Incorporation of unnatural amino acids for synthetic biology

Abstract

The challenge of synthetic biology lies in the construction of artificial cellular systems. This requires the development of modular "parts" that can be integrated into living systems to elicit an artificial, yet programmed, response or function. The development of methods to engineer proteins bearing unnatural amino acids (UAAs) provides essential components that may address this challenge. Here we review the emerging strategies for incorporating UAAs into proteins with the endgame of engineering artificial cells and organisms.

Vinyl Sulfone: A Multi-Purpose Function in Proteomics

“…This classical residue-specific modification chemistry, however, is rarely sufficiently selective to distinguish one residue within a sea of chemical functionality and for this reason more intricate approaches have been developed in recent times to introduce a unique chemical handle in the target protein that is orthogonal to the remainder of the proteome (Hackenberger & Schwarzer, 2008). Direct incorporation of non-canonical amino acids into proteins via the subversion of the biosynthetic machinery is an attractive means of introducing selectively new functionality by either a site-specific or residue-specific manner (Beatty & Tirrell, 2009;de Graaf et al, 2009;Johnson et al, 2010;Liu & Schultz, 2010;Voloshchuk & Montclare, 2010;Young & Schultz, 2010) that in combination with recent and notorious advances in bioorthogonal reactions (nucleophilic addition to carbonyl, 1,3-dipolar cycloaddition reactions, Diels-Alder reactions, olefin cross-metathesis reactions and palladium-catalyzed cross-coupling reactions) has allowed an exquisite level of selectivity in the covalent modification of proteins (Wiltschi & Budisa, 2008;Sletten & Bertozzi, 2009;Lim & Lin, 2010;Tiefenbrunn & Dawson, 2010). In spite that major technical challenges have been overcome, a prodigious amount of lab work and the concurrently optimization of a larger set of parameters is normally required for those advanced and selective methodologies in comparison with conventional organic reaction development.…”

Protein Engineering with Non-Natural Amino Acids

“…In its native context, the PylRS/tRNA evolved alongside the 20 canonical aaRSs and developed natural specificity for its cognate amino acid, making it an ideal pair for transfer into other expression hosts. Indeed, the PylRS/tRNA has been shown to be fully orthogonal in E. coli, yeast and mammalian cells (reviewed by Voloshchuk & Montclare, 2010). Furthermore, Pyl is naturally encoded by the amber stop codon, the most commonly reassigned codon to specify a NNAA, and thus requires no engineering to adapt it to this purpose.…”

“…A set of similar but not identical rules have been established for the N-terminal processing of NNAAs in recombinant interferon as well as other proteins (unpublished data). As a result, the N-terminal NNAA can be either retained or cleaved by choosing an appropriate 2nd residue ( Hendrickson et al, 2004;Voloshchuk & Montclare, 2010). In many cases, the intracellular level of wild-type aaRS may need to be augmented in order to compensate for the low activity of native aaRS towards the NNAA.…”

“…Employment of such hosts limits competition from the canonical amino acid for the reassigned sense codon, and improves the incorporation efficiency and yield of target proteins. For example, the MetRS has a relatively relaxed substrate binding pocket, and a number of Met analogs have been successfully incorporated into target proteins (reviewed by Hendrickson et al, 2004;Voloshchuk & Montclare, 2010). A special consideration worth mentioning when using Met analogs for protein engineering is the in vivo processing of NNAAs at the N-terminus of proteins.…”

Whole cell biotransformation for reductive amination reactions

“…Non-natural amino acids find application as synthons for chemical synthesis of active pharmaceutical ingredients (APIs) and as orthogonal modules for genetic code expansion in synthetic biology. 30 Enzyme-catalyzed amino acid production may proceed via ammonium addition to alkenes by ammonia lyases. 31 Commercial production of l-aspartate is based on aspartate ammonia lyase.…”