Valorization of tofu-processing wastewater for sustainable nattokinase production: insights from integrated multi-omics analysis.

Nattokinase (NK) is a fibrinolytic enzyme with considerable potential for applications in functional foods and biomedicine; however, its economical and efficient production remains challenging. In this study, tofu-processing wastewater (TPW) was employed as a low-cost nitrogen source for NK production, and integrated transcriptomic and metabolomic analyses were performed to characterize cellular responses and potential regulatory processes under different nitrogen sources. To evaluate the effects of different nitrogen sources on NK production, fermentation was conducted using tryptone (T), soybean peptone (SP), or TPW as the nitrogen source. Both TPW- and SP-based media supported higher NK production than the T-based medium. Transcriptomic analysis showed that the expression of aprN, which encodes nattokinase, was significantly higher under TPW conditions. TPW was characterized by relatively low levels of free amino acids and relatively high abundances of specific oligopeptides and proteins, and these nutritional characteristics were associated with changes in aprN expression and related metabolic responses. Integrated transcriptomic and metabolomic analyses further indicated coordinated changes in nutrient sensing, oligopeptide transport and metabolism, amino acid metabolism, and energy metabolism under TPW conditions. The relatively low levels of branched-chain amino acids (BCAAs) detected in TPW occurred concomitantly with changes in the expression of codY and aprN, suggesting that BCAA-associated nutrient sensing may be associated with the transcriptional regulation of aprN. Meanwhile, changes in the expression of nutrient-responsive genes, including kinA and kinB, suggested that Spo0A-associated regulatory processes may be involved in the cellular response to the nutritional characteristics of TPW. In addition, changes in the expression of several oligopeptide transport-related genes, together with correlations between specific oligopeptides and aprN expression, suggested that oligopeptide transport and metabolism may be associated with NK production under TPW conditions. Genes involved in respiratory-chain and energy metabolism also showed substantial changes in expression, suggesting alterations in cellular energy metabolism that may be associated with NK production. Overall, TPW as an alternative nitrogen source was associated with coordinated changes in nutrient sensing, regulatory processes, and multiple metabolic pathways in B. subtilis, which may collectively contribute to increased NK production. This study provides further insights into the molecular and metabolic responses of B. subtilis to TPW and provides a theoretical basis for TPW valorization, medium optimization, and the economical and efficient production of NK.