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

Abstract

The microbial transglutaminase (mTGase) from Streptomyces mobaraense is widely used in the food industry. However, recombinant production of mTGase is challenging because the mTGase is synthesized as an inactive zymogen, and needs to be activated by proteolytic processing. In this study, self-cleaving intein Ssp DnaB was applied to activate the mTGase in Corynebacterium glutamicum. Premature cleavage of intein Ssp DnaB also occurred, but instead of suppressing premature cleavage, this phenomenon was used to produce active mTGase in C. glutamicum. Both SDS-PAGE analysis and mTGase activity assays indicated that the premature cleavage of intein Ssp DnaB activated the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies further showed that the premature cleavage activated the mTGase intracellularly, in a highly specific manner. Moreover, the growth performance of C. glutamicum was not noticeably affected by the intracellular expression of active mTGase. Finally, the mTGase was produced in a 2 L bioreactor, with activity up to 49 U/mL, the highest intracellular mTGase activity ever reported. Using premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, we produced high levels of intracellular active mTGase. Moreover, this approach did not require any further processing steps, such as protease treatment or lengthy incubation, greatly simplifying the production of active mTGase. This efficient and simple approach has great potential for the large-scale industrial production of active mTGase.