Evidencing the role of lactose permease in IPTG uptake by Escherichia coli in fed-batch high cell density cultures.
The lac-operon and its components have been studied for decades and it is widely used as one of the common systems for recombinant protein production in Escherichia coli. However, the role of the lactose permease, encoded by the lacY gene, when using the gratuitous inducer IPTG for the overexpression of heterologous proteins, is still a matter of discussion. A lactose permease deficient strain was successfully constructed. Growing profiles and acetate production were compared with its parent strain at shake flask scale. Our results show that the lac-permease deficient strain grows slower than the parent in defined medium at shake flask scale, probably due to a downregulation of the phosphotransferase system (PTS). The distributions of IPTG in the medium and inside the cells, as well as recombinant protein production were measured by HPLC-MS and compared in substrate limiting fed-batch fermentations at different inducer concentrations. For the mutant strain, IPTG concentration in the medium depletes slower, reaching at the end of the culture higher concentration values compared with the parent strain. Final intracellular and medium concentrations of IPTG were similar for the mutant strain, while higher intracellular concentrations than in medium were found for the parent strain. Comparison of the distribution profiles of IPTG of both strains in fed-batch fermentations showed that lac-permease is crucially involved in IPTG uptake. In the absence of the transporter, apparently IPTG only diffuses, while in the presence of lac-permease, the inducer accumulates in the cytoplasm at higher rates emphasizing the significant contribution of the permease-mediated transport.
Departament d'Enginyeria Química, Unitat de Biocatàlisi Aplicada Associada al IQAC (CSIC), Universitat Autònoma de Barcelona, Bellaterra, Spain. alfred.fernandez@uab.cat
Alfred
A
Fernández-Castané
Claire E
CE
Vine
Glòria
G
Caminal
Josep
J
López-Santín
D001692:Biological Transport / Q000502:physiology; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development* / Q000378:metabolism; D005285:Fermentation; D007544:Isopropyl Thiogalactoside / Q000378:metabolism* / Q000493:pharmacokinetics; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D026901:Membrane Transport Proteins; D007544:Isopropyl Thiogalactoside; C021499:lactose permease; D010770:Phosphotransferases