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 speci c 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 speci c 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 e cient 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.

Microbial Transglutaminase Is a Very Frequently Used Food Additive and Is a Potential Inducer of Autoimmune/Neurodegenerative Diseases

“…When the Streptoverticillium mobaraense mTG gene was cloned into Lactococcus lactis, the bacterial mass increased significantly [81,82]. Newer bioengineered cloning of the mTG is successful in producing a higher yield and a more active form of the enzyme for a more cost-effective industrial application [83,84]. One wonders if a high mTG secreting bacteria will laterally transfer, by horizontal gene exchange, the mTG gene to the human microbiome, increasing its luminal yield and activity, thus perturbating luminal homeostasis [85].…”

“…Multiple issues are raised and the antibiotic resistance gene is of concern [85,[94][95][96]. In view of continuous efforts to bioengineer more cost-efficient mTG for industrial applications [8,26,[81][82][83][84]93] and in view of the all the detrimental effects of mTG and its trans-amidated complexes used for food processing (Figure 2), public health against the side effects of mTG should be a prime priority. The worldwide food and industrial safety regulatory authorities should reassess the updated observations; hence, consider the alleviation of the GRAS status and enforce the labelling of this heavily used processed food additive.…”

Efficient Production of a Thermostable Mutant of Transglutaminase by Streptomyces mobaraensis

“…24 Therefore, in heterologous hosts, S. mobaraensis TGase is generally expressed as a pro-TGase, which is then subjected to in vitro activation by different proteases. 19,25 A series of in vivo activations have been developed to achieve the active expression of S. mobaraensis TGase, such as coexpression with activating proteases 15 or pro-region, 26 designing selfcleaving pro-region, 27,28 and expression of the pro-enzyme in the host with activating proteases. 13 However, the active TGase is produced at a low level 29 or as intracellular proteins 28 in heterologous hosts.…”

“…19,25 A series of in vivo activations have been developed to achieve the active expression of S. mobaraensis TGase, such as coexpression with activating proteases 15 or pro-region, 26 designing selfcleaving pro-region, 27,28 and expression of the pro-enzyme in the host with activating proteases. 13 However, the active TGase is produced at a low level 29 or as intracellular proteins 28 in heterologous hosts. Recently, mutation breeding 30 and gene copy number optimization 30,31 have increased the TGase activity of S. mobaraensis to 40 U/mL.…”

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications

“…35 Although the combination of rational design and screening is an important option for further engineering of MTG, direct expression of the gene encoding mature MTG in the cytosol of host cells such as E. coli 32 is difficult, which hampers the construction of mutant libraries to tune its substrate specificity. 36 To overcome this limitation, a proteasemediated self-cleavable system 37 and an intein-mediated selfsplicing system 38 were designed for intracellular production of active MTG from its zymogen in E. coli and Corynebacterium glutamicum, respectively. High-throughput screening (HTS) of active MTG by post-translational proteolysis of zymogen mutants displayed on the yeast surface was investigated, 39 but it is still difficult to validate the substrate specificity of soluble MTG mutants by HTS.…”