Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli

Jacob A. Englaender, J. Andrew Jones, Brady F. Cress, Thomas E. Kuhlman, Robert J. Linhardt, Mattheos A.G. Koffas

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)710-720
Number of pages11
JournalACS synthetic biology
Issue number4
StatePublished - Apr 21 2017


  • cinnamic acid
  • episomal expression
  • flavonoid production
  • genomic integration
  • metabolic burden
  • violacein

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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