Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para-aminophenylalanine

Ali R Zomorrodi; Colin Hemez; Pol Arranz-Gibert; Terrence Wu; Farren J Isaacs; Daniel Segrè

doi:10.1016/j.isci.2022.104562

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para-aminophenylalanine

iScience. 2022 Jun 9;25(7):104562. doi: 10.1016/j.isci.2022.104562. eCollection 2022 Jul 15.

Authors

Ali R Zomorrodi^{1

2

3}, Colin Hemez^{4

5

6}, Pol Arranz-Gibert^{4

5}, Terrence Wu⁷, Farren J Isaacs^{4

5

6}, Daniel Segrè^{3

8

9

10}

Affiliations

¹ Mucosal Immunology and Biology Research Center, Pediatrics Department, Massachusetts General Hospital, Boston, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Bioinformatics Graduate Program, Boston University, Boston, MA, USA.
⁴ Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
⁵ Systems Biology Institute, Yale University, West Haven, CT, USA.
⁶ Department of Biomedical Engineering, Yale University, New Haven, CT, USA.
⁷ Yale West Campus Analytical Core, 600 West Campus Drive, West Haven, USA.
⁸ Department of Biology, Boston University, Boston, MA, USA.
⁹ Department of Biomedical Engineering, Boston University, Boston, MA, USA.
¹⁰ Biological Design Center, Boston University, Boston, MA, USA.

Abstract

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para-aminophenylalanine (pAF) in Escherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host's native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (∼20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

Keywords: Bioengineering; Bioinformatics; Metabolic engineering.