Biosynthesis of Heparosan A Bioengineered Heparin Precursor in Bacillus megaterium A Metabolic Engineering Approach

Abstract

quotHeparosan is a commercially expensive glycosaminoglycan (GAG), composed of repeating disaccharide units of GlcA and GlcNAc linked by and#945; (1and#8594;4) and and#946; (1and#8594;4) glycosidic bonds. Heparosan is a highly anionic and hydrophilic polysaccharide, attributed to the carboxy- late and hydroxyl groups. Traditionally, pharmaceutical grade heparin is extracted from animal tissues such as porcine intestine and bovine lung. In early 2008, Animal-derived heparin caused serious adverse reactions leading to hypotension and the death of nearly a hundred patients. Heparin contamination was identified to be caused by anaphylactic response due to the contaminant, over-sulfated chondroitin sulfate (OSCS) present in the raw heparin. The heparin contamination crisis prompted the FDA to devise strict regulations on the production and manufacturing of animal derived heparin. Chemoenzy- matic synthesis emerged as an attractive alternative to the conventional animal derived heparin. Chemoenzymatic synthesis of heparin requires the linear, unsulfated heparan sulfate backbone as a starting molecule. The heparan sulfate backbone is also known as heparosan. This thesis deals with the biosynthesis of heparosan polysaccharide, which is the first step of chemoenzymatic heparin synthesis process. newlineHeparosan production through microbial fermentation is a promising method to prepare chemoenzymatic heparin. Until now, E. coli K5, P. multocida type D and genetically modified microorganisms have been used to produce heparosan. Biosynthesis of hep- arosan in E. coli K5 is catalyzed by four proteins, KfiA, KfiB, KfiC and KfiD, present in serotype specific region 2 of kps loci. Though the heparosan production is high, E. coli K5 causes urinary tract infections in humans. E. coli K5 uses the heparosan capsule to masquerade against the host immune system, thus causing the infections. Moreover, newlineE. coli K5 produces exotoxins and endotoxins, which require extensive purification and subsequent increase in the processing cost. Above all, E. coli K5 genome encodes N-acety