Journal
ACS SYNTHETIC BIOLOGY
Volume 7, Issue 9, Pages 2170-2176Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00157
Keywords
chlorotyrosine; hydroxy acid; AGG codon; pyrrolysyl-tRNA synthetase; self-cleavage
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Funding
- JSPS KAKENHI [26291035, 16K21631]
- Grants-in-Aid for Scientific Research [16K21631, 26291035] Funding Source: KAKEN
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In the present study, we simultaneously incorporated two types of synthetic components into microbial transglutaminase (MTG) from Streptoverti-cillium 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 alpha-type enzyme at 60 degrees C. Next, this MTG variant was further modified by incorporating the a-hydroxy acid analogue of N-epsilon-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.
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