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.

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Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of "molecular surgery" to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical β-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual β-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties.Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level ("molecular surgery"), which can be adopted for the

The cover picture shows a journey from garlic to genetic code engineering: In this issue, Budisa and co‐workers report in vivo‐specific protein olefination with S‐allylcysteine (Sac), which is naturally abundant in garlic. Alternatively, it can also be biosynthesized in situ by supplementing the amino acid metabolism of E. coli with allylmercaptan. Sac is recognized and activated by the novel and unique pyrrolysyl‐tRNA synthetase‐based enzyme S‐allylcysteinyl‐tRNA synthetase (SacRS), which is capable of specifically accepting a small, polar amino acid instead of the long and bulky aliphatic natural substrate. In this way, a biochemical pathway for the production of proteins containing S‐allylcysteine as a minimal tag or label for chemical protein modification has been established. More information can be found in the communication by S. Panke, N. Budisa et al. on page 85 in Issue 1, 2017 (DOI: 10.1002/cbic.201600537).