title: Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO₂-based 3-hydroxypropionate production.
abstract: Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T(opt) 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (T(opt) 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695-7905.
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Hawkins
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695-7905.
authors.forename: Hong
authors.initials: H
authors.lastname: Lian
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695-7905.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Zeldes
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695-7905.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Loder
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602.
authors.forename: Gina L
authors.initials: GL
authors.lastname: Lipscomb
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Schut
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Keller
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695-7905. mkelly@ncsu.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D002245:Carbon Dioxide / Q000378:metabolism*; D020869:Gene Expression Profiling; D006358:Hot Temperature; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D020027:Pyrococcus furiosus / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D016943:Sulfolobaceae / Q000235:genetics
chemical_list: D002245:Carbon Dioxide; D019344:Lactic Acid; D008320:Maltose; C031601:hydracrylic acid
keywords: 3-hydroxypropionate; CO2 fixation; Metallosphaera sedula; Pyrococcus furiosus