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

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 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.