Engineering Prokaryotic Translation for Artificial Metalloenzyme Production

Type of Award: Catalyst
Date Awarded: September 2013
Award End Date: August 2015
Amount Awarded: $ 200,000.00
PI(s): Michael Jewett, PhD, NU; Jared Lewis, PhD, UChicago;

Abstract: The extraordinary synthetic capability of protein biosynthesis has driven extensive efforts to engineer translation systems to create proteins containing unnatural amino acids (UAAs). However, UAA incorporation efficiency is ca. 1000x lower than that of natural AAs due to the challenge of re-engineering a system of nearly 80 separate macromolecules. We will identify and eliminate bottlenecks in this system to enable incorporation of organometallic (M)UAAs into proteins. Specifically, we will study MUAA bioavailability, explore interactions between EF-Tu and MUAA-tRNAs, and evolve the entire translation system for maximal MUAA incorporation efficiency. Our work will expand understanding of the translation apparatus, provide a general platform to incorporate any UAA of interest, and facilitate direct expression of a new class of artificial metalloenzymes (ArMs). ArMs combine the selectivity and evolvability of enzymes with the bond forming capabilities of organometallic catalysts. We are particularly interested in non-biogenic metal complexes (e.g., Pd, Ir, and Rh) to promote site-specific C-H functionalization reactions. Such reactions are not currently possible using small molecule catalysts, and they could greatly expand the scope of metabolic engineering for chemical synthesis and enable new methods for direct interrogation of biomolecules in vivo.