title: Development of an efficient process intensification strategy for enhancing Pfu DNA polymerase production in recombinant Escherichia coli.
abstract: An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-β-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5% in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5% under the controlled dissolved oxygen concentration of 30% in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Hu
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Chun-Zhao
authors.initials: CZ
authors.lastname: Liu
mesh_terms: D019149:Bioreactors; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D007218:Industrial Microbiology; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D010100:Oxygen