Bacteria reprogrammed to make designer molecule used in pharmaceutical drugs

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 rapid drug manufacturing.

Envisioning an animal-free drug supply, scientists have – for the first time – reprogrammed a common bacterium to make a designer polysaccharide molecule used in pharmaceuticals and nutraceuticals. Published on March 2, 2021 in Nature Communicationresearchers modified E. coli to produce chondroitin sulfate, a drug best known as a dietary supplement to treat arthritis that currently comes from cow trachea.

Genetically modified E. coli is used to make a long list of medicinal proteins, but it has taken years to get bacteria to produce even the simplest of this class of related sugar molecules – called sulfated glycosaminoglycans – which are often used as medicines and nutraceuticals. .

“It’s a challenge to engineer E. coli to produce these molecules, and we had to make a lot of changes and balance those changes for the bacteria to grow well,” said Mattheos Koffas, lead 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 the design of biomolecular tools for the development of better drugs. Linhardt is a glycan expert and one of the world’s foremost authorities on anticoagulant heparin, a sulfated glycosaminoglycan currently derived from pig intestine.

Linhardt, who developed the first synthetic version of heparin, said 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 we make a gram, and it takes a month to make, and somebody calls us up 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 fermentation, you throw the modified organism into a vial, and you have the material, whether it’s a gram, 10 grams, or one kilogram. It’s the future.

“The ability to endow a simple bacterium with a biosynthetic pathway found only in animals is essential for synthesis at commercially relevant scales. Equally important is that the complex drug 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 needed to incorporate into the bacterium in order for it to produce chondroitin sulfate: introducing a gene cluster to produce an unsulfated polysaccharide precursor molecule, engineering the bacterium to make a sufficient in 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 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 into E. coli, they are no longer functional at all. You basically get nothing. So we had to do a lot of protein engineering to make it work.

The team first produced a structure of the enzyme, 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.

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

“This work is a milestone in the engineering and manufacturing of biologics and it opens up new avenues in several areas such as therapeutics and regenerative medicine which need a substantial supply of specific molecules whose production is lost. with aging and disease,” said Deepak Vashishth, director of CBIS. “Such advances originate and thrive in interdisciplinary environments made possible by the unique integration of knowledge and resources available at Rensselaer CBIS.”

Reference: “Complete biosynthesis of a sulphated chondroitin in Escherichia coliby 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

Alvin J. Chase