Production of Specific-molecular-weight Hyaluronan by Metabolically Engineered Bacillus Subtilis 168

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 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-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 2.20×10(3)-1.42×10(6)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.