Efficient biosynthesis of transglutaminase in Streptomyces mobaraensis via systematic engineering strategies

Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl transfer, and crosslinking reactions between Ƴ-carboxamide groups of peptides or protein-bound glutamine and the Ɛ-amino group of lysine. In this study, we demonstrated an efficient systematic engineering strategy to enhance the synthesis of TGase in a recombinant Streptomyces mobaraensis smL2020 strain in a 1000-L fermentor. Briefly, the enzymatic properties of the TGase TG_L2020 from S. mobaraensis smL2020 and TGase TG_LD from S. mobaraensis smLD were compared to obtain the TGase TG_LD with perfected characteristics for heterologous expression in a recombinant S. mobaraensis smL2020ΔTG without the gene tg. Through multiple engineering strategies, including promoter engineering, optimizing the signal peptides and recombination sites, and increasing copies of the expression cassettes, the final TG_LD activity in the recombinant S. mobaraensis smL2020ΔTG: (P-sp-protg-tg) (tgand BT1) reached 56.43 U/mL and 63.18 U/mL in shake flask and 1000-L fermentor, respectively, which was the highest reported to date. With the improvement of expression level, the application scope of TG_LD in the food industry will continue to expand. Moreover, the genetic stability of the recombinant strain maintained at more than 20 generations. These findings proved the feasibility of multiple systematic engineering strategies in synthetic biology and provided an emerging solution to improve biosynthesis of industrial enzymes.