Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids.

Applications of genetic code expansion (GCE) in live cells are widespread and continually emerging, yet they have been limited by their reliance on the supplementation of non-standard amino acids (nsAAs) to cell culturing media. Here, we report the design of a single engineered bacterial host that performs the steps of phenylalanine derivative semi-synthesis and site-specific incorporation within target proteins. Our platform pathway exhibits broad substrate specificity towards commercially ubiquitous, achiral building blocks of aryl aldehydes or carboxylic acids, producing the family of nsAAs that are most frequently used for GCE. We demonstrate biosynthesis of 12 distinct nsAAs, and we observe combined biosynthesis and incorporation for these chemistries using common orthogonal translation systems (OTSs) based on a fluorescent reporter assay. We additionally show that the combination of nsAA biosynthesis and GCE steps can extend the chemical reach of the intrinsic biological containment strategy of synthetic auxotrophy from reliance on nsAAs to instead reliance on low-cost and achiral building blocks. Our platform should aid industrial-scale manufacturing of proteins that contain nsAAs and democratize access to expensive or commercially unavailable chemistries for labs that lack separations or traditional synthesis expertise.