Objective: To investigate the impact of impaired electron transfer, caused by defects in the electron transport chain and hypoxia, on de novo pyrimidine synthesis in tumor cells and the underlying regulatory mechanisms. Methods: The effects of dihydroorotate dehydrogenase (DHODH) mitochondrial localization on its catalytic activity in synthesizing pyrimidine nucleotides were determined by immunofluorescence assays, cell viability assays, and Western blot. The effects of pyrimidine nucleotides on the growth of tumor cells with defects in mitochondrial ETC components were analyzed by cell viability assays and Western blot. The changes in pyrimidine nucleotide precursors and pyrimidine nucleotides when the final electron acceptor oxygen is scarce were detected by culturing different tumor cell lines under normoxia or hypoxia. The changes in signaling pathways within tumor cells when pyrimidine nucleotide synthesis is impaired were measured by Western blot and RNA-Seq. Results: The synthesis of pyrimidine nucleotides catalyzed by DHODH depends on its mitochondrial localization. Pyrimidine nucleotides are essential for the growth of tumor cells with defects in complex IV (cytochrome c oxidase). Hypoxia led to increased levels of pyrimidine nucleotide precursors and decreased levels of pyrimidine nucleotides in multiple tumor cell lines. The depletion of pyrimidine nucleotides activated the AMP-activated protein kinase (AMPK) signaling pathway in tumor cells. Conclusion: The synthesis of pyrimidine nucleotides is coupled with the electron transport chain and energy production in tumor cells, thereby influencing the AMPK signaling pathway and tumor cell growth.
Obejctive: Skin flap transplantation is an important means of surgical tissue defect repair, Platelet-rich plasma (PRP), as a bioactive agent containing a variety of growth factors, can significantly improve the microcirculation of skin flaps by promoting vascular reconstruction and tissue repair. This study explored the effect of PRP intervention on the survival of random flaps, by analyzing the expression of autophagy-related proteins and inflammatory factors, revealed that PRP promoted vascular reconstruction and tissue repair by regulating the autophagy-inflammatory network, provided a theoretical basis at the molecular mechanism level for optimizing the clinical treatment plan of PRP. Methods: A mouse model of random flaps was established. The therapeutic effect of PRP on the repair of random flaps was explored through monitoring the survival area of random flaps, histological staining, Western blot and real-time fluorescence quantitative PCR. Results: The results show that after PRP treatment, the survival rate of random flaps can be significantly increased, inhibited inflammation and promoted vascular reconstruction. Furthermore, PRP has the effect of activating autophagy in ischemic areas, and autophagy inhibitors (3-methyladenine, 3-MA) can reverse the effect of PRP. Conclusion: PRP can regulate autophagy and inflammation levels in the flap area, and 3-MA significantly reduces the effect of PRP in the flap area on the survival rate of the flap, thereby increasing the levels of inflammatory factors. In conclusion, the experimental results show that PRP significantly improves the survival rate of random flaps by activating autophagy, inhibiting inflammation, promoting vascular reconstruction, and reducing tissue necrosis.
Objective: Five-hydroxytryptophan (5-HTP), the direct precursor of serotonin (5-hydroxytryptamine, 5-HT), is of significant value in neuromodulation and drug development. However, 5-HTP biosynthesis is constrained by the low activity and poor stability of the key enzyme tryptophan hydroxylase (TPH). This study aimed to improve TPH performance and construct a cofactor-supply system to enhance whole-cell biosynthesis of 5-HTP. Methods: A thermostable TPH from Schistosoma mansoni (SmTPH) was heterologously expressed in Escherichia coli, and its specific activity and half-life at 37℃ were determined. A triple mutant, M14 (T270G/L267D/A140M), was obtained via semi-rational design; its activity and catalytic efficiency (kcat/Km) were measured, and a mechanistic analysis was performed. Subsequently, a tetrahydrobiopterin (BH4) biosynthesis and recycling/regeneration system was constructed in cells expressing M14. Shake-flask whole-cell biocatalysis was conducted under optimized conditions to quantify 5-HTP production. Results: SmTPH exhibited a specific activity of 0.3 U/mg and a half-life of 6.5 h at 37℃, and it significantly outperformed human TPH2. The engineered mutant M14 (T270G/L267D/A140M) increased activity by 182% compared with the wild type and improved kcat/Km to 6.05×10-3 L/(μmol·s). Mechanistic analysis indicated that enhanced structural flexibility and improved substrate-binding adaptability were the main contributors to the activity increase. After introducing BH4 biosynthesis and recycling, the whole-cell system produced 3.2 g/L of 5-HTP in shake flasks under optimal catalytic conditions. Conclusion: By expressing SmTPH in Escherichia coli, engineering the high-activity mutant M14, and coupling BH4 biosynthesis with recycling, this study enabled efficient whole-cell biosynthesis of 5-HTP. This provides technical support for industrial application.
Optogenetics, as a new interdisciplinary technology that integrates engineering idea, optical technology, and genetic principles, can respond to specific wavelengths of light to regulate biological processes. It has been widely applied in synthetic biology. Over the past two decades of research, optogenetic systems have been developed for different scenarios, which have unique advantages such as non-invasiveness, millisecond response speed, sub-micron spatial resolution, and multi-wavelength orthogonal regulation, providing a new solution for the precise regulation of gene expression. However, the requirements for optogenetic tools may vary greatly in different scenarios: some scenarios require light induction systems with the ability to support complex logic programming, while others require simpler genetic operations and a broader regulatory range. There are significant differences in the types of photoreceptors and system structures of the light-induced systems used in these two scenarios. Based on this, this article reviews the development history of optogenetics tools, their systematic classification into single and double components, their working principles, the latest progress, and their applications in synthetic biology. It explores the current challenges and possible future development directions of optogenetics, providing ideas for further accelerating its application in basic research and industrial practice.
Proteins are fundamental organic compounds that constitute cells and serve as the primary performers of life activities, most of which are accomplished through protein complexes. Protein-protein interactions play a crucial role in regulating cellular functions and signal transduction. The bimolecular fluorescence complementation (BiFC) system, which relies on the reconstruction of the split fluorescent protein fragments, enables accurate and reliable monitoring of protein interactions in living cells and in vivo, facilitating the understanding of the underlying molecular mechanisms of these interactions. This review will discuss the latest advancements in split fluorescent protein-based BiFC systems for detecting protein-protein interactions. We hope this review will serve as a reference for studying protein interactions and provide guidance for readers in selecting appropriate fluorescence complementation systems for their research.
Filamentous fungi are eukaryotic microorganisms characterized by their branched mycelial structures, which are widely distributed in nature and hold great potential for industrial applications. However, their complex genetic background significantly limits strain improvement and genome manipulation. The clustered regularly interspaced short palindromic repeats (CRISPR) system is an efficient genome-editing tool capable of precise DNA cleavage and modification. Its application in filamentous fungi has substantially improved genetic manipulation efficiency, providing robust technical support for metabolic engineering, functional genomics, and strain development. This review focuses on the principles, components, and expression mechanisms of CRISPR/Cas systems in filamentous fungi, summarizes recent research progress, and discusses the current challenges and future prospects of applying CRISPR/Cas technologies in these organisms.
Pyrroloquinoline quinone (PQQ), as a naturally occurring peptide-modified compound, represents the third class of coenzymes discovered following nicotinamide and riboflavin. The pyrroloquinoline ring within the PQQ molecular structure is responsible for its unique redox properties, which enable diverse and significant physiological functions such as antioxidant activity, anti-aging effects, and immune enhancement. This gives PQQ broad applications in fields such as medicine and healthcare. However, the large-scale production of PQQ currently faces numerous challenges, including complex and costly chemical synthesis steps, limited raw materials and low yields from natural extraction, and insufficient synthesis efficiency in microbial fermentation strains. These difficulties restrict its widespread application in the aforementioned areas.This paper reviews the biosynthetic pathway of PQQ and its metabolic regulatory mechanisms within microorganisms. It introduces research progress on PQQ synthesis in different microbial strains and proposes strategies such as mutagenesis breeding, metabolic engineering modification, and fermentation process optimization to enhance PQQ synthesis performance. Additionally, it provides recommendations and future perspectives on research strategies for constructing microbial cell factories for PQQ biosynthesis.
The nitrogen-nitrogen (N-N) bond, as a unique chemical linkage, serves as a core structural motif in numerous bioactive molecules, including natural products and synthetic pharmaceuticals. Compounds containing N-N bonds exhibit diverse pharmacological activities, such as antimicrobial, antiviral, and antitumor effects, highlighting their significant value in medicinal applications. However, the biosynthesis of N-N bonds faces inherent chemical challenges. Under physiological conditions, nitrogen atoms typically exhibit a nucleophilic character, making direct coupling between two nucleophilic nitrogen atoms thermodynamically and kinetically unfavorable. Recent advances integrating structural biology, computational chemistry, and synthetic biology have led to breakthroughs in elucidating enzymatic mechanisms of N-N bond formation. This review systematically summarizes enzymatic reactions involving nitrogen species at varying oxidation states, with a focus on the catalytic strategies employed by heme-dependent enzymes, non-heme metalloenzymes, and Cupin domain-containing proteins. Furthermore, we discuss future applications in drug development, synthetic biology, and environmental biotechnology. By comprehensively analyzing current research and emerging directions, this work aims to provide a theoretical foundation for designing novel biocatalysts and advancing their applications.
The discovery of plastic-degrading enzymes has laid the foundation for the development of technologies aimed at the biodegradation and recycling of plastic waste. However, the cost of employing enzymatic degradation as a method of plastic breakdown exceeds that of physical techniques, underscoring the necessity for the economical production of enzymes capable of degrading plastic. Here, the progress made in the recombinant expression of two representative bacterial PET-degrading enzymes, LCC and IsPETase, along with their superior mutants, was comprehensively reviewed. The recombinant expression ways for PET-degrading enzymes can be categorized into three distinct types: intracellular, secretory, and surface display (whole-cell catalysis). Intracellular expression is regarded as the preferred recombinant expression system for the characterization of novel enzymes or mutants. However, the high cost associated with lysing cells to obtain proteins limits its application in industrial-scale production. Secretory expression currently represents the primary method for industrial enzyme production; however, expression levels remain considerably below what is required for large-scale production. Surface display offers highly promising solutions to address issues such as the poor stability of free enzymes and enzyme recovery. Nevertheless, it is necessary to develop thermotolerant hosts and high-temperature-stable display systems to cope with the elevated temperatures required for PET degradation. Furthermore, optimization strategies for each expression system were analyzed, which could give theoretical and technical assistance for the efficient production of plastic-degrading enzymes.
CRISPR/Cas gene editing technology, as an emerging core technology in biological breeding, is an important engine for China to break through the bottleneck problem of seed sources.. Through patent bibliometric analysis, the study provided a macroscopic overview of the geographical layout, research and development entities, and technological distribution of crop CRISPR/Cas gene editing technology. In order to comprehensively understand the global competition pattern of CRISPR/Cas gene editing technology for crops, gain insight into the technological development gap in China, and provide data support and think tank suggestions for formulating the development strategy of the gene editing industry in China, the development overview of crop CRISPR/Cas gene editing technology, including regional layout, research and development entities, and technology distribution, was sorted out from a macro perspective through patent measurement analysis. Social network analysis and text mining methods were used to compare and analyze the differences in technological development between China and the world from fine-grained perspectives such as technology networks and technology themes. Evaluation indicators for technological gap were constructed from both quantity and quality dimensions to clarify the technological development gap between China and the world, and to analyze the shortcomings in innovation and industrialization of CRISPR/Cas gene editing technology in China. The results indicated that China was the second largest patent applicant country for crop CRISPR/Cas gene editing technology, with higher research and development enthusiasm and a tendency of emphasizing domestic patent layout and neglecting foreign patent layout. The main body of technology research and development was universities or research institutes, and the transformation and industrial application of technological achievements were insufficient, the overall technological advantages in terms of quantity and quality were not significant, and technology research and development mainly around CRISPR/Cas9 technology. Breadth and intersectionality of technology were not as good as those in the United States, and the proportion of applied research was relatively higher. There was still a gap in the underlying optimization of CRISPR system. Based on the above issues, suggestions were proposed for the innovation and industrial development of CRISPR/Cas gene editing technology in China.
This paper systematically reviews the progress of artificial intelligence (AI) applications in biomanufacturing, combining policy and strategy analyses with representative case studies. The focus is on five key technological areas: cellular system design and control, protein structure prediction and functional design, DNA synthesis optimization and intelligent gene editing, automated cellular manufacturing and intelligent execution platforms, and the intelligent integration of organ-on-a-chip and biosensing technologies. AI technologies have demonstrated significant advantages in improving design efficiency, enhancing system robustness, and promoting process automation. These advancements are accelerating the transformation of biomanufacturing from an empirical approach to an intelligent paradigm that integrates data-driven methods with predictive modeling. However, challenges remain, including data heterogeneity, limited model interpretability, complex system integration, and ethical concerns. Future research should emphasize the development of high-quality biological data resources, multimodal modeling techniques, cross-scale integration frameworks, and the establishment of robust ethical review and regulatory mechanisms to ensure the sustainable integration and practical application of AI in biomanufacturing.
Cell and gene therapy (CGT) has emerged as one of the most promising, innovative research and translational directions in the field of biomedicine. It holds the potential to address the clinical needs of patients with critical illnesses and gene mutation-associated rare diseases that are unresponsive to commonly used drug treatments, showcasing immense developmental potential and market prospects. In recent years, China has made significant strides in the development of cell and gene therapy, with technological innovation occurring frequently and the industrialization process accelerating continuously. However, challenges such as “unstable foundations”, “ineffective translation”, and “inefficient mechanisms” persist as challenges. It is imperative to further focus on key segments of the industrial chain, construct an all-encompassing innovation system, resolve development pain points and difficulties, optimize industrial policy coordination, strengthen industrial clustering and layout, and encourage open competition within the industry. These efforts aim to foster a policy environment more conducive to the innovative development and clinical translation of cell and gene therapy.
