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Enhanced biosynthesis of plasmid DNA from Escherichia coli VH33 using Box–Behnken design associated to aromatic amino acids pathway

Paper ID Volume ID Publish Year Pages File Format Full-Text
2913 142 2015 10 PDF Available
Title
Enhanced biosynthesis of plasmid DNA from Escherichia coli VH33 using Box–Behnken design associated to aromatic amino acids pathway
Abstract

•Addition of aromatic amino acids increases the target plasmid yield to 81.77 μg/mL.•Phenylalanine and tryptophan has a positive effect in pcDNA3–p53 plasmid DNA yield.•Aromatic amino acids supplementation slightly decreases maximum growth rate and raises the levels of plasmid copy number.•A first model for interaction of aromatic amino acids on plasmid DNA concentration is proposed.

Therapeutic applications of plasmid DNA have significantly advanced during the last years and future demand for plasmid DNA requires the development of efficient bioprocesses. Concerning these facts, our aim is to optimize the yield of a therapeutic plasmid pcDNA3–FLAG–p53 using a new bacterial strain, Escherichia coli VH33. This innovative research combines the potential of VH33, which uses a different glucose transport allowing efficient growth and lower acetate production, for pDNA biosynthesis with a validated factorial design. This work achieved higher plasmid yields of 81.77 μg pDNA/mL, when compared with other results for similar assays which reached about 40 μg pDNA/mL. The strategy is based on the change of fermentation media composition in terms of nutrients, by the development of an experimental design directed to aromatic amino acids pathway. The results revealed which combination should be applied in terms of nitrogen and carbon source requirements in order to obtain 36.69 μg pDNA/mg cell dry mass of specific yields and 6.01% of purity on lysates. Overall, the proposal model show the influence of tyrosine, phenylalanine and tryptophan on their pathway, providing necessary precursors to nucleotides’ network, as well as temperature shift (37–42 °C) to increase the plasmid copy number per cell.

Keywords
Biosynthesis; Plasmid DNA; E. coli VH33; Aromatic amino acids; Box–Behnken experimental design
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Enhanced biosynthesis of plasmid DNA from Escherichia coli VH33 using Box–Behnken design associated to aromatic amino acids pathway
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 98, 15 June 2015, Pages 117–126
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