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：
9276026
负责人：
FREDERICK R BLATTNER
金额：
$ 86.41万
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 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9276026
关键词：
Address Adopted Adoption Antibodies Antitoxins Attenuated Biological Biological Containment Biological Products Cytoplasmic Protein DNA Transposable Elements DNA analysis DNA sequencing Data Diphtheria Toxin Dose Drug Industry Engineering Equipment Escherichia coli Experimental Designs Failure Fermentation Funding Gelsolin Gene Proteins Genes Genetic Genetic Engineering Genome Genome engineering Genomics Goals IS Elements Industrialization Medicine Membrane Proteins Methods Microbial Biofilms Modeling Modification Monitor Mutation Organism Patients Peptide Hydrolases Performance Pharmaceutical Preparations Pharmacologic Substance Phase Problem Solving Procedures Process Production Prophages Proteins Pump Reaction Readiness Running Seeds Signal Transduction Stress System Technology Testing Therapeutic Time Toxin Toxoids Vaccines Work base c new commercialization cost cross reacting material 197 design experimental study flasks foot genome sequencing improved instrumentation nuclease plasmid DNA productivity loss programs prototype public health relevance repaired sensor software development

项目摘要

DESCRIPTION (provided by applicant): Scarab Genomics was founded to improve E coli as an industrial organism by genome engineering. These strains have stable genomes since all prophages, transposable and IS elements, and the error prone repair systems were removed. A recA- version has always been provided as an option. The goal of the Phase I project was to ascertain whether the changes already introduced are sufficient to realize extended or continuous fermentation. Data gathered in Phase I show that we have indeed supported that hypothesis. Data obtained using serial transfer with shake flasks met our criterion for production stability of 21 days, but only when the cultures were not induced. This suggested the use of a two tank system with seed and production tanks. To confirm this we performed actual fermentations using Scarab funds. The results show that stable production of a test protein, CRM197 can be extended at least 33 days with no loss of productivity. This is more than enough to justify a 10 fold lowering of the cost of production with continuous flow rather than fed batch procedures that have been used in for manufacturing in E. coli. Continuous cultures were also analyzed by DNA sequencing and this was able to detect contamination as well as mutations and rearrangements. This analysis also revealed that the Scarab strain competed well against contaminants in contrast to standard strains BL21. We are therefore proposing to take this system to the next level by developing a complete platform for continuous fermentation, called C-Flow. The FDA has recently encouraged adoption of continuous manufacturing in the pharmaceutical industry, and several large companies have recently signaled readiness to implement the change. Our aim is to attack the bottom line of E. coli fermentation by offering a simpler and cheaper process that will produce consistently higher quality bioproducts than fed batch fermentation. Our proposal is to fully characterize the C-flow system and work towards commercializing it by developing a prototype that fits in a standard 6 ft hood. One particularly useful feature will support optimization of fermentation parameters without the need to restart fermentation so the best possible performance can be quickly and inexpensively achieved by a user. Other products such as pDNA will be tested in the C-Flow system.

描述（由适用提供）：发现圣甲虫基因组学可以改善基因组工程的工业组织。这些菌株具有稳定的基因组，因为所有先知，可转座和是元素，并且去除了误差容易修复系统。始终提供了一个reca-版本作为选项。第一阶段项目的目标是确定已经引入的更改是否足以实现扩展或持续的发酵。在第一阶段收集的数据表明，我们确实支持了这一假设。使用奶昔瓶连续传输获得的数据符合我们的生产稳定性21天的标准，但仅当未诱导培养物时。这表明使用了带有种子和生产罐的两个水箱系统。为了确认这一点，我们使用圣甲虫基金进行了实际的发酵。结果表明，稳定的测试蛋白，CRM197至少可以延长33天而不会损失生产率。这足以证明以连续流量而不是喂养的生产成本的10倍降低合理的合理性。在大肠杆菌中用于制造的批量程序。还通过DNA测序分析了连续培养物，这能够检测到污染以及突变和重排。该分析还表明，与标准菌株BL21相比，圣甲虫菌株与污染物竞争良好。因此，我们建议通过开发完整的连续发酵平台将该系统提升到一个新的水平，称为C-Flow。 FDA最近鼓励在制药行业采用连续制造，而几家大型公司最近签署了准备进行更改的准备。我们的目的是通过提供一个更简单，廉价的过程来攻击大肠杆菌发酵的底线，该过程将产生比FED批处理发酵的质量更高的生物产品。我们的建议是充分表征C-Flow系统，并通过开发适合标准6英尺罩的原型来致力于商业化。一个特别有用的功能将支持发酵参数的优化，而无需重新启动发酵，因此用户可以快速且廉价地实现最佳性能。其他产品（例如pDNA）将在C-Flow系统中进行测试。