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

Englaender, Jacob A.
Jones, J. Andrew
Cress, Brady F.
Kuhlman, Thomas E.
Linhardt, Robert J.
Koffas, Mattheos A.G.
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli. J. Englaender, A. J. Jones, B. Cress, T. Kuhlman, R. J. Linhardt, M.A.G. Koffas, ACS Synthetic Biology, 6, 710−720, 2017.
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.
ACS Synthetic Biology, 6, 710−720
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The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
ACS Synthetic Biology