Skatole is a major component of the objectionable smell of manure and has an extremely low odor threshold, leading to severe odor problems and potential threats to animal and human health. Yet the molecular mechanisms underlying microbial skatole degradation remain poorly understood. To date, only a limited number of skatole monooxygenases have been reported; these enzymes belong to the Group E flavin-dependent monooxygenases and catalyze the initial oxidation of skatole, but the subsequent degradation pathways and associated genes remain unclear. In this study, we isolated a skatole-degrading strain Rhodococcus rhodochrous S3 from pig manure, elucidated a novel skatole degradation pathway and identified its key enzymes involved: SkoA is a Group B flavin-dependent monooxygenase responsible for oxidizing skatole to 3-methyloxindole (3-MOI); SkoB is a Baeyer-Villiger monooxygenase that catalyzes the conversion of 3-MOI to (4 R)-4-methyl-1,4-dihydro-3,1-benzoxazin-2-one (4-MDHBO); the esterase SkoC, encoded by a gene adjacent to skoB, hydrolyzes 4-MDHBO to form 2-carbamic acid-α-methylbenzyl alcohol (2-CMA), which is then completely degraded via decarboxylation and a series of oxygenation reactions through the catechol degradation pathway. Additionally, microbial distribution analysis revealed that SkoA-like skatole monooxygenases are widely distributed across Proteobacteria and Actinobacteria, indicating that these two phyla are the primary drivers of skatole degradation in nature. Together, these findings greatly improve our comprehension of microbial skatole degradation mechanisms and offers insights into the ecological distribution of skatole-degrading genes.

Enzymatic depolymerization is a potentially efficient and environmentally friendly solution for poly(butylene adipate-co-terephthalate) (PBAT) waste recycling and utilization. However, the depolymerization capacity of reported depolymerases against diverse, real-world commercial PBAT films remains unclear, hindering accurate evaluation of their utility. In this study, a mutant PBAT depolymerase (Tfcut-B3) with enhanced thermal stability and improved activity was obtained through directed evolution of Tfcut-DM. Tfcut-B3 completely depolymerized PBAT white film into terephthalic acid (TPA) within 48 h, yielding 2.0 and 1.3 times more TPA than Tfcut-DM and Tfcut-DM(Q132Y), respectively. Tfcut-B3 completely degraded 8/10 commercial PBAT-based films into TPA within 48 h, with the remaining two (PB-6 and PM-4) being broken down into small fragments; Tfcut-DM was unable to completely degrade any of the films. Tfcut-B3 overexpression and extracellular secretion in Bacillus subtilis was achieved using chromosomal integration; in flask fermentation, a Tfcut-B3 expression level of 274 mg/L was achieved through promoter and host optimization. Tfcut-B3 fermentation supernatant completely degraded up to 64.9 mg of PBAT white film within a 20 mL system at 72 h. These findings provide a theoretical basis for the large-scale application of PBAT depolymerases.

Although genes involved in isoprocarb (IPC) degradation, including the IPC hydrolase gene ipcH and the 2-isopropylphenol (IPP) hydroxylation gene cluster ippA1A2, have been identified in Rhodococcus sp. D-6, the transcriptional regulatory mechanism remains unclear. In this study, we characterized a PucR-type transcriptional regulator, IppR, which controls the inducible expression of the ippA1A2 cluster. Gene disruption and complementation analyses revealed that ippR was essential for activation of ippA1A2 expression in response to IPP in strain D-6. Quantitative reverse transcription-PCR (qRT-PCR) and promoter activity assays demonstrated that IPP was the effector molecule of IppR. Electrophoretic mobility shift assay (EMSA) showed that IppR bound to a 24-bp motif in the promoter region, with the CC/CG box within this motif identified as the critical site for IppR binding. Moreover, IppR residues likely involved in IPP binding include Val188, Leu192, Val218, Trp257, Trp259, His297, and Ala300, along with Lys382 and Thr384, which are predicted to be essential for DNA recognition. This study elucidates the regulatory mechanism of IPC degradation in strain D-6, and the identification of IppR expands the diversity of regulatory models within the PucR-type regulator.IMPORTANCECarbamate insecticides pose a potential threat to the environment and human health. Bacteria play an important role in the biodegradation of these compounds. Rhodococcus sp. D-6, which is capable of degrading isoprocarb (IPC), a representative carbamate insecticide, was isolated in our previous study. The expression of the IPC hydrolase gene ipcH was constitutive, while the expression of the ippA1A2 cluster responsible for hydroxylation of 2-isopropylphenol (IPP), the hydrolysis product of IPC, was inducible. The present study functionally characterized the PucR-type transcriptional regulator IppR, which activates the transcription of the ippA1A2 cluster to mediate IPP hydroxylation in strain D-6. This study advances our understanding of the regulatory mechanisms underlying IPC degradation.

Ibuprofen (IBU), a prevalent chiral pharmaceutical, was a common emerging contaminant in municipal wastewater. The mechanisms underlying its enantioselective microbial degradation and the horizontal gene transfer (HGT) of associated ipf genes remain poorly understood. Here, we reported Sphingopyxis sp. 550A, a bacterium capable of degrading both IBU enantiomers but exhibited a distinct preference for R-(-)-IBU, which preference was determined by the IpfF, an aromatic CoA ligase. Molecule docking analysis revealed that differential catalytic atomic distances govern enantioselective efficiency of IpfF. The ipfABDEFG genes demonstrated concentration-dependent genetic dynamics: low IBU stress (1 mg·L-1) promoted ipfABDEFG gene cluster transfer to other sphingomonads through HGT, while high stress (≥ 10 mg·L-1) induced toxic intermediate accumulation and IS6100-mediated gene loss to alleviate cellular toxicity. Leveraging these insights, we constructed a microbial co-culture of strain 550 A and Pseudomonas putida KT2440 for complete removal of high-concentration IBU and its toxic metabolite, 4-isobutylcatechol. This work provided a framework for understanding enzymatic enantioselectivity toward chiral pharmaceuticals and highlights the role of HGT in shaping bioremediation potential within engineered microbial communities.

Zearalenone (ZEN), an estrogenic mycotoxin commonly found in grains and feed, poses a severe threat to livestock production. The ZEN hydrolase ZenR, derived from Rhodococcus erythropolis HQ, exhibits high specific activity; however, its industrial application is constrained by limited thermostability. Through integrated computational design, consensus analysis, and molecular dynamics (MD) simulations, mutation hotspots were identified and the thermostable mutant ZenR9Mu was engineered after three rounds of iteration. ZenR9Mu demonstrates a 12.5°C increase in melting temperature (Tm) compared to the wild-type (WT) and retains 60% of its activity after incubation at 56°C for 2 min. MD simulations and intramolecular interaction analysis revealed that increased backbone rigidity, additional hydrogen bonds and salt bridges, and a reconfiguration of the hydrophobic network in the cap domain collectively contribute to thermostability. After a 3-min saturated steam pretreatment, ZenR9Mu in maize flour retained its activity and degraded ZEN from 1.12 μg/g to 0.31 μg/g within 10 min at 37°C, falling below the regulatory limit of 0.5 μg/g. This study provides a proven engineering framework for developing next-generation mycotoxin hydrolases and offers an effective enzymatic resource for ZEN detoxification in the feed industry.

Microbes drive global nitrogen cycling, yet the extent to which taxonomic identity is associated with functional potential across bacterial diversity remains poorly quantified. Using 73 472 representative bacterial genomes, we develop a quantitative framework integrating Information Gain analysis, functional classification, and molecular evolutionary analysis across six nitrogen cycling pathways and five taxonomic ranks. Association strength increases monotonically from phylum to genus level across all six pathways, with genus-level associations ranging from 39.5

The global use of bisphenol F (BPF) has led to widespread environmental contamination, posing significant threats to ecosystems and human health. However, the genes involved in BPF degradation remained unknown. In this study, a novel oxidase gene, bpfA , was obtained from strain Microbacterium sp. F2 through a four-step purification strategy. BpfA is classified as a member of the 4-phenol oxidizing (4PO) subfamily and contains a conserved FAD-binding domain (Asp152 and His393) as well as a Tyr-Tyr-Arg triad (Tyr93, Tyr474, Arg475). BpfA catalyzes the conversion of BPF to 4,4’-dihydroxydibenzophenone (DHBP) through a series of three consecutive reactions. In addition, BpfA exhibits catalytic activity towards 4-alkylphenols, such as vanillyl alcohol (VA), 4-n-propylguaiacol (4PG), and 4-(methoxymethyl)phenol (4MOP). BpfA homologs are widely distributed in the environment, particularly in soil. Bioinformatics-based taxonomic profiling revealed that BpfA homologs are widely distributed in metagenomes from cultivated land and forests, mainly belonging to Pseudomonadota and Actinomycetota. This study enhances our understanding of the microbial degradation mechanism of BPF, elucidates the catalytic function of the novel oxidase gene bpfA and its distribution pattern in the environment, and provides important insights into the evolutionary origin of BPF degradation genes and the targeted mining of environmental microbial resources. ### Competing Interest Statement The authors have declared no competing interest. National Key R&D Program of China, 2022YFA0912500 National Natural Science Foundation of China, 32170125, 32400085 Postdoctoral Fellowship Program of CPSF, GZC20231127

Isoprocarb (IPC), a widely used carbamate insecticide, causes environmental contamination and poses risks to humans and ecosystems. Rhodococcus sp. D-6, capable of utilizing IPC as the sole growth substrate, was isolated by our lab. Strain D-6 initiates IPC degradation through ester bond hydrolysis by the hydrolase IpcH, yielding 2-isopropylphenol (IPP). In this study, the catabolic pathway of IPP in strain D-6 was elucidated, and the ipp gene cluster responsible for its degradation was predicted by transcriptomic analysis. Key genes, including a novel two-component IPP monooxygenase gene ippA1A2 and a 2-isopropylhydroquinone dioxygenase gene ippB, are responsible for the successive conversion of IPP, resulting in the ring cleavage of IPP. IppA1, a group D flavin-dependent monooxygenase, along with its reductase component IppA2, hydroxylates IPP to 2-isopropylhydroquinone using reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) as cofactors. The optimal molar ratio of IppA1 to IppA2 was 3:1. The apparent Km and kcat values of IppA1 for IPP were 60.52 ± 1.27 µM and 70.07 ± 0.85 s-1, respectively. IppB, an extradiol dioxygenase, catalyzes 2-isopropylhydroquinone ring cleavage to generate 2-isopropyl-4-hydroxymuconic semialdehyde. The apparent Km and kcat values of IppB for 2-isopropylhydroquinone were 29.07 ± 1.54 μM and 147.65 ± 1.92 s-1, respectively. This study provides critical insights into the molecular mechanisms of IPP degradation, contributing to a comprehensive understanding of IPC degradation.IMPORTANCECarbamate insecticides kill pests by inhibiting the activity of acetylcholinesterase (AChE) and have been widely used in agriculture. Compared to the studies on the degradation mechanisms of carbofuran and carbaryl, little is known about IPC degradation. An IPC-degrading strain Rhodococcus sp. D-6 was isolated by our lab, and the hydrolase gene ipcH responsible for hydrolyzing IPC to IPP has been identified previously. This study further elucidates the IPP degradation pathway in strain D-6 and identifies the novel two-component IPP monooxygenase IppA1A2 and 2-isopropylhydroquinone dioxygenase IppB, which are respectively responsible for IPP hydroxylation and its subsequent ring cleavage. These findings enhance our understanding of the microbial degradation mechanism of IPC.

Phoxim, an organophosphorus insecticide (OP), poses risks to non-target organisms in the environment. However, the biodegradation mechanism of phoxim was not fully understood. In this study, a novel detoxification pathway for phoxim in Delftia lacustris PX-1 was identified. A gene cluster pho (phoABC1C2DEFGHI), located on the plasmid, was found to be involved in phoxim degradation. The genes phoA, phoB, and phoD, which were responsible for converting phoxim to benzoate, were functionally characterized in vitro. Notably, PhoA was highly abundant in global cultivated lands. Moreover, acute toxicity experiments showed that both phoxim and its metabolite, 2-hydroxyimino-2-phenylacetonitrile (2H2P), exhibited moderate toxicity to zebrafish, whereas strain PX-1 could detoxify both compounds through degradation. After exposure to 2 mg L-1 phoxim, the brain acetylcholinesterase (AChE) activity levels of zebrafish dramatically decreased by 31.28 % (P < 0.05) relative to unexposed controls. Interestingly, phoxim could promote the colonization of strain PX-1 in the zebrafish gut, which mitigated phoxim-induced damage to AChE activity in the zebrafish brain. In summary, our research reveals a novel phoxim detoxification mechanism and provides a theoretical foundation for the bioremediation of residual phoxim in the environment. IN BRIEF: This study reveals a novel metabolic pathway for phoxim biodegradation and elucidates the detoxification mechanism of strain PX-1 in zebrafish.

A Gram-stain-negative, strictly aerobic and motile bacterium capable of degrading trifloxystrobin, designated strain J-3 T , was isolated from activated sludge from Nanhu Wetland Park in Huaibei City, Anhui Province, PR China. Growth was observed at 0–1.0% NaCl (w/v; optimum: 0.5%) at 15–35 °C (optimum: 30 °C) and pH 5.0–8.0 (optimum: pH 7.5). Strain J-3 T could degrade 59.9% of 75.0 µM trifloxystrobin within 3 days of incubation. A novel esterase responsible for hydrolysing trifloxystrobin to trifloxystrobin acid, StrE, was identified in strain J-3 T ; it exhibited low similarity ( < 31%) with previously reported trifloxystrobin esterases, indicating its novelty. Phylogenetic analysis based on 16S rRNA gene sequences and genomes indicated that strain J-3 T was most closely related to strain Comamonas odontotermitis Dant 3-8 T . The 16S rRNA gene similarity between strain J-3 T and C. odontotermitis Dant 3-8 T was 97.7%. The average nucleotide identity and digital DNA–DNA hybridization values between strain J-3 T and strain C. odontotermitis Dant 3-8 T were 81.7% and 25.4%, respectively. The major fatty acids were C 16:0 , summed feature 3 (C 16:1 ω7 c and/or C 16:1 ω6 c) and summed feature 8 (C 18 : 1 ω7 c and/or C 18 : 1 ω6 c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The predominant respiratory quinone was Q-8. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic analysis, strain J-3 T was considered to represent a novel species within the genus Comamonas , for which the name Comamonas trifloxystrobinivorans sp. nov. is proposed with strain J-3 T (=KCTC 8781 T =MCCC 1K09559 T ) as the type strain.

Bisphenol F (BPF) is a component of synthetic epoxy resin and polycarbonate plastic. Its residue is frequently detected in water and sediments, posing a threat to aquatic organisms due to the endocrine disrupting effect of BPF. In this study, strain Microbacterium sp. F2, using BPF as the sole carbon source for growth and degrading 98.67 % of 0.10 mM BPF within 24 h, was isolated. Based on the identification of metabolites, strain F2 degraded BPF by the hydroxylation of the bridging carbon atom, and subsequently via oxidation and Baeyer-Villiger reaction to p-hydroxybenzoic acid (PHBA) and 1,4-hydroquinone (HQ), and both of them were further metabolized to provide strain F2 with the carbon source for growth. The half maximal inhibitory concentration (IC50) of BPF, 4,4 '-dihydroxybenzophenone (DHBP), 4-hydroxyphenyl 4-hydroxybenzoate (HPHB), HQ and PHBA against Chlorella ellipsoidea at 96 h were 64, 142.5, 205, 325 and 660 mu M, respectively. The toxicity of BPF and its metabolites to C. ellipsoidea showed a gradually decreasing trend. The median lethal concentration (LC50) of BPF, DHBP, HPHB, HQ, and PHBA against zebrafish at 96 h were 40.50, 70.31, 71.28, 72.44, and 74.94 mu M, respectively. There was no obvious difference in the toxicity of metabolites to zebrafish, but their toxicity was significantly lower than that of BPF. However, the complete mineralization of BPF by strain F2 achieved the detoxification of BPF. This study provides a potential strain for the bioremediation of BPF-polluted environment.

Bisphenol F, a widely used primary raw material in the production of polycarbonate and epoxy resins, is frequently detected in the environment and poses significant risks to ecosystems and human health. Microorganisms play an important role in bisphenol F degradation in the natural environment; however, the genetic determinants involved remain unknown. A flavoprotein oxidase BpfA from Microbacterium sp. strain F2 was identified in this study, which is responsible for the crucial steps of bisphenol F degradation involving its conversion to 4,4'-dihydroxybenzophenone through three consecutive reactions. BpfA phylogenetically clusters within the 4-phenol oxidizing subfamily of the vanillyl alcohol oxidase/para-cresol methylhydroxylase flavoprotein family. Three homologs in this subfamily-vanillyl alcohol oxidase VAO, eugenol oxidase EUGO, and flavoprotein oxidase FBO-shared over 35.0% identity with BpfA and demonstrated bisphenol F-degrading activity, yet the catalytic efficiency of BpfA against bisphenol F (508.1 mM-1 s-1) was significantly higher than that of vanillyl alcohol oxidase VAO (0.2 mM-1 s-1), eugenol oxidase EUGO (0.2 mM-1 s-1), and flavoprotein oxidase FBO (0.3 mM-1 s-1). Structural analysis indicated that strong active site hydrophobicity was likely the reason for this high catalytic efficiency. Bioinformatics-based taxonomic profiling revealed that candidate bisphenol F degraders carrying bpfA mainly belonged to the Pseudomonadota and Actinomycetota phyla, and were predominantly found in metagenomes from cultivated land and forests. This study elucidated the function and distribution pattern of bpfA, enhancing our understanding of microbial bisphenol F degradation in the environment.

3,5-Dichloroaniline (3,5-DCA) is extensively used in synthesizing dicarboximide fungicides, medical compounds and dyes. Due to its widespread use in agriculture and industry, 3,5-DCA is often detected in groundwater, wastewater, sediments and soil, posing great risk to animals and humans. However, the genes and enzymes involved in 3,5-DCA degradation remain unidentified. In this study, 3,5-DCA-degrading activated sludge was obtained through 90 days of alternating anaerobic-anoxic acclimation. Microbial community analysis and further study revealed that Dehalobacter sp. DH-1 (OTU10) can convert 3,5-DCA to 3-chloroaniline anaerobically. Pseudomonas sp. DCA-1 (OTU58) can degrade 3,5-DCA, 3-chloroaniline and 2,5-dichloroaniline aerobically. Two gene clusters, ddoA1A2A3A4 and ddoBCDE, responsible for 3,5-DCA degradation, were identified via transcriptome analysis and heterologous expression. The dioxygenase genes of cluster ddoA1A2A3A4 convert 3,5-DCA to 3,5-dichlorocatechol, while those of cluster ddoBCDE mineralize the latter. Ultimately, the iprodione-degrading gene cluster ipaH-ddaH-duaH (responsible for sequential hydrolysis of the dicarboximide fungicide iprodione to 3,5-DCA) was introduced into strain DCA-1 to construct a genetically engineered microorganism, named strain IHC-DCA-1, that is capable of completely degrading iprodione. This study reveals the degradation mechanism of 3,5-DCA and provides potential strains to remediate 3,5-DCA and iprodione in the environment.

Microbes are essential for global nitrogen cycling, yet the extent to which taxonomic identity constrains functional potential remains poorly quantified. Using 73,472 representative bacterial genomes, we establish a multi-scale quantitative framework revealing systematic, hierarchical relationships between taxonomic identity and nitrogen cycling functional potential. Hierarchical variance decomposition reveals that taxonomy explains 20–46% of functional variation across six nitrogen pathways, with the strongest constraints for dissimilatory nitrite reduction to ammonium (46.1%) and nitrogen fixation (44.2%), both negatively correlated with functional prevalence. K-means clustering identifies four class-level functional archetypes (Functionally Inactive, Diversified, Integrated, and Specialized) among 77 bacterial classes. 1,281 genera resolve into five ecological strategies differentiated by their nitrogen retention versus loss capabilities, exhibiting strong phylogenetic signals. Cross-scale validation demonstrates 78.7% genus-level conformity to class-level archetypes, confirming hierarchical functional organization. Molecular evolutionary analysis of 13 genes reveals sequence conservation as a dimension partially independent of pathway-level functional constraints. Paradoxically, the functionally most constrained pathway exhibits only moderate sequence conservation ( nrfA : 9th/13 genes), while a moderately constrained pathway shows exceptional conservation ( napA ). Four constraint-conservation patterns demonstrate that gene-specific structural and ecological factors generate evolutionary rate variation independently of taxonomic associations. Our results establish a hierarchical framework in which taxonomic constraints set baseline functional potential, ecological trade-offs shape strategy diversification, and molecular evolution modulates gene-level conservation patterns across biological scales. This framework establishes quantitative baselines that enable probabilistic inference of nitrogen cycling capabilities from taxonomic composition, with potential applications in amplicon-based community analysis, targeted cultivation, and biogeochemical modeling. Importance A fundamental challenge in microbial ecology is inferring functional potential from taxonomic data—a relationship widely assumed but never rigorously quantified. Resolving this is critical because while amplicon sequencing provides cost-effective taxonomic profiling, functional characterization requires expensive metagenomics, limiting large-scale biogeochemical studies. We provide the first systematic quantification demonstrating that taxonomic identity explains 20-46% of nitrogen cycling functional variation, operating hierarchically from class to genus level. Crucially, we reveal that taxonomic constraints operate at both functional distribution and molecular evolution levels as partially independent dimensions, indicating distinct evolutionary mechanisms. This work establishes quantitative foundations for taxonomy-based functional prediction, enabling researchers to extract functional insights from readily available taxonomic surveys. As reference databases expand, this framework will enhance predictive capabilities for nitrogen cycling and broader biogeochemical processes.

As the extensive use of polyurethane foam (PUF), the disposal of PUF waste poses significant environmental challenges. Enzyme-based recycling and bioremediation of PUF waste is regarded as a sustainable solution but still suffers from the lack of qualified enzymes. Here, a novel PUF-degrading bacterium, Pueribacillus sp. YX66, was isolated. An efficient polyurethane foam hydrolase (PufH), was identified from this strain, which showed less than 23 % sequence similarity to the previously reported PU depolymerases. Product analysis and molecular docking suggested that PufH specifically targeted the ester bonds within the soft segment of PUF. PufH exhibited remarkable activity against various commercial polyester-type PUFs, achieving a maximum depolymerization rate of 90 % within 48 h at 30 °C, significantly surpassing the performance of other PUF depolymerases. Moreover, PufH retained considerable activity at the temperature of 4-20 °C, enabling it to efficiently depolymerize PUF waste in soil within 14 days under ambient temperature. These findings underscore the potential of PufH as a promising biocatalyst in the recycling and bioremediation of PUF waste.

Pymetrozine boosts crop yields by managing insect pests, but the United States Environmental Protection Agency (USEPA) has labeled it a potential carcinogen for its detrimental effects on human reproduction and respiratory health. In this study, Pseudomonas guariconensis strain BYT-5, capable of degrading pymetrozine via a typical metabolic pathway as its sole carbon source for growth, was isolated. Genome sequencing and analysis of strain BYT-5 revealed the presence of the pymetrozine hydrolase gene pyzH and two nicotinic acid (NA) catabolic gene clusters, nic1 (nicAB1R1X1C1D1E1F1T1) and nic2 (nicR2X2C2D2E2F2T2B2). Verification of these genes was achieved through heterologous expression, gene knockout, and complementation assays. The gene pyzH initiated the breakdown of pymetrozine into 4-amino-6-methyl-4,5-dihydro-2H-[1,2,4]triazin-3-one (AMDT) and nicotinaldehyde, which was further oxidized to nicotinic acid (NA) by non-specific dehydrogenases. The genes nicAB1, nicC2, and nicX1 sequentially converted NA into 6-hydroxynicotinic acid (6HNA), 2,5-dihydroxypyridine (2,5-DHP), and N-formylmaleamic acid (NFM), respectively. The genes nicD2E2F2 was ultimately responsible for converting NFM to fumaric acid, entering the TCA cycle. Furthermore, the presence of diverse transposases around the pyzH, nic1, and nic2 clusters may facilitate BYT-5's acquisition of genes necessary for pymetrozine degradation. This study elucidates the molecular metabolic mechanism of pymetrozine at the molecular level.

Acetaminophen (APAP), an analgesic and antipyretic drug, is commonly detected in wastewater treatment plant (WWTP) effluents, surface water, and soil, indicating its status as an emerging environmental contaminant. In this study, we isolated a bacterium, Pseudomonas taiwanensis AP-1, capable of completely mineralizing APAP and utilizing it as the sole carbon source for growth. A newly identified metabolite, γ-glutamyl-4-aminophenol (γ-G4AP), was reported for the first time in the degradation of APAP by strain AP-1. Two amidases (ApaH1 and ApaH2), responsible for the conversion of APAP to 4-aminophenol (4-AP), were identified through a combination of genomic comparison, heterologous expression, and gene knockout. Notably, ApaH1 played a pivotal role in the degradation of APAP by strain AP-1. The catalytic triad of ApaH1 (K82-S161-S185) and ApaH2 (K85-S160-S184) were identified as by molecular docking and site-directed mutagenesis. Additionally, a gene cluster apd for the metabolism of 4-AP was also successfully identified in strain AP-1, consisting of the aniline dioxygenase gene cluster apdBCD1D2EF and the BT catabolic gene apdGH. Interestingly, the 4-AP metabolic gene cluster apd was highly conserved among other Pseudomonas strains capable of APAP degradation. Our results provide new insights into the mechanism of APAP biodegradation and strain AP-1 may be a promising bacterium for the bioremediation of APAP pollutions.

Strobilurin fungicides are a type of agricultural chemical commonly used to control fungal diseases in crops. However, the abuse of strobilurin fungicides leads to heavy residue in the environment, posing serious threats to ecosystems and human health. In this regard, the strains Hyphomicrobium sp. DY-1 and Hyphomicrobium sp. B1 could hydrolyze the strobilurin fungicides effectively (trifloxystrobin, azoxystrobin, pyraclostrobin, and picoxystrobin), generating the corresponding parent acid. Therefore, the present study sought to explore the molecular degradation mechanism of strobilurin fungicides by strain DY-1. The results showed that strain DY-1 exhibited a faster degradation rate for strobilurin fungicides than strain B1. Comparative genomic analysis and functional verification of both strains revealed that strain DY-1 possesses six strobilurin hydrolases (TriH, StrH, StrH1, StrH2, StrH3, and StrH4), whereas strain B1 possesses only three strobilurin hydrolases (TriH, StrH, and StrH1). All StrHs (StrH1, StrH2, StrH3, and StrH4) shared 50%–77% identity with StrH and contained conserved catalytic motif Ser-Glu-His. Further investigation into the distribution of strobilurin fungicide hydrolases revealed that strH and strH1 were aligned together and surrounded by transposases in the strains DY-1 and B1 and a genomic island containing strH and strH1 derived from the Hyphomicrobium genus. Overall, the function, diversity, and distribution of strobilurin hydrolases will provide insights into the microbial degradation of strobilurin fungicides.

Cotinine, the main metabolite of nicotine, was frequently detected in aquatic environment and pose a high risk to environmental safety and human health. Microbial degradation was considered as the most effective and environmental-friendly method for cotinine elimination. However, the microbial resources including bacterial consortia and pure cultures were limited. In this study, a synergistic consortium involved in cotinine degradation was successfully obtained and metagenomic analysis were investigated to reveal the key microorganisms responsible for cotinine and 6-hydroxy-3-succinoylpyridine (HSP) degradation, respectively. The genes encoding for cotinine hydroxylation (cotA1A2A3) and for HSP cleavage (hspB) were successfully amplified from the consortium C2 and functionally identified. Furthermore, a new cotinine-degrading pure culture strain, Terrabacter sp. cot-2, was isolated from the consortium C2. The degradation characterization and key intermediates of cotinine by strain cot-2 were analyzed. Then, the complete genome of strain cot-2 was determined and the gene cluster cot was demonstrated to be widely distributed in different environments. This study significantly broadened the knowledge on the degradation of the functional consortium and mechanism involved in cotinine biodegradation at molecular level, which also offering a promising solution to mitigate the impact of cotinine in aquatic ecosystems.

ABSTRACT 17 β -estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2. IMPORTANCE Steroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA , eanB1 , eanC , and eanD ) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.