Recombinant Bacillus subtilis for Enhanced Production of High Molecular Weight Hyaluronic Acid

Abstract This study aimed to synthesize hyaluronic acid (HA) using a non-pathogenic, recombinant bacterial host, specifically Bacillus subtilis 1A752. The hyaluronic acid synthase (HAS) gene, hasA, originally isolated from Streptococcus equi subsp. zooepidemicus, was introduced into B. subtilis 1A752. This host strain already harbors tuaD, gtaB, and gcaD genes, which are functional analogues to the Streptococcus genes hasB, hasC, and hasD, respectively. The hasA operon was amplified from genomic DNA of S. zooepidemicus through polymerase chain reaction (PCR) and subsequently cloned into B. subtilis 1A752 using recombinant DNA technology. The engineered B. subtilis strain was then utilized for HA production. Post-synthesis, HA was analyzed and deproteinized using Carbazole and Sevag methods, respectively. The purified HA was characterized using chromatographic techniques, and its molecular structure was confirmed through Nuclear Magnetic Resonance (NMR) and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Viscosimetric methods were employed to determine the HA's characterization and molecular weight. The results revealed that the engineered B. subtilis 1A752 strain could produce HA at a concentration of 195.45 mg. l-1, with molecular weights ranging from 1.7 to 2.7 MDa. Given its high molecular weight and non-pathogenic production source, the synthesized HA shows promising potential for applications in the pharmaceutical and biomedical industries.