Biochemical characterisation of the Glycyl Radical Enzyme Containing Microcompartment (GRM2) from Proteus mirabilis

在哺乳动物细胞培养、自然宿主(如植物、真菌)或化学合成中生产生物治疗蛋白、小分子药物和具有工业意义的化学中间体正成为一种越来越受欢迎的替代方案，后者可能速度慢、成本高。要在重组合成途径中获得高产品产量，需要平衡目标途径的每个酶反应的通量，以限制中间体的积累，特别是那些对宿主有毒的中间体。传统的策略包括调节单个酶的表达水平或限速酶的定向进化，并专注于平衡途径通量，但没有考虑其他关键因素，如扩散、运输、中间体的本地化以及产品的低成本纯化和回收。自然界通过将某些生化途径限制在不同的细胞器上，制定了应对其中许多问题的策略。在原核生物中，细菌微室(BMC)将与某些代谢过程相关的酶包裹在一个大的蛋白质壳中，使顺序作用的酶进入空间邻近，从而增加局部酶的浓度，保护细胞免受潜在的活性中间体的影响，并促进辅因子循环。在过去的十年中，在设计骨髓基质细胞以允许在细胞内构建具有新功能的骨髓基质细胞生物反应器方面取得了重大进展。该项目的总体目标是开发骨髓基质细胞作为合成生物学平台，用于空间组织酶，以提高在大肠杆菌中生产有价值的化合物。该项目将扩大我们对工程合成分区的基本原理、路径本地化的好处和局限性的理解，并为未来微分区衍生技术的合理设计提供信息。该项目与EPSRC生物过程工程领导力博士培训中心保持一致。它为细胞化学的重新设计提供了新的合成生物学方法，并将对经济高效和可持续的生物制造的发展产生影响。BMC工具和技术的开发将考虑到工业应用。这项工作得到了工业生物技术公司Ingenza的实物捐助的支持，Ingenza公司是应用合成生物学为生产化学品、生物制品、药品和生物燃料提供高效生物工艺的世界领先者。酶/途径的区隔可以为改进感兴趣的工业菌株提供一个有价值的策略。工业合作伙伴将提供感兴趣的酶/途径，并就工业希望如何应用BMC技术提供建议/咨询。

The production of bio-therapeutic proteins, small molecule drugs and chemical intermediates of industrial interest in recombinant hosts is becoming an increasingly popular alternative to production in mammalian cell culture, natural hosts (e.g. plants, fungi) or chemical synthesis, which can be slow and expensive. Achieving high product yields in recombinant synthetic pathways requires that the fluxes of each enzymatic reaction of the pathway of interest be balanced to limit the accumulation of intermediates, particularly those toxic to the host. Conventional strategies include the modulation of expression levels of individual enzymes or directed evolution of rate-limiting enzymes and have focused on balancing pathway flux, but don't consider other crucial factors such as diffusion, transport, localisation of intermediates and the cost-effective purification and recovery of products. Nature has developed strategies to deal with many of these issues by confining certain biochemical pathways to various organelles. In prokaryotes, bacterial microcompartments (BMCs) encapsulate enzymes associated with certain metabolic processes within a large protein shell, bringing sequentially acting enzymes into spatial proximity thus increasing local enzyme concentration, protecting the cell from potentially reactive intermediates and facilitating co-factor recycling. In the past decade significant progress has been made to engineer BMCs to permit the construction of BMC bioreactors with novel functions within the cell. The overall aim of this project is to develop BMCs as synthetic biology platforms for spatial organization of enzymes for enhanced production of valuable compounds in E. coli. The project will expand our understanding of the fundamentals of engineering synthetic compartmentalisation, the benefits and limitations of pathway localisation and inform the rational design of future microcompartment-derived technologies. The project is aligned with the EPSRC centre for Doctoral Training in Bioprocess Engineering Leadership. It provides new synthetic biology approaches to the redesign of cellular chemistry and will impact in the development of cost-effective and sustainable bio-based manufacture.BMC tools and technologies will be developed with industrial applications in mind. The work is supported by in-kind contribution from the industrial biotechnology company Ingenza, a world leader in the application of synthetic biology for the provision of efficient bioprocesses for the manufacture of chemicals, biologics, pharmaceuticals and biofuels. Compartmentalisation of enzymes/pathways may provide a valuable strategy for the improvement of industrial strains of interest. The industrial partner will offer enzymes/pathways of interest and advice/consultancy on how industry would like to see the BMC technology applied.