Computational design and construction of an Escherichia coli strain engineered to produce a non-standard amino acid

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 hosts native metabolism to improve pAF production. Next, we explored different ways of imposing these flux interventions experimentally and found that the upregulation of flux in chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers ([~]20 fold) while maintaining a reasonable pAF yield-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.