A DELAYED TOXIN EXPRESSION SYSTEM FOR IMPROVED BIOMANUFACTURING PRODUCTIVITY

Abstract

The field of biomanufacturing is rapidly expanding with the use of genetically modified organisms. The advantages that genetically modified organisms have in being able to produce larger quantities of product or even entirely new pathways explains their rapid uptake in the field. These genetically modified organisms are not without weakness however, as their higher production leads to higher burden on the cell. Due to this weakness later generations of cells in a fermentation run lose productivity which results in a loss of millions of dollars in product at the industrial scale. To address this problem a delayed toxin expression system was designed that allows for the killing of older generations of cells that are experiencing a loss of productivity while having a constant stream of fresh younger generation of cells. This stops the productivity from falling off over time and increases the amount of product that is made in each fermentation run. The delayed toxin expression system was designed using E. coli pause sites to delay a T7 polymerase from expressing the target protein. The T7 polymerase was bound to a mutagenizer that slowly deactivated the pause sites by mutating their sequences. This delayed toxin expression design acts like a tunable fuse that eliminates cells after their productivity begins to drop off.