title: Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol.
abstract: 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.
authors.affiliation: Joint Graduate Group in Bioengineering at UC Berkeley and UCSF, 306 Stanley Hall, Berkeley, CA 94720, United States.
authors.forename: Sergey
authors.initials: S
authors.lastname: Boyarskiy
authors.affiliation: Department of Chemistry, University of California Berkeley, 201 Gilman Hall, Berkeley, CA 94720, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Davis López
authors.affiliation: Department of Molecular and Cell Biology, University of California Berkeley, 142 LSA #3200, Berkeley, CA 94720, United States.
authors.forename: Niwen
authors.initials: N
authors.lastname: Kong
authors.affiliation: Joint Graduate Group in Bioengineering at UC Berkeley and UCSF, 306 Stanley Hall, Berkeley, CA 94720, United States; Department of Chemistry, University of California Berkeley, 201 Gilman Hall, Berkeley, CA 94720, United States; Department of Chemical and Biomolecular Engineering, University of California Berkeley, 201 Gilman Hall, Berkeley, CA 94720, United States. Electronic address: dtercek@berkeley.edu.
authors.forename: Danielle
authors.initials: D
authors.lastname: Tullman-Ercek
mesh_terms: D020001:1-Butanol / Q000302:isolation & purification / Q000378:metabolism*; D002470:Cell Survival / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D020001:1-Butanol; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli
keywords: AcrB; Dynamic gene regulation; Membrane protein overexpression; N-butanol production