Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Abstract

The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits ar ange of biological activities. It is also as mall bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report as imple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploitedt he exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyltRNA synthetase (SacRS) capable of specifically acceptingt he small, polar amino acid instead of its long andb ulky aliphatic natural substrate. We succeeded in generating an ovel and inexpensive strategy for the incorporation of af unctionally versatile amino acid. This will help in the conversiono fo rthogonal translation from as tandard technique in academic research to industrial biotechnology.

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

“…coli‐ endogenous, PLP‐dependent O ‐acetylserine sulfhydrylase (OASS; Table , entry 4). By screening a mutant library of Methanosarcina mazei tRNA synthetase, SaC was incorporated into the model protein GFP by SCS, and the resulting engineered protein was immobilized onto a thiol‐functionalized poly(ethylene glycol) hydrogel …”

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

“…[88] Although chemical synthesis is still the main source of ncAAs,t heir market availability,p rice,a nd purity are serious bottlenecks for industrial biotechnology.Amore useful, broadly applicable chemical handle would be an ncAA that is produced intracellularly from low-cost chemical precursors added to the growth medium. Fore xample,E xner et al [44] reported very recently an engineered PylRS variant capable of activating and loading the corresponding tRNAw ith S-allylcysteine (Sac) generated in situ from acheap precursor allylmercaptan by using the cysteine biosynthesis pathway.T he biological abundance and biosynthetic production pathway of Sac greatly reduces the cost of protein production, potentially making industrial-scale applications feasible.…”

“…To ensure sufficient intracellular concentrations,t he ncAA analogues are supplied in excess in the culture and this may become expensive,inparticular for scale-up fermentations.However, this issue was recently addressed by engineering the expression host with metabolic pathways for the intracellular production of ncAAs. [44,75,88] Similarly, E. coli strains have been engineered for the SCS production of proteins containing Tyr, [177] Phe, [178] and Pyl [179] analogues.W ithout doubt, an expanded genetic code in combination with metabolic engineering would further diversify the chemical arsenal for the large-scale production of useful xenoproteins,w hich are today still the exclusive domain of proof-of-principle experiments.…”

Efforts and Challenges in Engineering the Genetic Code

“…In the most common approach, amino acid uptake is artificially controlled by feeding auxotrophs with NCAAs [33,34,35] (Figure 2a). Attempts to control NCAA synthesis have involved supplying organisms with NCAA precursors [57,58,59,60,61,62] (Figure 2b), which is also known as metabolic engineering [63,64]. In one example, the precursor l -β-thieno [3,2-b]pyrrolyl ([3,2]Trp) was fed to a tryptophan (Trp)-auxotrophic E. coli capable of synthesizing [3,2]Tpa (a Trp analog) to generate mutants that could propagate on l -β-(thieno [3,2-b]pyrrolyl)alanine ([3,2]Tpa) [57] (Figure 2b).…”