Heparin is an anionic polysaccharide that is widely used as a clinical anticoagulant. This glycosaminoglycan is prepared from animal tissues in metric ton quantities. Animal‐sourced heparin is also widely used in the preparation of low molecular weight heparins that are gaining in popularity as a result of their improved pharmacological properties. The recent contamination of pharmaceutical heparin together with concerns about increasing demand for this life saving drug and the fragility of the heparin supply chain has led the scientific community to consider other potential sources for heparin. This review examines progress toward the preparation of engineered heparins through chemical synthesis, chemoenzymatic synthesis, and metabolic engineering.

Heparin is ah ighly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de-Nacetylation, chemical N-sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. Aseries of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of an on-radioactive NMR assayf or glucuronosyl-C5-epimerase,a nd background-free quantification of in vivo half-life following administration to rabbits.W e anticipate that this approach can be extended to produce other isotope-enriched glycosaminoglycans.

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis–Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3′-phosphoadenosine 5′-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis–Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.

Heparin is ah ighly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de-Nacetylation, chemical N-sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. Aseries of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of an on-radioactive NMR assayf or glucuronosyl-C5-epimerase,a nd background-free quantification of in vivo half-life following administration to rabbits.W e anticipate that this approach can be extended to produce other isotope-enriched glycosaminoglycans.

Background Heparosan 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. Results We 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. Conclusion We 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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1187-9) contains supplementary material, which is available to authorized users.