Strain Engineering and Pathway Enhancement of Bacillus subtilis for Efficient Hyaluronic Acid Production

Hyaluronic acid (HA) is a biopolymer with broad biomedical and pharmaceutical uses, yet microbial HA production is limited by strain safety, and process inefficiencies; to address these challenges we engineered a biosafe Bacillus subtilis chassis by integrating catalytic genes identified from the BRENDA database. Two constructs on the pHT01 shuttle vector were prepared, a dual-gene plasmid ( hasA –NX02_04625) and a single gene plasmid ( hasA ), and following transformation and molecular confirmation recombinant colonies were screened in different media to identify optimal production conditions. HA was quantified by the CTAB assay, its structure validated by FTIR, and molecular weight distribution characterized by GPC. Cloning and molecular validation were successful, HA synthesis was detected only in engineered strains and not in wild type controls, and the dual-gene strain produced significantly more HA than the single gene strain, reaching up to 1.2 g/L in shake flask cultures. Preliminary bioreactor cultivation of the dual gene strain in the selected medium yielded 0.62 g/L HA. These results indicate that engineered B. subtilis is a biosafe, scalable platform for HA production, offering an industrially viable alternative to pathogenic hosts and highlighting a promising source for HA synthesis applicable to pharmaceutical, biomedical, and cosmetic industries.