Functional Incorporation of Unnatural Amino Acids into Proteomes

Orthogonal translation machinery and engineered genomes can now be used to readily introduce non-canonical amino acids into cellular proteomes. This raises the question of what amino acids should be introduced, for what functionality? We have focused our efforts on two amino acids that have both structural and metabolic functionality, selenocysteine and L-DOPA.

Selenocysteine is normally incorporated via machinery that has evolved to introduce it across from opal codons programmed to interact with a so-called SECIS element in the RNA and with SelB, an EF-Tu equivalent specific for selenocysteine. We have removed these constraints by reprogramming an amber suppressor tRNA to be charged with selenocysteine. We have introduced this new, orthogonal translation machinery into the â??Amberlessâ?? E. coli, and have carried out long term evolution experiments (LTEE) to direct highly efficient incorporation of selenocysteine. These experiments were facilitated by the development of a conditionally essential protein, NMC-A beta-lactamase, that was completely dependent upon selenocysteine for function. We will detail results of the LTEEs that suggest genetic adaption of the evolved strain increases fitness and enables improved production of proteins containing the 21st amino acid.

More recently, we have engineered translational machinery for the incorporation of the redox active non-canonical amino acid L-DOPA. An evolved aminoacyl-tRNA synthetase enables highly specific incorporation of L-DOPA into proteins in E. coli. We have also shown that L-DOPA can form novel crosslinks with cysteine, which may provide the basis for the development of essential, crosslinked proteins that will â??addictâ?? strains to the amino acid. Furthermore, we have optimized an aromatic detoxification pathway for the biosynthesis of L-DOPA. These discrete components form the building blocks for establishing a parallel metabolism in E. coli and will allow the development of stable and independent organisms with expanded genetic codes.