Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

• Biosynthetic pathways for GlcUA and GlcNAc were optimized in Bacillus subtilis.
• Leech hyaluronidase (LHAase) was expressed and secreted to 1.62×106 U mL−1.
• Hyaluronan titer increased significantly (19.38 g L−1) with hyaluronidase expression.
• Low-molecular-weight hyaluronans were produced by controllable expression of LHAase.

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L−1 to 3.16 g L−1, with a molecular weight range of 1.40×106–1.83×106 Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×106 U mL−1), the production of HA was substantially increased from 5.96 g L−1 to 19.38 g L−1. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×103–1.42×106 Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.