title: Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.
abstract: The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, United States. Electronic address: mthorger@uga.edu.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Thorgersen
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, United States. Electronic address: gpries@uga.edu.
authors.forename: Gina L
authors.initials: GL
authors.lastname: Lipscomb
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, United States. Electronic address: gerti@uga.edu.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Schut
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States. Electronic address: rmkelly@ncsu.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, United States. Electronic address: adams@bmb.uga.edu.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D019843:Archaeal Proteins; D003066:Coenzyme A Ligases; D017353:Gene Deletion; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019344:Lactic Acid; C031601:hydracrylic acid; D003066:Coenzyme A Ligases
keywords: 3-Hydroxypropionate; Acetyl-CoA synthetase; Electrofuels; Metallosphaera sedula