title: ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways.
abstract: The ability to fine tune gene expression has created the field of metabolic pathway optimization and balancing where a variety of factors affecting flux balance are carefully modulated to improve product titers, yields, and productivity. Using a library of isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein biosynthesis involves a complex five-gene pathway that is an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediates and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the total initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829 ± 46 mg/L; a 2.6-fold improvement in titer and a 30-fold improvement in productivity from previous literature reports.
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Victoria R
authors.initials: VR
authors.lastname: Vernacchio
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Lachance
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Lebovich
authors.affiliation: Department of Chemistry, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Fu
authors.affiliation: Department of Chemistry, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Abhijit N
authors.initials: AN
authors.lastname: Shirke
authors.affiliation: Department of Chemistry, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Victor L
authors.initials: VL
authors.lastname: Schultz
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Brady
authors.initials: B
authors.lastname: Cress
authors.affiliation: 1] Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA [2] Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy NY 12180, USA [3] Department of Chemistry, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: 1] Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA [2] Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy NY 12180, USA.
authors.forename: Mattheos A G
authors.initials: MA
authors.lastname: Koffas
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007211:Indoles / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D031161:Pseudoalteromonas / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D007211:Indoles; C063155:violacein