Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered Escherichia coli

• Published in: Biotechnology letters 2016-04, Vol.38 (4), p.651-657
• Main Authors: Ramírez, Elisa A, Velázquez, Daniela, Lara, Alvaro R
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Batch Cell Culture Techniques; Biochemistry; biomass; Biomedical and Life Sciences; Biotechnology; Carbon; Carbon sources; Culture; culture media; Density; Deoxyribonucleic acid; DNA; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli Proteins - genetics; glucan 1,4-alpha-glucosidase; Glucose; Glucose - metabolism; Life Sciences; Metabolic Engineering; Metabolites; Microbiology; Mutation; Original Research Paper; Plasmid DNA; Plasmids; Plasmids - genetics; polysaccharides; Strain; Vaccines, DNA - biosynthesis
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-015-2017-8

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.