Combinatorial optimization of ribosomal binding site, promoter and genomic locus for high-yield production of a thermostable transglutaminase in Streptomyces mobaraensis.

Abstract

Streptomyces mobaraensis transglutaminase (smTG) is an important food-processing biocatalyst, yet its industrial potential is limited by low yield and thermal instability. Here, we report the multi-level engineering of S. mobaraensis smY2022 for high-level production of a thermostable variant, TGm2. Ab initio design of the ribosomal binding site (RBS) based on RBS Calculator prediction yielded RBS10, which improved TGm2 yield by 22%. Extending the spacer between the SD sequence and start codon to 6 bases (RBS10-1) further increased yield by 19%. Engineering the native smTG promoter by restoring upstream regulatory sites (two AdpA-binding sites and one LytR-binding site) produced PsmY2022-2, enhancing expression by 145% compared to the truncated promoter. The optimized cassette (PsmY2022-2:RBS10-1:TGm2) was integrated into the smTG locus, yielding strain smY2022-TGm2-S (72.5 U/mL). Genomic locus screening identified a high-expression site (K7I03_09295); integrating a second copy at this locus generated strain smY2022-TGm2-S-09295, achieving a record activity of 102.9 U/mL. This work provides an efficient high-yielding strain and a combinatorial engineering strategy for industrial enzyme production in Streptomyces.