Engineering Halomonas bluephagenesis TD01 as a Robust Chassis for the Sustainable Production of Hyaluronic Acid

This study evaluates the development of Halomonas bluephagenesis TD01 as a novel, sustainable microbial platform for the production of hyaluronic acid (HA). Three distinct hyaluronan synthase genes (sezHasA and spHasA—Class I from the Streptococcal group—and pmHasA) were heterologously expressed and compared, with the Class II synthase from Pasteurella multocida (pmHasA) emerging as the superior variant in rich media 60-LBG, achieving significantly higher titers of 0.88 g/L and molecular weight (M_w) of 1.15 MDa (Mega Daltons). Using a combination of Plackett–Burman design and response surface methodology (RSM), the fermentation process was optimized, identifying initial pH, nitrogen source, and NaCl concentration as critical factors. These optimizations led to a maximum HA yield from 0.88 to 2.38 g/L (265% improvement) and M_w from 1.15 to 9.67 MDa. Furthermore, the study demonstrates precise tuning of HA molecular weight, ranging from 2.04 MDa to 9.67 MDa in a modified medium (40LBG-Y), by modulating L-arabinose induction levels. The structural integrity of the purified HA was confirmed via ESI-MS and ¹H-NMR. These findings establish H. bluephagenesis TD01 as a robust Next-Generation Industrial Biotechnology (NGIB) chassis for the scalable and customizable production of HA with a minimal cost and high-molecular-weight HA for medical applications.