In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

Abstract

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.

An Engineered Escherichia coli Strain with Synthetic Metabolism for in‐Cell Production of Translationally Active Methionine Derivatives

“…A list of potentially interesting nucleophiles were tested in vitro as substrate for cg OAHSS and a number of them produced stable l ‐homoserine derivatives (Table S4). For example, incorporation into proteins of ncAAs carrying a thioallyl group, such as S ‐allyl‐L‐homocysteine can be of great interest [53] . At a biotechnology application level, the benefit of alkene moieties present in thioallyl residues introduced into recombinant protein concerns the photochemical surface modification by both thiol‐ene click reaction [54,55] and photoirradiation [56] .…”

“…In Figure 5A, the deconvoluted mass of Ahalabeled B* protein (before dOG conjugation) is shown to be 10247. 53 [2Met] and GFP2M grown according to our Aha production/incorporation protocol. The nominal molecular weight of GFP is 26.7 kDa.…”

“…For example, incorporation into proteins of ncAAs carrying a thioallyl group, such as S-allyl-L-homocysteine can be of great interest. [53] At a biotechnology application level, the benefit of alkene moieties present in thioallyl residues introduced into recombinant protein concerns the photochemical surface modification by both thiol-ene click reaction [54,55] and photoirradiation. [56] These photochemistry click reactions are highly attractive bio-conjugation methods because of the low harm for proteins in living systems.…”

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

“…accomplished the intracellular biosynthesis and simultaneous protein incorporation of the methionine analogue S ‐allylhomoscysteine (SahC, Table , entry 5) by SPI in E . coli . Although both SaC and SahC bear the same functional group and were synthesized in vivo by supplementing microbial cultures with allyl mercaptan, the amino acid substrate is homoserine, which originates from the tricarboxylic acid cycle (Scheme C).…”

Noncanonical Amino Acids in Synthetic Biosafety and Post‐translational Modification Studies

“…In an optimized condition, azide and azoles (e. g., pyrazole, triazole, tetrazole, thiols and selenol) were efficiently used by OASS to synthesize β‐substituted ncAAs [27] . Recently, a similar strategy was used to synthesize S‐allyl cysteine in E. coli [28] . By applying an evolved PylRS/tRNA pair, researchers successfully incorporated the synthesized ncAA into the model protein, EGFP.…”