Rewiring Amino Acid Flux for Efficient Transglutaminase Production in Streptomyces mobaraensis

Transglutaminase (TGase) from Streptomyces mobaraensis is widely used in food processing to improve the texture properties of food products, but its broader application is constrained by low titers. Herein, through a quantitative analysis of the intracellular amino acid pool, we identified an insufficient supply of several amino acids as the primary bottleneck for TGase overproduction. To address this limitation, we overexpressed the biosynthetic pathways of seven critically insufficient amino acids, which increased the TGase titer by 46.3% (from 41.0 to 60.0 U/mL). Additionally, repressing the expression of nonessential high-abundance proteins reduced the metabolic competition for amino acids, boosting the TGase titer by 57.5% (from 40.0 to 63.0 U/mL). Combining both strategies yielded a maximum titer of 66.9 U/mL, which is a 62.4% improvement over the starting strain. This study provides a foundation for efficient TGase production and highlights precursor flux manipulation as a general strategy for enzyme overproduction.