(325g) Biosynthesis and Site-Specific Incorporation of Non-Standard Amino Acids within Proteins Starting from Readily Accessible Precursors

Expansion of the genetic code has allowed synthetic biologists to design proteins with novel functions or properties via incorporation of non-standard amino acids (nsAAs). However, typical application of genetic code expansion requires external supplementation of the nsAA to the cell, often at high concentrations. Additionally, chemical synthesis of enantiomerically pure nsAAs is often expensive and thus may be cost prohibitive at industrial scale. Biosynthesis is a valuable alternative to produce nsAAs intracellularly with high enantioselectivity. However, many biosynthetic routes designed so far are restricted in chemical scope or may be thermodynamically unfavorable for nsAA production. Herein, we design a biosynthetic pathway comprised of promiscuous enzymes for the biosynthesis and incorporation of a range of nsAAs from readily accessible precursors in an engineered strain of E. coli. We demonstrate the biosynthesis of nsAAs from supplemented aldehydes or their associated carboxylic acids, where the latter are chosen as precursors because of their ubiquity in metabolism, low toxicity, and low cost. We show that we can couple this platform biosynthetic pathway to orthogonal translation machinery for the site-specific incorporation of multiple biosynthesized nsAAs. Our research seeks to demonstrate a platform for accessing nsAAs with a range of functional group chemistries in vivo, thereby addressing a current hurdle in the application of genetic code expansion technologies.