Engineering bacterial hosts cells for robust growth at high external osmolarities

由于它们的相对简单和快速的生长速度，细菌已被设计用于生产各种散装化学品，从有机酸和二醇到乙醇和丁醇等燃料。随着它们作为生物技术平台的使用，人们对控制细菌生长速度的兴趣越来越大。在生物生产过程中，细菌生长的调节是对外部渗透压变化的响应，这是一种非常常见但却鲜为人知的现象。在生物技术应用过程中，代谢产物和底物饲料的高水平排泄不断增加培养基浓度，可能导致细胞生长受限和体积生产力低下。同样，一些较便宜的介质，用于降低制造成本，特别是在清洁水有限的地区，渗透压高。以前发现外渗透压的增加会影响细菌的渗透压。基于这些发现，人们一直认为渗透压的变化会导致生长速率的降低。我们最近结合了单细胞研究的尖端显微镜技术，表明情况并非如此。当外部渗透压增加时，大肠杆菌的渗透压恢复到休克前的初始值，而细胞在休克后立即继续缓慢生长（Biophys J，正在审查中）。因此，渗透压不能解释生长的减缓。相反，我们对细胞能量学的初步测量表明，生长的减少是由于维持较高的细胞质浓度所需的持续能量负担。这一发现为控制细胞生长和产量开辟了几种新的可能性。在这个项目中，学生将接受跨学科技能的培训，以探索一系列方法，对生物技术应用的外部浓度变化做出更理想的反应。该项目的结构提供了几种方法，因此有不同程度的风险，以确保项目的成功。该项目的目标是：(1)确定在恢复和随后的生长过程中使用不同渗透调节成分的能量成本。该学生将对大肠杆菌菌株进行基因工程改造，使其具有特定的渗透调节成分组合，并使用先进的显微镜技术来测量随后生长过程中细胞能量学变化的程度。我们令人鼓舞的初步数据表明，这将产生具有较高外部渗透压下生长特征响应的简化菌株。(2)设计合成途径，使大肠杆菌能够利用积累的渗透压作为替代糖源进行生产。该学生将利用在较高的外部渗透压下（如海藻糖）生长过程中增加的渗透溶解液产量来设计合成电路，使大肠杆菌能够利用积累的海藻糖进行化合物生产。(3)光为大肠杆菌的质子动力提供能量，以提高可用能级。该学生将表达一种光动力泵，变形紫质，并测试用绿光人工提高细胞能量是否能在高外部渗透压下促进生长和产量。(4)将Aim 1-3中的方法应用于英力士专有细菌。

Due to their relative simplicity and rapid growth rates bacteria have been engineered to produce a variety of bulk chemicals, from organic acids and diols to fuels such as ethanol and butanol. In line with their use as platforms for biotechnology there has been an increased interest in gaining control over bacterial growth rates. A very common, yet poorly understood, modulation of bacterial growth during bio production occurs in response to changes in external osmolarities. High-level excretion of metabolites and substrate feed during biotechnological applications constantly increases medium concentration and can result in limited cell growth and low volumetric productivity. Similarly, some of the less expensive media, used to decrease fabrication costs particularly in the areas where clean water is limited, are high in osmolarity.Increases in the external osmolarity were previously found to effect bacterial osmotic pressure. Based on these findings it was long thought that the change in osmotic pressure leads to decreased growth rates. We recently combined cutting-edge microscopy techniques for single cell studies to show that this is not the case. Upon an increase in external osmolarity osmotic pressure of Escherichia coli recovers to the initial pre-shock value, while the cells continue to grow slower immediately after (Biophys J, under review). Therefore the osmotic pressure does not explain the slowdown in growth. Instead, our preliminary measurements of cellular energetics indicate that the reduction in growth is due to the sustained energetic burden needed to maintain higher cytoplasmic concentration. The finding opens several novel possibilities of controlling cellular growth and production yields.In this project the student will be trained in a versatile set of interdisciplinary skills needed to explore a range of approaches to achieve more desirable responses to changes in external concentrations for biotechnology applications. The project has been structured to offer several approaches, and thus contingency options, with varying degrees of risk to ensure project success. The project aims are:(1) Determine the energetic costs of employing different osmoregulatory components during recovery and subsequent growth.The student will genetically engineer E.coli strains with a given combination of osmoregulatory components and use advanced microscopy techniques to measure the extent of changes in cellular energetics during subsequent growth. Our encouraging preliminary data indicated that this will generate simplified strains with a characteristic response to growth at higher external osmolarity.(2) Design synthetic pathways that enable E. coli to use accumulated osmolites as alternative sugar sources for production.The student will utilize increased osmolyte production during growth at higher external osmolarities, such as treahlose, to design synthetic circuit that allow E.coli to use accumulated treahlose for compound production.(3) Light power the proton motive force of E. coli to elevate available energy levels.The student will express a light-powered pump, proteorhodopsin, and test if artificially boosting cellular energetics with green light can enhance growth and production yields at high external osmolarity.(4) Apply the approaches from Aim 1-3 to INEOS proprietary bacteria.