Characterization of a low mutation rate E. coli in extended fermentation

描述(申请人提供)：本申请旨在测试一种新的金黄色葡萄球菌菌株MDS42pdu，用于商业发酵生产生物制药、氨基酸和生物燃料。它是为极低的点突变和插入序列转座而设计的，特别是在重组蛋白生产的应激条件下。我们建议在这里研究对大规模发酵的一个基本问题的影响：发酵罐中细菌的达尔文进化导致生产力损失。在培养中发生的随机突变可以产生摆脱产物形成负担的细胞，这些具有选择性的优势。很快，这些就会取代传统文化，降低或消除生产力。简而言之，文化在退化。突变的选择也会降低产品的纯度。我们希望减缓或消除这种性能下降，以提高目前补料批次方法的稳定性、质量和效率，并可能支持未来更连续的发酵方案。拟议的研究使用扩展培养物的周期性总基因组测序来比较普通生产菌株和新的低突变菌株，以确定降解前的培养生产力周期是否延长，以及我们是否可以确定与性能退化有关的突变类型。这两个目标建议在摇瓶中对连续转移的培养物进行定期全基因组测序，以廉价地量化低突变菌株在各种情况下的表现，随后将使用连续流动发酵进行更详细的研究。我们预计，使用普通大肠杆菌菌株的对照培养将比MDS42pdu更快地变为非生产性。如果找到这个预期的结果，我们将确定在新的生产系统中出现问题之前的最大世代数。如果生产寿命大大延长，我们将认为这是我们的补料分批发酵产品商业化的一个重要里程碑。测序还将有助于确定其他突变机制是否可能是活跃的，这些机制可能在未来的工作中被有益地灭活。极长的生产培养寿命将表明该菌株连续发酵过程的可行性。

DESCRIPTION (provided by applicant): This application aims to test a new Scarab E. coli strain, MDS42pdu, for commercial fermentation to produce biopharmaceuticals, amino acids and biofuels. It is designed for a very low rate of point mutations and Insertion Sequence transposition, especially in the stress conditions of recombinant protein production. We propose here to study the impact on a fundamental problem of large scale fermentation: Darwinian evolution of bacteria in the fermenter toward loss of productivity. Random mutations occurring in culture can produce cells freed from the burden of product formation and these have a selective advantage. Soon these overtake the culture and reduce or eliminate productivity. In short, cultures deteriorate. Selection of mutations can also undermine product purity. We would like to slow or eliminate this degradation of performance to improve stability, quality and efficiency of current fed batch methods and possibly, to support a more continuous fermentation protocol in the future. The proposed studies use periodic total genomic sequencing of extended cultures to compare ordinary production strains with the new low mutation strain to see if the period of culture productivity before degradation is extended, and whether we can identify the types of mutations involved in performance degradation. The two aims propose periodic whole genome sequencing of serially transferred cultures in shake flasks to quantify inexpensively the performance of the low mutation strain in a wide variety of cases, to be followed by more detailed study using continuous flow fermentation. We anticipate that control cultures using ordinary E. coli strains will become non productive considerably more quickly than MDS42pdu. If this anticipated result is found, we will determine the maximum number of generations before problems emerge in the new production sytem. If a greatly extended productive lifetime is achieved we will consider it a major milestone toward commercialization of our product for fed batch fermentation. Sequencing will also help determine whether additional mutational mechanisms may be active that could be beneficially inactivated in future work. An extremely long productive culture life will indicate the feasibility of a continuous fermentation process usig this strain.