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（由申请人提供）：Scarab Genomics 的成立是为了通过基因组工程改进大肠杆菌作为工业生物体，因为所有原噬菌体、转座和 IS 元件以及容易出错的修复系统都被删除。第一阶段项目的目标是确定已经引入的变化是否足以实现扩展或持续发酵。第一阶段收集的数据表明我们确实支持了使用该假设获得的数据。使用摇瓶的连续转移满足我们 21 天的生产稳定性标准，但仅当培养物未被诱导时才如此。为了证实这一点，我们使用 Scarab 基金进行了实际发酵。结果表明，测试蛋白 CRM197 的稳定生产可以延长至少 33 天，而不会损失生产力，这足以证明采用连续流而不是补料可将生产成本降低 10 倍。还通过 DNA 测序对用于生产的连续培养物进行了分析，这能够检测污染以及突变和重排。该分析还表明，相比之下，圣甲虫菌株能够很好地对抗污染物。因此，我们建议通过开发一个完整的连续发酵平台（称为 C-Flow）将该系统提升到一个新的水平，FDA 最近鼓励在制药行业采用连续生产，并且几家大公司也已经这样做了。最近表示准备实施这一变革，我们的目标是通过提供一种更简单、更便宜的工艺来突破大肠杆菌发酵的底线，该工艺将持续生产比补料分批发酵更高质量的生物产品。系统并致力于通过开发适合标准 6 英尺罩的原型来实现其商业化，其中一项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经济地实现最佳性能。其他产品如pDNA 将在 C-Flow 系统中进行测试。