RRC ID 50031
著者 Ihling N, Bittner N, Diederichs S, Schelden M, Korona A, Höfler GT, Fulton A, Jaeger KE, Honda K, Ohtake H, Büchs J.
タイトル Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains.
ジャーナル Biotechnol Prog
Abstract Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018.
巻・号 34(2)
ページ 315-327
公開日 2018-3-1
DOI 10.1002/btpr.2600
PMID 29314728
MeSH 3-Hydroxyacyl CoA Dehydrogenases / genetics 3-Hydroxyacyl CoA Dehydrogenases / metabolism Bacillus subtilis Bacterial Proteins / genetics Bacteriological Techniques / instrumentation* Bacteriological Techniques / methods Culture Media / chemistry Culture Media / pharmacology DNA-Directed RNA Polymerases Escherichia coli / drug effects Escherichia coli / genetics Escherichia coli / metabolism* Isopropyl Thiogalactoside / pharmacology Microorganisms, Genetically-Modified Online Systems Oxygen / analysis* Oxygen / metabolism Protein Engineering Recombinant Proteins / genetics Recombinant Proteins / metabolism* Thermus thermophilus / genetics Viral Proteins
IF 2.334
引用数 3
リソース情報
遺伝子材料 Thermus thermophilus expression plasmid TEx05G09 (THR002153)