Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids

Abstract

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine o-succinyltransferase (metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with p-benzoylphenylalanine (pBzF), increases the melting temperature of E. coli metA by 21°C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Tackling Achilles’ Heel in Synthetic Biology: Pairing Intracellular Synthesis of Noncanonical Amino Acids with Genetic‐Code Expansion to Foster Biotechnological Applications

“…In general terms, ncAAs can be incorporated into recombinant proteins by both in vivo and in vitro methods . Granted both processes offer certain benefits; however, in vivo approaches have the advantage of simpler culture conditions that are easily scalable and result in lower production costs.…”

“…Synthetic biology has long been established as a robust methodology to engineer proteins with novel chemical and physical properties, beyond those that can be provided by the natural repertoire of the standard 20 amino acids . By modifying the genetic‐code machinery, scientists have been able to encode the efficient incorporation of more than 200 noncanonical amino acids (ncAAs) to produce novel and exciting proteins and peptides .…”

“…By modifying the genetic‐code machinery, scientists have been able to encode the efficient incorporation of more than 200 noncanonical amino acids (ncAAs) to produce novel and exciting proteins and peptides . Besides providing valuable insight into the molecular‐dynamic forces that shaped the chemistry of life, these new biomaterials have extraordinary potential for a myriad of biotechnological processes, such as protein immobilization and stabilization, proteomics, drug discovery, drug delivery, medical diagnosis, industrial biocatalysis, bioremediation, bioplastics, and many others . More recently, scientists have begun to explore the possibility of completely reprogramming the genetic code in a single microbial organism, thus creating a new form of life centered on an alternative biochemical alphabet .…”

“…A possible solution to this problem is to develop a biocatalytic approach for the synthesis of the required ncAAs coupled with genetic‐code expansion within the same host cell. In this methodology, the host organism is provided with the metabolic route, cofactors, and carbon source to synthesize the desired ncAA intracellularly, as well as the genetically reassigned translation machinery to incorporate the biogenerated ncAA into the target protein …”

Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis

“…Seeking chemically stable covalent bonds to replace reversible disulfide bonds for protein stabilization poses a novel challenge. Incorporating noncanonical amino acid (ncAA) into enzymes can improve enzymatic properties, such as thermostability [26] . Unnatural thioether linkages can be formed as a result of the cross-linking reaction between a cysteine and a noncanonical O -2-bromoethyl tyrosine (O2beY), introducing unique properties of redox-stability and irreversibility [27] .…”