Quiescent Microbial Cell Factories

人们普遍认识到，大宗商品化学品和燃料的生产必须从石化产品转向可持续的原料基地。然而，越来越明显的是，开发燃料和化学品的生物制造工艺将需要能够提高工艺生产率、稳健性和经济竞争力的技术改进。改进的生物制造的一个关键组成部分是改进的细菌细胞工厂的发展，这构成了这一应用的重点。发展细菌细胞工厂的一个普遍接受的标准是，它必须以最少的副产品积累将最大数量的原材料直接转化为所需的最终产品。虽然许多研发工作都致力于消除不良代谢物的共同产生，但主要的副产品，即微生物生长积累的细胞生物量，往往被认为是这一过程的必然结果而被忽视。然而，通过将产品形成与生长分离，保持生物量恒定，同时允许在较长时间内将底物转化为产品，将提高效率。这种方法已被用于转基因菌株生产某些氨基酸，但并不普遍适用于大多数代谢工程策略，在这些策略中，需要生长以维持细菌细胞的代谢健康。该项目的重点是使用剑桥大学遗传学系萨默斯实验室开发的静止细胞(Q-Cell)技术。Q细胞是一种不生长但具有新陈代谢活性的细胞工厂，由大肠杆菌产生。具有特定基因修饰的细菌宿主可以通过添加吲哚作为化学触发器而被诱导进入这种状态。在实验室研究中，Q细胞的生产力比传统的大肠杆菌培养物高出10倍，在没有生物质产生的情况下，资源被更有效地引导到产品中。此外，在静止开始后，中心碳代谢的途径通量仍然很高，提供了一个不断再生的代谢物池，可以被转移到产品中。Q细胞在与工业应用相关的条件下的性能将在这个项目中进行评估。在剑桥大学遗传学系，将在实验室范围内改进该系统，比较一系列化学触发因素诱导静止的效果。还将评估环境因素(温度)对系统有效运行的作用。希望这些改变可以避免对细菌宿主菌株进行基因改造，从而提高系统的易用性和效率。剑桥大学的调查将在摇瓶培养中进行，可能还会在小型发酵罐中进行。然而，至关重要的是要确定在这些条件下的良好表现是否会扩大到工业条件。这就是工业伙伴CPI的作用至关重要的地方。CPI将首先对目前形式的Q-Cell系统的实用性进行严格测试。由于剑桥的工作表明可能的改进，这些将被纳入CPI方案。

It is widely recognised that the manufacture of commodity chemicals and fuels must switch from petrochemicals to a sustainable feedstock base. However it is also increasingly evident that development of processes for the bio-manufacture of fuels and chemicals will require technology improvements capable of increasing process productivity, robustness and economic competitiveness. A key component of improved bio-manufacture is the development of improved bacterial cell factories, which constitutes the focus of this application.A universally accepted criterion for a bacterial cell factory development is that it must convert the maximum amount of raw material directly to the desired end product with minimum by-product accumulation. While much R&D effort is dedicated to eliminating the co-production of undesirable metabolites, the primary by-product, cell biomass accumulated due to microbial growth, is often dismissed as an inevitable consequence of the process. However, efficiency would be increased by uncoupling product formation from growth, keeping biomass constant while allowing the conversion of substrate to product for an extended period of time. This approach has been exploited in the production of certain amino acids by genetically modified strains but is not generally applicable for the majority of metabolic engineering strategies where growth is required to maintain the metabolic health of the bacterial cell.The focus of this project is the use of quiescent cell (Q-Cell) technology developed in the Summers laboratory in the Department of Genetics at Cambridge University. Q-Cells are a non-growing but metabolically-active cell factory generated from the bacterium Escherichia coli. The bacterial host, possessing a specific genetic modification, can be induced into this state by the addition of indole as a chemical trigger of quiescence. In laboratory studies the productivity of Q-cells is up to 10-fold greater than conventional E. coli cultures, resources being channelled more efficiently into product in the absence of biomass generation. Moreover, pathway flux in central carbon metabolism remains high after the onset of quiescence, providing a constantly regenerated pool of metabolites that can be diverted into product.The performance of Q-cells under conditions relevant to industrial application will be evaluated in this project. At Cambridge University Department of Genetics, work will be undertaken to improve the system on the laboratory scale, comparing the efficacy of a range of chemical triggers to induce quiescence. There will also be an assessment of the role of environmental factors (temperature) on the efficient operation of the system. It is hoped that these changes might avoid the need for genetic modification of the bacterial host strain, thus increasing the ease of use of the system as well as increasing its efficiency. The investigations at Cambridge will be conducted in shake-flask culture and possibly in small-scale fermenters. However it is essential to determine whether good performance under these conditions will scale up to industrial conditions. This is where the role of the industrial partner CPI is crucial. CPI will initially conduct rigorous testing of the utility of the Q-Cell system in its present form. As work in Cambridge suggests potential improvements these will be incorporated into the CPI programme.