(798g) Metabolic Pathway Inactivation Via Post Translation Modifications

Metabolic engineering relies on recombinant DNA technology to optimize a biological system toward the production of commodity chemicals. Generally, this technology can be classified into three methods: overexpression of product-producing pathways, knockouts of competing pathways, and rewiring of regulatory networks. One important feature of these technologies is that they take place only on the transcriptional and translation synthesis level. This is problematic because biomass-producing enzymes cannot be knocked out lest the organism doesn’t grow. Even when synthesis is modulated on the transcriptional or translation level, protein stability ensures continued biomass anabolism. Protein half-lives are long, with only 2% to 7% degraded under half an hour in E. coli. Other studies indicate generally long half-lives for the majority of proteins in E. coli (>20 hours).

We have established a tool to inactivate proteins directly. Our technology has been successfully applied to two unique proteins in E. coli: mCherry and beta-galactosidase. We are investigating the techology on enzymes citrate synthase and
isocitrate dehydrogenase. Inactivation is fast and efficient.

Implications of this technology mean that metabolic engineers can reach unprecedented yields, where resources are not wasted toward the cell’s biomass, and potentially longer fed batch processes where chemical catalysis continues without cell accumulation problems such as oxygen transfer.