Metabolic engineering of non-pathogenic Escherichia coli strains for the controlled production of low molecular weight heparosan and size-specific heparosan oligosaccharides

https://doi.org/10.1016/j.bbagen.2020.129765Get rights and content

Highlights

  • Non-pathogenic E. coli strains are engineered to produce heparin precursor, heparosan.

  • Low molecular weight heparosan polysaccharide (~5 KDa) chains are produced first time.

  • Heparosan oligosaccharides, DP4 to DP10, are directly produced from E. coli strains.

  • Optimized conditions for the controlled production of size-specific oligosaccharides

  • N-sulfation, C5-epimerization, and 6-O sulfation of oligosaccharides are accomplished.

Abstract

Background

Heparin, a lifesaving blood thinner used in over 100 million surgical procedures worldwide annually, is currently isolated from over 700 million pigs and ~200 million cattle in slaughterhouses worldwide. Though animal-derived heparin has been in use over eight decades, it is a complex mixture that poses a risk for chemical adulteration, and its availability is highly vulnerable. Therefore, there is an urgent need in devising bioengineering approaches for the production of heparin polymers, especially low molecular weight heparin (LMWH), and thus, relying less on animal sources. One of the main challenges, however, is the rapid, cost-effective production of low molecular weight heparosan, a precursor of LMWH and size-defined heparosan oligosaccharides. Another challenge is N-sulfation of N-acetyl heparosan oligosaccharides efficiently, an essential modification required for subsequent enzymatic modifications, though chemical and enzymatic N-sulfation is effectively performed at the polymer level.

Methods

To devise a strategy to produce low molecular weight heparosan and heparosan oligosaccharides, several non-pathogenic E. coli strains are engineered by transforming the essential heparosan biosynthetic genes with or without the eliminase gene (elmA) from pathogenic E. coli K5.

Results

The metabolically engineered non-pathogenic strains are shown to produce ~5 kDa heparosan, a precursor for low molecular weight heparin, for the first time. Additionally, heparosan oligosaccharides of specific sizes ranging from tetrasaccharide to dodecasaccharide are directly generated, in a single step, from the recombinant bacterial strains that carry both heparosan biosynthetic genes and the eliminase gene. Various modifications, such as chemical N-sulfation of N-acetyl heparosan hexasaccharide following the selective protection of reducing end GlcNAc residue, enzymatic C5-epimerization of N-sulfo heparosan tetrasaccharide and complete 6-O sulfation of N-sulfo heparosan hexasaccharide, are shown to be feasible.

Conclusions

We engineered non-pathogenic E. coli strains to produce low molecular weight heparosan and a range of size-specific heparosan oligosaccharides in a controlled manner through modulating culture conditions. We have also shown various chemical and enzymatic modifications of heparosan oligosaccharides.

General significance

Heparosan is a precursor of heparin and the methods to produce low molecular weight heparosan is widely awaited. The methods described herein are promising and will pave the way for potential large scale production of low molecular weight heparin anticoagulants and bioactive heparin oligosaccharides in the coming decade.

Introduction

Glycosaminoglycans (GAGs) are structurally diverse, highly anionic linear polysaccharides. They play critical roles in various developmental, physiological, and pathological processes [1,2]. GAGs are comprised of repeating disaccharide units that differ in terms of sugar building blocks, anomeric linkages, and modifications [3]. Heparan sulfate (HS) and heparin are two members of the GAG family that regulate numerous biological processes such as cell proliferation and differentiation, cell-cell interactions and adhesion, receptor-mediated interaction with proteins and pathogens, angiogenesis, blood coagulation, neuronal development, and cancer metastasis among others [1,3]. Though heparin is predicted to confer benefits in treating many human diseases, it has been primarily exploited therapeutically for its anticoagulant activity in various medical procedures since the 1940s [4]. Both HS and heparin have similar disaccharide repeating units of a D-glucosamine (GlcN) α-(1 → 4) linked to a hexuronic acid, D-glucuronic acid (GlcA) or L-Iduronic Acid (IdoA). The GlcN residues can carry modifications such as N-acetyl (GlcNAc) or N-sulfate (GlcNS), 3-O- and 6-O-sulfate groups whereas the GlcA/IdoA residues may carry a 2-O-sulfate group. These modifications are orchestrated in the Golgi apparatus by a large number of biosynthetic enzymes and their isoforms: N-Deacetylase/N-sulfotransferase (NDST), C5-epimerase, 2-O-sulfotransferase (2OST), 3-O-sulfotransferase (3OST), and 6-O-sulfotransferase (6OST) [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]]. These modifications contribute to high polydispersity and dictate heparin-protein interactions, which in turn fine-tune diverse biological functions. However, this enormous structural diversity also creates significant hurdles to study the structure-function relationships of heparin-protein interactions [2].

At present, clinically used low molecular weight heparin anticoagulants are obtained from unfractionated heparin polymers extracted from animal sources, mostly from bovine lungs and porcine intestines [15]. Despite extensive purification and characterization of heparin extracts, animal sources still pose several risks, including heparin adulteration, as happened during the 2008 heparin crisis [16,17]. Also, risks such as deadly African Swine Fever may cause reduction of pig population worldwide leading to a potentially severe shortage of heparin anticoagulants [18]. Though chemical synthesis of antithrombotic heparin polymer is challenging, this approach was implemented in the large scale production of heparin pentasaccharide that is currently available as a commercial drug [19,20]. Chemoenzymatic synthesis, on the other hand, involves the use of various biologically-sourced biosynthetic enzymes to rapidly assemble specific heparin structures [[21], [22], [23]]. Though the chemoenzymatic approach offers great promise in the production of larger heparin structures, there are several challenges involved, including the cost-effective production of heparosan, the backbone precursor structure of heparin. Heparosan polysaccharide consists of repeating disaccharide units of [−GlcA-β-(1 → 4)-GlcNAc-α-(1 → 4)-], isolated from the bacterium E. coli K5 [24]. However, this E. coli strain is a human pathogen and can cause severe urinary tract infections. This bacterium has a K5 biosynthetic gene cluster and cloning the second region of the cluster, specifically the region KfiABCD, in non-pathogenic bacterial strains was shown to be sufficient to produce recombinant heparosan polymers [[25], [26], [27], [28], [29], [30]]. Until now, published reports suggest only the production of very high molecular weight heparosan, ranging from ~25,000 Da to ~150,000 Da, which may not be suitable as a substrate in the production of heparin structures for clinical usage, as unfractionated heparin has lower predictable pharmacokinetic properties compared to LMWH. Thus, there is an urgent need for the production of low molecular weight heparosan (~3 to 5 kDa) that will reduce one major hurdle in the quest for production of biotechnological low molecular weight heparins for clinical usage. In the current study, four essential heparosan biosynthetic genes KfiA (encodes GlcNAc transferase enzyme), KfiB (encodes a polymerase factor), KfiC (encodes GlcA transferase enzyme) and KfiD (encodes UDP-Glucose dehydrogenase enzyme) were cloned in different orders within several expression vectors and transformed into three non-pathogenic E. coli strains [BL21(DE3), HT115(DE3), Shuffle T7 Express B] to create several distinct recombinant strains. By systematically studying different cloning orders, host organisms, culture media compositions, and length of culture times, we are able to, most importantly, control the chain length of heparosan produced, ranging from high molecular weight heparosan (>150 kDa) to low molecular weight heparosan (~5 kDa). Furthermore, the elmA gene, found in E. coli K5, which encodes the eliminase enzyme that cleaves heparosan polysaccharide into smaller oligosaccharides, was cloned [31,32]. An earlier study has shown that induction of both biosynthetic gene cluster and elmA together can generate various oligosaccharide sizes without an ability to control the range of oligosaccharides produced [33]. Finally, this report describes an approach, through a combined expression of the heparosan biosynthetic and eliminase gene products under two different induction systems, to control the chain length of heparosan oligosaccharides generated from the metabolically engineered non-pathogenic strains, ranging from tetrasaccharide to dodecasaccharide, in a single step.

Section snippets

Production of low-molecular and high-molecular-weight heparosan polymers

The genes required for heparosan biosynthesis were cloned from the region 2 (KfiABCD, Fig. 1) of the heparosan biosynthetic gene cluster of E. coli K5. It is highly desirable to control the recombinant heparosan polymer chain length while improving the yield. Therefore, at first, we investigated whether cloning the genes in distinct orders in different host organisms could help optimize the production of recombinant heparosan of various molecular weights. To achieve this, the genes were

Conclusions

Heparosan is an essential starting material for the production of heparin-like structures to study structure-function relationships and for preparation of recombinant LMWH anticoagulants. By cloning heparosan biosynthetic gene clusters in various orders from E. coli K5 into different non-pathogenic E. coli strains, BL21(DE3), HT115(DE3), and Shuffle T7 Express B, five distinct strains were metabolically engineered to prepare recombinant heparosan polysaccharides with yields up to ~480 mg/L of

Vectors and bacterial strains

E. coli K5 and DH5α were obtained from American Type Culture Collection (ATCC), and E. coli HT115 was a kind gift of Dr. Sanchez Alvarado (Stowers Institute for Medical Research). E. coli BL21(DE3) and chemically competent E. coli Shuffle T7 Express B (a mutated BL21 DE3 strain) were obtained from New England Biolabs. Chemically competent E.coli cells were prepared by the Rubidium chloride method and were used for sub-cloning and plasmid constructions. Plasmids, pETDuet-1, pRSFDuet-1, and

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by NHLBI sponsored Programs of Excellence in Glycosciences Grant, HL107152.

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