(641d) Metabolic Engineering of Microorganisms for the Efficient Synthesis of Polysaccharides

Heparin and chondroitin sulfate (CS) are glycosaminoglycans with many biological and physiological functions that contribute to their widespread utilization as anticoagulants and anti-inflammatory drugs. These sulfated polysaccharides are primarily extracted and purified from animal tissues, where factors such as source material, manufacturing processes, and the presence of contaminants impact overall safety and biological and pharmacological efficacy. As part of ongoing efforts to separate the food chain from the drug chain, we are engineering microbial metabolism in order to produce chondroitin sulfate in Escherichia coli, and heparosan, a valuable precursor to heparin, in Bacillus megaterium.

The engineered production of non-sulfated chondroitin in moderate quantities (~1 g/L) in various microorganisms has been reported, but no industrial scale biotechnological process using E. coli to produce CS has been established to date. We are engineering E. coli for the production of CS through a biotransformation scheme, where the microbial non-sulfated chondroitin backbone is sulfated in vitro using multiple E. coli strains to generate the required components. The biotransformation scheme involves expression of several active biosynthetic pathway enzymes and requires examination of their specificity and stability. To this end, chondroitin 6-sulfotransferase, which catalyzes the transfer of sulfate to position-6 of N-acetylgalactosamine residues of chondroitin, was expressed by E. coli in active form. Together with non-sulfated chondroitin produced by co-expression of E. coli strain K4 genes kfoA, kfoC, and kfoE in the non-pathogenic strain E. coli BL21, two important components of the biotransformation scheme are made available for in vitro CS production. This proof of principle represents an important checkpoint on the path toward de novo biosynthesis of chondroitin sulfate from simple carbon sources like glucose and glycerol in a single microbial host.

Furthermore, although heparosan production in engineered Bacillus subtilis has been previously reported, the larger GRAS organism, Bacillus megaterium, is a more attractive alternative for industrial scale production since it possesses the intrinsic favorable properties of low protease activity and high secretion capability. To exploit these attributes, we have engineered B. megaterium for heparosan production. The T7 RNA polymerase (T7 RNAP) expression system for B. megaterium, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of heparosan synthase (PmHS2). Specifically, we show that B. megaterium MS941 cells co-transformed with pT7-RNAP and pPT7-pmHS2 plasmids are capable of producing heparosan upon induction with xylose, providing an alternative, safe source of heparosan as a precursor for heparin production.