Systematic optimization of fed-batch fermentation for enhanced heparosan production in engineered Corynebacterium...

Heparin, a highly sulfated glycosaminoglycan derived from heparan sulfate (HS), is widely employed as a clinical anticoagulant and antithrombotic agent. Low-molecular-weight heparin (LMWH, 4-5 kDa), a derivative of heparin, provides improved safety, ease of administration, and reduced need for monitoring, making it the preferred anticoagulant in modern medicine. However, LMWH produced through animal extraction or chemical synthesis are associated with inherent limitations (e.g., batch variability, contamination risk, cost, scalability). Enzymatic chemo-synthesis modification of heparosan, a heparin precursor polysaccharide, has recently emerged as a promising strategy for producing heparin-based therapeutics. In this study, we optimized the fed-batch fermentation process to enhance the biosynthesis of LMWH in engineered Corynebacterium glutamicum. Compared to batch fermentation, fed-batch cultivation significantly improved both cell biomass and heparosan titer. Among the strategies tested, variable-rate feeding achieved the highest production, resulting in a 3.93-fold increase. Optimization of agitation speed identified 500 rpm as the most favorable condition, maintaining dissolved oxygen (DO) at 15-30 % and leading to a maximum heparosan titer of 8.02 g/L. Maintaining glucose levels within 1-10 g/L effectively supported cell growth while maximizing heparosan biosynthesis. The product was subsequently purified through a multi-step process, achieving 98.4 % apparent purity.