Chondroitin sulfate is a sulfated glycosaminoglycan widely used for osteoarthritis and joint health, with additional anti-inflammatory, anticoagulant, and anticancer activities that broaden its pharmaceutical and nutraceutical value.
At present, commercial production relies almost entirely on extraction from animal cartilage, including bovine, porcine, and shark sources. This method faces growing challenges such as limited raw material supply, long production cycles, quality variability, and safety concerns related to allergens and zoonotic pathogens.
Although chemical synthesis is possible, it is costly and environmentally intensive. These limitations have driven interest in microbial biosynthesis as a sustainable alternative. However, achieving efficient and site-specific sulfation in microbes remains a major bottleneck, often requiring a compromise between product yield and sulfation level.
A study (DOI: 10.1016/j.bidere.2025.100062) published in BioDesign Research on 2 December 2025 by Jiazhang Lian’s team, Zhejiang University, establishes a robust yeast-based platform that overcomes the long-standing trade-off between yield and sulfation, enabling sustainable, high-level production of high-quality chondroitin sulfate without reliance on animal sources.
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By systematically redesigning multiple metabolic modules inside Komagataella phaffii, the researchers achieved both high product yield and high sulfation quality, two goals that have long been difficult to reach at the same time.
Three genes
The researchers first established de novo chondroitin biosynthesis in Komagataella phaffii by introducing three heterologous genes—kfoC and kfoA from Escherichia coli K4 and tuaD from Bacillus subtilis—and systematically comparing promoter types and gene architectures. Constitutive versus methanol-inducible promoters and polycistronic versus individual expression cassettes were evaluated to identify an optimal configuration.
This combinatorial design revealed that independent expression of the three enzymes under constitutive promoters was most effective, yielding 927 mg/L chondroitin in strain P02, a 2.06-fold improvement over a single-cassette design, with an average molecular weight of ~265 kDa.
Building on this high-level backbone platform, the team then introduced and optimized a chondroitin 4-O-sulfotransferase (CHST11) to enable chondroitin sulfation. Multiple enzyme sources, soluble fusion tags, and promoters were tested, showing that human CHST11 fused to a thioredoxin (TrxA) tag and driven by the ADH2 promoter achieved the highest initial sulfation (~2.17%), confirming functional CSA production by HPLC and LC–MS.
Low sulfation ceiling
To overcome the low sulfation ceiling of the wild-type enzyme, the researchers increased gene copy number and applied protein engineering, demonstrating that substitution with an engineered CHST11 mutant (SMp) markedly enhanced catalytic performance. A single SMp copy increased sulfation to 12.1%, while multi-copy genomic integration led to a strong gene-dosage effect, culminating in strain PM06 with 45.0% sulfation and CSA titers exceeding 1.2 g/L.
Recognizing that sulfate donor availability had become limiting, the team next optimized PAPS metabolism through targeted pathway modifications. While most strategies had modest or negative effects, co-overexpression of endogenous PPK and BPNT in the high-performance background further increased sulfation to ~48%.
Finally, the optimized strain PM06 was subjected to high-density fed-batch fermentation under carbon-restricted conditions, avoiding sulfate supplementation. This process sustained robust growth and product formation, delivering 7.13 g/L CSA with a stable sulfation degree of 48.4% and a molecular weight of ~234 kDa, demonstrating that coordinated pathway engineering and process optimization can simultaneously achieve high titer and high sulfation in microbial CSA production.
Robust non-animal platform
This work establishes yeast as a robust, non-animal platform for producing high-quality chondroitin sulfate. Such a system could stabilize global supply chains, reduce reliance on animal agriculture, and lower safety risks associated with animal-derived materials.
For manufacturers, microbial production offers consistent quality and easier regulatory control. For consumers and patients, it promises cleaner, more sustainable products.
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