Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum

Abstract Background The microbial transglutaminase (mTGase) from Streptomyces mobaraense has been widely used in the food industry. Recombinant production of mTGase is tricky because the mTGase is synthesized as an inactive zymogen, which needs to be activated by proteolytic processing. Self-cleaving inteins have been applied to activate the zymogen in a simple and highly specific manner as compared with proteolytic processing. However, self-cleaving inteins suffer from the inherent problem of premature cleavage. Moreover, self-cleaving inteins normally require an additional step of long time incubation to induce the cleavage. These two inherent problems limit self-cleaving inteins for their potential application in the production of mTGase.Results In this study, the premature cleavage of intein Ssp DnaB was observed in Corynebacterium glutamicum when the Ssp DnaB was used to activate mTGase precursor protein. Rather than suppressing it, the premature cleavage was applied to produce active mTGase in C. glutamicum. The SDS-PAGE analysis and the mTGase activity assay indicated that the premature cleavage of intein Ssp DnaB was successfully applied to activate the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies demonstrated that the premature cleavage activated the mTGase intracellularly in a highly specific manner. Finally, in a jar fermentor, the intracellular mTGase activity was up to 49 U/mL, which was the highest intracellular mTGase activity ever reported.Conclusions An efficient and simple approach with great potential for large-scale industrial production of active mTGase was presented in this study. This approach employed premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, resulting in high-level intracellular production of active mTGase. Moreover, this approach did not require any further processing steps such as protease treatment or long time incubation, greatly simplifying the production of active mTGase.