Genome-wide analysis and identification of Carotenoid Cleavage Oxygenase (CCO) gene family in coffee (coffee arabica) under abiotic stress.

The coffee industry holds importance, providing livelihoods for millions of farmers globally and playing a vital role in the economies of coffee-producing countries. Environmental conditions such as drought and temperature fluctuations can adversely affect the quality and yield of coffee crops.Carotenoid cleavage oxygenases (CCO) enzymes are essential for coffee plants as they help break down carotenoids contributing to growth and stress resistance. However, knowledge about the CCO gene family in Coffee arabica was limited. In this study identified 21 CCO genes in Coffee arabica (C. arabica) revealing two subfamilies carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxy carotenoid dioxygenases (NCED) through phylogenic analysis. These subfamilies exhibited distribution patterns in terms of gene structure, domains, and motifs. The 21 CaCCO genes, comprising 5 NCED and 16 CCD genes were found across chromosomes. Promoter sequencing analysis revealed cis-elements that likely interact with plant stress-responsive, growth-related, and phytohormones, like auxin and abscisic acid. A comprehensive genome-wide comparison, between C. arabica and A. thaliana was conducted to understand the characteristics of CCO genes. RTqPCR data indicated that CaNCED5, CaNCED6, CaNCED12, and CaNCED20 are target genes involved in the growth of drought coffee plants leading to increased crop yield, in a conditions, with limited water availability. This reveals the role of coffee CCOs in responding to abiotic stress and identifies potential genes useful for breeding stress-resistant coffee varieties.

NinaB and BCO Collaboratively Participate in the ß-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and ß, ß-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro ß-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with ß-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its ß-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the ß-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically ß-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Knockout of the Chlorophyll a Oxygenase Gene OsCAO1 Reduces Chilling Tolerance in Rice Seedlings.

Chilling stress is one of the main abiotic factors affecting rice growth and yield. In rice, chlorophyllide a oxygenase encoded by OsCAO1 is responsible for converting chlorophyllide a to chlorophyllide b, playing a crucial role in photosynthesis and thus rice growth. However, little is known about the function of OsCAO1 in chilling stress responses. The presence of the cis-acting element involved in low-temperature responsiveness (LTR) in the OsCAO1 promoter implied that OsCAO1 probably is a cold-responsive gene. The gene expression level of OsCAO1 was usually inhibited by low temperatures during the day and promoted by low temperatures at night. The OsCAO1 knockout mutants generated by the CRISPR-Cas9 technology in rice (Oryza sativa L.) exhibited significantly weakened chilling tolerance at the seedling stage. OsCAO1 dysfunction led to the accumulation of reactive oxygen species and malondialdehyde, an increase in relative electrolyte leakage, and a reduction in antioxidant gene expression under chilling stress. In addition, the functional deficiency of OsCAO1 resulted in more severe damage to chloroplast morphology, such as abnormal grana thylakoid stacking, caused by low temperatures. Moreover, the rice yield was reduced in OsCAO1 knockout mutants. Therefore, the elevated expression of OsCAO1 probably has the potential to increase both rice yield and chilling tolerance simultaneously, providing a strategy to cultivate chilling-tolerant rice varieties with high yields.

Genome wide analysis of carotenoid cleavage oxygenases (CCO) gene family in Arachis hypogaea (peanut) under biotic stress.

Carotenoid cleavage oxygenases (CCOs) enzymes play a vital role in plant growth and development through the synthesis of apocarotenoids and their derivative. These chemicals are necessary for flower and fruit coloration, as well as the manufacture of plant hormones such as abscisic acid (ABA) and strigolactones, which control a variety of physiological processes. The CCOs gene family has not been characterized in Arachis hypogaea. Genome mining of A. hypogaea identifies 24 AhCCO gene members. The AhCCO gene family was divided into two subgroups based on the recent study of the Arabidopsis thaliana CCO gene family classification system. Twenty-three AhCCO genes, constituting 95.8% of the total, were regulated by 29 miRNAs, underscoring the significance of microRNAs (miRNAs) in governing gene expression in peanuts. AhCCD19 is the only gene that lacks a miRNA target site. The physicochemical characteristics of CCO genes and their molecular weights and isoelectric points were studied further. The genes were then characterized regarding chromosomal distribution, structure, and promoter cis-elements. Light, stress development, drought stress, and hormone responsiveness were discovered to be associated with AhCCO genes, which can be utilized in developing more resilient crops. The investigation also showed the cellular location of the encoded proteins and discovered that the peanut carotenoid oxygenase gene family's expansion was most likely the result of tandem, segmental, and whole-genome duplication events. The localization expresses the abundance of genes mostly in the cytoplasm and chloroplast. Expression analysis shows that AhCCD7 and AhCCD14 genes show the maximum expression in the apical meristem, lateral leaf, and pentafoliate leaf development, while AhNCED9 and AhNCED13 express in response to Aspergillus flavus resistance. This knowledge throws light on the evolutionary history of the AhCCO gene family and may help researchers better understand the molecular processes behind gene duplication occurrences in plants. An integrated synteny study was used to find orthologous carotenoid oxygenase genes in A. hypogaea, whereas Arabidopsis thaliana and Beta vulgaris were used as references for the functional characterization of peanut CCO genes. These studies provide a foundation for future research on the regulation and functions of this gene family. This information provides valuable insights into the genetic regulation of AhCCO genes. This technology could create molecular markers for breeding programs to develop new peanut lines.

A rationally designed miniature of soluble methane monooxygenase enables rapid and high-yield methanol production in Escherichia coli.

Soluble methane monooxygenase (sMMO) oxidizes a wide range of carbon feedstocks (C1 to C8) directly using intracellular NADH and is a useful means in developing green routes for industrial manufacturing of chemicals. However, the high-throughput biosynthesis of active recombinant sMMO and the ensuing catalytic oxidation have so far been unsuccessful due to the structural and functional complexity of sMMO, comprised of three functionally complementary components, which remains a major challenge for its industrial applications. Here we develop a catalytically active miniature of sMMO (mini-sMMO), with a turnover frequency of 0.32 s-1, through an optimal reassembly of minimal and modified components of sMMO on catalytically inert and stable apoferritin scaffold. We characterise the molecular characteristics in detail through in silico and experimental analyses and verifications. Notably, in-situ methanol production in a high-cell-density culture of mini-sMMO-expressing recombinant Escherichia coli resulted in higher yield and productivity (~ 3.0 g/L and 0.11 g/L/h, respectively) compared to traditional methanotrophic production.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.

Identification of YUC genes associated with leaf wrinkling trait in Tacai variety of Chinese cabbage.

Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) stands as a widely cultivated leafy vegetable in China, with its leaf morphology significantly influencing both quality and yield. Despite its agricultural importance, the precise mechanisms governing leaf wrinkling development remain elusive. This investigation focuses on 'Wutacai', a representative cultivar of the Tacai variety (Brassica campestris L. ssp. chinensis var. rosularis Tsen et Lee), renowned for its distinct leaf wrinkling characteristics. Within the genome of 'Wutacai', we identified a total of 18 YUCs, designated as BraWTC_YUCs, revealing their conservation within the Brassica genus, and their close homology to YUCs in Arabidopsis. Expression profiling unveiled that BraWTC_YUCs in Chinese Cabbage exhibited organ-specific and leaf position-dependent variation. Additionally, transcriptome sequencing data from the flat leaf cultivar 'Suzhouqing' and the wrinkled leaf cultivar 'Wutacai' revealed differentially expressed genes (DEGs) related to auxin during the early phases of leaf development, particularly the YUC gene. In summary, this study successfully identified the YUC gene family in 'Wutacai' and elucidated its potential function in leaf wrinkling trait, to provide valuable insights into the prospective molecular mechanisms that regulate leaf wrinkling in Chinese cabbage.

Flavin-containing monooxygenase (FMO): Beyond xenobiotics.

Flavin-containing monooxygenases (FMOs), traditionally known for detoxifying xenobiotics, are now recognized for their involvement in endogenous metabolism. We recently discovered that an isoform of FMO, fmo-2 in Caenorhabditis elegans, alters endogenous metabolism to impact longevity and stress tolerance. Increased expression of fmo-2 in C. elegans modifies the flux through the key pathway known as One Carbon Metabolism (OCM). This modified flux results in a decrease in the ratio of S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH), consequently diminishing methylation capacity. Here we discuss how FMO-2-mediated formate production during tryptophan metabolism may serve as a trigger for changing the flux in OCM. We suggest formate bridges tryptophan and OCM, altering metabolic flux away from methylation during fmo-2 overexpression. Additionally, we highlight how these metabolic results intersect with the mTOR and AMPK pathways, in addition to mitochondrial metabolism. In conclusion, the goal of this essay is to bring attention to the central role of FMO enzymes but lack of understanding of their mechanisms. We justify a call for a deeper understanding of FMO enzyme's role in metabolic rewiring through tryptophan/formate or other yet unidentified substrates. Additionally, we emphasize the identification of novel drugs and microbes to induce FMO activity and extend lifespan.

Overview of indigo biosynthesis by Flavin-containing Monooxygenases: History, industrialization challenges, and strategies.

Indigo is a natural dye extensively used in the global textile industry. However, the conventional synthesis of indigo using toxic compounds like aniline, formaldehyde, and hydrogen cyanide has led to environmental pollution and health risks for workers. This method also faces growing economic, sustainability, and environmental challenges. To address these issues, the concept of bio-indigo or indigo biosynthesis has been proposed as an alternative to aniline-based indigo synthesis. Among various enzymes, Flavin-containing Monooxygenases (FMOs) have shown promise in achieving a high yield of bio-indigo. However, the industrialization of indigo biosynthesis still encounters several challenges. This review focuses on the historical development of indigo biosynthesis mediated by FMOs. It highlights several factors that have hindered industrialization, including the use of unsuitable chassis (Escherichia coli), the toxicity of indole, the high cost of the substrate L-tryptophan, the water-insolubility of the product indigo, the requirement of reducing reagents such as sodium dithionite, and the relatively low yield and high cost compared to chemical synthesis. Additionally, this paper summarizes various strategies to enhance the yield of indigo synthesized by FMOs, including redundant sequence deletion, semi-rational design, cheap precursor research, NADPH regeneration, large-scale fermentation, and enhancement of water solubility of indigo.

Isolation of a facultative methanotroph Methylocystis iwaonis SD4 from rice rhizosphere and establishment of rapid genetic tools for it.

Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.

Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Nitrous oxide respiration in acidophilic methanotrophs.

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.

[Cloning and functional analysis of AcFMO from onion during alliine biosynthesis].

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.

Hif-1α expression targets the TMA/Fmo3/TMAO axis to participate in gallbladder cholesterol stone formation in individuals living in plateau regions.

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.

Structure-Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion.

Structure-guided engineering of a CHMO from Amycolatopsis methanolica (AmCHMO) was performed for asymmetric sulfoxidation activity and stereoselectivity toward omeprazole sulfide. Initially, combinatorial active-site saturation test (CASTing) and iteratively saturation mutagenesis (ISM) were performed on 5 residues at the "bottleneck" of substrate tunnel, and MT3 was successfully obtained with a specific activity of 46.19 U/g and R-stereoselectivity of 99 % toward OPS. Then, 4 key mutations affecting the stereoselectivity were identified through multiple rounds of ISM on residues at the substrate binding pocket region, resulting MT8 with an inversed stereoselectivity from 99 % (R) to 97 % (S). MT8 has a greatly compromised specific activity of 0.08 U/g. By introducing additional beneficial mutations, MT11 was constructed with significantly increased specific activity of 2.29 U/g and stereoselectivity of 97 % (S). Enlarged substrate tunnel is critical to the expanded substrate spectrum of AmCHMO, while reshaping of substrate binding pocket is important for stereoselective inversion. Based on MD simulation, pre-reaction states of MT3-OPSproR, MT8-OPSproS, and MT11-OPSproS were calculated to be 45.56 %, 17.94 %, and 28.65 % respectively, which further confirm the experimental data on activity and stereoselectivity. Our results pave the way for engineering distinct activity and stereoselectivity of BVMOs toward bulky prazole thioethers.

N-Cα Bond Cleavage Catalyzed by a Multinuclear Iron Oxygenase from a Divergent Methanobactin-like RiPP Gene Cluster.

DUF692 multinuclear iron oxygenases (MNIOs) are an emerging family of tailoring enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Three members, MbnB, TglH, and ChrH, have been characterized to date and shown to catalyze unusual and complex transformations. Using a co-occurrence-based bioinformatic search strategy, we recently generated a sequence similarity network of MNIO-RiPP operons that encode one or more MNIOs adjacent to a transporter. The network revealed >1000 unique gene clusters, evidence of an unexplored biosynthetic landscape. Herein, we assess an MNIO-RiPP cluster from this network that is encoded in Proteobacteria and Actinobacteria. The cluster, which we have termed mov (for methanobactin-like operon in Vibrio), encodes a 23-residue precursor peptide, two MNIOs, a RiPP recognition element, and a transporter. Using both in vivo and in vitro methods, we show that one MNIO, homologous to MbnB, installs an oxazolone-thioamide at a Thr-Cys dyad in the precursor. Subsequently, the second MNIO catalyzes N-Cα bond cleavage of the penultimate Asn to generate a C-terminally amidated peptide. This transformation expands the reaction scope of the enzyme family, marks the first example of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPPs.

Loss of flavin-containing monooxygenase 3 modulates dioxin-like polychlorinated biphenyl 126-induced oxidative stress and hepatotoxicity.

Dioxin-like pollutants (DLPs), such as polychlorinated biphenyl 126 (PCB 126), are synthetic chemicals classified as persistent organic pollutants. They accumulate in adipose tissue and have been linked to cardiometabolic disorders, including fatty liver disease. The toxicity of these compounds is associated with activation of the aryl hydrocarbon receptor (Ahr), leading to the induction of phase I metabolizing enzyme cytochrome P4501a1 (Cyp1a1) and the subsequent production of reactive oxygen species (ROS). Recent research has shown that DLPs can also induce the xenobiotic detoxification enzyme flavin-containing monooxygenase 3 (FMO3), which plays a role in metabolic homeostasis. We hypothesized whether genetic deletion of Fmo3 could protect mice, particularly in the liver, where Fmo3 is most inducible, against PCB 126 toxicity. To test this hypothesis, male C57BL/6 wild-type (WT) mice and Fmo3 knockout (Fmo3 KO) mice were exposed to PCB 126 or vehicle (safflower oil) during a 12-week study, at weeks 2 and 4. Various analyses were performed, including hepatic histology, RNA-sequencing, and quantitation of PCB 126 and F2-isoprostane concentrations. The results showed that PCB 126 exposure caused macro and microvesicular fat deposition in WT mice, but this macrovesicular fatty change was absent in Fmo3 KO mice. Moreover, at the pathway level, the hepatic oxidative stress response was significantly different between the two genotypes, with the induction of specific genes observed only in WT mice. Notably, the most abundant F2-isoprostane, 8-iso-15-keto PGE2, increased in WT mice in response to PCB 126 exposure. The study's findings also demonstrated that hepatic tissue concentrations of PCB 126 were higher in WT mice compared to Fmo3 KO mice. In summary, the absence of FMO3 in mice led to a distinctive response to dioxin-like pollutant exposure in the liver, likely due to alterations in lipid metabolism and storage, underscoring the complex interplay of genetic factors in the response to environmental toxins.

Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Deciphering the diurnal rhythm regulating mechanism of flavin-containing monooxygenase 3 in mouse liver.

Circadian genes play an important role in the field of drug metabolism. Flavin-containing monooxygenase 3 is a well-known phase I enzyme which participates in metabolism of many exogenous and endogenous substances, especially production of trimethylamine N-oxide. Here, we aimed to decipher diurnal rhythms of flavin-containing monooxygenase 3 expression and activity, and explore the regulation mechanism by clock genes. Our results showed that its mRNA and protein exhibited robust diurnal rhythms in mouse liver and cell lines. Consistently, significant alterations were observed for in vitro microsomal N-oxidation rates of procainamide, which kept in line with its protein expression at different time in wild-type and reverse erythroblastosis virus α knockout mice. Further, flavin-containing monooxygenase 3 was negatively regulated by E4 promoter-binding protein 4 in AML12 and Hepa1-6 cells, while it was positively influenced by reverse erythroblastosis virus α and brain and muscle ARNT-like protein-1. Moreover, luciferase reporter assays and electrophoretic mobility shift assays showed E4 promoter-binding protein 4 inhibited the transcription of flavin-containing monooxygenase 3 by binding to a D-box1 element (-1606/-1594 bp), while brain and muscle ARNT-like protein-1 positively activated the transcription via direct binding to three E-boxes (-863/-858 bp, -507/-498 bp, and -115/-104 bp) in this enzyme promoter. Taken together, this study would be helpful to reveal the mechanism of clock-controlled drug metabolism and facilitate the practice of chrono-therapeutics.

Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.