Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon Reassignments

Abstract

In the present study, we simultaneously incorporated two types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense to enhance the utility of this industrial enzyme. The first amino acid, 3-chloro-l-tyrosine, was incorporated into MTG in response to in-frame UAG codons to substitute for the 15 tyrosine residues separately. The two substitutions at positions 20 and 62 were found to each increase thermostability of the enzyme, while the seven substitutions at positions 24, 34, 75, 146, 171, 217, and 310 exhibited neutral effects. Then, these two stabilizing chlorinations were combined with one of the neutral ones, and the most stabilized variant was found to contain 3-chlorotyrosines at positions 20, 62, and 171, exhibiting a half-life 5.1-fold longer than that of the wild-type enzyme at 60 °C. Next, this MTG variant was further modified by incorporating the α-hydroxy acid analogue of N ε-allyloxycarbonyl-l-lysine (AlocKOH), specified by the AGG codon, at the end of the N-terminal inhibitory peptide. We used an Escherichia coli strain previously engineered to have a synthetic genetic code with two codon reassignments for synthesizing MTG variants containing both 3-chlorotyrosine and AlocKOH. The ester bond, thus incorporated into the main chain, efficiently self-cleaved under alkaline conditions (pH 11.0), achieving the autonomous maturation of the thermostabilized MTG. The results suggested that synthetic genetic codes with multiple codon reassignments would be useful for developing the novel designs of enzymes.

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

“…By contrast, AlocLysOH, which is an α-hydroxy acid, was incorporated slightly more efficiently by the pairs involving MmBocLysRS2 than the CMa pair. It is likely that the amino acid substitutions in MmBoLysRS2 increased its affinity with the hydroxy acid [ 13 ].…”

“…Lastly, the tRNA Pyl recognition by PylRS does not involve the anticodon moiety and tRNA Pyl can be engineered to read any other codons than UAG without impairing aminoacylation [ 7 , 8 , 9 ]. Thus, multiple PylRS variants were recently developed to genetically encode more than 100 synthetic amino acids [ 2 , 4 ] including α-hydroxy acids [ 10 , 11 , 12 , 13 ].…”

Versatility of Synthetic tRNAs in Genetic Code Expansion

“…The genetic code naturally expanded to include Sec and Pyl, through the recoding of UGA and UAG, respectively. However, Sec can be efficiently inserted at sense codons [ 44 , 45 ] and improving incorporation in a SECIS-independent manner is achieved through UAG suppression [ 26 , 28 , 29 , 31 ]. Anticodon mutations are sufficient to recode sense and stop codons with Sec and Pyl, since cognate SerRS and PylRS do not utilize identity elements in the anticodon loop of tRNA Sec and tRNA Pyl .…”

“…Therefore, Sec pathway components are removed to prevent interaction with the OTS. Recent work in Escherichia coli has focused on improving the Sec incorporation efficiency and discovering EF-Tu compatible tRNA Sec variants for selenoprotein expression without the requirement for SECIS in the coding sequence [ 26 , 27 , 28 , 29 , 30 , 31 ].…”

“…As a result of increasing ncAA-tRNA concentrations, the cellular levels of the PylRS•tRNA Pyl pairs and components of the Sec pathway must be manipulated to out-compete host tRNAs or release factors for the targeted codon, while maintaining cellular fitness. Furthermore, altering the stoichiometry of the Sec-OTS components is important for the efficiency and homogeneity of selenoprotein production [ 26 , 29 ]. Thus, in addition to mutagenesis approaches to improving OTS interactions, the expression levels of each individual component are critical.…”