Abstract
Changes in the physiology of Escherichia coli cells producing a recombinant α-glucosidase were studied in glucose limited fed-batch fermentations. High α-glucosidase formation is connected to growth inhibition and loss of culturability. Although some metabolic functions are maintained, the ability for replication is apparently not only impaired by competition of recombinant product synthesis to the formation of cellular house-keeping proteins, but is related to continued damage of the chromosomal DNA, which is concluded from electron microscopical analysis and from the behaviour of the SOS response repressor protein LexA. Although, from the decrease of LexA, we propose an SOS signal, the cells are unable to induce the SOS response, due to the high synthesis of α-glucosidase and the concurrent inhibition of the protein synthesis system.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Afflerbach H., Schroder O., and Wagner R. Effects of the Escherichia coli DNA-binding protein H-NS n rRNA synthesis in vivo. Mol. Microbiol. 28 (1998): 641–653.
Andersson L., Yang S., Neubauer P., and Enfors S. O. Impact of plasmid presence and induction on ellular responses in fed-batch cultures of Escherichia coli. J. Biotechnol. 46, (1996): 255–263.
Aris A., Corchero J. L., Benito A., Carbonell X., Viaplana E., and Villaverde A. The expression of recombinant genes from bacteriophage λ strong promoters triggers the SOS response in Escherichia oli. Biotechnol. Bioeng. 60 (1998): 551–559.
Bhattacharya S. K., and Dubey A. K. Effects of dissolved oxygen and oxygen mass transfer on verexpression of target gene in recombinant Escherichia coli. Enzyme Microb. Technol. 20 (1997): 55–360.
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein tilising the principle of protein dye binding. Anal. Biochem. 72 (1976): 248–254.
Brinkmann U., Mattes R. E., and Buckel P. High-level expression of recombinant genes in Escherichia oli is dependent on the availability of the dnaYgene product. Gene 85 (1989): 109–114.
Chou C. H., Bennett G. N., and San K. Y. Genetic manipulation of stationary-phase genes to enhance ecombinant protein production in Escherichia coli. Biotechnol. Bioeng. 50 (1996): 636–64.
Cserjan-Puschman M., Kramer W., Duerrschmid E., Striedner G., and Bayer K. Metabolic approaches for he optimisation of recombinant fermentation processes. Appl. Microbiol. Biotechnol. 53 (1999): 43–50.
Dedhia N., Richins R., Mesina A., and Chen W. Improvement in recombinant protein production in pGpp-deficient Escherichia coli. Biotechnol. Bioeng. 53 (1997): 379–386.
Dersch P., Schmidt K., and Bremer E. Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-inding protein H-NS is subjected to growth-phase control and autoregulation. Mol. Microbiol. 8 1993): 875–889.
Dong H., Nilsson L., and Kurland C. G. Gratuitous overexpression of genes in Escherichia coli leads to rowth inhibition and ribosome destruction. J. Bacteriol. 177 (1995): 1497–1504.
Falconi M., Brandi A., La Teana A., Gualerzi C. O., and Pon C. L. Antagonistic involvement of FIS and-NS proteins in the transcriptional control oihns expression. Mol. Microbiol. 19 (1996): 965–975.
Goff S.A., and Goldberg A. L. An increased content of protease La, the Ion gene product, increases rotein degradation and blocks growth in Escherichia coli. J. Biol. Chem. 262 (1987): 4508–4515.
Goff S. A., and Goldberg A. L. Production of abnormal proteins in Escherichia coli stimulates transcription of lon and other heat shock genes. Cell 41 (1985): 587–595.
Hecker M., Schroeter A., Trader K., and Mach F. Role of relA mutation in the survival of amino acid-starved Escherichia coli. Arch. Microbiol. 143 (1986): 400–402.
Holme T., Arvidson S., Lindholm B., and Pavlu B. Enzymes: laboratory-scale production. Process Biochem. 5 (1970): 62–66.
Jürgen B., Lin H.Y., Riemschneider S., Scharf C., Neubauer P., Schmid R., Hecker M., and Schweder T. Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations. Biotechnol. Bioeng. 70 (2000): 217–224.
Kellenberger E., and Ryter A. Modern developments in electron microscopy. In Bacteriology, Siegel B. M., Ed., Academic Press Inc.: London, 1964.
Kopetzki E., Schumacher G., and Buckel P. Control of formation of active soluble or inactive insoluble baker’s yeast α-glucosidase PI in Escherichia coli by induction and growth conditions. Mol. Gen. Genet. 216(1989): 149–155.
Kosinski M. J., and Bailey J. E. Temperature and induction effects on the degradation rate of an abnormal β-galactosidase in Escherichia coli. J. Biotechnol. 18 (1991): 55–68.
Kosinski M. J., Rinas U., and Bailey J. E. Proteolytic response to the expression of an abnormal β- galactosidase in Escherichia coli. Appl. Microbiol. Biotechnol. 37 (1992): 335–341.
Kurland C. G., and Dong H. Bacterial growth inhibition by overproduction of protein. Mol. Microbiol. 21 (1996): 1–4.
Laemmli U. K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227 (1970): 680–685.
Lin H.Y., and Neubauer P. Effects of insufficient mixing in bioreactors: Influence of controlled glucose oscillations on a recombinant fed-batch process of Escherichia coli. J. Biotechnol. 79 (2000): 27–37.
Lin H.Y., Mathiszik B., Xu B., Enfors S. O., and Neubauer P. Determination of the maximum specific uptake capacities for glucose and oxygen in glucose limited fed-batch fermentations of Escherichia coli. Biotechnol. Bioeng. (2001) In press.
Meyer S., Noissomit-Rizzi N., Reuss M., and Neubauer P. Optimized analysis of intracellular adenosine and guanosine nucleotides in Escherichia coli. Anal. Biochem. 271 (1999): 43–52.
Reynolds E. S. The use of lead citrate of high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17 (1963): 208–213.
Rinas U. Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli. Biotechnol. Prog. 12 (1996): 196–200.
Ryter A., Kellenberger E., Birch-Andersen A., and Maaløe O. Etude au microscope électronique de plasmas contenant de l’acide désoxyribonucléique. I. Les nucleotides des bacteries en croissance active. Zschr. Naturforsch. Teil B 13 (1958): 597–605.
Sassanfar M., and Roberts J. Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J. Mol. Biol. 212 (1990): 79–96.
Schweder T., Lin H. Y., Jürgen B., Riemschneider S., Khalameyzer V., Gupta A., and Neubauer P. Overexpression of recombinant proteins in clpP-deficient Escherichia coli strains. Manuscript, submitted.
Seeger A., Schneppe B., McCarthy J.-E.-G., Deckwer W.-D., and Rinas U. Comparison of temperature- and isopropyl-β-D-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Enzyme Microb. Technol. 10 (1995): 947–953.
Shimizu N., Fukuzono S., Fujimori K., Nishimura N., and Odawara Y. Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring. J. Ferm. Technol. 66 (1988): 187–191.
Spurio R., Durrenberger M., Falconi M., La Teana A., Pon C. L., and Gualerzi C. O. Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy. Mol. Gen. Genet. 231 (1992): 201–211.
Teich A., Lin H. Y., Andersson L., Meyer S., and Neubauer P. Amplification of ColEl related plasmids in recombinant cultures of Escherichia coli after IPTG induction. J. Biotechnol. 64 (1998): 197–210.
Teich A., Meyer S., Lin H. Y., Andersson L., Enfors S. O., and Neubauer P. Growth rate related concentration changes of the starvation response regulators σs and ppGpp in glucose limited fed-batch and continuous cultures of Escherichia coli. Biotechnol. Prog. 15 (1999): 123–129.
van Workum M., van Dooren S. J., Oldenburg N., Molenaar D., Jensen P. R., Snoep J. L., and Westerhoff H. V. DNA supercoiling depends on the phosphorylation potential in Escherichia coli. Mol. Microbiol. 20 (1996): 351–360.
Vind J., Sorensen M. A., Rasmussen M. D., and Pedersen S. Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes: Expression from reporter genes does not always reflect functional mRNA levels. J. Mol. Biol. 231 (1993): 678–688.
Walker G. C. The SOS response of Escherichia coli. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Neidhardt F. C., Curtiss III R., Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger H. E., Eds., ASM: Washington, D.C. (1996): 1400–1616.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Lin, H.Y. et al. (2001). Cellular Responses to Strong Overexpression of Recombinant Genes in Escherichia Coli . In: Merten, OW., et al. Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology. Springer, Dordrecht. https://doi.org/10.1007/978-94-015-9749-4_5
Download citation
DOI: https://doi.org/10.1007/978-94-015-9749-4_5
Publisher Name: Springer, Dordrecht
Print ISBN: 978-90-481-5756-3
Online ISBN: 978-94-015-9749-4
eBook Packages: Springer Book Archive