Computationally assisted design of in situ activatable recombinant microbial transglutaminase variants

Microbial transglutaminase (MTG, EC: 2.3.2.13) from Streptomyces mobaraense catalyzes the formation of isopeptide bonds between proteinaceous glutamine and lysine residues. In addition, MTG catalyzes the conjugation of various amines to glutamine residues. These characteristics make it a valuable tool for usage in different industrial applications from medicine to food production. Native MTG is secreted as an inactive zymogen by Streptomyces mobaraense and proteolytically activated in the extracellular space. The zymogen consists of the mature polypeptide and an N-terminal pro-peptide, which is crucial for correct folding and keeping MTG inactive until its activity is needed. The expression as an inactive zymogen presents some difficulties for the recombinant production of highly active MTG, as the removal of the pro-peptide to activate MTG is a neccessary processing step. This extra step complicates purification, increases costs and can introduce unwanted side-effects, such as overdigestion of MTG by non-native proteases. To overcome these challenges, various approaches, e.g., the introduction of self-cleaving pro-peptide variants or the introduction of destabilizing mutations in the pro-peptide have been published. To simplify the production of active recombinant MTG, we systematically searched for mutations in the pro-peptide sequence, that would lead to protease-independent MTG-activation. By in silico site-saturation mutagenesis of selected pro-peptide residues and comparison of Rosetta ΔΔG predictions, we obtained destabilizing and stabilizing mutations. Several of these pro-peptide mutations were selected and the pro-peptide sequence was combined with the highly active and thermostable mature MTG variants TG16 and TG2A via Golden Gate Cloning. The resulting twelve single and two double mutants per MTG variant were expressed, purified and tested for protein stability, activity at a broad range of temperatures and ability to crosslink β-casein. Moreover, we present several MTG variants, that can be readily produced, easily purified and activated, as well as deactivated in situ.