Engineering in vivo sulfation in Escherichia coli for the complete biosynthesis of sulfated glycosaminoglycans

Abstract

In this thesis, we attempt to address the demand for animal-free GAGs by engineering single microbial cell factories for complete, one-step GAG biosynthesis. Specifically, we focus on production of chondroitin sulfate (CS), a type of GAG with important applications in human and veterinary medicine for the treatment of osteoarthritis and general joint health. Towards this, we first engineer E. coli for accumulation of the universal sulfate donor for biological sulfation - PAPS. PAPS (3'-phosphoadenosine-5'-phosphosulfate) is an activated, organic form of sulfate used as the sulfo- group donor by sulfotransferase enzymes. In the first part of the thesis, we demonstrate PAPS accumulation in E. coli by employing a combination of metabolic engineering strategies. The resulting engineered E. coli strain shows over a 1000-fold increase in intracellular PAPS concentrations compared to undetectable amounts in the wildtype strain.

The PAPS-accumulating E. coli developed was then further engineered to produce CS in the second part of the work. Here, we focus on synthesis of 4-O-sulfated CS (CS-A). Assembly of metabolic steps involved in the biosynthesis of all required precursors enabled in vivo CS production for the very first time in a microbial system. We also investigate the effects of manipulating PAPS levels, sulfotransferase activity, induction conditions and GAG export on the sulfation levels of the product. Our studies indicate that CS-A with different sulfation levels can be produced microbially. Moreover, we also show achievement of high 4-O-sulfation levels (~55%) comparable to animal-sourced CS-A (~70%). The microbial CS-A obtained from our engineered strain also has a distinct advantage since it only consists of 4-O-sulfated CS-A unlike animal CS-A (which contain other CS types like 6-O-sulfated CS). Overall, this is the first demonstration of a simple, one-step microbial production of a sulfated GAG and marks an important milestone in the animal-free production of these molecules.

Biological sulfation is a relatively under-explored process in the microbial engineering community. This is especially disproportionate to the huge class of sulfated biologics with commercial importance. Engineering sulfation capabilities in cell factories like E. coli can bring us a step closer to microbial production of valuable sulfated biomolecules like glycosaminoglycans (GAGs). GAGs form an important class of pharmaceuticals widely used in western medicine. However, most GAGs are still manufactured by extraction from animal tissues, an industry plagued with contamination crises, quality control issues, heterogeneity and unsustainability. Animal-free methods for GAG production are yet to emerge as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. At its current state, this domain can be beneficially impacted by microbial metabolic engineering. Engineering bacteria for GAG biosynthesis will facilitate one-step, scalable production with good control over sulfation levels in contrast to extraction from animal sources.