title: Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli.
abstract: Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Englaender
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.|Department of Chemistry, Hamilton College , Clinton, New York 13323, United States.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Brady F
authors.identifier: 0000-0002-2948-2846
authors.initials: BF
authors.lastname: Cress
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Carl. R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Kuhlman
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.|Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.|Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Mattheos A G
authors.initials: MAG
authors.lastname: Koffas
mesh_terms: D000642:Ammonia-Lyases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D002934:Cinnamates / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D056426:Genetic Loci; D007211:Indoles / Q000032:analysis / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D008780:Methyltransferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D000642:Ammonia-Lyases; D002934:Cinnamates; D029968:Escherichia coli Proteins; D007211:Indoles; D008164:Luminescent Proteins; D008780:Methyltransferases; D011938:Rec A Recombinases; D000097573:Red Fluorescent Protein; C029010:cinnamic acid; C457299:GidB protein, E coli; C023208:L-tyrosine ammonia-lyase; C063155:violacein
keywords: cinnamic acid; episomal expression; flavonoid production; genomic integration; metabolic burden; violacein