ChemBioChem

Indigo is widely used in dyes, medicines and semiconductors materials due to its excellent dyeing efficiency, antibacterial, antiviral, anticancer, anti‐corrosion, and thermostability properties. Here, a biosynthetic pathway for indigo was designed, integrating two enzymes (EcTnaA, MaFMO) into a higher L‐tryptophan‐producing the strain Escherichia coli TRP. However, the lower catalytic activity of MaFMO was a bottleneck for increasing indigo titers. To overcome this limitation, the enzyme activity of MaFMO was enhanced through mechanism‐guided rational design. The optimal mutant obtained in this study, MaFMOD197E, whose kcat/Km was 1.34 times that of the wild type, and its specific activity was 2.36 times that of the wild type. In addition, the expression levels of EcTnaA and MaFMOD197E were regulated by optimizing the promoters and increasing the copy number to generate the strain E. coli IND‐13. Finally, in the optimal fermentation conditions (220 rpm, 0.05% Tween‐80), the strain E. coli IND‐13 achieved the indigo titer of 568.52 mg/L in a 5‐L bioreactor, with the yield and productivity of 2.62 mg/g and 12.96 mg/L/h (the highest to date), respectively. The results presented here can lay a foundation for further construction of cell factories for indigo and its derivatives with industrial application potential.

In this study, we employed a novel fluorescent probe, RO7304924—which selectively targets cannabinoid 2 receptor (CB2R)—to assess the lateral mobility of CB2R within the plasma membrane of Chinese hamster ovary cells stably expressing a functional, untagged receptor variant. Utilizing confocal fluorescence recovery after photobleaching (FRAP), we quantified the diffusion coefficient and mobile fraction of CB2R, thereby demonstrating the efficacy of RO7304924 as an innovative tool for elucidating the dynamics of this major endocannabinoid‐binding G protein‐coupled receptor. Our present findings highlight the potential of combining advanced ligand‐based fluorescent probes with FRAP for future investigations into the biochemical details of CB2R mobility in living cells, and its impact on receptor‐dependent cellular processes.

Engineering of nonribosomal peptide synthetases (NRPSs) could transform the production of bioactive natural product derivatives. A number of recent reports have described the engineering of NRPSs without marked reductions in yield. Comparative analysis of evolutionarily related NRPSs can provide insights regarding permissive fusion sites for engineering where recombination may occur during evolutionary processes. Studies involving engineering of NRPSs using these recombination sites showed that they have great potential. Moreover, we highlight recent advances in engineering of NRPSs using CRISPR‐associated protein 9 (Cas9)‐based gene editing technology. The use of Cas9 facilitates the editing of even larger biosynthetic gene clusters (BGCs) close to or over 100 kb in size by precisely targeting and digesting DNA sequences at specific sites. This technology combined with growing understanding of potential fusion sites from large‐scale informatics analyses will accelerate the scalable exploration of engineered NRPS assembly lines to obtain bioactive natural product derivatives in high yields.

Methylation modification is a critical regulatory mechanism in epigenetics and plays a significant role in various biological processes. N6‐methyladenosine (m⁶A) is the most common modification found in RNA. The fat mass and obesity‐associated protein (FTO) facilitate the demethylation of m⁶A in RNA, and its abnormal expression is closely linked to the development of several diseases. As a result, FTO has the potential to serve as an important biomarker for clinical disease diagnosis. Despite its significance, there has been a lack of comprehensive reviews addressing advancements in detection methods for the demethylase FTO. This review provides an overview of the progress in FTO detection methods, ranging from traditional approaches to innovative techniques, with a particular emphasis on recently reported advancements. These novel detection methods can be categorized into strategies based on enzymes, functional nucleic acids (FNA), and conformational changes. We summarize the principles and applications of these detection methods and discuss the current challenges and prospects in this field.

In clinical practice, thymopentin (TP‐5) is a commonly utilized immunomodulatory peptide drug. The relatively short half‐life of TP‐5, however, significantly limits its applicability in immunotherapy. Inspired by the structure of the TLR2 ligand lipopeptide Pam3CSK4, fatty acid‐modified TP‐5 peptides were designed and synthesized in this study. Utilizing its amphiphilicity, they were sonicated to assemble into nanoparticles with the diameters of approximately 100 nm. Compared with TP‐5, TP‐5 monopalmitate‐modified nanoparticle has immune‐activating properties both in vivo and in vitro. It markedly increased TNF‐α secretion from RAW264.7 cells and aided in the maturation of DCs. The immunogenicity of OVA model antigen was increased in vivo when capsulated by TP‐5 lipopeptide nanoparticle, which considerably slowed the growth of B16‐OVA melanoma. This fatty acid‐modified TP‐5 assembled nanoparticle offers a straightforward and useful delivery system for the design of innovative nanovaccine for cancer immunotherapy.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by pruritus and impaired skin barrier function. Advances in drug delivery systems have transformed AD treatment by enhancing drug stability, bioavailability, and targeted delivery. Drug delivery systems such as liposomes, hydrogels, and microneedles enable deeper skin penetration, prolonged drug retention, and controlled release, reducing side effects and treatment frequency. Liposomes improve drug absorption and stability, while hydrogels offer high water content and responsive drug release. Microneedles facilitate painless, localized drug delivery, enhancing patient compliance. These systems address the limitations of traditional therapies like topical corticosteroids and systemic immunosuppressants, which are associated with adverse effects and poor patient adherence. Recent innovations include Janus kinase (JAK) inhibitors and biologics targeting immune pathways, demonstrating significant efficacy in reducing inflammation and symptoms. Drug delivery systems offer a safer, more efficient alternative for delivering these advanced therapies. By improving therapeutic outcomes and patient experience, drug delivery systems represent a crucial advancement in AD management.

DNA double crossover (DX) motifs including DAE (double crossover, antiparallel, even spacing) and DAO (double crossover, antiparallel, odd spacing) are well‐known monolayered DNA building blocks for construction of 2D DNA arrays and tubes in nanoscale and microscale. Compared to the 3D architectures of DNA origami and single‐stranded DNA bricks to build nanoscale 3D bundles, tessellations, gears, castles, etc., designs of double‐ and multi‐layers of DX motifs for 3D architectures are still limited. Herein, we report two types of double‐layered tiles derived from DAE motifs with single‐stranded circular 42‐ and 64‐nt oligonucleotides as scaffold strands. Further tiling of the tiles generated planar 3D crystalline domains and curved tubes, correspondingly. Finally, we applied the chiral index theory to derive the unit tube parameters of six E‐tiling (inter‐tile distance of even spacing) tubes and analyzed the causation of difference between these tubes.

Alzheimer's disease (AD) is a significant neurodegenerative disorder primarily affecting individuals over the age of 65. It is characterized by impairments in memory, thinking, analytical judgment, visuospatial recognition, and mood. In recent years, the development of protein and peptide drugs targeting amyloid‐beta (Aβ) has gained momentum, with several therapies entering clinical trials and even receiving marketing approval. Novel functional protein and peptide drugs, as the first‐generation immunotherapeutic agents for neurodegenerative diseases, have pioneered cellular immunotherapy for AD. However, the currently available drugs are associated with toxicity issues, which can lead to serious complications such as cerebral haemorrhage or edema. Consequently, this study examines the potential for a new generation of Aβ‐targeting drugs to mitigate the side effects of existing treatments and offers innovative perspectives for the advancement of therapies for AD.

BTG13, a non‐heme iron‐dependent enzyme with a distinctive coordination environment of four histidines and a carboxylated lysine, has been found to catalyze the cleavage of the C4a−C10 bond in anthraquinone. Contrary to typical dioxygenase mechanisms, our quantum mechanical/molecular mechanical (QM/MM) calculations reveal that BTG13 functions more like a monooxygenase. It selectively inserts an oxygen atom into the C10−C4a bond, creating a lactone species that subsequently hydrolyzes, leading to the formation of a ring‐opened product. This discovery highlights the unique catalytic properties of BTG13 and expands our understanding of non‐heme iron enzyme mechanisms.

Nucleic acid, as a carrier of genetic information, has been widely employed as a building block for the construction of versatile nanostructures with pre‐designed sizes and shapes through complementary base pairing. With excellent programmability, addressability, and biocompatibility, nucleic acid nanostructures are extensively applied in biomedical researches, such as bio‐imaging, bio‐sensing, and drug delivery. Notably, the original gene‐encoding capability of the nucleic acids themselves has been utilized in these structurally well‐defined nanostructures. In this review, we will summarize the recent progress in the design of double‐stranded DNA and mRNA‐encoded nanostructures for various biological applications, such as gene regulation, gene expression, and mRNA transcription. Furthermore, the challenges and future opportunities of genetically encoded nucleic acid nanostructures in biomedical applications will be discussed.

Recent advances in computational methods like AlphaFold have transformed structural biology, enabling accurate modeling of protein complexes and driving applications in drug discovery and protein engineering. However, predicting the structure of systems involving weak, transient, or dynamic interactions, or of complexes with disordered regions, remains challenging. Nuclear Magnetic Resonance (NMR) spectroscopy offers atomic‐level insights into biomolecular complexes, even in weakly interacting and dynamic systems. Paramagnetic NMR, in particular, provides long‐range structural restraints, easily exceeding distances over 25 Å, making it ideal for studying large protein complexes. Advances in chemical tools for introducing paramagnetic tags into proteins, combined with progress in electron paramagnetic resonance (EPR) spectroscopy, have enhanced the method's utility. This perspective article discusses paramagnetic NMR approaches for analyzing biomolecular complexes in solution and in the solid state, emphasizing quantities like pseudocontact shifts, residual dipolar couplings, and paramagnetic relaxation enhancements. Additionally, dynamic nuclear polarization offers a promising method to amplify NMR signals of large complexes, even in complex environments. The integration of AlphaFold protein structure prediction with paramagnetic NMR holds great potential for advancing our understanding of biomolecular interactions.

Nucleotide sugars (NSs) are essential building blocks for the enzymatic assembly of glycans. In this study, we established a preparation and recycling avenue to the biocatalysts for the enzymatic synthesis of NSs. This approach involves fusing two enzymes into a bifunctional chimera and using elastin‐like polypeptides (ET64) as a purification tag, which allows for easy recovery of these biocatalysts without the need for chromatography. We successfully constructed and obtained five bifunctional fusion enzymes (GalK‐USP‐ET64, GlmU‐NahK‐ET64, ManC‐NahK‐ET64, FKP‐ET64, and NanA‐CSS‐ET64) for the synthesis of five common NSs (UDP‐Gal, UDP‐GlcNAc, GDP‐Man, GDP‐Fuc, and CMP‐Neu5Ac). These enzymes were obtained using the Inverse Transition Cycling (ITC) process in yields ranging from 60 to 124 mg per liter of fermentation. The enzymatic synthesis of NSs was carried out on a scale from hundreds of milligrams to multiple grams using these biocatalysts. Furthermore, we investigated the reusability of these biocatalysts by recycling them from the reaction solution using the ITC process. The recycling of GalK‐USP‐ET64, GlmU‐NahK‐ET64, FKP‐ET64, and NanA‐CSS‐ET64 was effectively achieved for 15, 13, 3, and 4 times, respectively. These biocatalysts could be used not only for the enzymatic synthesis of NSs but also for the chemoenzymatic synthesis of glycan biomolecules when coupled with glycosyltransferases.

The evolution of antitumor drug development has transitioned from single‐agent chemotherapy to targeted therapy, immunotherapy, and more recently, multispecific drugs. These innovative drugs target multiple cellular or molecular pathways simultaneously, offering a more comprehensive anticancer approach and addressing some of the limitations inherent in traditional monotherapies. However, preclinical assessment of multispecific drugs remains challenging, as conventional tumor models often lack the necessary complexity to accurately reflect the interactions between various cell types and targets. Patient‐derived immunocompetent tumor organoids (PDITOs), which incorporate both tumor cells and immune cells, present a promising platform for the evaluation of these drugs. Beyond their use in drug evaluation, PDITOs can also be utilized in personalized drug screening and predicting patient‐specific treatment outcomes, thus advancing both multispecific drug development and precision medicine. This perspective discusses the current landscape of multispecific drug development and the methodologies for constructing PDITOs. It also addresses the associated challenges and introduces the concept of employing these organoids to optimize the evaluation and rational design of multispecific drug therapies.

The ubiquitin (Ub) ligase E6AP, which is encoded by the UBE3A gene, has been associated with several human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease‐relevant substrates of E6AP is still limited. The formation of a thioester complex between Ub and the catalytic Cys residue of E6AP represents an essential intermediate step in E6AP‐mediated ubiquitination. As potential substrates have to come into close proximity of the thioester bond to be ubiquitinated, we reasoned that a stable E6AP‐Ub conjugate should represent a suitable affinity matrix for the identification of E6AP substrates. In addition, we employed a Ub variant equipped with a diazirine (Ub‐DEA), as the resulting E6AP‐Ub‐DEA conjugate enables to covalently trap substrate proteins via photo‐crosslinking (PCL). We validated the applicability of our approach in PCL‐assisted affinity enrichment coupled to mass spectrometry (PCL‐AE‐MS) experiments. The results obtained indicate that PCL‐AE‐MS is indeed suited to identify substrates of E6AP and, presumably, of other enzymes of the Ub‐conjugating system forming thioester complexes with Ub.

The resistance of cancer cells to apoptosis poses a significant challenge in cancer therapy, driving the exploration of alternative cell death pathways such as pyroptosis, known for its rapid and potent effects. While initial efforts focused on chemotherapy‐induced pyroptosis, concerns about systemic inflammation highlight the need for precise activation strategies. Photothermal therapy emerges as a promising non‐invasive technique, minimizing pyroptosis‐related side effects by targeting tumors spatially and temporally. However, accurately pinpointing tumors to avoid collateral damage remains a challenge. Thus, we utilize NIR‐II fluorescence imaging to achieve precise PTT‐induced pyroptosis activation in glioma. A polymer semiconductor‐based PTT agent was developed with high optical stability, integrated with mesoporous silica to enhance its biocompatibility. These nanoparticles, stabilized through PEG modification and targeted with cRGD peptides, effectively induced pyroptosis in vitro. Furthermore, this design facilitated precise tumor imaging guidance and subsequent pyroptosis activation in vivo, presenting a promising strategy for glioma therapy with minimized adverse effects.

Reactive oxygen species (ROS) play crucial roles in both cell signaling and defense mechanisms. Hypochlorous acid (HOCl), a strong oxidant, aids the immune response by damaging pathogens. In this study, we developed two pyridinium‐based fluorophores PSSM and PSSE for selective hypochlorite detection. Out of these two fluorescent probes, PSSM shows a strong turn‐on emission via a photoinduced electron transfer (PeT) mechanism, excellent mitochondrial localization, and rapid response to HOCl with high selectivity among reactive oxygen species by achieving a detection limit of 2.41 μM. It successfully detects both exogenous and endogenous HOCl in live cells, enabling the study of HOCl's role at the organelle level. Structural analysis of PSSM via thioether oxidation confirmed by HPLC, NMR and HRMS further supports its specificity. Confocal imaging and flow cytometry studies further highlights its utility in investigating oxidative stress, positioning this fluorophore as a valuable tool for monitoring HOCl imbalances in biological systems.

The α‐helix is an abundant and functionally important element of protein secondary structure, which has motivated intensive efforts toward chemical strategies to stabilize helical folds. One such method is the incorporation of non‐canonical backbone composition through an additional methyl substituent at the Cα atom. Examples of monomers include the achiral 2‐aminoisobutyric acid (Aib) with geminal dimethyl substitution and chiral analogues with one methyl and one non‐methyl substituent. While Aib and chiral Cα‐Me residues are both established helix promoting moieties, their comparative ability in this regard has not been quantitatively investigated. Addressing this gap would help to inform the use of these building blocks in the construction of peptide and protein mimetics as well as provide fundamental insights into consequences of backbone methylation on folding. Here, we report a quantitative comparison of the impacts of Aib and chiral αMe residues on the high‐resolution folded structure and folding thermodynamics of a small helical protein. These results reveal a synergistic stabilizing effect arising from the presence of Cα methylation in conjunction with a Cα stereocenter.

The heme enzymes of the cytochrome P450 superfamily (CYPs) catalyse the selective hydroxylation of unactivated C−H bonds in organic molecules. There is great interest in applying these enzymes as biocatalysts with a focus on self‐sufficient CYP ‘fusion’ enzymes, comprising a single polypeptide chain with the electron transfer components joined to the heme domain. Here we elucidate the function of the self‐sufficient CYP116B46 fusion enzyme, from the thermophilic bacterium Tepidiphilus thermophilus. We demonstrate that it efficiently hydroxylates aromatic organic acids, exemplified by oxidation of 2‐hydroxyphenylacetic acid to homogentisic acid (2,5‐dihydroxyphenylacetic acid), an important metabolite in bacterial catabolism. In line with the thermophilic nature of the source bacterium, activity increased at higher temperatures, (50 °C), with a catalytic preference for NADPH over NADH. While self‐sufficient fusion enzymes simplify biocatalysis; engineered peroxygenase activity is also a key advance in the application of these enzymes as biocatalysts as it eliminates the need for electron transfer partner proteins and nicotinamide cofactors. We demonstrate that a T278E mutation in the heme domain of CYP116B46, confers peroxygenase activity. This engineered peroxygenase enzyme is stable to elevated temperatures and catalytic concentrations of hydrogen peroxide, with an observed optimal activity resulting in a total turnover number of ~650.

In this work, we studied six Ruthenium(II)‐diphosphine compounds containing different mercapto ligands (N−S), with general formula [Ru(N−S)(dppm)2]Cl (dppm=1,1‐bis(diphenylphosphino)methane). These compounds were characterized by several techniques (NMR [¹H, ³¹P(¹H), and ¹³C], HRMS, IR, UV‐Vis and XRD) and their purity confirmed by elemental analysis. DLS experiments revealed low diameters and polydispersity indexes, and positive log P values in n‐octanol/PBS indicated their preference for the organic phase. In general, these compounds are stable in different media over 48 h. Cytotoxicity experiments revealed promising IC50 values on A549 breast cancer cells, 0.48 μM and 0.80 μM for [Ru(mtz)(dppm)2]Cl (1) and [Ru(mmi)(dppm)2]Cl (2), respectively (mtz and mmi are 2‐mercapto‐2‐thiazoline and mercapto‐1‐methylimidazole in their deprotonated form, respectively). Clonogenic and migration experiments indicated their antiproliferative and anti‐migratory capacity. ICP‐MS results indicated their cellular accumulation in the nucleus, with little amounts in mitochondria. No covalent DNA binding was observed by ICP‐MS. JC‐1 and cell Mito Stress test confirmed mitochondrial dysfunction, which was verified by mitochondrial membrane potential uncoupling and drastic alterations in the oxygen consumption rate. Taken together, our results provide crucial insights regarding the anticancer potential of ruthenium(II)‐phosphine compounds.

Diabetes is a metabolic disorder characterized by insufficient endogenous insulin production or impaired sensitivity to insulin. In recent years, a class of incretin‐based hypoglycemic drugs, glucagon‐like peptide‐1 receptor agonists (GLP‐1RAs), have attracted great attention in the management of type 2 diabetes mellitus (T2DM) due to their benefits, including stable glycemic control ability, a low risk of hypoglycemia, and weight reduction for patients. However, like other peptide drugs, GLP‐1RAs face challenges such as instability, susceptibility to enzymatic degradation, and immunogenicity, which severely limit their clinical application. In recent years, various strategies have been developed to improve the bioavailability and therapeutic efficacy of GLP‐1RAs, including structural modification and carrier‐mediated delivery. This article briefly introduces the research and application status of several common GLP‐1RAs and their limitations. Taking exendin‐4 as an example, we focus on the research progress of improving bioavailability and therapeutic efficacy based on structural modification and carrier delivery strategies, aiming to provide reference for the development of new GLP‐1RAs treatment systems.

Smart shape‐memory DNA hydrogels, which can respond to various types of external stimuli and undergo macroscopic shape deformations, have shown great potential in various applications. By constructing free‐standing films, the deformation and response properties of these hydrogels can be further enhanced, and visualized deformation can be achieved. However, DNA hydrogels that can exhibit rapid and high‐degree shape deformations, such as the inverse shape deformations, are still lacking. Herein, free‐standing oligoadenine strand‐functionalized polyacrylamide hydrogel films were developed that can exhibit reversible and high degree of inverse shape deformation upon cyclic pH changes. The oligoadenine strands exhibit a pH‐stimulated reversible conformational transition between a flexible single‐stranded state and parallel duplex A‐motif structures, resulting in their role change in the film from negatively charged side chains to “head‐to‐head” crosslinking structures, driving a high degree of inverse shape deformation with a relative bending angle change of 223.7 % of the film, which is more than 5 times that of a film driven by pH‐responsive i‐motif structures, facilitating the development of bilayer hydrogel film actuators with potential in flexible sensors and robots.

Pancreatic ductal adenocarcinoma (PDAC) is marked by significant desmoplastic reactions, or the accumulation of excessive extracellular matrices. PDAC stroma has abnormally high stiffness, which alters cancer cell behaviors and creates a barrier for effective drug delivery. Unfortunately, clinical trials using a combination of chemotherapy and matrix‐degrading enzyme have led to disappointing results, as the degradation of stromal tissue likely accelerated the dissemination of cancer cells. High matrix stiffness has been shown to activate cancer‐associated fibroblasts (CAFs), increasing their interaction with pancreatic cancer cells (PCCs) through promoting proliferation, migration, and resistance to chemotherapy. With the advance of biomaterials science and engineering, it is now possible to design chemically defined matrices to understand the role of stiffness in activating pancreatic CAFs and how this may alter cancer cell migration. Here, we developed a norbornene‐based click hydrogel system with independently tunable stiffness and cell adhesive ligand to evaluate stiffness‐induced activation of CAFs and migration of PCCs. Our results show that matrix stiffness did not alter matrix deposition from CAFs but affected nuclear localization of Yes‐associated protein (YAP). Our results also verify the role of CAFs on promoting PCC migration and an elevated substrate stiffness further increased PCC motility.