Sulfated glycosaminoglycans (GAGs) represent the bulk of sulfated biologics that are of pharmaceutical/nutraceutical interest today. Key biological roles of specifically sulfated GAG oligosaccharides continue to be uncovered. As a consequence, GAG extraction from animal tissues will no longer suffice to meet world demands. Several on-going studies aim at developing efficient and scalable chemical and chemoenzymatic methods to produce these compounds. However, the most important questions for metabolic engineers working with E. coli are: 1. Can we achieve total in vivo microbial synthesis of sulfated GAGs? 2. Will metabolic engineering be more economically advantageous than chemical or chemoenzymatic synthesis? Certain strains of E. coli possess the ability to biosynthesize unsulfated GAG backbones. These have been well-studied and optimized for GAG production. However, E. coli is not adept at sulfation of biomolecules. Sulfation requires the universal sulfate donor, 3’-phosphoadenosine-5’-phosphosulfate (PAPS) as well as appropriate sulfotransferase enzymes to transfer sulfate moiety onto the GAG backbone. In vitro biotransformation of unsulfated GAGs using PAPS and sulfotransferase circumvents this issue. However, the scalability of that process is still in question, since PAPS is both expensive and unstable. In this work, we explore the metabolic pathway for PAPS synthesis in E. coli and identify strain designs that facilitate PAPS accumulation. We use this to step-up the state-of-art chemo-enzymatic biotransformation method to use PAPS from the cell lysate of these different E. coli constructs. We also use this system and its comparison to other in vitro biotransformation systems to identify the bottlenecks for total in vivo synthesis of sulfated GAGs in E. coli. We show for the first time that with this system, we have achieved the synthesis of chondroitin sulfate A without the dependence on the expensive synthesis of PAPS cofactor.

Engineering sulfate donor accumulation in Escherichia coli: Improved in vitro chondroitin sulfate biotransformation and a step towards in vivo microbial production

2019 Spring National Meeting

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