ObjectivesTo 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.ResultsThe 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.ConclusionsThe 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. |