Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Abstract

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. 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. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. 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 a sulfated GAG and marks an important step in the animal-free production of these molecules.

Heterologous production of chondroitin

“…Those authors showed that the glycosylated and non-glycosylated forms of the enzyme had similar activities. More recently, the soluble expression of human C4OST enzyme was also achieved in E. coli K4 and in E. coli K-12 MG1655 [160] .…”

Synthetic biology as driver for the biologization of materials sciences

“…In another example, the production of more complex sulfated glycosaminoglycans was engineered into E. coli . To this aim, the Koffas group introduced three metabolic pathways into cells to produce the three required components of chondroitin sulfate synthesis — chondroitin, a sulfate donor, and a sulfotransferase [ 58 ]. This study provides a simple synthesis route as an alternative to the conventional isolation of chondroitin sulfate from animal tissues.…”

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

“…Therefore, in the past few years research efforts have been focused on the improvement of the production process, as well as on the design of better heterologous hosts to produce GAGs. Recent advances in the microbial production of GAGs using non-pathogenic hosts (mostly prokaryotic organisms) have been reported [12][13][14][15][16][17][18][19].…”