Steroids with anti-inflammatory, anti-allergic, endocrine-regulating, and other pharmaceutical activities represent the second most widely used class of drugs worldwide, following antibiotics. Their industrial production primarily relies on Mycobacteria-mediated biotransformation of sterols into key intermediates, followed by chemical or enzymatic modifications. While the sterol metabolic pathways in Mycobacteria have been intensively studied, the identification and functional characterization of key enzymes, particularly cytochrome P450 enzymes (CYPs or P450s) and their cognate redox partners, remain incomplete. Here, we heterologously expressed 24 P450s, 10 ferredoxin reductases (FdRs), and 12 ferredoxins (Fdxs) from Mycobacterium neoaurum ZC-1 in Escherichia coli. In vitro biochemical experiments identified five P450 enzymes (CYP125A76, CYP125A77, CYP125A78, CYP142A12, and CYP124A1) capable of catalyzing sterol side-chain terminal oxidation. Screening 120 redox partner combinations revealed FdR4662/Fdx4443 as the optimal cognate redox partners for all five P450 enzymes. With this redox partner pair, CYP142A12 achieved a conversion ratio of 89% for 4-cholesten-3-one with NADH as the preferred cofactor. Structural analyses indicate that the electron-transfer efficiency is primarily governed by electrostatic complementarity around the Fe-S cluster, the redox-center distance between the Fe-S cluster and heme-iron, and the FAD-to-cluster distance within the FdR-Fdx complex. These findings highlight the critical role of redox partner selection in enhancing P450 catalytic efficiency and provide a solid foundation for engineering high-efficiency industrial strains to improve steroid biomanufacturing and reduce production costs.

Medium- and long-chain dicarboxylic acids (M/LCDAs) are key monomers for the synthesis of nylons and high-performance engineering plastics. Compared to traditional chemical methods, microbial synthesis offers advantages such as environmental friendliness and high regioselectivity. However, its industrial application remains limited by bottlenecks, including low mass transfer efficiency on hydrophobic substrates, instability of key oxidase systems, and cellular metabolic imbalances. This review summarizes recent strategies leveraging enzyme engineering, systems metabolic engineering, and diverse synthetic biology approaches to overcome current limitations in the biosynthesis of M/LCDAs. We specifically highlight mechanisms for enhancing the transmembrane transport of hydrophobic substrates and the mining of novel transporters. Furthermore, we elaborate on protein engineering efforts targeting key enzymes (e.g. cytochrome P450s), covering rational design, fusion expression, and novel dimerization techniques. At the systems level, we discuss metabolic network regulation achieved through the construction of the reverse β-oxidation cycle (r-BOX) and the reprogramming of cofactor regeneration and energy metabolism. Finally, future perspectives on integrating AI-aided design and waste valorization are proposed to provide theoretical guidance for the efficient and sustainable biomanufacturing of M/LCDAs.

C21-hydroxylation is a crucial step in the synthesis of corticosteroids. For instance, C21-hydroxylase catalyzes the conversion of progesterone (PRO) into 11-deoxycorticosterone (DOC, the precursor of cortisol and aldosterone). Through structure-guided rational design combined with focused rational iterative site-directed mutagenesis (FRISM), we engineered the wild-type CYP154C5 from Nocardia farcinica to obtain the highly efficient variant M6a (F92A/V291L/L294I/Q239K/F180W/Q398M). This variant demonstrated a complete regioselectivity shift from C16α to C21 hydroxylation (98% selectivity) of PRO with high catalytic efficiency (>99% conversion). To further enhance catalytic performance, we engineered a redox fusion variant (M6a-RhFRED L3) that demonstrated the highest catalytic activity, achieving a 1.43-fold improvement compared to the M6a-RhFRED. Additionally, M6a-RhFRED L3 catalyzed the C21-hydroxylation of three steroid analogs (dydrogesterone, 16-dehydroprogesterone, and pregna-4,6-diene-3,20-dione), with selectivity reaching 98% and conversion rates exceeding 60%. Molecular docking and molecular dynamics (MD) simulations revealed that the PRO substrate undergoes conformational rearrangement in the M6a active site. This structural reorganization underscores the critical role of key residues in modulating regioselectivity, particularly the shift from C16α to C21 hydroxylation. This study not only provides an efficient biocatalyst for steroids C21-hydroxylation but also offers valuable insight for the rational engineering of the P450 enzymes.

The selective conversion of chemically inert linear alkanes into alpha,omega-dicarboxylic acids remains a significant challenge in sustainable chemistry. alpha,omega-Dicarboxylic acids are key building blocks for high-performance polymers and pharmaceuticals, their industrial production relies on energy-intensive, poorly selective multi-step oxidation processes. These limitations stem from the intrinsic inertness of inactivated primary C-H bonds and the difficulty of achieving regioselective dual-terminal C-H bond oxyfunctionalization under mild conditions. Consequently, progress in the conversion of chemically inert linear alkanes into alpha,omega-dicarboxylic acids hinges on two central challenges: enabling highly regioselective terminal C-H oxidation and developing sustainable transformation pathways. To address these challenges, we developed a multi-enzyme cascade driven by internal cofactor cycling that enables one-pot deep oxidation of dodecane to dodecanedioic acid under mild conditions. In the first stage, a hydrogen-borrowing-based NADH regeneration strategy enables selective oxidative C-H functionalization, converting dodecane to dodecanoic acid. Coupling this step with a second hydrogen-borrowing cycle and in situ elimination of H2O2 further drives terminal oxidation, ultimately affording 1.49 mM dodecanedioic acid. In addition, nonane, decane, undecane, and tridecane were successfully converted into their corresponding alpha,omega-dicarboxylic acids. Overall, this study establishes a green biocatalytic paradigm for the deep transformation of inert alkanes using molecular oxygen as the sole oxidant, enabled by internal cofactor cycling and in situ reactive oxygen management. This strategy maximizes atom economy and provides an efficient and sustainable route for upgrading linear alkanes into value-added products.

Avermectins (AVMs) are a class of 16-membered ring macrolides produced by Streptomyces avermitilis. Renowned for their potent insecticidal and acaricidal properties, AVMs are widely used as environmentally friendly biopesticides. Although the biosynthetic gene aveE encoding a cytochrome P450 monooxygenase was identified 30 years ago, its exact catalytic function and mechanism have remained elusive due to a lack of biochemical characterization. Here, we overcome the long-standing challenge in soluble and functional protein expression of AveE in Escherichia coli and reconstitute the in vitro activity of this P450 enzyme using surrogate redox partner proteins. Time-course studies reveal monohydroxylation at the C8a position of the substrates as the initial step, subsequently leading to tetrahydrofuran (THF) ring formation. Isotopic labeling experiments provide significant insight into the catalytic mechanism of AveE, revealing the cooperation of the C8a,C6-diol pathway and the C6-nucleophilic attack pathway. This study not only presents an effective strategy for heterologous expression of difficult P450 enzymes in E. coli but also elucidates the exact process of THF ring formation during AVM biosynthesis.

Benzyl and phenylpropanoid acids are widely used in organic synthesis of fine chemicals, such as pharmaceuticals and condiments. However, biocatalysis of these acids has received less attention than chemical synthesis. One of the main challenges for biological production is the limited availability of alcohol dehydrogenases and aldehyde dehydrogenases. Environmental microorganisms are potential sources of these enzymes. In this study,129 alcohol dehydrogenases and 42 aldehyde dehydrogenases from Corynebacterium glutamicum, Pseudomonas aeruginosa, and Bacillus subtilis were identified and explored with various benzyl and phenylpropanoid alcohol and aldehyde substrates, among which four alcohol dehydrogenases and four aldehyde dehydrogenases with broad substrate specificity and high catalytic activity were obtained. Moreover, a cascade whole-cell catalytic system including ADH-90, ALDH-40, and the NAD(P)H oxidase LreNox was established, which showed high efficiency in converting cinnamyl alcohol and p-methylbenzyl alcohol into the respective carboxylic acids. Remarkably, this biocatalytic system can be easily scaled up to gram-level production, facilitating preparation purposes.

Polycarbonate (PC) is known to suffer from damage over time, particularly in low-temperature settings besides its inherent flammability, where it can develop notches and lose its impact toughness. To address these issues, we synthesized schiff-based polysiloxane containing the benzene substituted with methoxy (referred to as PSOMe-X, X being the number of methoxy substitution on the benzene ring). Interestingly, by merely increasing the number of methoxy substitutions in PSOMe-X, the PC/PSOMe-X composites demonstrated significantly improved comprehensive performance. The PC/PSOMe-3 composite successfully passed the UL-94 (vertical burning test for Flammability of Plastic Materials for Parts in Devices and Appliances developed by Underwriters Laboratories) V0 rating, and the limiting oxygen index (LOI) was increased to 29.9 %. This improvement was accompanied by a notable reduction in the peak heat release rate (PHRR) by 64 % and total smoke generation (TSP) by 18 %. Meanwhile the notched impact strength of the PC/PSOMe-3 composite was improved by 190 % at room temperature and 72 % at -25 degrees C. The mechanism for improved flame retardancy and impact tougheness for PC/ PSOMe-X composites were unveiled.

In this work, we synthesized a series of compounds, denoted as xCTAB@AMP, by incorporating varying different molar fractions of cetyltrimethylammonium bromide (CTAB) into ammonium phosphomolybdate (AMP). The effects of CTAB modification on the surface characteristics, morphology and hygroscopicity of AMP were studied. Moreover, we explored the combined flame-retardant impact between xCTAB@AMP and aluminum diethylphosphinate (ADP) when incorporated into epoxy resin (EP), as well as the resulting composite's mechanical properties, thermal stability and antibacterial properties. The EP composite containing 50 molar percent CTAB-modified AMP (EP/50CTAB@AMP/ADP) demonstrated remarkable flame retardancy, achieving a UL-94 V-0 rating and increasing limiting oxygen index (LOI) to 30.0%. This formulation significantly lowered the peak heat release rate (PHRR) to 452 kW/m2, a 65% reduction to that of EP, and the total heat release (THR) to 68 MJ/m2, a 24% decrease to that of EP. Additionally, compared to EP, the peak smoke production rate (PSPR) of this composite was decreased by 30% (0.28 m2/s), the total smoke production (TSP) was reduced by 25% (30.2 m2), and peak carbon monoxide release rate (PCOP) was diminished by 34% (0.038 g/s). The combination of 50CTAB@AMP and ADP in the EP matrix exhibited an exceptional synergistic flame-retardant effect. Concurrently, the CTAB modification bolstered the interfacial interactions between AMP and the EP matrix, which enhanced the mechanical properties of EP/AMP/ADP composites. As a result, the tensile strength and elongation at break of the EP/50CTAB@AMP/ADP composite increased by 13% and 15%, respectively, compared to the EP/AMP/ADP. Moreover, the 50CTAB@AMP maintained its inherent antibacterial activity, which endowed the EP/50CTAB@AMP/ADP composite with a potent inhibitory effect against Staphylococcus aureus, a common pathogenic bacterium.

Widespread use of polyethylene terephthalate (PET) plastics and their recycling challenges have led to substantial accumulation of PET wastes in global environments, with inevitable consequences for their entry into the food chains. Recent studies have increasingly documented the ingestion of microplastics by humans through food and beverages. However, the fate of these microplastics within the gastrointestinal tract, particularly the role of the human gut microbiota, remains inadequately understood. To address this knowledge gap, we employed a bioinformatics workflow integrated with functional verification to investigate the PET digestion/degradation capabilities of intestinal microorganisms. This approach identified a novel PET hydrolase—HGMP01 from the human gut metagenome, which exhibits the capacity to hydrolyze PET nanoparticles. Moreover, comprehensive exploration for HGMP01 homologues in the human gut metagenome and metatranscriptome unveil their distribution in diverse intestinal microorganisms. This study provides biochemical evidence for an unforeseen role of human gut microbiome in plastic digestion, thus holding substantial implications for human health.

Cytochrome P450 enzymes (CYPs or P450s) and ferredoxins (Fdxs) are ubiquitously distributed in all domains of life. Bacterial P450s are capable of catalyzing various oxidative reactions with two electrons usually donated by Fdxs. Particularly in Streptomyces, there are abundant P450s that have exhibited outstanding biosynthetic capacity of bioactive metabolites and great potential for xenobiotic metabolisms. However, no systematic study has been conducted on physiological functions of the whole cytochrome P450 complement (CYPome) and ferredoxin complement (Fdxome) of any Streptomyces strain to date, leaving a significant knowledge gap in microbial functional genomics. Herein, we functionally analyze the whole CYPome and Fdxome of Streptomyces venezuelae ATCC 15439 by investigating groups of single and sequential P450 deletion mutants, single P450 overexpression mutants, and Fdx gene deletion or repression mutants. Construction of an unprecedented P450-null mutant strain indicates that none of P450 genes are essential for S. venezuelae in maintaining its survival and normal morphology. The non-housekeeping Fdx1 and housekeeping Fdx3 not only jointly support the cellular activity of the prototypic P450 enzyme PikC, but also play significant regulatory functions. These findings significantly advance the understandings of the native functionality of P450s and Fdxs as well as their cellular interactions.

Indigoids, a class of bis-indoles, have long been applied in dyeing, food, and pharmaceutical industries. Recently, interest in these 'old' molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties. However, these indigo derivatives are difficult to access through chemical synthesis. In this study, we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution. A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors. Among the prepared indigoid organic photocatalysts, a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation, making them promising candidates for the development of new organic electronic devices. Thus, we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids, offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.

For improving the flame-retardant performance of epoxy thermosets without sacrificing other properties, a novel aromatic imine-containing DOPO-based bisphenol, DOPO-AI, was synthesized. Diglycidyl ether bisphenol F epoxy resin (DGEBF) reacted with DOPO-AI first, followed by curing with 4, 4 '-diaminodiphenylmethane (DDM). When the mass of DOPO-AI was 25% of DGEBF, the resulting epoxy thermoset EP/20DOPO-AI exhibited the V-0 rating with the limiting oxygen index of 35.4%. In the cone calorimetry test, the peak heat release rate and total heat release of EP/20DOPO-AI showed the reduction of 26% and 27%, respectively, compared to those of DGEBF/DDM (EP). The mechanism of DOPO-AI in reducing heat release and increasing char-form ability of EP/ DOPO-AI was studied. Interestingly, the mechanical and thermal mechanical properties of EP/20DOPO-AI were improved, although the crosslinking density was much lower than that of EP. The tensile strength and storage modulus of EP/20DOPO-AI were increased by 13% and 5%, while the glass transition temperature and the elongation at break were kept, compared with those of EP. The high rigidity of DOPO and aromatic imine was the reason for the improvement of the mechanical properties of EP/20DOPO-AI. Furthermore, EP/20DOPO-AI showed a small reduction in thermal stability, compared with that of EP.

Abstract Synthetic photoelectrochemistry (PEC), the fusion of photoredox catalysis (PRC), and synthetic organic electrochemistry (SOE) provide a powerful way for the selective C(sp 2 )/C(sp 3 )H functionalization via single‐electron transfer (SET). Compared with PRC and SOE, PEC shows stronger redox ability under mild conditions and significant atomic and energy economy. The combination of PEC with hydrogen atom transfer (HAT) makes the inert CH activation possible. Herein, the corresponding work is reviewed by the classification of the formation of CO, CC, CN, and CP bonds.

Phytochemical research on an extract of Notopterygium incisum yielded twenty-four compounds (1-24), including four new compounds 7, 10, 13 and 17. The structures of new compounds were elucidated by spectroscopic analysis, and the absolute configuration of 5 were assigned via Mosher's method. In addition, compounds 2, 9, 19 and 24 were analyzed by single crystal X-ray analysis for the first time. The new compound 7 exhibited remarkable cytotoxic activity against the human pancreatic cancer cell lines PANC-1, CAPAN-2, CFPAC-1, and SW1990 with IC50 values of 6.25 +/- 0.54, 7.74 +/- 1.12, 6.68 +/- 1.11, and 5.35 +/- 0.33 mu M, respectively. The primary mechanistic study indicated that 7 caused cell death probably due to induced defects in the mitotic process. Moreover, compounds 2 and 7-9 effectively protected against H2O2-induced neurotoxicity in the SHSY5Y cells, and 1, 3, 6, 10, 11, 16 and 19 exhibited strong DPPH scavenging ability.

Herein, we describe an environmentally benign enzymatic approach for the preparation of indigoid from indole derivatives. A series of beneficial P450BM3 mutants were obtained using a stepwise approach involving site-directed, random, and combinatory hot-site mutations. Using H2O2 as the terminal oxidant and N-(omega-imidazolyl)-hexanoyl-(L)-phenylalanine as a co-catalyst, the quadruple-mutant F87G/T268V/F77I/E140D efficiently catalyzed the 3-hydroxylation of indole and subsequent autooxidation to indigo with higher efficiency than the flavin-containing monooxygenase, PTDH-mFMO, which was the best oxidizing enzyme for indigo synthesis reported to date. Following this procedure, 12 indigoid compounds were prepared using indole derivatives with various substituents as starting materials with moderate to high isolated yields (31.3-79.5%). The catalytic efficiencies (k(cat)/K-m) of the beneficial mutants for typical indole substrates ranged from 182-2849 mM(-1) . min(-1), almost 2- to 712-fold higher than those of the reported P450 enzymes. This study provides a new enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives.

BACKGROUND:Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides.RESULTS:In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides.CONCLUSIONS:Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Most P450s require redox partners for the electron transfer during catalysis. However, little information is available on cognate redox partners for P450s, which greatly limits P450 function exploration and practical application. Thus, the stategy of building various hybrid P450 catalytic systems with surrogate redox partner has often adopted to engineer P450 biocatalysts. In this study, we compare three pairs of frequently-used surrogate redox partner SelFdx1499/SelFdR0978, Adx/AdR and Pdx/PdR and in terms of their electron transfer properties. The three selected bacterial Class I P450s include PikC, P450sca-2 and CYP-sb21, which are responsible for production of high-value-added products. Here we show that SelFdx1499/SelFdR0978 is the most promising redox partner compared to Adx/AdR and Pdx/PdR. The results provide insights into the domination for P450-redox partner interactions in modulating the catalytic activity of P450s. This study not only produces a more active biocatalyst but also suggests a general chose for a universal reductase which would facilitate engineering of P450 catalyst.

A series of epoxy resins containing aromatic imine are synthesized via the Duff reaction by using various bisphenols, which show significantly reduced heat release capacity (HRC) and total heat release (THR) after curing. Compared to diglycidyl ether bisphenol A/4,4-diaminodiphenyl methane (DGEBA/DDM), the HRC and THR of phenolphthalein-based epoxy resin containing aromatic imine/DDM (BPP-E/DDM) are reduced by 87% (464 J/g K vs 58 J/g K) and 62% (30.2 kJ/g vs 11.4 kJ/g), respectively. Furthermore, the BPP-E/DDM exhibits a UL-94 V-0 rating (1.6 mm) and a limiting oxygen index of 48% (3.2 mm). Compared to DGEBA/DDM in the cone calorimeter test, the peak heat release rate, THR, peak smoke produce rate, and total smoke production of BPP-E/DDM were reduced by 91% (1006 kW/m2 vs 87 kW/m2), 51% (89 MJ/m2 vs 44 MJ/m2), 89% (0.36 m2/s vs 0.09 m2/s), and 79% (38 m2 vs 8 m2), respectively. The influences of aromatic imine and the chemical structures between two benzene rings in the bisphenols on HRC and THR of the resultant epoxy thermosets are investigated. The mechanism studies show that the presence of aromatic imine in the epoxy resins completely changes the decomposition process to generate the products containing aromatic N-heterocycle, which contributes greatly to the improved char-forming ability and flame retardancy. Meanwhile the thermosets bearing carbonyl, 3-phthalidylene, and sulfonyl between two benzene rings in the bisphenols show better char-forming ability and flame retardancy among the resultant epoxy resins containing aromatic imine, due to more polycyclic aromatic hydrocarbons produced in the condensed phase.

Presently, pregnancy test strip (PTS) is one of the most widely used and advanced biomedical testing products. In this study, a portable, sensitive, and low-cost biosensor based on PTS was developed for detecting pathogenic bacteria. In the present study, PTS combined with metal-organic frameworks (MOFs) and hybridization chain reaction (HCR) was used for visual point-of-care (POC) detection of Escherichia coli O157:H7. A single-stranded capture probe (CP) was immobilized onto the surface of magnetic beads (MBs). The CP and an E. coli O157:H7-specific aptamer (AP) formed a double-stranded structure on the surface of MBs. The AP preferentially bound to E. coli O157:H7, thereby exposing some CP and causing their release. Subsequently, the single-stranded CP hybridized with biotin-labeled double-stranded DNA polymers by HCR through the 5-end sequence of the reporter probe (RP). Numerous MOFs encapsulated with human chorionic gonadotropin (hCG) and modified streptavidin (SA) were then introduced and could be directly and visually detected by PTSs. Under optimal conditions, the method can detect E. coli O157:H7 with a detection limit of 530 CFU/mL. Thus, this method has a high potential for cost-effective, portable, and rapid determination of E. coli O157:H7, especially in resource-limited environments.

In this work, we demonstrated a new method for rapidly forming a three-dimensional network porous char layer during the combustion of PP composite foam, based on the combination of intumescent flame retardant (IFR) with expandable graphite (EG) and carbon nanotubes (CNTs). The IFR was composed of pentaerythritol (PER) and ammonium polyphosphate (APP) with a mass ratio of 1:2. Compared to PP composite foams with 40% IFR (PP/40IFR), the PP/EG/CNTs/IFR composite foams we designed could preserve the original foam shape and show excellent flame retardancy after combustion due to the formation of a complete porous char layer. It was found that the EG aggregates violently expanded during combustion and offset the shrink of cellular structure, which can blocks the heat transfer from outside to inside and protect the inner material from the fire. At the same time, the CNTs could bind expanded EG aggregates tightly through the interaction between CNTs and EGs. Very interestingly, the IFR was found to form char residue on the surface of CNTs/expanded EG aggregates to not only improve the compactness of the porous char layer but also promote thermal oxidation resistance of this char layer. Owing to the synergistic effect of the IFR with expanded EG and CNTs, the flame retardancy of PP/EG/ CNTs/IFR composite foams was dramatically improved, including delayed the flame spread and completely inhibited the droplet phenomenon, and reached the level of flame retardant HF-1 in the standard level combustion test. Moreover, the PP/10EG/5CNTs/25IFR composite foams prepared by this work also presented excellent ablation resistance in the case of a large-scale flame.