Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

Wang, Wenya; Englaender, Jacob A.; Xu, Peng; Mehta, Krunal K.; Suwan, Jiraporn; Dordick, Jonathan S.; Zhang, Fuming; Yuan, Qipeng; Linhardt, Robert J.; Koffas, Mattheos

doi:10.1007/s12010-013-0415-8

Cited by 13 publications

(7 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A significant reduction in endotoxin levels (2.5 log) was recorded for chitosan‐purified heparosan while SAX purified heparosan still contained a high endotoxin load. This endotoxin reduction is important, as use of chitosan in the bioengineered heparin process can circumvent the need for a change in enzyme expression systems to mitigate endotoxin risk arising due to use of E. coli based expression systems . Further characterization of this process, however, is necessary to ascertain selectivity.…”

Section: Resultsmentioning

confidence: 99%

“…This endotoxin reduction is important, as use of chitosan in the bioengineered heparin process can circumvent the need for a change in enzyme expression systems to mitigate endotoxin risk arising due to use of E. coli based expression systems. [49][50][51][52] Further characterization of this process, however, is necessary to ascertain selectivity. The purified products, appearing as a single peak, suggest a higher molecular weight of heparosan compared to ammonium sulfate purified heparosan derived from the same fermentation broth.…”

Section: Characterization Of Heparosan Products Purified Using Sax Chmentioning

confidence: 99%

See 1 more Smart Citation

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

Bhaskar

Hickey

et al. 2015

Biotechnol Progress

Self Cite

View full text Add to dashboard Cite

The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical Ndeacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatographymass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Characterization Of Heparosan Products Purified Using Sax Chmentioning

confidence: 99%

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

Bhaskar

Hickey

et al. 2015

Biotechnol Progress

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, its ability to adapt to varying environmental conditions as well as its classification as toxic free GRAS has contributed tremendously to its success in the industrial platform (Baysal and Yıldız, 2017). B. subtilis as an endotoxin free host amplified its utilization in the production of sterile recombinant and therapeutic proteins expression as compared to E. coli which could have potential contamination due to the lipopolysaccharide endotoxins (Wang et al, 2013). For instance, B. subtilis and Bacillus megaterium were the preferred hosts over E. coli in the production of bioengineered heparin in order to diminish toxin contamination (Wang et al, 2013).…”

Section: Microbial Cell Factoriesmentioning

confidence: 99%

A Review of the Microbial Production of Bioactive Natural Products and Biologics

et al. 2019

View full text Add to dashboard Cite

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

show abstract

“…Metabolically engineered CHO cells might be useful for producing high-value “designer heparins” with special properties allowing their high-cost, small-scale production. Metabolic engineering of prokaryotes (without a Golgi), such as E. coli , posses even greater challenges [96,107]. While the metabolic engineering of a single strain of engineered bacteria to produce heparin is far off, there are a number of intermediate steps that could be introduced sooner using a biotransformation approach.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Bioengineered heparins and heparan sulfates

Suflita

Linhardt

2016

Advanced Drug Delivery Reviews

Self Cite

107

View full text Add to dashboard Cite

Heparin and heparan sulfates are closely related linear anionic polysaccharides, called glycosaminoglycans, which exhibit a number of important biological and pharmacological activities. These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of animal cells. While heparan sulfate is a widely distributed membrane and extracellular glycosaminoglycan, heparin is found primarily intracellularly in the granules of mast cells. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role heparan sulfate plays in normal and pathophysiology. The chemical synthesis of these glycosaminoglycans is largely precluded by their structural complexity. Today, we depend on livestock animal tissues for the isolation and the annual commercial production of hundred ton quantities of heparin used in the manufacture of anticoagulant drugs and medical device coatings. The variability of animal-sourced heparin and heparan sulfates, their inherent impurities, the limited availability of source tissues, the poor control of these source materials and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans, driven by both therapeutic applications and as probes to study their natural functions. This review focuses on the complex biology of these glycosaminoglycans in human health and disease, and the use of recombinant technology in the chemoenzymatic synthesis and metabolic engineering of heparin and heparan sulfates.

show abstract

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

Cited by 13 publications

References 15 publications

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

A Review of the Microbial Production of Bioactive Natural Products and Biologics

Bioengineered heparins and heparan sulfates

Contact Info

Product

Resources

About