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

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新陈代谢/生理发生重大变化，以产生理想的产品，包括药物、化学品、维生素、生物燃料等。最终，合成生物学可能导致构建新的新陈代谢途径，甚至新的生命形式。合成生物学领域的一大进步将是在细胞中制造隔间以容纳特定代谢过程的能力。这种定制细胞器的发展将允许对这一过程进行更好的控制和调节，将封闭的过程从细胞新陈代谢中的任何负面影响中分离出来。我们已经证明了通过1，2-丙二醇利用(PDU)操纵子的代谢工程在大肠杆菌中合成这种细胞器是可能的，PDU操纵子是一种含有大约20,000个多肽亚基的复合体，分子质量约为300丙二醛。最近，我们已经证明，通过组成细胞器外壳(外壳)的5个基因产物的协调过量生产，有可能制造空的细菌微室(BMC)。我们还证明了通常被包裹在BMC中的蛋白质是针对这个空泡的，并且其他非囊泡蛋白可以通过将它们融合到靶蛋白上而被结合到BMC中。通过用GFP标记蛋白质，可以对细胞器进行活细胞成像，并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，以便制造定制的生物反应器。这样一种方法将提供对区隔的新陈代谢优势的见解。将对壳蛋白进行工程设计，以帮助合理设计具有更广泛底物专一性的半透膜壳。壳蛋白在细胞器中的分子内定位将通过抗体和蛋白分解过程的组合进行研究。关于BMC中壳蛋白分子间排列的详细信息将通过使用一系列不同的GFP-壳蛋白融合研究和FRET对BMC的分析而获得。通过这种方式，我们将能够生成细胞器的模型，这将与许多其他实验方法相结合，将允许研究蛋白质是如何被靶向并结合到BMC中的。细胞器中蛋白质交换的速率和并入细胞器的顺序将不仅通过使用活细胞成像来定义，还将通过使用FRAP来定义。关于细胞器如何在细胞内保持的细节将使用一系列定向细胞骨架突变体进行研究。Ras类型的GTP酶PduV在细胞器动力学中的作用也将被探讨。本申请中概述的研究结合了基础科学和令人兴奋的应用机会。该项目正好属于BBSRC的职权范围，它促进了对复杂生物过程的基本了解，并直接涉及合成生物学优先领域。

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.