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Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

Paper ID Volume ID Publish Year Pages File Format Full-Text
31566 44819 2012 7 PDF Available
Title
Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor
Abstract

As a precursor of bioengineered heparin, heparosan is currently produced from Escherichia coli K5, which is pathogenic bacteria potentially causing urinary tract infection. Thus, it would be advantageous to develop an alternative source of heparosan from a non-pathogeneic strain. In this work we reported the biosynthesis of heparosan via the metabolic engineering of non-pathogenic E. coli BL21 as a production host. Four genes, KfiA, KfiB, KfiC and KfiD, encoding enzymes for the biosynthesis of heparosan in E. coli K5, were cloned into inducible plasmids pETDuet-1 and pRSFDuet-1 and further transformed into E. coli BL21, yielding six recombinant strains as follows: sA, sC, sAC, sABC, sACD and sABCD. The single expression of KfiA (sA) or KfiC (sC) in E. coli BL21 did not produce heparosan, while the co-expression of KfiA and KfiC (sAC) could produce 63 mg/L heparosan in shake flask. The strain sABC and sACD could produce 100 and 120 mg/L heparosan, respectively, indicating that the expression of KfiB or KfiD was beneficial for heparosan production. The strain sABCD could produce 334 mg/L heparosan in shake flask and 652 mg/L heparosan in 3-L batch bioreactor. The heparosan yield was further increased to 1.88 g/L in a dissolved oxygen-stat fed-batch culture in 3-L bioreactor. As revealed by the nuclear magnetic resonance analysis, the chemical structure of heparosan from recombinant E. coli BL21 and E. coli K5 was identical. The weight average molecular weight of heparosan from E. coli K5, sAC, sABC, sACD, and sABCD was 51.67, 39.63, 91.47, 64.51, and 118.30 kDa, respectively. This work provides a viable process for the production of heparosan as a precursor of bioengineered heparin from a safer bacteria strain.

► Single expression of KfiA or KfiC did not produce heparosan in Escherichia coli BL21. ► Additional expression of KfiB or KfiD was beneficial for heparosan production. ► Heparosan yield reached 1.88 g/L in 3-L bioreactor by co-expressing four kfi genes. ► Structure of heparosan from E. coli K5 and recombinant E. coli BL21 was identical. ► Molecular weight of heparosan strongly depends on the expression of four genes.

Keywords
Bioengineered heparin; Heparosan; Escherichia coli K5; Metabolic engineering; Molecular weight; Chemical structure
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Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 5, September 2012, Pages 521–527
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us