Bacteria reprogrammed to make a design molecule used in pharmaceutical drugs

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

The process paves the way for safe, ethical and timely drug manufacturing.

Envisioning an animal-free drug supply, scientists – for the first time – reprogrammed a common bacteria to make a design polysaccharide molecule used in pharmaceuticals and nutraceuticals. Posted on March 2, 2021 in Nature Communication, researchers modified E. coli to produce chondroitin sulfate, a drug best known as a dietary supplement to treat arthritis that currently originates in the trachea of ​​cows.

Genetically engineered E. coli is used to create a long list of medicinal proteins, but it has taken years for bacteria to produce even the simplest of this class of linked sugar molecules – called sulfated glycosaminoglycans – which are often used as drugs and nutraceuticals. .

“It’s a challenge to design E. coli to produce these molecules, and we had to make a lot of changes and balance those changes in order for the bacteria to grow well,” said Mattheos Koffas, senior researcher and professor of chemical engineering and organic in Rensselaer. Polytechnic Institute. “But this work shows that it is possible to produce these polysaccharides using E. coli without animals, and the procedure can be extended to produce other sulfated glycosaminoglycans.”

At Rensselaer, Koffas worked with Jonathan Dordick, professor of chemical and biological engineering, and Robert Linhardt, professor of chemistry and chemical biology. All three are members of the Center for Biotechnology and Interdisciplinary Studies. Dordick is a pioneer in the use of enzymes for the synthesis of materials and in the design of biomolecular tools for the development of better drugs. Linhardt is a glycans expert and one of the world’s leading authorities on the anticoagulant heparin, a sulfated glycosaminoglycan currently derived from the pig intestine.

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

“If we make chondroitin sulfate chemoenzymatically and make a gram of it, and it takes a month, and somebody calls us and says, ‘Well, now I need 10 grams’ , we’re going to have to spend another month to get 10 grams, ”Linhardt said. “Whereas with fermentation you throw the modified organism into a flask and you have the material, whether it’s a gram, 10 grams or a kilogram. It’s the future.

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

Koffas described three major steps the team had to integrate into the bacteria in order for it to produce chondroitin sulfate: introducing a group of genes to produce an unsulfated polysaccharide precursor molecule, designing the bacteria to provide a sufficient supply of an energetically expensive sulfur donor molecule, and introducing a sulfurtransferase enzyme to place the sulfur donor molecule on the unsulfated polysaccharide precursor molecule.

The introduction of a functional sulfotransferase enzyme has posed a particularly difficult challenge.

“Sulfotransferases are made by much more complex cells,” Koffas said. “When you take them out of a complex eukaryotic cell and put them in E. coli, they’re not functional at all. Basically, you don’t get anything. So we had to do a lot of protein engineering to make it work. “

The team first produced a structure for the enzyme, then used an algorithm to help identify any mutations they might make in the enzyme to produce a stable version that would work in E. coli.

Although modified E. coli produce a relatively low yield – on the order of micrograms per liter – they thrive under ordinary laboratory conditions, offering strong proof of concept.

“This work is an important step in the engineering and manufacturing of biological products and opens up new avenues in several fields such as therapeutics and regenerative medicine which require a substantial supply of specific molecules whose production is lost with aging and diseases, ”said Deepak Vashishth, director of CBIS. “Such advancements arise and thrive in interdisciplinary environments made possible through the unique integration of knowledge and resources available at Rensselaer CBIS.

Reference: “Complete biosynthesis of a sulphated chondroitin in Escherichia coli»By Abinaya Badri, Asher Williams, Adeola Awofiranye, Payel Datta, Ke Xia, Wenqin He, Keith Fraser, Jonathan S. Dordick, Robert J. Linhardt and Mattheos AG Koffas, March 2, 2021, Nature Communication.
DOI: 10.1038 / s41467-021-21692-5


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