Dan Meng

As an isoflavone metabolite with diverse physiological activities, the development of efficient and sustainable manufacturing technologies for (S)-equol holds significant importance. This study focuses on the semi-rational design of daidzein reductase (DZNR), the first key enzyme in the (S)-equol biotransformation pathway. Through multiple sequence alignment and three-dimensional structural analysis, two critical residues, Gly30 and Ala105, were identified in DZNR. A library of single and combinatorial mutants was constructed and screened, yielding the double variant DZNR30S+105S with substantially enhanced catalytic performance. In a whole-cell biocatalytic system, the recombinant E. coli (Escherichia coli) strain harboring this combinatorial mutant achieved a yield of 238.3 mg/L (S)-equol at a substrate concentration of 1 mM daidzein, demonstrating markedly improved catalytic efficiency. Upon increasing the daidzein concentration to 2 mM, the reaction reached equilibrium within 5 h, producing 384.6 mg/L (S)-equol, which highlights the mutant’s excellent potential for high-substrate-concentration applications. This study not only provides novel mechanistic insights into DZNR catalysis but also successfully establishes a DZNR variant with enhanced activity, offering an efficient biocatalytic component for the industrial-scale biomanufacturing of (S)-equol and thereby advancing the development of green biosynthesis technologies for this valuable compound.

Herein , an electrochemical/photoelectrochemical (EC/PEC) dual-mode sensing platform based on carbon dots/liquid exfoliated graphene (CDs/LEG) nanocomposites was developed in this work. The CDs/LEG heterogeneous effectively exploited the excellent photoelectric activity of CDs and the high conductivity of LEG. Ferrocene-labelled aptamer (Fc-Apt) and methylene blue-labelled complementary DNA (MB-cDNA) were introduced as dual-signal probes. Upon aflatoxin B1 (AFB₁) binding, it competitively displaced MB-cDNA, restoring Fc-Apt hairpin conformation and causing MB to move away from the electrode surface while Fc approached it. This conformational change significantly increased the I_Fc to I_MB peak current ratio (I_Fc/I_MB), enhancing the EC signals; meanwhile, the aptamer complex immobilized on the electrode surface reduced the PEC signals through spatial site-blocking and light-shielding effects. The dual-mode responded synergistically to form a unique self-validation mechanism, which showed excellent linearity over the AFB₁ concentration range of 0.01-100 ng/mL, with detection limits (LOD) of 1.356 pg/mL (EC mode) and 0.087 pg/mL (PEC mode). Graphical Abstract

Ciliary dysfunction results in multiorgan developmental diseases, collectively known as ciliopathies. The B9D1-B9D2-MKS1protein complex maintains the gatekeeper function at the ciliary transition zone (TZ). However, the function of B9 proteins and the mechanisms underlying why different variants in the same B9 gene cause different ciliopathies are not fully understood. Here, we investigated the function of B9 proteins and revealed 2 critical functions. First, the B9 complex interacted with and anchored TMEM67 to the TZ membrane. Disruption of the B9-TMEM67 complex reduced posttranslational modifications of axonemal microtubules due to deregulation of tubulin-modifying enzymes within cilia. Second, B9 proteins localized to centrioles prior to ciliogenesis, where they facilitated the initiation of ciliogenesis. In addition, we identified B9D2 variants in a cohort of patients with Joubert syndrome. We found that Joubert syndrome–associated B9D2 variants primarily affected axonemal microtubule modifications without disrupting ciliogenesis, whereas the Meckel syndrome–associated B9D2 variant disrupted both ciliogenesis and axonemal microtubule modifications. Thus, besides its role as a gatekeeper for ciliary membrane proteins, the B9 complex also controls axonemal microtubule posttranslational modifications and early stages of ciliogenesis, providing insights into the distinct pathologies arising from different variants of the same gene.

GPR120 is a free fatty acid receptor capable of signalling excess fatty acids. GPR120 can be activated by various types of free fatty acids, causing intracellular signal transduction and exerting energy regulation, immune homeostasis, and neuronal functions. It has been suggested that Trp198 may be an important residue in the recognition and activation of GPR120 by fatty acid ligands, but direct experimental evidence is lacking. In this study, a GPR120-based bilayer gold nanoparticle biosensor (Trp198→Pro) was constructed by genetically manipulating Trp198 on GPR120 by replacing it with proline for the determination of linkage variability between 14 naturally occurring fatty acid ligands and mutant receptors. The results showed that both before and after amino acid substitution the GPR120 bilayer nanogold receptor sensor responded to all 14 natural fatty acid ligands. And the linkage transformation constants of crotonic acid, dodecanoic acid, oleic acid, linoleic acid, α-linolenic acid, and DHA decreased after Trp198 was replaced by Pro. To further reveal its molecular recognition mechanism, molecular simulation docking experiments were performed on GPR120 and 14 fatty acid ligand compounds before and after amino acid substitutions, respectively. The results showed that before and after the amino acid substitutions, the binding conformational affinity values of GPR120 docked with the ligands were negative, implying that these fatty acid ligands can spontaneously bind to the active pocket of GPR120 without absorbing external energy. Upon replacement of Trp198 by Pro, the active pocket of GPR120 at the optimal docking site with the fatty acid ligand is altered, leading to changes in the amino acid residues that exert the interaction. The above results demonstrate that Trp198 indeed plays an important role in the recognition of fatty acid ligands on GPR120. The present study provides direct quantitative evidence for the roles played by different amino acid residues in receptor-ligand recognition and interaction. At the same time, it provides new ideas for the study of other receptor-ligand-linked metastable mechanisms and kinetic laws.

Rice protein is a high-quality plant-based protein source that is gluten-free, with high biological value and low allergenicity. However, the low solubility of rice protein not only affects its functional properties such as emulsification, gelling, and water-holding capacity but also greatly limits its applications in the food industry. Therefore, it is crucial to modify and improve the solubility of rice protein. In summary, this article discusses the underlying causes of the low solubility of rice protein, including the presence of high contents of hydrophobic amino acid residues, disulfide bonds, and intermolecular hydrogen bonds. Additionally, it covers the shortcomings of traditional modification methods and the latest compound improvement methods, compares various modification methods, and puts forward the best sustainable, economical, and environmentally friendly method. Finally, this article lists the uses of modified rice protein in dairy, meat, and baked goods, providing a reference for the extensive application of rice protein in the food industry.