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.

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.

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.

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.

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.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

[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.

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.

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.

Direct Methane Oxidation by Copper- and Iron-Dependent Methane Monooxygenases.

Methane is a potent greenhouse gas that contributes significantly to climate change and is primarily regulated in Nature by methanotrophic bacteria, which consume methane gas as their source of energy and carbon, first by oxidizing it to methanol. The direct oxidation of methane to methanol is a chemically difficult transformation, accomplished in methanotrophs by complex methane monooxygenase (MMO) enzyme systems. These enzymes use iron or copper metallocofactors and have been the subject of detailed investigation. While the structure, function, and active site architecture of the copper-dependent particulate methane monooxygenase (pMMO) have been investigated extensively, its putative quaternary interactions, regulation, requisite cofactors, and mechanism remain enigmatic. The iron-dependent soluble methane monooxygenase (sMMO) has been characterized biochemically, structurally, spectroscopically, and, for the most part, mechanistically. Here, we review the history of MMO research, focusing on recent developments and providing an outlook for future directions of the field. Engineered biological catalysis systems and bioinspired synthetic catalysts may continue to emerge along with a deeper understanding of the molecular mechanisms of biological methane oxidation. Harnessing the power of these enzymes will necessitate combined efforts in biochemistry, structural biology, inorganic chemistry, microbiology, computational biology, and engineering.

Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency.

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2 s-1) compared to free cofactor (kdec=1.8 s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s-1. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.

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.

Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

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.

Rare but impaired flavin-containing monooxygenase 3 (FMO3) variants reported in a recently updated Japanese mega-databank of genome resources.

Genetic variants of human flavin-containing monooxygenase 3 (FMO3) were investigated using an updated Japanese population panel containing 54,000 subjects (the previous panel contained 38,000 subjects). One stop codon mutation and six amino acid-substituted FMO3 variants were newly identified in the updated databank. Of these, two substituted variants (p.Thr329Ala and p.Arg492Trp) were previously identified in compound haplotypes with p.[(Glu158Lys; Glu308Gly)] and were associated with the metabolic disorder trimethylaminuria. Three recombinant FMO3 protein variants (p.Ser137Leu, p.Ala334Val, and p.Ile426Val) expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75-125 %) as wild-type FMO3 (117 min-1); however, the recombinant novel FMO3 variant Phe313Ile showed moderately decreased FMO3 catalytic activity (∼20 % of wild-type). Because of the known deleterious effects of FMO3 C-terminal stop codons, the novel truncated FMO3 Gly184Ter variant was suspected to be inactive. To easily identify the four impaired FMO3 variants (one stop codon mutation and three amino-acid substitutions) in the clinical setting, simple confirmation methods for these FMO3 variants are proposed using polymerase chain reaction/restriction fragment length polymorphism or allele-specific PCR methods. The updated whole-genome sequence data and kinetic analyses revealed that four of the seven single-nucleotide nonsense or missense FMO3 variants had moderately or severely impaired activity toward trimethylamine N-oxygenation.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1-6) in tree shrews.

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1-5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1-6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85-90 % and 81-89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1-6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1-6, which are orthologous to human FMOs (FMO1-5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

Genome-wide analysis of carotenoid cleavage oxygenases and identification of ripening-associated DzNCED5a in durian (Durio zibethinus) fruit.

Durian (Durio zibethinus L.), popularly known as the "King of fruits," holds significant economic importance in Southeast Asia, including Thailand. During its ripening process, the phytohormone abscisic acid (ABA) content has been reported to increase. However, a comprehensive understanding of ABA's specific role in durian fruit ripening remains elusive. Furthermore, little is known about the molecular aspects of the carotenoid cleavage pathway in this iconic fruit. Therefore, we performed genome-wide identification of the carotenoid cleavage oxygenase (CCO) family in durian. This family includes the nine-cis-epoxycarotenoid dioxygenases (NCEDs) responsible for ABA production and the carotenoid cleavage dioxygenases exhibiting diverse substrate specificities. Through phylogenetic analysis, we classified 14 CCOs in durian into 8 distinct subfamilies. Notably, each DzCCO subfamily displayed a conserved motif composition. Cis-acting element prediction showed that cis-elements related to plant hormones and environmental stress responses were distributed in the DzCCO promoter. In addition, transcriptome analysis was performed to examine the expression pattern during the fruit development and ripening stages. Interestingly, DzNCED5a, a ripening-associated gene, exhibited the highest expression level at the ripe stage, outperforming other CCOs. Its expression markedly correlated with increased ABA contents during the ripening stages of both the "Monthong" variety and other durian cultivars. Transiently expressed DzNCED5a in Nicotiana benthamiana leaves confirmed its function in ABA biosynthesis. These findings highlight the involvement of DzNCED5a in ABA production and its potential importance in durian fruit ripening. Overall, this study provides insights into the significance of CCOs in durian fruit ripening.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.