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

低突变率大肠杆菌在延长发酵中的表征

基本信息

批准号：
8455785
负责人：
FREDERICK R BLATTNER
金额：
$ 28.26万
依托单位：
SCARAB GENOMICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8455785
关键词：
Agreement Amino Acids Animal Feed Antibodies Bacteria Biological Assay Biological Products Biomanufacturing Businesses Categories Cell Line Cells Chemical Industry Cloning Computers Consult Cytolysis DNA DNA Insertion Elements Data Development Engineering Escherichia coli Evaluation Evolution Fee-for-Service Plans Fermentation Flagella Future Gel Generations Genetic Genome Genomic Segment Genomics Growth Hormones IS Elements Immunoglobulin Fragments Infant formula Lead Legal patent Length Licensing Life Literature Lymphokines Mails Marketing Measures Methods Molecular Weight Mutation Organism Peptide Hydrolases Peptides Performance Pharmacologic Substance Phase Plasmids Point Mutation Process Production Productivity Prophages Proteins Protocols documentation Publications Reader Recombinant Proteins Recombinants Research Route Sales Sampling Solutions Staging Stress Swimming Testing Threonine Time Work commercialization design experience feeding flasks genome sequencing improved meetings payment plasmid DNA productivity loss public health relevance web site

项目摘要

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.

描述（由申请人提供）：本申请旨在测试一种新的圣甲虫大肠杆菌菌株 MDS42pdu，用于商业发酵生产生物制药、氨基酸和生物燃料。它专为极低发生率的点突变和插入序列转座而设计，特别是在重组蛋白生产的应激条件下。我们在此建议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。培养物中发生的随机突变可以产生摆脱产物形成负担的细胞，并且这些细胞具有选择性优势。很快这些就会取代文化并降低或消除生产力。简而言之，文化在退化。突变的选择也会破坏产品的纯度。我们希望减缓或消除这种性能下降，以提高当前分批补料方法的稳定性、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定期全基因组测序来比较普通生产菌株与新的低突变菌株，以了解降解之前的培养物生产力周期是否延长，以及我们是否可以识别与性能下降相关的突变类型。这两个目标建议对摇瓶中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性能，然后使用连续流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物将比 MDS42pdu 更快地变得无效。如果找到了这个预期结果，我们将确定新生产系统出现问题之前的最大代数。如果实现了大大延长的生产寿命，我们将认为这是我们的分批补料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处于活跃状态，而这些机制可能在未来的工作中被有效地灭活。极长的生产培养寿命表明使用该菌株进行连续发酵过程的可行性。