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Alpha-ketoglutarate-dependent dioxygenases (αKGDs) have recently emerged as useful biocatalysts for C-H oxidation and functionalization. In this work, we characterized a new αKGD from aculene biosynthesis, AneA, which displays broad promiscuity toward a number of substrates with different ring systems. Unexpectedly, AneA was found to be capable of both desaturation and hydroxylation and require an amino ester motif on its substrate for productive catalysis. Insights gathered from the functional characterization and substrate-activity profiling of AneA enabled the development of a chemoenzymatic strategy toward several complex sesquiterpenoids., (© 2024 Wiley-VCH GmbH.)

Sulfur is an essential element for life. Bacteria can obtain sulfur from inorganic sulfate; but in the sulfur starvation-induced response, Pseudomonads employ two-component flavin-dependent monooxygenases (TC-FMOs) from the msu and sfn operons to assimilate sulfur from environmental compounds including alkanesulfonates and dialkylsulfones. Here, we report binding studies of oxidized FMN to enzymes involved within the P. fluorescens enzymatic pathway responsible for converting dimethylsulfone (DMSO 2 ) to sulfite. In this catabolic pathway, SfnG serves as the initial TC-FMO for sulfur assimilation, which is investigated in detail by solving the 2.6-Å resolution crystal structure of unliganded SfnG and the 1.75-Å resolution crystal structure of the SfnG ternary complex containing FMN and DMSO 2 . We find that SfnG adopts a (β/α) 8 barrel fold with a distinct quaternary configuration from other tetrameric class C TC-FMOs. To probe the unexpected tetramer arrangement, structural heterogeneity is assessed by chromatography and light scattering to confirm ligand binding correlates with a tetramer. Binding of FMN and DMSO 2 accompanies ordering of the active site, with DMSO 2 bound on the si -face of the flavin. A previously unobserved protein backbone conformation is found within the oxygen-binding site on the re -face of the flavin. Functional assays and the positioning of ligands with respect to the oxygen-binding site are consistent with use of an N5-(hydro)peroxyflavin pathway. Biochemical endpoint assays and docking studies reveal SfnG breaks the C-S bond of a range of dialkylsulfones., Competing Interests: Competing interests statement:The authors declare no competing interest.

The molecular profile of paracancerous tissue has been reported to be prognostic for recurrence in patients with different cancers. This study investigated the clinical significance of the protein expression of gamma-butyrobetaine hydroxylase 1 (BBOX1) in tumor and paracancerous tissues of hepatocellular carcinoma (HCC) patients. This study assessed the role of BBOX1 mRNA in tumor and paracancerous tissues in HCC via bioinformatics analysis. The protein levels of BBOX1 in paired tumor and paracancerous tissues from 83 HCC patients were determined by immunohistochemistry. The associations among BBOX1 protein levels, clinicopathological characteristics and the survival probability of HCC patients were also analyzed. The results revealed that BBOX1 mRNA expression was significantly lower in HCC tissues than in noncancerous tissues. HCC patients with low BBOX1 mRNA expression had a significantly poorer overall survival than those with high BBOX1 expression did. Immunohistochemical analysis of BBOX1 protein expression further confirmed that its levels were markedly lower in HCC tissues than in paracancerous tissues. High BBOX1 expression in paracancerous tissues correlated with poor overall survival and disease-free survival in HCC patients. These findings suggest that the paracancerous BBOX1 expression might be a credible indicator for overall survival or disease-free survival in HCC patients., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Lytic polysaccharide monooxygenases (LPMOs) form a copper-dependent family of enzymes classified under the auxiliary activity (AA) superfamily. The LPMOs are known for their boosting of polysaccharide degradation through oxidation of the glycosidic bonds that link the monosaccharide subunits. This oxidation has been proposed to be dependent on either O 2 or H 2 O 2 as cosubstrate. Theoretical investigations have previously supported both mechanisms, although this contrasts with recent experiments. A possible explanation is that the theoretical results critically depend on how the Cu active site is modeled. This has also led to different results even when employing only H 2 O 2 as cosubstrate. In this paper, we investigate both the O 2 - and H 2 O 2 -driven pathways, employing Ls AA9 as the underlying LPMO and a theoretical model based on a quantum mechanics/molecular mechanics (QM/MM) framework. We ensure to consistently include all residues known to be important by using extensive QM regions of up to over 900 atoms. We also investigate several conformers that can partly explain the differences seen in previous studies. We find that the O 2 -driven reaction is unfeasible, in contrast with our previous QM/MM calculations with smaller QM regions. Meanwhile, the H 2 O 2 -driven pathway is feasible, showing that for Ls AA9, only H 2 O 2 is a viable cosubstrate as proposed experimentally.

5-Methyldeoxycytidine (5mC) is a major epigenetic marker that regulates cellular functions in mammals. Endogenous lipid peroxidation can convert 5mC into 3,N4-etheno-5-methylcytosine (ϵ5mC). ϵ5mC is structurally similar to the mutagenic analog 3,N4-ethenocytosine (ϵC), which is repaired by AlkB family enzymes in the direct reversal repair (DRR) pathway and excised by DNA glycosylases in the base excision repair (BER) pathway. However, the repair of ϵ5mC has not been reported. Here, we examined the activities against ϵ5mC by DRR and BER enzymes and TET1-3, enzymes that modify the 5-methyl group in 5mC. We found that the etheno modification of 5mC blocks oxidation by TET1-3. Conversely, three human homologs in the AlkB family, ALKBH2, 3 and FTO were able to repair ϵ5mC to 5mC, which was subsequently modified by TET1 to 5-hydroxymethylcytosine. We also demonstrated that ALKBH2 likely repairs ϵ5mC in MEF cells. Another homolog, ALKBH5, could not repair ϵ5mC. Also, ϵ5mC is not a substrate for BER glycosylases SMUG1, AAG, or TDG. These findings indicate DRR committed by ALKBH2, 3 and FTO could reduce the detrimental effects of ϵ5mC in genetics and epigenetics and may work together with TET enzymes to modulate epigenetic regulations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry. Thus, an effective strategy for streamlining the process from enzyme identification to implementation is essential to expanding biocatalysis. Here, we present a strategy combining bioinformatics-guided enzyme mining and ancestral sequence reconstruction (ASR) to resurrect enzymes for biocatalytic synthesis. Specifically, we achieve an enantioselective synthesis of azaphilone natural products using two ancestral enzymes: a flavin-dependent monooxygenase (FDMO) for stereodivergent oxidative dearomatization and a substrate-selective acyltransferase (AT) for the acylation of the enzymatically installed hydroxyl group. This cascade, stereocomplementary to established chemoenzymatic routes, expands access to enantiomeric linear tricyclic azaphilones. By leveraging the co-occurrence and coevolution of FDMO and AT in azaphilone biosynthetic pathways, we identified an AT candidate, CazE, and addressed its low solubility and stability through ASR, obtaining a more soluble, stable, promiscuous, and reactive ancestral AT (AncAT). Sequence analysis revealed AncAT as a chimeric composition of its descendants with enhanced reactivity likely due to ancestral promiscuity. Flexible receptor docking and molecular dynamics simulations showed that the most reactive AncAT promotes a reactive geometry between substrates. We anticipate that our bioinformatics-guided, ASR-based approach can be broadly applied in target-oriented synthesis, reducing the time required to develop biocatalytic steps and efficiently access superior biocatalysts.

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production., (© 2024. The Author(s).)

Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O -linked disaccharide composed of 3-α-epimycarose and 2- O -methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.

Objective: Colorectal cancer (CRC) is a diverse and multifaceted disease characterized by genetic and epigenetic changes that contribute to tumor initiation and progression. CRC pathophysiology has been linked to the deregulation of the Wnt signaling pathway and the ten-eleven translocation (TET) DNA demethylases. This study aimed to evaluate the expression level of selective miRNAs (miR-200 and miR-494), TET1, and Wnt1 in colorectal polyps, actual colorectal tumors, and normal adjacent tissues. We also evaluated the effect of 5-aza cytidine on the expression level of TET1 and wnt1 in the HT29 cell line., Materials and Methods: In this study, we assessed TET1 and Wnt1 expression in 5-azacytidine-treated HT29 cells, a demethylating agent commonly used in cancer therapy. Additionally, we enrolled 114 individuals who underwent radical surgical colon resection, including 47 with cancerous tissues and 67 with polyps. We utilized qRT-PCR to measure miR-200, miR-494, TET1, and Wnt1 mRNA levels in colorectal polyps, actual colorectal tumors, and normal adjacent tissues., Results: Our study revealed that TET1 expression was notably lower in both polyps and CRC tissue compared to adjacent normal tissue, with higher TET1 expression in tumors than polyps. We also observed significant differences in miR-200 and miR-494 expression in tumor samples compared to adjacent normal tissue. Our in vitro experiments revealed that 5-azacytidine administration increased TET1 and decreased Wnt1 expression in CRC cell lines. This suggests that DNA-demethylating drugs may have a therapeutic role in modifying TET1 and Wnt signaling in the development of CRC., Conclusions: Overall, our findings shed light on the intricate interactions between TET1, Wnt1, and specific miRNAs in colorectal cancer (CRC) and their potential implications for diagnosis and treatment., (© 2024. The Author(s).)

Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72 h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8 µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72 h and a 5.5-fold increase at 24 h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2 g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20 µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8 µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57 g/L compared to 9.1 ± 0.23 g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Gibberellin (GAs) is an important plant hormone that plays a key role in plant growth and development. Gibberellin 2-oxidase (GA2ox) catalyzes the inactivation of biologically active GA or their direct precursors. In this study, five GA2ox genes were isolated from the wild type Poa pratensis 'Baron', named PpGA2ox3, PpGA2ox4, PpGA2ox5, PpGA2ox8, and PpGA2ox9. Phylogenetic tree analysis showed that PpGA2ox3, PpGA2ox4, PpGA2ox5, and PpGA2ox8 belong to class I GA2ox genes, while PpGA2ox9 belongs to class III GA2ox genes. They expressed in all tissues of Poa pratensis, in each plant tissue and growth stage, the expression patterns were different. After GA 3 spraying treatment, the expression of each gene showed different patterns. Subcellular localization showed that PpGA2ox3 was located in chloroplasts, while PpGA2ox5 and PpGA2ox9 were located in the cytoplasm. When PpGA2ox3 and PpGA2ox9 were overexpressed in Arabidopsis thaliana, they all led to a typical dwarf phenotype, as well as low plant height, small leaves and late flowering. Similarly, when they overexpressed in P. pratensis, the transgenic plants also exhibited a dwarf phenotype with a lower leaf length/width ratio. Hormone analysis suggested that these dwarfing traits might be caused by a decrease in GA 4 content. These studies indicated that the PpGA2ox gene family played an important role in studying the mechanism of plant dwarfism and also had the potential to become important genes for the breeding of P. pratensis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shu-Xia Yin reports financial support was provided by Foundation for Innovative Research Groups of the National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

5-hydroxyvalerate (5-HV) is a crucial C5 platform chemical with versatile applications, yet its efficient production remains a challenge. The Raip, gabT, and yahK genes were integrated into the E. coli LE genome, deleted gabD, and enhanced gabP expression, resulting in the QluMG strain. Additionally, the impact of ethanol and H 2 O 2 on 5-HV production was investigated. Further enhancement was achieved by incorporating an NADPH supplementation system, resulting in the QluMG strain. In the 5 L fermenter, the QluMGD strain produced 21.7 g/L of 5-HV from 50 g/L glucose, with a conversion rate of 43.4 %. The successful integration of the RaiP pathway into the E. coli genome significantly enhanced 5-HV production. The QluMG strain achieved the highest reported yield from glucose in engineered E. coli to date. This study provides a new strategy for the efficient production of 5-HV and other chemicals using 5-HV as a precursor, demonstrating potential for industrial application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, we developed a wearable nanozyme-enzyme electrochemical biosensor that enablies sweat lactate monitoring. The biosensor comprises a flexible electrode system prepared on a polyimide (PI) film and the Janus textile for unidirectional sweat transport. We obtained favorable electrochemical activities for hydrogen peroxide reduction by modifying the laser-scribed graphene (LSG) electrode with cerium dioxide (CeO 2 )-molybdenum disulphide (MoS 2 ) nanozyme and gold nanoparticles (AuNPs). By further immobilisation of lactate oxidase (LOx), the proposed biosensor achieves chronoamperometric lactate detection in artificial sweat within a range of 0.1-50.0 mM, a high sensitivity of 25.58 μA mM -1 cm -2 and a limit of detection (LoD) down to 0.135 mM, which fully meets the requirements of clinical diagnostics. We demonstrated accurate lactate measurements in spiked artificial sweat, which is consistent with standard ELISA results. To monitor the sweat produced by volunteers while exercising, we conducted on-body tests, showcasing the wearable biosensor's ability to provide clinical sweat lactate diagnosis for medical treatment and sports management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Herein, a ccRCC targeting nanodrug is designed to enhance chemodynamic therapy (CDT) as well as activate cuproptosis and tumor immunotherapy via ccRCC cell membrane modifying CuO@Gd 2 O 3 yolk-like particles (CGYL) loaded with lactate oxidase (LOx) (mCGYL-LOx). Benefiting from the homologous targeting effect of Renca cell membranes, the mCGYS-LOx can be effectively internalized by Renca cells, open the "gate", and then release LOx and copper (Cu) ions. LOx can catalyze excessive lactate in Renca cells into H 2 O 2 , following that the produced H 2 O 2 is further converted by Cu ions to the highly toxic ·OH, contributing to tumor CDT. Meanwhile, the excessive Cu ions effectively trigger tumor cuproptosis. These synergistic effects induce the release of damage associated molecular patterns (DAMPs) and activate immunogenic cell death (ICD), leading to DC maturation and infiltration of immune effector cells. Moreover, LOx-mediated lactate consumption downregulates the expression of PD-L1, crippling tumor immune escape. In addition, the mCGYL-LOx improves T 1 -weighted MRI signal, allowing for accurate diagnosis of ccRCC. This study demonstrates that the mCGYL-LOx has great potential for improving therapy of ccRCC via the synergistic actions of CDT and cuproptosis as well as immunotherapy., (© 2024 Wiley‐VCH GmbH.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Exploring the structural basis of membrane proteins is significant for a deeper understanding of protein functions. In situ analysis of membrane proteins and their dynamics, however, still challenges conventional techniques. Here we report the first attempt to immobilize membrane protein complexes on surface-enhanced Raman scattering (SERS)-active supports, titanium dioxide-coated silver (Ag@TiO 2 ) nanoparticles. Biocompatible immobilization of microsomal monooxygenase complexes is achieved through lipid fission and fusion. SERS activity of the Ag@TiO 2 nanoparticles enables in situ monitoring of protein-protein electron transfer and enzyme catalysis in real time. Through SERS fingerprints of the monooxygenase redox centers, the correlations between these protein-ligand interactions and reactive oxygen species generation are revealed, providing novel insights into the molecular mechanisms underlying monooxygenase-mediated apoptotic regulation. This study offers a novel strategy to explore structure-function relationships of membrane protein complexes and has the potential to advance the development of novel reactive oxygen species-inducing drugs for cancer therapy.

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from Marasmius rotula ( Mro UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of Mro UPO, a UPO gene from Marasmius wettsteinii ( Mwe UPO-1), that has a similar reaction profile to Mro UPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant Mwe UPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.

Indolocarbazoles are natural products with a broad spectrum of bioactivity. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. We characterise the pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C-5/C-5'-dihydroxylated indolocarbazole, which we assign as arcyriaflavin F. We also show that a flavin-dependent monooxygenase AcfX catalyses the C-5/C-5' dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Antonis Karantonis reports financial support was provided by Hellenic Foundation for Research and Innovation. Evangelos Topakas reports financial support was provided by European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bioactive dimeric (pre-)anthraquinones are ubiquitous in nature and are found in bacteria, fungi, insects, and plants. Their biosynthesis via oxidative phenol coupling (OPC) is catalyzed by cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well-known, the respective enzymes in mushroom-forming fungi (Basidiomycota) are unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A 1 and B 1 of the mushroom Cortinarius odorifer. The biosynthesis of these unsymmetrically 7,10'-homo-coupled dihydroanthracenones was heterologously reconstituted in the mold Aspergillus niger. Methylation of the parental monomer atrochrysone to its 6-O-methyl ether torosachrysone by the O-methyltransferase CoOMT1 precedes the regioselective homocoupling to phlegmacin, catalyzed by the enzyme CoUPO1 annotated as an "unspecific peroxygenase" (UPO). Our results reveal an unprecedented UPO reaction, thereby expanding the biocatalytic portfolio of oxidative phenol coupling beyond the commonly reported enzymes. The results show that Basidiomycota use peroxygenases to selectively couple aryls independently of and convergently to any other group of organisms, emphasizing the central role of OPC in natural processes., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Optimal plant height is crucial in modern agriculture, influencing lodging resistance and facilitating mechanized crop production. Upland cotton (Gossypium hirsutum) is the most important fiber crop globally; however, the genetic basis underlying plant height remains largely unexplored. In this study, we conducted a genome-wide association study to identify a major locus controlling plant height (PH1) in upland cotton. This locus encodes gibberellin 2-oxidase 1A (GhPH1) and features a 1133-bp structural variation (PAV PH1 ) located approximately 16 kb upstream. The presence or absence of PAV PH1 influences the expression of GhPH1, thereby affecting plant height. Further analysis revealed that a gibberellin-regulating transcription factor (GhGARF) recognizes and binds to a specific CATTTG motif in both the GhPH1 promoter and PAV PH1 . This interaction downregulates GhPH1, indicating that PAV PH1 functions as a distant upstream silencer. Intriguingly, we found that DWARF53 (D53), a key repressor of the strigolactone (SL) signaling pathway, directly interacts with GhGARF to inhibit its binding to targets. Moreover, we identified a previously unrecognized gibberellin-SL crosstalk mechanism mediated by the GhD53-GhGARF-GhPH1/PAV PH1 module, which is crucial for regulating plant height in upland cotton. These findings shed light on the genetic basis and gene interaction network underlying plant height, providing valuable insights for the development of semi-dwarf cotton varieties through precise modulation of GhPH1 expression., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The tomato (Solanum lycopersicum) anthocyanin reduced (are) mutant harbors a mutation in FLAVANONE 3-HYDROXYLASE (F3H), resulting in impaired flavonol antioxidant biosynthesis. The are mutant has reduced pollen performance and seed set relative to the VF36 parental line, phenotypes that are accentuated at elevated temperatures. Transformation of are with the wild-type F3H gene, or chemical complementation with flavonols, prevented temperature-dependent reactive oxygen species (ROS) accumulation in pollen and restored the reduced viability, germination, and tube elongation of are to VF36 levels. Overexpression of F3H in VF36 prevented temperature-driven ROS increases and impaired pollen performance, revealing that flavonol biosynthesis promotes thermotolerance. Although stigmas of are had reduced flavonol and elevated ROS levels, the growth of are pollen tubes was similarly impaired in both are and VF36 pistils. RNA-seq was performed at optimal and stress temperatures in are, VF36, and the F3H overexpression line at multiple timepoints across pollen tube elongation. The number of differentially expressed genes increased over time under elevated temperatures in all genotypes, with the greatest number in are. These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that lead to reproductive failure., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Unspecific peroxygenase from Agrocybe aegerite (AaeUPO) is a remarkable catalyst for the oxyfunctionalization of non-activated C-H bonds under mild conditions. It exhibits comparable activity to P450 monooxygenase but offers the advantage of using H 2 O 2 instead of a complex electron transport chain to reductively activate O 2 . Here, we demonstrate the successful oxidation of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using sol-gel encapsulated AaeUPO. Remarkably, cyclohexane serves both as a solvent and a substrate in this system, which simplifies product isolation. The ratio of cyclohexanone to cyclohexanol using this approach is remarkably higher compared to the oxidation using free AaeUPO in aqueous media using acetonitrile as a cosolvent. The utilization of sol-gel encapsulated AaeUPO offers a promising approach for oxyfunctionalization reactions and improves the chances for this enzyme to be incorporated in the same pot with other chemical transformations., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Tumor immunotherapy has gained more and more attention in tumor treatment. However, the accumulation of lactic acid in tumor tissue inhibits the response of immune cells to form an immunosuppressive microenvironment (ISME). To reverse the ISME, an acid-responsive nanoplatform (termed as MLLN@HA) is reported for synergistically enhanced tumor immunotherapy. MLLN@HA is constructed by the co-loading of lactate oxidase (LOX) and DNA repair inhibitor (NU7441) in a manganese-doped layered double hydroxide (Mn-LDH), and then modified with hyaluronic acid (HA) for tumor-targeted delivery. After endocytosis by tumor cells, MLLN@HA decomposes and releases LOX, NU7441 and Mn 2+ ions in the acidic tumor microenvironment. The released LOX catalyzes the conversion of lactic acid into hydrogen peroxide (H 2 O 2 ), which not only alleviates the ISME, but also provides reactants for the Mn 2+ -mediated Fenton-like reaction to enhance chemodynamic therapy (CDT). Released NU7441 prevents CDT-induced DNA damage from being repaired, thereby increasing double-stranded DNA (dsDNA) fragments within tumor cells. Importantly, the released Mn 2+ ions enhance the sensitivity of cyclic GMP-AMP synthase (cGAS) to dsDNA fragments, and activate the stimulator of interferon genes (STING) to induce an anti-tumor immune response. Such an orchestrated immune-boosting strategy ultimately achieves effective tumor growth inhibition and prevents tumor lung metastasis. STATEMENT OF SIGNIFICANCE: To improve the efficacy of tumor immunotherapy, an innovative acid-responsive MLLN@HA nanoplatform was developed for synergistically enhanced tumor immunotherapy. The MLLN@HA actively targets to tumor cells through the interaction of HA with CD44, and then degrades to release LOX, NU7441 and Mn 2+ ions in the acidic tumor microenvironment. The released LOX generates H 2 O 2 for the Mn 2+ -mediated Fenton reaction and reverses the ISME by consuming lactate. NU7441 prevents DNA damage repair, leading to an increased concentration of free DNA fragments, while Mn 2+ ions activate the cGAS-STING pathway, enhancing the systemic anti-tumor immune response. The orchestrated immune-boosting nanoplatform effectively inhibits tumor growth and lung metastasis, presenting a promising strategy for cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

S-omeprazole and R-rabeprazole are important proton pump inhibitors (PPIs) used for treating peptic disorders. They can be biosynthesized from the corresponding sulfide catalyzed by Baeyer-Villiger monooxygenases (BVMOs). During the development of BVMOs for target sulfoxide preparation, stereoselectivity and overoxidation degree are important factors considered most. In the present study, LnPAMO-Mu15 designed previously and TtPAMO from Thermothelomyces thermophilus showed high (S)- and (R)-configuration stereoselectivity respectively towards thioethers. TtPAMO was found to be capable of oxidating omeprazole sulfide (OPS) and rabeprazole sulfide (RPS) into R-omeprazole and R-rabeprazole respectively. However, the overoxidation issue existed and limited the application of TtPAMO in the biosynthesis of sulfoxides. The structural mechanisms for adverse stereoselectivity between LnPAMO-Mu15 and TtPAMO towards OPS and the overoxidation of OPS by TtPAMO were revealed, based on which, TtPAMO was rationally designed focused on the flexibility of loops near catalytic sites. The variant TtPAMO-S482Y was screened out with lowest overoxidation degree towards OPS and RPS due to the decreased flexibility of catalytic center than TtPAMO. The success in this study not only proved the rationality of the overoxidation mechanism proposed in this study but also provided hints for the development of BVMOs towards thioether substrate for corresponding sulfoxide preparation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plants must balance light capture for photosynthesis with protection from potentially harmful ultraviolet (UV) radiation. Photoprotection is mediated by concerted action of photoreceptors, but the underlying molecular mechanisms are not fully understood. In this study, we provide evidence that UV RESISTANCE LOCUS 8 (UVR8) UV-B, phytochrome red, and cryptochrome blue-light photoreceptors converge on the induction of FERULIC ACID 5-HYDROXYLASE 1 (FAH1) that encodes a key enzyme in the phenylpropanoid biosynthesis pathway, leading to the accumulation of UV-absorbing sinapate esters in Arabidopsis (Arabidopsis thaliana). FAH1 induction depends on the basic leucine zipper transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG that function downstream of all 3 photoreceptors. Noticeably, mutants with hyperactive UVR8 signaling rescue fah1 UV sensitivity. Targeted metabolite profiling suggests that this phenotypic rescue is due to the accumulation of UV-absorbing metabolites derived from precursors of sinapate synthesis, namely, coumaroyl glucose and feruloyl glucose. Our genetic dissection of the phenylpropanoid pathway combined with metabolomic and physiological analyses show that both sinapate esters and flavonoids contribute to photoprotection with sinapates playing a major role for UV screening. Our findings indicate that photoreceptor-mediated regulation of FAH1 and subsequent accumulation of sinapate "sunscreen" compounds are key protective mechanisms to mitigate damage, preserve photosynthetic performance, and ensure plant survival under UV., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Asthma is characterized by aberrant airway smooth muscle (ASM) proliferation, which increases the thickness of the ASM layer within the airway wall and exacerbates airway obstruction during asthma attacks. The mechanisms that drive ASM proliferation in asthma are not entirely elucidated. Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC. In this study, TET1 activity and protein were enhanced in asthmatic human ASM cell cultures. Moreover, the concentration of 5-hmC was higher in asthmatic ASM cells than in nonasthmatic ASM cells. Knockdown (KD) of TET1, but not TET2, reduced the concentration of 5-hmC in asthmatic cells. Because the cytoskeletal protein nestin controls cell proliferation by modulating mTOR, we evaluated the effects of TET1 KD on this pathway. TET1 KD reduced nestin expression in ASM cells. In addition, TET1 inhibition alleviated the platelet-derived growth factor-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. TET1 inhibition also attenuated the proliferation of ASM cells. Taken together, these results suggest that TET1 drives ASM proliferation via the nestin-mTOR axis.

In animals, limiting oxygen upregulates the hypoxia-inducible factor (HIF) and promotes a metabolic shift towards glycolysis. Factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that regulates HIF function by reducing its interaction with histone acetyl transferases. HIF levels are negatively regulated by the HIF prolyl hydroxylases (PHDs) which, like FIH, are 2-oxoglutarate (2OG) oxygenases. Genetic loss of FIH promotes both glycolysis and aerobic metabolism. FIH has multiple non-HIF substrates making it challenging to connect its biochemistry with physiology. A structure-mechanism guided approach identified a highly potent in vivo active FIH inhibitor, ZG-2291, the binding of which promotes a conformational flip of a catalytically important tyrosine, enabling the selective inhibition of FIH over other Jumonji C subfamily 2OG oxygenases. Consistent with genetic studies, ZG-2291 promotes thermogenesis and ameliorates symptoms of obesity and metabolic dysfunction in ob/ob mice. The results reveal ZG-2291 as a useful probe for the physiological functions of FIH and identify FIH inhibition as a promising strategy for obesity treatment., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Exosomes are mediators of intercellular communication. Cancer cell-secreted exosomes allow exosome donor cells to promote cancer growth, as well as metastasis., Methods: Here, exosomes were isolated from the serum of non-small cell lung cancer (NSCLC) patients and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. NSCLC cell proliferation and migration were assessed using CCK-8, 5-ethynyl-2'-deoxyuridine (EdU) and Transwell assays. H1299 tumor formation and pulmonary metastasis were examined in a xenograft model in nude mice., Results: We found that exosomes derived from NSCLC (NSCLC-Exos) promoted NSCLC cell migration and proliferation, and that NSCLC-Exo-mediated malignant progression of NSCLC was mediated by miR-199b-5p. Inhibition of miR-199b-5p decreased the effects of NSCLC-Exos on NSCLC malignant progression. HIF1AN was identified as a downstream target of miR-199b-5p. Furthermore, overexpression of HIF1AN reversed the effects of miR-199b-5p on NSCLC malignant progression., Conclusion: In summary, our findings demonstrated that exosomal-specific miR-199b-5p promoted proliferation in distant or neighboring cells via the miR-199b-5p/HIF1AN axis, resulting in enhanced tumor growth., Competing Interests: Declaration of Competing Interest All the authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster., (© 2024 Wiley‐VCH GmbH.)

Enzymes are making a significant impact on chemical synthesis. However, the range of chemical products achievable through biocatalysis is still limited compared to the vast array of products possible with organic synthesis. For instance, azoxy products have rarely been synthesized using enzyme catalysts. In this study, we discovered that fungal unspecific peroxygenases are promising catalysts for synthesizing azoxy products from simple aniline starting materials. The catalytic features (up to 48,450 turnovers and a turnover frequency of 6.7 s -1 ) and substrate transformations (up to 99% conversion with 98% chemoselectivity) highlight the synthetic potential. We propose a mechanism where peroxygenase-derived hydroxylamine and nitroso compounds spontaneously (non-enzymatically) form the desired azoxy products. This work expands the reactivity repertoire of biocatalytic transformations in the underexplored field of azoxy compound formation reactions., (© 2024. The Author(s).)

2-ketoglutarate (2-KG)/Fe 2+ -dependent dioxygenases can catalyze the highly specific regio- and stereoselective functionalization of C(sp 3 )-H bond of complex compounds under mild reaction conditions. Hyoscyamine 6β-hydroxylase (H6H), a member of these dioxygenases, catalyzes two consecutive oxidation reactions in the synthesis of scopolamine. The first reaction is the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the second is epoxidation of 6β-hydroxyhyoscyamine. This paper introduces the catalytic mechanism, substrate scope, and application of H6H and evaluates the possibility of this enzyme as a biocatalyst for the functionalization of C(sp 3 )-H bond in complex compounds with different structural characteristics via hydroxylation or epoxidation, providing a theoretical basis for modification and application of this enzyme.

Background: Tea is an important cash crop and buds are its main product. To elucidate the molecular mechanism of the sprouting time of tea plants, 'Yuchunzao', which was an early sprouting tea cultivar, was studied. 'Echa 1', sprout one week later than 'Yuchunzao' in spring, was used as the control., Results: A total of 26 hormonal compounds and its derivatives in tea plants were qualified by using Ultra Performance Liquid Chromatography-Tandem mass spectrometry (UPLC-MS/MS). The result showed that GA 20 , GA 3 and ICA were significantly different in 'Yuchunzao' than in 'Echa 1', with GA 20 and GA 3 up-regulated and ICA down-regulated. Based on the Illumina platform, transcriptome analysis revealed a total of 5,395 differentially expressed genes (DEGs). A diterpenoid biosynthesis related gene, gibberellin 2-oxidase 1 (CsGA2ox1), was downregulated in 'Yuchunzao' compared to 'Echa 1'. CsGA2ox1 regulate the transformation of GA different forms in plants. The relative expression of CsGA2ox1 showed an adverse trend with the content of GA 20 and GA 3 . Our results suggest that down regulation of CsGA2ox1 resulted in the accumulation of GA 3 and GA 20 , and then promoted sprout of 'Yuchunzao'., Conclusion: This study provides theoretical basis of tea plants sprout and guides the tea breeding in practice., (© 2024. The Author(s).)

Epigenetic regulation plays a role in Parkinson's disease (PD), and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) catalyzes the first step in DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine. We investigated whether TET1 binds to the promoter of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and regulates its expression, thereby controlling oxidative stress in PD. TRPV1 was identified as an oxidative stress-associated gene in the GSE20186 dataset including substantia nigra from 14 patients with PD and 14 healthy controls and the Genecards database. Lentiviral vectors were used to manipulate Trpv1 expression in rats, followed by 6-hydroxydopamine hydrochloride (6-OHDA) injection for modeling. Behavioral tests, immunofluorescence, Nissl staining, western blot assays, DHE fluorescent probe, biochemical analysis, and ELISA were conducted to assess oxidative stress and neurotoxicity. Trpv1 expression was significantly reduced in the brain tissues of 6-OHDA-treated Parkinsonian rats. Trpv1 alleviated behavioral dysfunction, oxidative stress, and dopamine neuron loss in rats. TET1 mediated TRPV1 hydroxymethylation to promote its expression, and Trpv1 inhibition reversed the mitigating effect of Tet1 on oxidative stress and behavioral dysfunction in PD. TRPV1 activated the AMPK signaling by promoting AMPK phosphorylation to alleviate neurotoxicity and oxidative stress in SH-SY5Y cells. Tet1-mediated Trpv1 hydroxymethylation modification promotes the Ampk signaling activation, thereby eliciting neuroprotection in 6-OHDA-treated Parkinsonian rats. These findings provide experimental evidence that targeting the TET1/TRPV1 axis may be neuroprotective for PD by acting on the AMPK signaling., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aspartyl/asparaginyl hydroxylase (AspH) catalyzes the post-translational hydroxylations of vital human proteins, playing an essential role in maintaining their biological functions. Single-point mutations in the Second Coordination Sphere (SCS) and long-range (LR) residues of AspH have been linked to pathological conditions such as the ophthalmologic condition Traboulsi syndrome and chronic kidney disease (CKD). Although the clinical impacts of these mutations are established, there is a critical knowledge gap regarding their specific atomistic effects on the catalytic mechanism of AspH. In this study, we report integrated computational investigations on the potential mechanistic implications of four mutant forms of human AspH with clinical importance: R735W, R735Q, R688Q, and G434V. All the mutant forms exhibited altered binding interactions with the co-substrate 2-oxoglutarate (2OG) and the main substrate in the ferric-superoxo and ferryl complexes, which are critical for catalysis, compared to the wild-type (WT). Importantly, the mutations strongly influence the energetics of the frontier molecular orbitals (FMOs) and, thereby, the activation energies for the hydrogen atom transfer (HAT) step compared to the WT AspH. Insights from our study can contribute to enzyme engineering and the development of selective modulators for WT and mutants of AspH, ultimately aiding in treating cancers, Traboulsi syndrome and, CKD., (© 2024 Wiley-VCH GmbH.)

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 °C), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95 % conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Six new angucycline structures, including spirocyclione A ( 1 ), which contains an unusual oxaspiro[5.5]undecane architecture, and its ring-A-cleaved product spirocyclione B ( 2 ), were discovered by heterologous expression of a type II polyketide biosynthetic gene cluster captured from a marine actinomycete strain Streptomyces sp. HDN155000. Three flavoprotein monooxygenases are confirmed to be responsible for the oxidative carbon skeleton rearrangements in the biosynthesis of compounds 1 and 2 . The obtained compounds showed promising cytotoxicity against different types of cancer cells.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium . The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N -acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2 H 2 O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.

Dihydroquercetin is a vital flavonoid compound with a wide range of physiological activities. However, factors, such as metabolic regulation, limit the heterologous synthesis of dihydroquercetin in microorganisms. In this study, flavanone 3-hydroxylase (F3H) and flavanone 3'-hydroxylase (F3'H) were screened from different plants, and their co-expression in Saccharomyces cerevisiae was optimized. Promoter engineering and redox partner engineering were used to optimize the corresponding expression of genes involved in the dihydroquercetin synthesis pathway. Dihydroquercetin production was further improved through multicopy integration pathway genes and systems metabolic engineering. By increasing NADPH and α-ketoglutarate supply, the catalytic efficiency of F3'H and F3H was improved, thereby effectively increasing dihydroquercetin production (235.1 mg/L). Finally, 873.1 mg/L dihydroquercetin titer was obtained by fed-batch fermentation in a 5-L bioreactor, which is the highest dihydroquercetin production achieved through de novo microbial synthesis. These results established a pivotal groundwork for flavonoids synthesis.

In the quest for innovative cancer therapeutics, paclitaxel remains a cornerstone in clinical oncology. However, its complex biosynthetic pathway, particularly the intricate oxygenation steps, has remained a puzzle in the decades following the characterization of the last taxane hydroxylase. The high divergence and promiscuity of enzymes involved have posed significant challenges. In this study, we adopted an innovative approach, combining in silico methods and functional gene analysis, to shed light on this elusive pathway. Our molecular docking investigations using a library of potential ligands uncovered TB574 as a potential missing enzyme in the paclitaxel biosynthetic pathway, demonstrating auspicious interactions. Complementary in vivo assays utilizing engineered S. cerevisiae strains as novel microbial cell factory consortia not only validated TB574's critical role in forging the elusive paclitaxel intermediate, T5αAc-1β,10β-diol, but also achieved the biosynthesis of paclitaxel precursors at an unprecedented yield including T5αAc-1β,10β-diol with approximately 40 mg/L. This achievement is highly promising, offering a new direction for further exploration of a novel metabolic engineering approaches using microbial consortia. In conclusion, our study not only furthers study the roles of previously uncharacterized enzymes in paclitaxel biosynthesis but also forges a path for pioneering advancements in the complete understanding of paclitaxel biosynthesis and its heterologous production. The characterization of T1βOH underscores a significant leap forward for future advancements in paclitaxel production using heterologous systems to improve cancer treatment and pharmaceutical production, thereby holding immense promise for enhancing the efficacy of cancer therapies and the efficiency of pharmaceutical manufacturing., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Among the enzymes derived from fungus that act on polysaccharides, lytic polysaccharide monooxygenase (LPMOs) has emerged as a new member with complex reaction mechanisms and high efficiency in dealing with recalcitrant crystalline polysaccharides. This study reported the characteristics, structure, and biochemical properties of a novel LPMO from Talaromyces sedimenticola (namely MaLPMO9K) obtained from the Mariana Trench. MaLPMO9K was a multi-domain protein combined with main body and a carbohydrate-binding module. It was heterologously expressed in E. coli for analyzing peroxidase activity in reactions with the substrate 2,6-DMP, where H 2 O 2 serves as a co-substrate. Optimal peroxidase activity for MaLPMO9K was observed at pH 8 and 25 °C, achieving the best V max value of 265.2 U·g -1 . In addition, MaLPMO9K also demonstrated the ability to treat cellulose derivatives, and cellobiose substrates without the presence of reducing agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Esomeprazole is the most popular proton pump inhibitor for treating gastroesophageal reflux disease. Previously, a phenylacetone monooxygenase mutant LnPAMOmu15 (LM15) was obtained by protein engineering for asymmetric synthesis of esomeprazole using pyrmetazole as substrate. To scale up the whole cell asymmetric synthesis of esomeprazole and reduce the cost, in this work, an Escherichia coli whole-cell catalyst harboring LM15 and formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) were constructed through optimized gene assembly patterns. CRISPR/Cas9 mediated insertion of P trc promoter in genome was done to enhance the expression of key genes to increase the cellular NADP supply in the whole cell catalyst, by which the amount of externally added NADP + for the asymmetric synthesis of esomeprazole decreased to 0.05 mM from 0.3 mM for reducing the cost. After the optimization of reaction conditions in the reactor, the scalable synthesis of esomeprazole was performed using the efficient LM15-BstFDH whole-cell as catalyst, which showed the highest reported space-time yield of 3.28 g/L/h with 50 mM of pyrmetazole loading. Isolation procedure was conducted to obtain esomeprazole sodium of 99.55 % purity and > 99.9 % ee with 90.1 % isolation yield. This work provides the basis for production of enantio-pure esomeprazole via cost-effective whole cell biocatalysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes. TET1 missense mutations are previously reported in many types of leukemia. Here, the human induced pluripotent stem cell line MURAi001-A was generated from skin fibroblasts derived from a 56-year-old female patient carrying the TET1 gene mutation c.4404A > G (p.I1468M), who had a history of ovarian germ cell tumor. The MURAi001-A cell line demonstrated embryonic-like characteristics as it expressed specific stemness markers, differentiated into the three germ layers, and retained normal karyotyping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cellulose-binding domains (CBDs) play a vital role in cellulose degradation by enzymes. Despite the strong ability of brown-rot fungi to degrade cellulose in wood, they have been considered to lack or have a low number of enzymes with CBD. Here, we report the C-terminal domain of a lytic polysaccharide monooxygenase from the brown-rot fungus Gloeophyllum trabeum (GtLPMO9A-2) functions as a CBD, classified as a new family of carbohydrate-binding module, CBM104. The amino acid sequence of GtCBM104 shows no similarity to any known CBDs. A BLAST search identified 84 homologous sequences at the C-terminus of some CAZymes, mainly LPMO9, in basidiomycetous genomes. Binding experiments revealed GtCBM104 binds selectively to native crystalline cellulose (cellulose I), but not to artificially modified crystalline or amorphous cellulose, while the typical fungal CBD (CBM1) bound to all cellulosic materials tested. The adsorption efficiency of GtCBM104 to cellulose I was >20-times higher than that of CBM1. Adsorption tests and microscopic observations strongly suggested that GtCBM104 binds to the hydrophilic regions of cellulose microfibrils, while CBM1 recognizes the hydrophobic surface. The discovery of GtCBM104 strongly suggests that the contribution of CBD to the cellulose enzymatic degradation mechanism of brown-rot fungi is much larger than previously thought., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing ShF5H1 under the control of the C4H (cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that ShF5H1 overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the ShF5H1 overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.

The degradation of phenylacetate (PA) was investigated as a model to explore aromatic compound breakdown in the fungal system. Fungal strains capable of utilizing PA as their sole carbon source were isolated using a minimal solid medium supplemented with 0.5 % PA. Subsequent cultivation in minimum liquid medium revealed that selected fungal strains, including Trametes versicolor TV0876 and TV3295, Paecilomyces hepiali PH4477, and Akanthomyces muscarius AM1091, efficiently removed PA within 24 h. HPLC analysis of culture supernatants from various fungal strains revealed a time-dependent accumulation of 2-hydroxyphenylacetate (2-HPA) and 4-hydroxyphenylacetate (4-HPA), two key major metabolic products primarily found in ascomycetes and basidiomycetes, respectively. This suggests that the first hydroxylation of PA is catalyzed by two distinct hydroxylases, one for each fungal group. Furthermore, fungal species that make 4-HPA also produce phenylethanol (PE), indicating a distinct catabolic mechanism to remove PA by direct reduction of PA to PE. A. muscarius AM1091, identified as the most efficient PA degrader in this study, was studied further to determine the biochemical pathway of PA degradation. RNA-Seq and RT-PCR analyses of AM1091 revealed two oxidative enzyme genes, CYP1 and DIO4, upregulated in the presence of PA. Targeted disruption utilizing preassembled Cas9-gRNA ribonucleoprotein complexes and homologous DNAs harboring the URA3 gene as an auxotrophic marker resulted in the cyp1 and dio4 mutant strains. The cyp1 mutant was incapable of converting PA to 2-HPA, indicating its involvement in the C2 hydroxylation, whereas the dio4 mutant was unable to degrade 2,5-dihydroxyphenylacetate (2,5-DHPA), resulting in the accumulation of 2,5-DHPA. Our findings indicate that A. muscarius AM1091 degrades PA through the activities of CYP1 and DIO4 for the C2 hydroxylation and subsequent ring-opening reactions, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Human cortical neurons (hCNs) exhibit high dendritic complexity and synaptic density, and the maturation process is greatly protracted. However, the molecular mechanism governing these specific features remains unclear. Here, we report that the hominoid-specific gene TBC1D3 promotes dendritic arborization and protracts the pace of synaptogenesis. Ablation of TBC1D3 in induced hCNs causes reduction of dendritic growth and precocious synaptic maturation. Forced expression of TBC1D3 in the mouse cortex protracts synaptic maturation while increasing dendritic growth. Mechanistically, TBC1D3 functions via interaction with MICAL1, a monooxygenase that mediates oxidation of actin filament. At the early stage of differentiation, the TBC1D3/MICAL1 interaction in the cytosol promotes dendritic growth via F-actin oxidation and enhanced actin dynamics. At late stages, TBC1D3 escorts MICAL1 into the nucleus and downregulates the expression of genes related with synaptic maturation through interaction with the chromatin remodeling factor ATRX. Thus, this study delineates the molecular mechanisms underlying human neuron development., Competing Interests: Declaration of interests The authors have no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (k cat /K m ) of 6.6×10 -2 mg -1 ml min -1 and 1.8×10 -2 mg -1 ml min -1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C 1 /C 4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: • Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. • Cello-oligosaccharides strongly downregulated the LPMO1 expression. • Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3., (© 2024. The Author(s).)

Polyacrylamide (PAM) is a high-molecular-weight polymer with extensive applications. However, the inefficient natural degradation of PAM results in environmental accumulation of the polymer. Biodegradation is an environmentally friendly approach in the field of PAM treatment. The first phase of PAM biodegradation is the deamination of PAM, forming the product poly(acrylic acid) (PAA). The second phase of PAM biodegradation involves the cleavage of PAA into small molecules, which is a crucial step in the degradation pathway of PAM. However, the enzyme that catalyzes the degradation of PAA and the molecular mechanism remain unclear. Here, a novel monooxygenase PCX02514 is identified as the key enzyme for PAA degradation. Through biochemical experiments, the monooxygenase PCX02514 oxidizes PAA with the participation of NADPH, causing the cleavage of carbon chains and a decrease in the molecular weight of PAA. In addition, the crystal structure of the monooxygenase PCX02514 is solved at a resolution of 1.97 Å. The active pocket is in a long cavity that extends from the C-terminus of the TIM barrel to the protein surface and exhibits positive electrostatic potential, thereby causing the migration of oxygen-negative ions into the active pocket and facilitating the reaction between the substrates and monooxygenase PCX02514. Moreover, Arg10-Arg125-Ser186-Arg187-His253 are proposed as potential active sites in monooxygenase PCX02514. Our research characterizes the molecular mechanism of this monooxygenase, providing a theoretical basis and valuable tools for PAM bioremediation.