摘要：SummaryIntroducing 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 nsAApara-aminophenylalanine (pAF) inEscherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model ofE. colimetabolism 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.Graphical abstractDisplay OmittedHighlights•Sought to optimizepara-aminophenylalanine (pAF) production and growth inE. coli•Identified interventions in the host native metabolism using genome-scale models•Constructed multiple mutant strains involving gene knockouts and/or overexpressions•Flux modification in chorismate biosynthesis pathway significantly raised pAF titerBioengineering; Metabolic engineering; Bioinformatics
