Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. They have extensive uses as pharmaceuticals (anticoagulants and in regenerative medicine) as well as nutraceuticals. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. We will demonstrate the development of a number of synthetic biology approaches, both cell-based and cell-free for the complete biosynthesis of chondroitin sulfate (CS) as well as deuterated heparin. We engineer E. coli to produce all three required components for CS production-chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of sulfated GAGs and marks an important step in the animal-free production of these molecules. Enzyme engineering of other chondroitin sulfotransferases allowed further modification of the chemistry of the produced polysaccharides.
