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.

Isoflavonoids, such as calycosin-7-glucoside, possess significant pharmaceutical value but are typically sourced from plants, where production is limited by slow growth and complex isolation process. Here, we engineered Saccharomyces cerevisiae for heterologous production of calycosin-7-glucoside. First, via the introduction of isoflavone 4′-O-methyltransferase from Pueraria montana var. lobata (PlOMT9), isoflavone-3′-hydroxylase from Astragalus membranaceus (AmI3′H), and calycosin 7'-O-glucosyltransferase from A. membranaceus (AmUCGT) into our previously constructed daidzein-producing strain, we achieved de novo biosynthesis of calycosin-7-glucoside for the first time. Then, we disrupted the endogenous glucoside hydrolase (EXG1 knockout), enhanced UDP-glucose supply (UGP1 overexpression), and screened a glycosyltransferase (AmUGT88E29 from A. membranaceus) with higher catalytic activity to substantially improve the production of our target compound. Using LC-MS-based metabolomics, we analyzed pathway intermediates and metabolic flux distribution, revealing PlOMT9 as the major bottleneck. Subsequent optimization of PlOMT9 gene copy number further enhanced bioproduction of calycosin-7-glucoside, achieving a final titer of 0.22 ​mg/L. This study establishes a yeast-based platform for high-value isoflavonoid biosynthesis and provides a foundation for future pathway optimization.