title: Enhancement of plasmid DNA yields during fed-batch culture of a fruR-knockout Escherichia coli strain.
abstract: Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be optimized to attain higher plasmid yields. To date, nearly all production optimization efforts are focused on media or fermentation process design. Although there has been a simple empirical evaluation of the available host strains, there is a lack of systematic effort at engineering these host strains for improved plasmid DNA production. In view of this, we engineered DH5alpha WT (wild-type) cells carrying a DNA vaccine plasmid by knocking out the fruR (fructose repressor) [also known as the Cra (catabolite repressor activator)] global regulator gene and evaluated the growth and plasmid yields of these P+ (plasmid-bearing) fruR cells (fruR-knockout cells) during fed-batch cultures with exponential feeding. The P+ fruR cells showed a more rapid accumulation of plasmid DNA towards the end of the fed-batch cultures compared with the P+ WT cells. As a result, the specific plasmid yield of the P+ fruR cells was 21% higher than that of the P+ WT cells [19.2 versus 15.9 mg/g DCW (dry cell weight)]. These results demonstrate that, from an initial high-yielding fermentation process, the knockout of the fruR global regulator gene in E. coli DH5alpha further improves plasmid yields during fed-batch culture.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, 06-01 Centros, Singapore. dave ow@bti.a-star.edu.sg
authors.forename: Dave Siak-Wei
authors.initials: DS
authors.lastname: Ow
authors.forename: Miranda Gek-Sim
authors.initials: MG
authors.lastname: Yap
authors.forename: Steve Kah-Weng
authors.initials: SK
authors.lastname: Oh
mesh_terms: D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D010957:Plasmids / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000235:genetics*; D013050:Spectrometry, Fluorescence
chemical_list: D029968:Escherichia coli Proteins; C489416:FruR protein, E coli; D012097:Repressor Proteins