Garrote-Sánchez Emilio, Moya Andrés, Gil Rosario
Evolutionary Genetics, Institute for Integrative Systems Biology (I2SysBio), University of Valencia and Spanish Research Council, Valencia, Spain.
Genomic and Health Area, Foundation for the Promotion of Sanitary and Biomedical Research of the Valencia Region, Valencia, Spain.
Front Bioeng Biotechnol. 2025 Mar 27;13:1527084. doi: 10.3389/fbioe.2025.1527084. eCollection 2025.
Genetically enhanced microorganisms have wide applications in different fields and the increasing availability of omics data has enabled the development of genome-scale metabolic models (GEMs), which are essential tools in synthetic biology. Bartonella quintana str. Toulouse, a facultative intracellular parasite, presents a small genome and the ability to grow in axenic culture, making it a potential candidate for genome reduction and synthetic biology applications. This study aims to reconstruct and analyze the metabolic network of B. quintana to optimize its growth conditions for laboratory use.
A metabolic reconstruction of B. quintana was performed using genome annotation tools (RAST and ModelSEED), followed by refinement using multiple databases (KEGG, BioCyc, BRENDA). Flux Balance Analysis (FBA) was conducted to optimize biomass production, and in-silico knockouts were performed to evaluate growth yield under different media conditions. Additionally, experimental validation was carried out by testing modified culture media and performing proteomic analyses to identify metabolic adaptations.
FBA simulations identified key metabolic requirements, including 2-oxoglutarate as a crucial compound for optimal growth. In-silico knockouts of transport genes revealed their essentiality in nutrient uptake. Experimental validation confirmed the role of 2-oxoglutarate and other nutrients in improving bacterial growth, though unexpected decreases in viability were observed under certain supplemented conditions. Proteomic analysis highlighted differential expression of proteins associated with cell wall integrity and metabolic regulation.
This study represents a step toward developing as a viable chassis for synthetic biology applications. The reconstructed metabolic model provides a comprehensive understanding of 's metabolic capabilities, identifying essential pathways and growth limitations. While metabolic predictions align with experimental results in key aspects, further refinements are needed to enhance model accuracy and optimize growth conditions.
基因工程改造的微生物在不同领域有着广泛应用，组学数据的日益丰富推动了基因组规模代谢模型（GEMs）的发展，而GEMs是合成生物学中的重要工具。五日热巴尔通体图卢兹菌株是一种兼性细胞内寄生虫，其基因组较小且能够在无细胞培养基中生长，这使其成为基因组精简和合成生物学应用的潜在候选对象。本研究旨在重建并分析五日热巴尔通体的代谢网络，以优化其在实验室条件下的生长条件。
利用基因组注释工具（RAST和ModelSEED）对五日热巴尔通体进行代谢重建，随后使用多个数据库（KEGG、BioCyc、BRENDA）进行优化。进行通量平衡分析（FBA）以优化生物量生产，并进行虚拟基因敲除以评估不同培养基条件下的生长产量。此外，通过测试改良培养基并进行蛋白质组学分析以识别代谢适应性来进行实验验证。
FBA模拟确定了关键的代谢需求，包括2-氧代戊二酸是最佳生长的关键化合物。对转运基因的虚拟基因敲除揭示了它们在营养物质摄取中的必要性。实验验证证实了2-氧代戊二酸和其他营养物质在促进细菌生长中的作用，尽管在某些补充条件下观察到了存活率意外下降的情况。蛋白质组学分析突出了与细胞壁完整性和代谢调节相关蛋白质的差异表达。
本研究朝着将五日热巴尔通体开发成为合成生物学应用的可行底盘迈出了一步。重建的代谢模型全面了解了五日热巴尔通体的代谢能力，确定了基本途径和生长限制。虽然代谢预测在关键方面与实验结果一致，但仍需要进一步改进以提高模型准确性并优化生长条件。
