E. coli has been reprogrammed to produce a chondroitin sulfate, a complex sugar. [Rensselaer Polytechnic Institute]

Chondroitin sulfate is an important structural component of cartilage and is naturally found in connective tissues in the human body and in animal cartilage. It is part of a protein molecule that helps give cartilage its elastic properties and is thought to have an anti-inflammatory effect, which can help reduce swelling in the joints. It has been used as a dietary supplement for treating osteoarthritis. Currently, it is sourced from cow trachea. Envisioning an animal-free drug supply, researchers at Rensselaer Polytechnic Institute have modified E. coli to produce chondroitin sulfate.

Their study, “Complete biosynthesis of a sulfated chondroitin in Escherichia coli,” was published in Nature Communications.

“Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues,” the researchers wrote. “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, the requirement for multiple stages, and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG.”

“It’s a challenge to engineer E. coli to produce these molecules, and we had to make many changes and balance those changes so that the bacteria will grow well,” admitted Mattheos Koffas, PhD, lead researcher and a professor of chemical and biological engineering at Rensselaer Polytechnic Institute. “But this work shows that it is possible to produce these polysaccharides using E. coli in animal-free fashion, and the procedure can be extended to produce other sulfated glycosaminoglycans.”

At Rensselaer, Koffas worked with fellow professor of chemical and biological engineering, Jonathan Dordick, PhD, and Robert Linhardt, PhD, a professor of chemistry and chemical biology.

Linhardt, who developed the first synthetic version of heparin, explained engineering E. coli to produce the drug has many advantages over the current extractive process or even a chemoenzymatic process.

“If we prepare chondroitin sulfate chemoenzymatically, and we make one gram, and it takes a month to make, and someone calls us and says, ‘Well, now I need 10 grams,’ we’re going to have to spend another month to make 10 grams,” Linhardt said. “Whereas, with the fermentation, you throw the engineered organism in a flask, and you have the material, whether it’s one gram, or 10 grams, or a kilogram. This is the future.”

“The ability to endow a simple bacterium with a biosynthetic pathway only found in animals is critical for synthesis at commercially relevant scales. Just as important is that the complex medicinal product that we produced in E. coli is structurally the same as that used as the dietary supplement,” said Dordick.

The researchers focused on three major steps to produce chondroitin sulfate: introducing a gene cluster to produce an unsulfated polysaccharide precursor molecule, engineering the bacteria to make an ample supply of an energetically expensive sulfur donor molecule, and introducing a sulfurtransferase enzyme to put the sulfur donor molecule onto the unsulfated polysaccharide precursor molecule.

“The sulfotransferases are made by much more complex cells,” Koffas said. “When you take them out of a complex eukaryotic cell and put them into E. coli, they’re not functional at all. You basically get nothing. So we had to do quite a bit of protein engineering to make it work.”

The researchers produced a structure of the enzyme, and then used an algorithm to help identify mutations they could make to the enzyme to produce a stable version that would work in E. coli.

“This work is a milestone in engineering and manufacturing of biologics and it opens new avenues in several fields such as therapeutics and regenerative medicine that need a substantial supply of specific molecules whose production is lost with aging and diseases,” added Deepak Vashishth, PhD, director of the Rensselaer Polytechnic Institute Center for Biotechnology & Interdisciplinary Studies (CBIS).

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