Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase.

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.

Molecular cloning of TPM3 gene in qinchuan cattle and its effect on myoblast proliferation and differentiation.

Tropomyosin 3 (TPM3) plays a significant role as a regulatory protein in muscle contraction, affecting the growth and development of skeletal muscles. Despite its importance, limited research has been conducted to investigate the influence of TPM3 on bovine skeletal muscle development. Therefore, this study revealed the role of TPM3 in bovine myoblast growth and development. This research involved conducting a thorough examination of the Qinchuan cattle TPM3 gene using bioinformatics tools to examine its sequence and structural characteristics. Furthermore, TPM3 expression was evaluated in various bovine tissues and cells using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the coding region of TPM3 spans 855 bp, with the 161st base being the T base, encoding a protein with 284 amino acids and 19 phosphorylation sites. This protein demonstrated high conservation across species while displaying a predominant α-helix secondary structure despite being an unstable acidic protein. Notably, a noticeable increase in TPM3 expression was observed in the longissimus dorsi muscle and myocardium of calves and adult cattle. Expression patterns varied during different stages of myoblast differentiation. Functional studies that involved interference with TPM3 in Qinchuan cattle myoblasts revealed a very significantly decrease in S-phase cell numbers and EdU-positive staining (P < 0.01), and disrupted myotube morphology. Moreover, interference with TPM3 resulted in significantly (P < 0.05) or highly significantly (P < 0.01) decreased mRNA and protein levels of key proliferation and differentiation markers, indicating its role in the modulation of myoblast behavior. These findings suggest that TPM3 plays an essential role in bovine skeletal muscle growth by influencing myoblast proliferation and differentiation. This study provides a foundation for further exploration into the mechanisms underlying TPM3-mediated regulation of bovine muscle development and provides valuable insights that could guide future research directions as well as potential applications for livestock breeding and addressing muscle-related disorders.

Precise Spectral Overlap-Based Donor-Acceptor Pair for a Sensitive Traffic Light-Typed Bimodal Multiplexed Lateral Flow Immunoassay.

Bimodal-type multiplexed immunoassays with complementary mode-based correlation analysis are gaining increasing attention for enhancing the practicability of the lateral flow immunoassay (LFIA). Nonetheless, the restriction in visually indistinguishable multitargets induced by a single fluorescent color and difficulty in single acceptor ineffectual fluorescence quenching due to the various spectra of multiple different donors impede the further execution of colorimetric-fluorescence bimodal-type multiplexed LFIAs. Herein, the precise spectral overlap-based donor-acceptor pair construction strategy is proposed by regulating the size of the nanocore, coating it with an appropriate nanoshell, and selecting a suitable fluorescence donor with distinct colors. By in situ coating Prussian blue nanoparticles (PBNPs) on AuNPs with a tunable size and absorption spectrum, the resultant APNPs demonstrate efficient fluorescence quenching ability, higher colloidal stability, remarkable colorimetric intensity, and an enhanced antibody coupling efficiency, all of which facilitate highly sensitive bimodal-type LFIA analysis. Following integration with competitive-type immunoreaction, this precise spectral overlap-supported spatial separation traffic light-typed colorimetric-fluorescence dual-response assay (coined as the STCFD assay) with the limits of detection of 0.013 and 0.152 ng mL-1 for ractopamine and clenbuterol, respectively, was proposed. This work illustrates the superiority of the rational design of a precise spectral overlap-based donor-acceptor pair, hinting at the enormous potential of the STCFD assay in the point-of-care field.

Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.

Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.

Postamplifying Cas12a Activation through Hybridization Chain Reaction-Triggered Fluorescent Nanocluster Formation for Ultrasensitive Nucleic Acid Detection.

CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.

A tailored series of engineered yeasts for the cell-dependent treatment of inflammatory bowel disease by rational butyrate supplementation.

Intestinal microbiota dysbiosis and metabolic disruption are considered essential characteristics in inflammatory bowel disorders (IBD). Reasonable butyrate supplementation can help patients regulate intestinal flora structure and promote mucosal repair. Here, to restore microbiota homeostasis and butyrate levels in the patient's intestines, we modified the genome of Saccharomyces cerevisiae to produce butyrate. We precisely regulated the relevant metabolic pathways to enable the yeast to produce sufficient butyrate in the intestine with uneven oxygen distribution. A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients, and the strongest can reach 1.8 g/L title of butyrate. Next, this series of strains was used to co-cultivate with gut microbiota collected from patients with mild-to-moderate ulcerative colitis. After receiving treatment with engineered strains, the gut microbiota and the butyrate content have been regulated to varying degrees depending on the synthetic ability of the strain. The abundance of probiotics such as Bifidobacterium and Lactobacillus increased, while the abundance of harmful bacteria like Candidatus Bacilloplasma decreased. Meanwhile, the series of butyrate-producing yeast significantly improved trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice by restoring butyrate content. Among the series of engineered yeasts, the strain with the second-highest butyrate synthesis ability showed the most significant regulatory and the best therapeutic effect on the gut microbiota from IBD patients and the colitis mouse model. This study confirmed the existence of a therapeutic window for IBD treatment by supplementing butyrate, and it is necessary to restore butyrate levels according to the actual situation of patients to restore intestinal flora.

Enzyme Engineering Renders Chlorinase the Activity of Fluorinase.

Organofluorine compounds have attracted substantial attention owing to their wide application in agrochemistry. Fluorinase (FlA) is a unique enzyme in nature that can incorporate fluorine into an organic molecule. Chlorinase (SalL) has a similar mechanism as fluorinase and can use chloride but not fluoride as a substrate to generate 5'-chloro-deoxyadenosine (5'-ClDA) from S-adenosyl-l-methionine (SAM). Therefore, identifying the features that lead to this selectivity for halide ions is highly important. Here, we engineered SalL to gain the function of FlA. We found that residue Tyr70 plays a key role in this conversion through alanine scanning. Site-saturation mutagenesis experiments demonstrated that Y70A/C/S/T/G all exhibited obvious fluorinase activity. The G131S mutant of SalL, in which the previously thought crucial residue Ser158 for fluoride binding in FlA was introduced, did not exhibit fluorination activity. Compared with the Y70T single mutant, the double mutant Y70T/W129F increased 5'-fluoro-5-deoxyadenosine (5'-FDA) production by 76%. The quantum mechanics (QM)/molecular mechanics (MM) calculations suggested that the lower energy barriers and shorter nucleophilic distance from F- to SAM in the mutants than in the SalL wild-type may contribute to the activity. Therefore, our study not only renders SalL the activity of FlA but also sheds light on the enzyme selectivity between fluoride versus chloride.

Harnessing aptamers for the biosensing of cell surface glycans - A review.

Cell surface glycans (CSGs) are essential for cell recognition, adhesion, and invasion, and they also serve as disease biomarkers. Traditional CSG recognition using lectins has limitations such as limited specificity, low stability, high cytotoxicity, and multivalent binding. Aptamers, known for their specific binding capacity to target molecules, are increasingly being employed in the biosensing of CSGs. Aptamers offer the advantage of high flexibility, small size, straightforward modification, and monovalent recognition, enabling their integration into the profiling of CSGs on living cells. In this review, we summarize representative examples of aptamer-based CSG biosensing and identify two strategies for harnessing aptamers in CSG detection: direct recognition based on aptamer-CSG binding and indirect recognition through protein localization. These strategies enable the generation of diverse signals including fluorescence, electrochemical, photoacoustic, and electrochemiluminescence signals for CSG detection. The advantages, challenges, and future perspectives of using aptamers for CSG biosensing are also discussed.

Boosting N2O Catalytic Decomposition by the Synergistic Effect of Multiple Elements in Cobalt-Based High-Entropy Oxides.

Nitrous oxide (N2O) has a detrimental impact on the greenhouse effect, and its efficient catalytic decomposition at low temperatures remains challenging. Herein, the cobalt-based high-entropy oxide with a spinel-type structure (Co-HEO) is successfully fabricated via a facile coprecipitation method for N2O catalytic decomposition. The obtained Co-HEO catalyst displays more remarkable catalytic performance and higher thermal stability compared with single and binary Co-based oxides, as the temperature of 90% N2O decomposition (T90) is 356 °C. A series of characterization results reveal that the synergistic effect of multiple elements enhances the reducibility and augments oxygen vacancy in the high-entropy system, thus boosting the activity of the Co-HEO catalyst. Moreover, density functional theory (DFT) calculations and the temperature-programmed surface reaction (TPSR) with isotope labeling demonstrate that N2O decomposition on the Co-HEO catalyst follows the Langmuir-Hinshelwood (L-H) mechanism with the promotion of abundant oxygen vacancies. This work provides a fundamental understanding of the synergistic catalytic effect in N2O decomposition and paves the way for the novel environmental catalytic applications of HEO.

Global regulator IrrE on stress tolerance: a review.

Stress tolerance is a vital attribute for all living beings to cope with environmental adversities. IrrE (also named PprI) from Deinococcus radiodurans enhances resistance to extreme radiation stress by functioning as a global regulator, mediating the transcription of genes involved in deoxyribonucleic acid (DNA) damage response (DDR). The expression of IrrE augmented the resilience of various species to heat, radiation, oxidation, osmotic stresses and inhibitors, encompassing bacterial, fungal, plant, and mammalian cells. Moreover, IrrE was employed in a global regulator engineering strategy to broaden its applications in stress tolerance. The regulatory impacts of heterologously expressed IrrE have been investigated at the molecular and systems level, including the regulation of genes, proteins, modules, or pathways involved in DNA repair, detoxification proteins, protective molecules, native regulators and other aspects. In this review, we discuss the regulatory role and mechanism of IrrE in the antiradiation response of D. radiodurans. Furthermore, the applications and regulatory effects of heterologous expression of IrrE to enhance abiotic stress tolerance are summarized in particular.

Anti-CRISPR with non-protein substances.

Therapeutics based on clustered regularly interspaced short palindromic repeats (CRISPR) have gained significant attention as a promising synthetic biology technique, but there are concerns about the potential for persistent activation of CRISPR-associated protein (Cas) and subsequent off-target effects. This forum focuses on advances in anti-CRISPR studies based on non-protein substances in the hope of developing effective anti-CRISPR strategies to mitigate these concerns.

Unleashing the capacity of Rhodococcus for converting lignin into lipids.

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus' metabolic pathways converting them to lipids are analyzed from a "beginning-to-end" view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus' utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. The possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

A single-cell transcriptomic atlas characterizes age-related changes of murine cranial stem cell niches.

The craniofacial bones provide structural support for the skull and accommodate the vulnerable brain tissue with a protective cavity. The bone tissue undergoes constant turnover, which relies on skeletal stem cells (SSCs) and/or mesenchymal stem cells (MSCs) and their niches. SSCs/MSCs and their perivascular niche within the bone marrow are well characterized in long bones. As for cranial bones, besides bone marrow, the suture mesenchyme has been identified as a unique niche for SSCs/MSCs of craniofacial bones. However, a comprehensive study of the two different cranial stem cell niches at single-cell resolution is still lacking. In addition, during the progression of aging, age-associated changes in cranial stem cell niches and resident cells remain uncovered. In this study, we investigated age-related changes in cranial stem cell niches via single-cell RNA sequencing (scRNA-seq). The transcriptomic profiles and cellular compositions have been delineated, indicating alterations of the cranial bone marrow microenvironment influenced by inflammaging. Moreover, we identified a senescent mesenchymal cell subcluster and several age-related immune cell subclusters by reclustering and pseudotime trajectory analysis, which might be closely linked to inflammaging. Finally, differentially expressed genes (DEGs) and cell-cell communications were analyzed during aging, revealing potential regulatory factors. Overall, this work highlights the age-related changes in cranial stem cell niches, which deepens the current understanding of cranial bone and suture biology and may provide therapeutic targets for antiaging and regenerative medicine.

Application of Induced Pluripotent Stem Cells in Malignant Solid Tumors.

In the past decade, induced pluripotent stem cells (iPSCs) technology has significantly progressed in studying malignant solid tumors. This technically feasible reprogramming techniques can reawaken sequestered dormant regions that regulate the fate of differentiated cells. Despite the evolving therapeutic modalities for malignant solid tumors, treatment outcomes have not been satisfactory. Recently, scientists attempted to apply induced pluripotent stem cell technology to cancer research, from modeling to treatment. Induced pluripotent stem cells derived from somatic cells, cancer cell lines, primary tumors, and individuals with an inherited propensity to develop cancer have shown great potential in cancer modeling, cell therapy, immunotherapy, and understanding tumor progression. This review summarizes the evolution of induced pluripotent stem cells technology and its applications in malignant solid tumor. Additionally, we discuss potential obstacles to induced pluripotent stem cell technology.

Enhanced single-base mutation diversity by the combination of cytidine deaminase with DNA-repairing enzymes in yeast.

The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted mutagenesis system named MutaT7trans to significantly increase mutation rate and generate mutations across all four nucleotides in yeast. We constructed different DNA-repairing enzyme-PmCDA1-T7 RNA polymerase (T7 RNAP) fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-repairing enzymes. With this mutagenesis tool, the proportion of non-C â†’ T mutations was 10-11-fold higher than the cytidine deaminase-based evolutionary tools, and the transversion mutation frequency was also elevated. The mutation rate of the target gene was significantly increased to 5.25 × 10-3 substitutions per base (s. p. b.). We also demonstrated that MutaT7trans could be used to evolve the CrtE, CrtI, and CrtYB gene in the ß-carotene biosynthesis process and generate different types of mutations.

MooSeeker: A Metabolic Pathway Design Tool Based on Multi-Objective Optimization Algorithm.

Recently, metabolic pathway design has attracted considerable attention and become an increasingly important area in metabolic engineering. Manual or computational methods have been introduced to retrieve the metabolic pathway. These methods model metabolic pathway design as a single-objective optimization problem with the weighted sum of a variety of criteria as the final score. While these methods have demonstrated promising results, the majority of current methods do not account for comparisons and competition among criteria. Here, we propose MooSeeker, a metabolic pathway design tool based on the multi-objective optimization algorithm that aims to trade off all the criteria optimally. The metabolic pathway design problem is characterized as a multi-objective optimization problem with three objectives including pathway length, thermodynamic feasibility and theoretical yield. In order to digitize the continuous metabolic pathway, MooSeeker develops the encoding strategy, BioCrossover and BioMutation operators to search for the candidate pathways. Finally, MooSeeker outputs the Pareto optimal solutions of the candidate metabolic pathways with three criterion values. The experiment results show that MooSeeker is capable of constructing the experimentally validated pathways and finding the higher-performance pathway than the single-objective-based methods.

Artificial mixed microbial system for polycyclic aromatic hydrocarbons degradation.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants with major risks to human health. Biological degradation is environmentally friendly and the most appealing remediation method for a wide range of persistent pollutants. Meanwhile, due to the large microbial strain collection and multiple metabolic pathways, PAH degradation via an artificial mixed microbial system (MMS) has emerged and is regarded as a promising bioremediation approach. The artificial MMS construction by simplifying the community structure, clarifying the labor division, and streamlining the metabolic flux has shown tremendous efficiency. This review describes the construction principles, influencing factors, and enhancement strategies of artificial MMS for PAH degradation. In addition, we identify the challenges and future opportunities for the development of MMS toward new or upgraded high-performance applications.

Genome engineering on size reduction and complexity simplification: A review.

BACKGROUND: Genome simplification is an important topic in the field of life sciences that has attracted attention from its conception to the present day. It can help uncover the essential components of the genome and, in turn, shed light on the underlying operating principles of complex biological systems. This has made it a central focus of both basic and applied research in the life sciences. With the recent advancements in related technologies and our increasing knowledge of the genome, now is an opportune time to delve into this topic. AIM OF REVIEW: Our review investigates the progress of genome simplification from two perspectives: genome size reduction and complexity simplification. In addition, we provide insights into the future development trends of genome simplification. KEY SCIENTIFIC CONCEPTS OF REVIEW: Reducing genome size requires eliminating non-essential elements as much as possible. This process has been facilitated by advances in genome manipulation and synthesis techniques. However, we still need a better and clearer understanding of living systems to reduce genome complexity. As there is a lack of quantitative and clearly defined standards for this task, we have opted to approach the topic from various perspectives and present our findings accordingly.

Ethylenediamine pretreatment simultaneously improved carbohydrate hydrolysis and lignin valorization.

Lignocellulosic biomass (LCB) is the promising feedstock for value-added products, which would contribute to the bioeconomy and sustainable development. The efficient pretreatment is still required in the biorefinery of LCB. To make a simultaneous utilization of carbohydrates and lignin, a novel easy-recycled ethylenediamine (EDA) pretreatment was designed and evaluated in the present study. The results highlighted that this pretreatment yielded 96% glucose and 70% xylose in enzymatic hydrolysis. It simultaneously promoted the depolymerization of lignin into small molecules and functionalized the yielded lignin with Schiff base and amide structures. These animated-lignins showed a pH-responsive behavior and the excellent flocculation capacity by reducing more than 90% turbidity of kaolin suspensions. Therefore, easy-recycled EDA pretreatment hold the promise to simultaneously enhance the enzymatic hydrolysis of carbohydrates and endowed the new functionality of lignin toward downstream valorization, which improved the process feasibility and potentially enable the sustainability of LCB utilization.