Abstract
BackgroundHeparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. In addition to its utility as the starting material for this important anticoagulant and anti-inflammatory drug, heparosan is a versatile compound that possesses suitable chemical and physical properties for making a variety of high-quality tissue engineering biomaterials, gels and scaffolds, as well as serving as a drug delivery vehicle. The selected production host was the Gram-positive bacterium Bacillus megaterium, which represents an increasingly used choice for high-yield production of intra- and extracellular biomolecules for scientific and industrial applications.ResultsWe have engineered the metabolism of B. megaterium to produce heparosan, using a T7 RNA polymerase (T7 RNAP) expression system. This system, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of Pasteurella multocida 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 alternate, safe source of heparosan. Productivities of ~ 250 mg/L of heparosan in shake flasks and ~ 2.74 g/L in fed-batch cultivation were reached. The polydisperse Pasteurella heparosan synthase products from B. megaterium primarily consisted of a relatively high molecular weight (MW) heparosan (~ 200–300 kD) that may be appropriate for producing certain biomaterials; while the less abundant lower MW heparosan fractions (~ 10–40 kD) can be a suitable starting material for heparin synthesis.ConclusionWe have successfully engineered an asporogenic and non-pathogenic B. megaterium host strain to produce heparosan for various applications, through a combination of genetic manipulation and growth optimization strategies. The heparosan products from B. megaterium display a different range of MW products than traditional E. coli K5 products, diversifying its potential applications and facilitating increased product utility.
Highlights
Heparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin
liquid chromatography mass spectrometry (LCMS) disaccharide analysis of heparosan produced in the supernatant of the selected colonies indicated the presence of a single disaccharide with mass-to-charge ratio (m/z) of 572, corresponding to uronic acid/N-acetyl hexosamine and consistent with the uniform repeating structure of heparosan: [→4) β-d-glucuronic acid (GlcA) (1→4) N-acetyl-α-d-glucosamine (GlcNAc) (1→)]n
In this study we explored the use of the Gram-positive endotoxin-free host organism B. megaterium for producing heparosan capsular polysaccharide (CPS)
Summary
Heparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. This polysaccharide is the natural precursor of heparan sulfate (HS), as well as heparin, a widely used drug [2, 3] that is primarily employed in surgery to stop vein thrombosis and administrated in other medical procedures [4]. Both kfiA and kfiC are required for polymerization activity, since they are mono-action transferases, respectively encoding for N-acetylglucosaminyltransferase and D-glucuronyltransferase [10, 11]
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