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

 描述(申请人提供)：圣甲虫基因组公司成立，旨在通过基因组工程将大肠杆菌作为工业有机体进行改良。这些菌株具有稳定的基因组，因为所有的前驱体、转座子和IS元件都被移除了，而且容易出错的修复系统被移除了。RecA版本一直都是作为选项提供的。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延长或连续发酵。在第一阶段收集的数据表明，我们确实支持这一假设。用摇瓶连续转移获得的数据满足我们21天的生产稳定性标准，但只有在没有诱导培养的情况下才能获得。这建议使用带有种子罐和生产罐的双罐系统。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，试验蛋白CRM197的稳定生产可延长至少33天而不损失产量。这足以证明连续生产而不是喂料生产成本降低10倍是合理的。 已用于在大肠杆菌中生产的批处理程序。连续培养也通过DNA测序进行分析，这能够检测到污染以及突变和重排。这项分析还表明，与标准菌株BL21相比，Scarab菌株对污染物的竞争能力很强。因此，我们建议将这一系统提升到一个新的水平，开发一个完整的连续发酵平台，称为C-Flow。FDA最近鼓励制药行业采用连续生产，几家大公司最近也表示准备实施这一变化。我们的目标是通过提供一种更简单、更便宜的工艺来生产比补料分批发酵始终更高质量的生物制品，从而攻克大肠杆菌发酵的底线。我们的建议是充分描述C-Flow系统的特征，并通过开发适合标准6英尺引擎盖的原型来实现其商业化。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，如PDNA，将在C-Flow系统中进行测试。

 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.