Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol

• A genetic network that optimizes membrane protein expression is created in E. coli.
• Controlling expression of a butanol-secreting pump gives higher n-butanol titers.
• The system is tunable and generalizable to other toxic membrane proteins.

Microorganisms can be engineered to produce a variety of biofuels and commodity chemicals. The accumulation of these products, however, is often toxic to the cells and subsequently lowers production yields. Efflux pumps are a natural mechanism for alleviating toxicity through secretion of the product; unfortunately, pump overexpression also often inhibits growth. Tuning expression levels with inducible promoters is time-consuming and the reliance on small-molecule inducers is cost-prohibitive in industry. We design an expression regulation system utilizing a native Escherichia coli stress promoter, PgntK, to provide negative feedback to regulate transporter expression levels. We test the promoter in the context of the efflux pump AcrB and its butanol-secreting variant, AcrBv2. PgntK-driven AcrBv2 confers increased tolerance to n-butanol and increased titers of n-butanol in production. Furthermore, the system is responsive to stress from toxic overexpression of other membrane-associated proteins. Our results suggest a use for feedback regulation networks in membrane protein expression.

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