Engineering compartmentalized utilization of glycerol with glucose for high-level chondroitin production in...

Chondroitin sulfate (CS) is an important glycosaminoglycan widely used in biomedical and nutraceutical fields. However, its commercial production remains dependent on extraction from animal tissues. Microbial synthesis of its unsulfated precursor, chondroitin, followed by enzymatic sulfation, presents a sustainable alternative. Nevertheless, low chondroitin titer remains a major bottleneck for efficient biosynthesis. This study established an efficient Corynebacterium glutamicum cell factory for enhanced chondroitin production. The base chassis strain CgC25 was engineered by deleting lactic and acetic acid synthesis pathways and blocking glycolysis. To compensate for metabolic impairments, an optimized glycerol utilization pathway was implemented via an RBS library, establishing a parallel carbon metabolism system. Redox homeostasis was also reinforced to alleviate metabolic stress. The final strain CgC40 achieved a titer of 12.37 ± 0.51 g/L with a yield of 102.94 ± 4.90 mg/g carbon source, increased by 24.20% and 30.07%, respectively. This titer constitutes the highest level reported to date. Multi-omics analysis further confirmed the enhanced chondroitin synthesis and a corresponding reduction in TCA cycle activity. This work demonstrates an effective metabolic reprogramming strategy that coordinated carbon co-utilization to enhance bioproduction efficiency, providing a broadly applicable platform for synthesizing diverse high-value compounds.