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 H2O2 serves as a co-substrate. Optimal peroxidase activity for MaLPMO9K was observed at pH 8 and 25 °C, achieving the best Vmax 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.

The environmental benefits of utilizing protease as a biocatalyst for wool shrink-resist finishing have been widely recognized. However, the efficacy of individual protease treatment is unsatisfactory due to its incapability towards the outermost cuticle layer of wool fibers that contains hydrophobic fatty acids. In order to weaken the structural integrity of the highly cross-linked scales and promote the enzymatic anti-felting, sodium sulfite and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were employed in combination with papain, respectively, aiming at obtaining a low shrinkage without unacceptable fiber damages. Based on the synergistic effect of papain and TCEP, the edges of wool scales were slightly destroyed by the reduction of disulfide bonds, accompanied by enzymatic hydrolysis of the keratin component. Through the controlled reduction and hydrolysis of wool scales, satisfactory anti-felting result was achieved without causing severe damage to the fiber interiors. In the presence of 0.25 g/L TCEP and 25 U/mL papain, the area shrinkage of wool fabric decreased to approximately 6 %, with a low strength loss of less than 8 %. Meanwhile, the dyeing behavior of the wool fabric under low-temperature conditions was dramatically improved, leading to decreased energy consumption during production. The present work provides an alternative for eco-friendly finishing of wool fabrics, which can be applied commercially.

Establishing a multi-enzyme synergistic lignocellulosic biodegradation system using lytic polysaccharide monooxygenase (LPMO) and polyphenol oxidases is vital for efficiently utilizing plant biomass waste, ultimately benefiting the carbon cycle and promoting environmental protection. Single-residue mutations of LPMO can improve the efficiency of lignocellulosic biomass degradation. However, the activity of mutant-type LPMO in relation to lignin-diverted reducing agents has not been sufficiently explored. In this study, laccase and tyrosinase were initially investigated and their optimal conditions and impressive thermal stability were revealed, indicating their potential synergistic abilities with LPMO in lignocellulose biodegradation. When utilizing gallic acid as a reducing agent, the activities of LPMOs were increased by over 10%, which was particularly evident in mutant-type LPMOs after the addition of polyphenol oxidases. In particular, the combination of tyrosinase with either 4-hydroxy-3-methoxyphenylacetone or p-coumaric acid was shown to enhance the efficacy of LPMOs. Furthermore, the highest activity levels of wild-type LPMOs were observed with the addition of laccase and 3-methylcatechol. The similarities between wild and mutant LPMOs regarding their activities in lignin-diverted phenolic compounds and reducing agents are almost identical, suggesting that the single-residue mutation of LPMO does not have a detrimental effect on its performance. Above all, this study indicates that understanding the performance of both wild and mutant types of LPMOs in the presence of polyphenol oxidases and various reducing agents constitutes a key link in the industrialization of the multi-enzyme degradation of lignocellulose.

Dyes pose great threats to the aquatic environment and human health. Fe0-based Fenton-like systems have been widely employed for the degradation of organic dyes. However, the regulation of degradability and recyclability was still unclear. In this study, Rhodamine B (RhB) was served as the model pollutant, hydroxylamine hydrochloride was selected as the RA, the natural photocatalysis system demonstrated stable operation. RA, as performance enhancement agent, was firstly reported in micro/nano-Zero-Valent Iron@Biochar (m/nZVI@BC) based SPC-RA system. Carrier size-fractionated m/nZVI@BC was fabricated by one-step carbothermal method. As a result, RA synergistically interacted with SPC, and the reaction time reduced from 15 min to 4 min. In the 0.010 g m/nZVI@BC-mediated SPC-RA system, over 95% of RhB (100 mg·L-1, 1041.667 mg·g-1) was successfully degraded. The maximum degradation ability could still exceed 1g·g-1 via 5 times repeated applications. Meanwhile, the loss of degradability, caused by halving SPC concentration could be compensated by RA dosage measurement. The entire degradation process was predominantly dominated by free radicals (•OH> 1O2> •O2-> •CO3-). Reactive oxidizing species (ROSs) were primarily excited by α-Fe0, Fe3C and N sites of biochar (BC). Light and BC carrier dedicated slight influence. These discoveries shed a light on the activity and recyclability regulation of catalytic material, aligning with the principles of green chemistry and cleaner production. This study demonstrates a novel approach to efficient management of solid waste disposal, reuse of waste biomass, advanced treatment of dye-containing wastewater, pollution control in aquatic environments.

Morphology-transformational self-assembly of peptides allows for manipulation of the performance of nanostructures and thereby advancing the development of biomaterials. Acceleration of the morphological transformation process under a biological microenvironment is important to efficiently implement the tailored functions in living systems. Herein, we report redox-regulated in situ seed-induced assembly of peptides via design of two co-assembled bola-amphiphiles serving as a redox-resistant seed and a redox-responsive assembly monomer, respectively. Both of the peptides are able to independently assemble into nanoribbons, while the seed monomer exhibits stronger assembling propensity. The redox-responsive monomer undergoes morphological transformation from well-defined nanoribbons to nanoparticles. Kinetics studies validate the role of the assembled inert monomer as the seeds in accelerating the assembly of the redox-responsive monomer. Alternative addition of oxidants and reductants into the co-assembled monomers promotes the redox-regulated assembly of the peptides facilitated by the in situ-formed seeds. The reduction-induced assembly of the peptide could also be accelerated by in situ-formed seeds in cancer cells with a high level of reductants. Our findings demonstrate that through precisely manipulating the assembling propensity of co-assembled monomers, the in situ seed-induced assembly of peptides could be achieved. Combining the rapid assembly kinetics of the seed-induced assembly with the common presence of redox agents in a biological microenvironment, this strategy potentially offers a new method for developing biomedical materials in living systems.

Dimeric prodrug nanoassemblies (DPNAs) stand out as promising strategies for improving the efficiency and safety of chemotherapeutic drugs. The success of trisulfide bonds (-SSS-) in DPNAs makes polysulfide bonds a worthwhile focus. Here, we explore the comprehensive role of tetrasulfide bonds (-SSSS-) in constructing superior DPNAs. Compared to trisulfide and disulfide bonds, tetrasulfide bonds endow DPNAs with superlative self-assembly stability, prolonged blood circulation, and high tumor accumulation. Notably, the ultra-high reduction responsivity of tetrasulfide bonds make DPNAs a highly selective \"tumor bomb\" that can be ignited by endogenous reducing agents in tumor cells. Furthermore, we present an \"add fuel to the flames\" strategy to intensify the reductive stress at tumor sites by replenishing exogenous reducing agents, making considerable progress in selective tumor inhibition. This work elucidates the crucial role of tetrasulfide bonds in establishing intelligent DPNAs, alongside the combination methodology, propelling DPNAs to new heights in potent cancer therapy.

Ruminants are essential for global food security, but these are major sources of the greenhouse gas methane. Methane yield is controlled by the cycling of molecular hydrogen (H2), which is produced during carbohydrate fermentation and is consumed by methanogenic, acetogenic, and respiratory microorganisms. However, we lack a holistic understanding of the mediators and pathways of H2 metabolism and how this varies between ruminants with different methane-emitting phenotypes. Here, we used metagenomic, metatranscriptomic, metabolomics, and biochemical approaches to compare H2 cycling and reductant disposal pathways between low-methane-emitting Holstein and high-methane-emitting Jersey dairy cattle. The Holstein rumen microbiota had a greater capacity for reductant disposal via electron transfer for amino acid synthesis and propionate production, catalyzed by enzymes such as glutamate synthase and lactate dehydrogenase, and expressed uptake [NiFe]-hydrogenases to use H2 to support sulfate and nitrate respiration, leading to enhanced coupling of H2 cycling with less expelled methane. The Jersey rumen microbiome had a greater proportion of reductant disposal via H2 production catalyzed by fermentative hydrogenases encoded by Clostridia, with H2 mainly taken up through methanogenesis via methanogenic [NiFe]-hydrogenases and acetogenesis via [FeFe]-hydrogenases, resulting in enhanced methane and acetate production. Such enhancement of electron incorporation for metabolite synthesis with reduced methanogenesis was further supported by two in vitro measurements of microbiome activities, metabolites, and public global microbiome data of low- and high-methane-emitting beef cattle and sheep. Overall, this study highlights the importance of promoting alternative H2 consumption and reductant disposal pathways for synthesizing host-beneficial metabolites and reducing methane production in ruminants.

BACKGROUND: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant.
RESULTS: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate.
CONCLUSIONS: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Microbially mediated NO3--N and Cr(VI) reduction is being recognized as an eco-friendly and cost-effective remediation strategy. Iron sulfide mineral, as a natural inorganic electron donor, has a strong influence on NO3--N and Cr(VI) transformation, respectively. However, little is known about the simultaneous nitrate and chromium removal performance and underlying mechanism in an iron sulfide mineral-involved mixotrophic biofilter. This study demonstrated that the NO3--N and Cr(VI) removal efficiencies were stable at 62 ± 8% and 56 ± 10%, and most of them were eliminated in the 0-100-mm region of the biofilter. Cr(VI) was reduced to insoluble Cr(III) via microbial and chemical pathways, which was confirmed by the SEM-EDS morphology and the XPS spectra of biofilm and pyrite particles. SO42- was as a main byproduct of pyrite oxidation; however, the bacterial SO42- reduction synchronously occurred, evidenced by the variations of TOC and SO42- concentrations. These results suggested that there were complicated and intertwined biochemical relations between NO3--N/Cr(VI)/SO42-/DO (electron acceptors) and pyrite/organics (electron donors). Further investigation indicated that both the maximal biomass and greatest denitrifiers\' relative abundances in microbial sample S1 well explained why the pollutants were removed in the 0-100-mm region. A variety of denitrifiers such as Pseudoxanthomona, Acidovorax, and Simplicispira were enriched, which probably were responsible for both NO3--N and Cr(VI) removal. Our findings advance the understanding of simultaneous nitrate and chromium removal in pyrite-involved mixotrophic systems and facilitate the new strategy development for nitrate and chromium remediation.

Particulate methane monooxygenase (pMMO) plays a critical role in catalyzing the conversion of methane to methanol, constituting the initial step in the C1 metabolic pathway within methanotrophic bacteria. However, the membrane-bound pMMO\'s structure and catalytic mechanism, notably the copper\'s valence state and genuine active site for methane oxidation, have remained elusive. Based on the recently characterized structure of membrane-bound pMMO, extensive computational studies were conducted to address these long-standing issues. A comprehensive analysis comparing the quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulated structures with cryo-EM data indicates that both the CuC and CuD sites tend to stay in the Cu(I) valence state within the membrane environment. Additionally, the concurrent presence of Cu(I) at both CuC and CuD sites leads to the significant reduction of the ligand-binding cavity situated between them, making it less likely to accommodate a reductant molecule such as durohydroquinone (DQH2). Subsequent QM/MM calculations reveal that the CuD(I) site is more reactive than the CuC(I) site in oxygen activation, en route to H2O2 formation and the generation of Cu(II)-O•- species. Finally, our simulations demonstrate that the natural reductant ubiquinol (CoQH2) assumes a productive binding conformation at the CuD(I) site but not at the CuC(I) site. This provides evidence that the true active site of membrane-bound pMMOs may be CuD rather than CuC. These findings clarify pMMO\'s catalytic mechanism and emphasize the membrane environment\'s pivotal role in modulating the coordination structure and the activity of copper centers within pMMO.