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）：此申请旨在测试新的棒棒大肠杆菌菌株MDS42PDU，以生产生物药物，氨基酸和生物燃料的商业发酵。它设计用于非常低的点突变率和插入序列换位，尤其是在重组蛋白产生的应力条件下。我们在这里建议研究对大规模发酵的基本问题的影响：发酵罐在发酵罐中降低生产力的进化。在培养物中发生的随机突变会产生释放产物形成负担的细胞，并且具有选择性的优势。很快，这些超越了文化并降低或消除生产力。简而言之，文化恶化。突变的选择也会破坏产品纯度。我们想减慢或消除这种性能的退化，以提高当前美联储方法的稳定性，质量和效率，并可能支持将来更连续的发酵协议。拟议的研究使用扩展培养物的定期总基因组测序将普通生产菌株与新的低突变菌株进行比较，以查看是否扩大了降解之前的培养基生产率，以及我们是否可以识别性能降解所涉及的突变类型。这两个目的提出了在摇瓶中串行转移培养物的定期全基因组测序，以廉价地量化低突变菌株在多种情况下的性能，然后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控制培养物将比MDS42PDU更快地变得更快。如果找到了预期的结果，我们将确定新生产系统中出现问题之前的最大世代数。如果实现了巨大的生产寿命，我们将认为这是我们产品商业化的主要里程碑，以进行美联储发酵。测序还将有助于确定是否可能在未来的工作中有益地灭活的其他突变机制。生产性较长的文化生活将表明连续发酵过程的可行性USIG这种菌株。