Abstract
Objectives
To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production.
Results
The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l−1. Both strains reached similar cell densities ranging from 5 to 8.8 g l−1 under the different conditions. The highest pDNA yields on biomass (YpDNA/X) for both strains were obtained when 3 U enzyme l−1were used. Under these conditions, 35 ± 3 mgof pDNA l−1 were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl−1 after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains.
Conclusions
The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.
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Acknowledgments
This work was supported by CONACyT Grants 183911 and 264460.
Supporting information
Supplementary Fig. 1—Glucose concentration in shake-flasks under culture conditions using 1 (triangles), 2 (squares) or 3 (circles) U of glucoamylase l−1 in EBP medium without cells.
Supplementary Fig. 2—Final pH of the cultures of strains DH5α (red bars) and VH33 Δ(recA deoR) (green bars) using different amounts of enzyme. Error bars show the experimental error between duplicates.
Supplementary data 1: Sequence of the pdna produced by VH33 Δ(recAdeoR).
Supplementary data 2: Sequence of the pdna produced by DH5α.
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Ramírez, E.A., Velázquez, D. & Lara, A.R. Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered Escherichia coli . Biotechnol Lett 38, 651–657 (2016). https://doi.org/10.1007/s10529-015-2017-8
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DOI: https://doi.org/10.1007/s10529-015-2017-8