PIR formation from 5-OH PIR in HLM. ALDH2 knockdown in HepG2 cells by siRNA decreased 5-COOH PIR formation by 61%. Significance Statement This study clarified that 5-COOH PIR formation from 5-OH PIR proceeds via a two-step oxidation reaction catalyzed by ADH4 and disulfiram-sensitive enzymes, including ALDH2. Inter-individual differences in the expression levels or functions of these enzymes could cause variations in the pharmacokinetics of PIR.

Redox Biocatalysis in Lidocaine-based Hydrophobic Deep Eutectic Solvents: Non-conventional Media Outperform Aqueous Conditions.

Redox biocatalysis is an important pillar of the chemical industry. Yet, the enzymes' nature restricts most reactions to aqueous conditions, where the limited substrate solubility leads to unsustainable diluted biotranformations. Non-aqueous media represent a strategic solution to conduct intensified biocatalytic routes. Deep eutectic solvents (DESs) are designable solvents that can be customized to meet specific application needs. Within the large design space of combining DES components (and ratios), hydrophobic DESs hold the potential to be both enzyme-compatible - keeping the enzymes' hydration -, and solubilizers for hydrophobic reactants. We explored two hydrophobic DESs, lidocaine/oleic acid, and lidocaine/decanoic acid, as reaction media for carbonyl reduction catalyzed by horse liver alcohol dehydrogenase, focusing on the effect of water contents and on maximizing substrate loadings. Enzymes remained highly active and stable in the DESs with 20 wt.% buffer, whereas the reaction performance in DESs outperformed the pure buffer system with hydrophobic substrates (e.g., cinnamaldehyde to form the industrially relevant cinnamyl alcohol), with a 2-fold higher specific activity. Notably, the cinnamaldehyde reduction was for the first time performed at 800 mM (~100 g[[EQUATION]]L-1) with full conversion, which opens up new avenues to industrial applications of hydrophobic DESs for enzyme catalysis.

Genome-wide identification of alcohol dehydrogenase (ADH) gene family in oilseed rape (Brassica napus L.) and BnADH36 functional verification under salt stress

BackgroundAlcohol dehydrogenase (ADH) is an enzyme that binds to zinc, facilitating the interconversion of ethanol and acetaldehyde or other corresponding alcohols/aldehydes in the pathway of ethanol fermentation. It plays a pivotal role in responding to environmental stress. However, the response of the ADH family to abiotic stress remains unknown in rapeseed.ResultIn this study, we conducted a comprehensive genome-wide investigation of the ADH family in rapeseed, encompassing analysis of their gene structure, replication patterns, conserved motifs, cis-acting elements, and response to stress. A total of 47 ADH genes were identified within the rapeseed genome. Through phylogenetic analysis, BnADHs were classified into four distinct clades (I, II, IV, V). Prediction of protein domains revealed that all BnADH members possessed a GroES-like (ADH_N) domain and a zinc-bound (ADH_zinc_N) domain. Analysis of promoter sequences demonstrated that BnADHs contained numerous cis-acting elements associated with hormone and stress responses, indicating their widespread involvement in various biological regulatory processes. Expression profiling under different concentrations of salt stress treatments (0%, 0.4%, 0.8%, 1.0% NaCl) further highlighted the significant role played by the BnADH family in abiotic stress response mechanisms. Overexpression of BnADH36 in rapeseed significantly improved the salt tolerance of rapeseed.ConclusionThe features of the BnADH family in rapeseed was comprehensively characterized in this study, which could provide reference to the research of BnADHs in abiotic stress response.

Cannabidiol (CBD) modulates the transcriptional profile of ethanol-exposed human dermal fibroblast cells.

Cannabidiol (CBD) is abundant in the Cannabis sativa plant and exhibits complex immunomodulatory, anxiolytic, antioxidant, and antiepileptic properties. Several studies suggest that CBD could be used for different purposes in alcohol use disorder (AUD) and alcohol-related injuries to the brain and the liver. In this study, we focused on analyzing transcriptional alterations in human dermal fibroblasts (HDFs) cell line challenged simultaneously with ethanol and CBD as an ethanol-protective agent. We aimed to expose the genes and pathways responsible for at least some of the CBD effects in those cells that can be related to the AUD. Transcriptome analysis was performed using HDFs cell line that expresses both cannabinoid receptors and can metabolize ethanol through alcohol dehydrogenase activity. Fibroblasts are also responsible for the progression of liver fibrosis, a common comorbidity in AUD. With the use of a cellular test, we found that CBD at the lowest applied concentration (0.75μM) was able to stimulate depressed metabolism and reduce the level of apoptosis of cells treated with different concentrations of ethanol to the level observed in the control cells. Similar observations were made at the transcriptome level, in which cells treated with ethanol and CBD had similar expression profiles to the control cells. CBD also affects several genes connected with extracellular matrix formation (especially its collagen constituent), which can have potential implications for, e.g., fibrosis process.

Isolation and degradation characterization of a 1, 4-dioxane-degrading bacterial strain

To address the potential pollution caused by the carcinogen 1, 4-dioxane in aquatic environments, we isolated a highly efficient 1, 4-dioxane-degrading bacterial strain, designated as DXTK-010, from the groundwater contaminated by 1, 4-dioxane. According to the morphological characteristics, the phylogenetic tree established based on the 16S rRNA gene sequence, and the whole genome sequence, we identified DXTK-010 as Aminobacter aminovorans. This strain demonstrated robust degradation capacity within a temperature range of 20 ℃ to 37 ℃ and a pH range of 5.0 to 8.0. Furthermore, single-factor experiments indicated the optimal degradation conditions at 30 ℃ and pH 7.5. Under the optimal conditions, the strain completely degraded 200 mg/L of 1, 4-dioxane within 24 h, achieving a maximum degradation rate of 9.367 mg/(L·h). The Monod equation was adopted to fit the degradation kinetics of 1, 4-dioxane at different initial concentrations, which revealed a maximum specific degradation rate of 0.224 mg 1, 4-dioxane/(mg protein·h), a half-saturation constant (Ks) of 41.350 mg/L, and a cell yield of 0.130 mg protein/(mg 1, 4-dioxane). Whole genome sequencing revealed a circular chromosome and three plasmids within DXTK-010. Functional gene annotation and analysis underscored the significance of the propane monooxygenase gene cluster and alcohol dehydrogenase gene in facilitating the efficient degradation of 1, 4-dioxane by this strain. DXTK-010 outperformed the existing degraders for 1, 4-dioxane, expanding the strain resources for the bioremediation of 1, 4-dioxane pollution. This study provides a theoretical basis for the practical application of DXTK-010 in the remediation of 1, 4-dioxane pollution.

Magnetic protein aggregates generated by supramolecular assembly of ferritin cages - a modular strategy for the immobilization of enzymes.

Efficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. This study aimed to introduce a novel immobilization strategy using in vivo-produced magnetic protein aggregates (MPAs). MPA production was achieved by expressing gene fusions of the yellow fluorescent protein variant citrine and ferritin variants, including a magnetically enhanced Escherichia coli ferritin mutant. Cellular production of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, driven by citrine-dependent dimerization of ferritin cages. Magnetic properties were confirmed using neodymium magnets. A bait/prey strategy was used to attach alcohol dehydrogenase (ADH) to the MPAs, creating catalytically active MPAs (CatMPAs). These CatMPAs were purified via magnetic columns or centrifugation. The fusion of the mutant E. coli ferritin to citrine yielded fluorescent, insoluble protein aggregates, which are released upon cell lysis and coalesce into MPAs. MPAs display magnetic properties, as verified by their attraction to neodymium magnets. We further show that these fully in vivo-produced protein aggregates can be magnetically purified without ex vivo iron loading. Using a bait/prey strategy, MPAs were functionalized by attaching alcohol dehydrogenase post-translationally, creating catalytically active magnetic protein aggregates (CatMPAs). These CatMPAs were easily purified from crude extracts via centrifugation or magnetic columns and showed enhanced stability. This study presents a modular strategy for the in vivo production of MPAs as scaffold for enzyme immobilization. The approach eliminates the need for traditional, expensive carriers and simplifies the purification process by leveraging the insoluble nature and the magnetic properties of the aggregates, opening up the potential for novel, streamlined applications in biocatalysis.