Synthetic biology approaches to compartmentalisation in bacteria and the construction of novel bioreactors

细菌分区的合成生物学方法和新型生物反应器的构建

• 批准号: BB/H013180/1
• 负责人: Martin Warren
• 金额: $ 111.44万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH013180%2F1
• 关键词: Synthetic; biology; approaches; compartmentalisation; bacteria

Synthetic biology has emerged from the development of techniques that allow for significant alterations in the metabolism/physiology of the cell to produce desirable products, including drugs, chemicals, vitamins, biofuels etc. Ultimately synthetic biology could lead to the construction of new metabolic pathways and even new life forms. A major advance in the area of synthetic biology would be the ability to make compartments in the cell to house specific metabolic processes. The development of such bespoke organelles would allow for greater control and regulation of the process, detaching the encased process from any negative influences within cellular metabolism. We have demonstrated that it is possible to synthesise such organelles in E. coli by the metabolic engineering of the 1,2-propanediol utilizing (pdu) operon, a complex that contains around 20,000 polypeptide subunits and has a molecular mass of approximately 300 mDa. More recently, we have shown that it is possible to make empty bacterial microcompartments (BMCs) by the coordinated overproduction of 5 gene products that compose the shell (outer casing) of the organelle. We have also demonstrated that proteins normally encased within the BMC are targeted to this empty vesicle and that other non-vesicle proteins can be incorporated to the BMC by fusing them onto target proteins. By labelling the proteins with GFP it has been possible to undertake live cell imaging of the organelles and evidence for movement between organelles via filaments has been observed. We now wish to extend this study by directing specific enzymes to the BMC in order to make bespoke bioreactors. Such an approach will provide insights into the metabolic advantage of compartmentalisation. Engineering of the shell proteins will be undertaken to help in the rational design of a semi permeable shell with a broader substrate specificity. The intramolecular orientation of the shell proteins within the organelle will be investigated by a combination of antibodies and proteolytic processing. Detail on the intermolecular arrangement of shell proteins within the BMC will be obtained from studies using a range of different GFP-shell protein fusions and the analysis of the BMC by FRET. In this way we will be able to generate a model of the organelle and this, in combination with a number of other experimental approaches, will permit an investigation into how proteins are targeted and incorporated into the BMC. The rate of protein exchange in the organelle and the order of incorporation into the organelle will be defined not only using live cell imaging by also by employing FRAP. Detail on how the organelle is held within the cell will be investigated using a range of directed cell cytoskeleton mutants. The role of a Ras-type GTPase, PduV, in organelle dynamics will also be probed. The research outlined in this application combines a mixture of basic science with exciting applied opportunities. The project falls squarely in the remit of the BBSRC by advancing the fundamental understanding of complex biological processes and directly addresses the synthetic biology priority area.

合成生物学已经从技术的发展中出现，这些技术允许细胞的代谢/生理学发生重大改变，以产生所需的产品，包括药物，化学品，维生素，生物燃料等。合成生物学领域的一个重大进展将是在细胞中制造隔间以容纳特定代谢过程的能力。这种定制的细胞器的发展将允许更好地控制和调节该过程，使封闭的过程免受细胞代谢中的任何负面影响。我们已经证明，在大肠杆菌中合成这种细胞器是可能的。通过代谢工程化利用1，2-丙二醇（pdu）操纵子（一种含有约20，000个多肽亚基且具有约300 mDa的分子量的复合物）来转化大肠杆菌。最近，我们已经表明，它是可能的，使空的细菌微区室（BMC）的协调过量生产的5个基因产物组成的外壳（外壳）的细胞器。我们还证明了通常包裹在BMC内的蛋白质被靶向到该空囊泡，并且其他非囊泡蛋白质可以通过将它们融合到靶蛋白上而被并入BMC。通过用GFP标记蛋白质，已经可以进行细胞器的活细胞成像，并且已经观察到细胞器之间通过细丝移动的证据。我们现在希望通过将特定的酶定向到BMC来扩展这项研究，以制造定制的生物反应器。这种方法将提供对区室化的代谢优势的见解。将进行壳蛋白的工程化，以帮助合理设计具有更广泛底物特异性的半渗透壳。壳蛋白在细胞器内的分子内取向将通过抗体和蛋白水解处理的组合来研究。BMC内壳蛋白分子间排列的细节将从使用一系列不同GFP-壳蛋白融合物的研究和通过FRET分析BMC获得。通过这种方式，我们将能够生成一个细胞器的模型，这与许多其他实验方法相结合，将允许研究蛋白质如何被靶向并整合到BMC中。细胞器中蛋白质交换的速率和掺入细胞器的顺序将不仅使用活细胞成像还通过采用FRAP来定义。细胞器是如何在细胞内举行的细节将使用一系列定向细胞骨架突变体进行研究。一个Ras型的GTdirt，PduV，在细胞器动力学的作用也将被探讨。本申请中概述的研究将基础科学与令人兴奋的应用机会相结合。该项目福尔斯完全属于BBSRC的职权范围，通过推进对复杂生物过程的基本理解，并直接解决合成生物学的优先领域。

项目成果

Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments.

生物工程对细菌微剖间的大小，形状，组成和刚性的影响。

• DOI: 10.1038/srep36899
• 发表时间: 2016-11-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Mayer MJ;Juodeikis R;Brown IR;Frank S;Palmer DJ;Deery E;Beal DM;Xue WF;Warren MJ
• 通讯作者: Warren MJ