Programmable Control of Acetate Metabolism Escherichia coli

Acetate production in Escherichia coli fermentations reduces the total yield and quality of recombinant protein products. Acetate production occurs due to metabolic overflow in the presence of excess glucose, is increased when oxygen levels are low, and is facilitated by the phosphate acetyltransferase (pta) gene. We have engineered a genetic program that measures levels of glucose, oxygen, and acetate in the cell, combines the information into two Boolean AND gates, and produces an output that reduces acetate accumulation in batch cultures of E. coli. The output is composed of three modules, each regulating the expression of pta at different levels of the central dogma. A CRISPRi guide RNA module reduces the transcription of the gene, an interfering RNA that reduces translation, and a LON protease that degrades the enzyme. Together, these outputs enable real-time control of pta expression in response to conditions that lead to acetate formation in E. coli.