Enhanced Production of Transglutaminase in Streptomyces mobaraensis through Random Mutagenesis and Site-Directed Genetic Modification

Abstract

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the −10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.

Improved Productivity of Streptomyces mobaraensis Transglutaminase by Regulating Zymogen Activation

“…S. mobaraensis smY2109 and smY2019∆ tg were used to study TGase and TAP, respectively ( Yin et al, 2021 ). The plasmid pET-22b (+) and Escherichia coli BL21 (DE3) were used for expressing pro-TGase.…”

“…In contrast to the enzymes extracted from animals and plants, Streptomyces mobaraensis TGase is a Ca 2+ -independent enzyme and easier to be produced on a large scale ( Ando et al, 1989 ). Although Streptomyces TGases have been expressed in a variety of heterologous hosts, only S. mobaraensis TGase is approved as generally recognized as safe (GRAS) ( Yin et al, 2021 ). These advantages benefit the TGase application and make S. mobaraensis fermentation become the main source of commercial TGase products ( Santhi et al, 2017 ).…”

“…Since the discovery of Streptomyces TGase in 1989, isolating novel TGase-producing strains ( Ceresino et al, 2018 ) and screening high-yielding mutant strains have long been used for enhancing TGase production ( Jiang et al, 2017 ; Yin et al, 2021 ). On the other hand, the media composition and fermentation process were critical for TGase biosynthesis ( Akbari et al, 2021 ).…”

“…Compared to genetic modification, these “non-genetic” strategies are much easier to be accepted for food industries. To date, the highest TGase activity (19.7 U/mL) in S. mobaraensis was achieved by random mutagenesis based on atmospheric and room-temperature plasma mutagenesis and flow cytometry technology ( Yin et al, 2021 ). However, the fermentation period of Streptomyces TGase usually reached 72–96 h or even over 10 days ( Akbari et al, 2021 ).…”

“…In this study, we first investigated the effects of three inorganic nitrogen sources on the fermentation period of smY 2019 (a robust variant of S. mobaraensis DSM40587) ( Yin et al, 2021 ), determining that NH 4 + could improve TGase activation. Based on a reliable measure for TAP activity using a TGase-deficient S. mobaraensis strain smY2019∆ tg , the changes of TAP activity during fermentation were precisely characterized.…”