Establishing Komagataella phaffii as a cell factory for efficient production of chondroitin sulfate

Chondroitin sulfate (CS) is a vital sulfated glycosaminoglycan with essential physiological functions and broad applications in pharmaceuticals and nutraceuticals. Commercial CS production currently relies on extraction from animal tissues, which suffers from raw material scarcity, long production cycles, and safety concerns. Here, an efficient microbial platform for de novo biosynthesis of chondroitin sulfate A (CSA) was established in Komagataella phaffii. The chondroitin biosynthetic pathway was first reconstructed and optimized through rearrangement of three heterologous genes and promoter selection, achieving 927 mg/L unsulfated chondroitin. Functional expression of a chondroitin-4-O-sulfotransferase (CHST11) enabled the biosynthesis of 750.5 mg/L CSA with a sulfation degree of 2.6 %. Replacement of wild-type CHST11 with its engineered mutant (SMp) significantly enhanced sulfation to 12.1 %. Subsequent multi-copy genomic integration of the SMp expression cassette further increased the sulfation degree to 45.0 % while maintaining a high CSA titer of 1.10 g/L in shake flasks. Enhancement of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) supply, the key cofactor for sulfation, further improved sulfation to 48.0 %. Finally, the optimized strain PM06 achieved a CSA titer of 7.13 g/L with a sulfation degree of 48.4 % in a 5-L fed-batch fermentation, representing the highest microbial CSA production reported to date. This study demonstrates the successful establishment of K. phaffii as a robust cell factory for high-level and high-sulfation production of CSA. The modular engineering strategy described here provides a generalizable framework for balancing multi-enzyme pathways and offers an efficient, non-animal-derived route for the sustainable industrial production of CS.