Summary
Escherichia coli TG1 (pHRW500) permanently expressed the human interferon α1 gene (ifnα1) directed by the tryptophan promoter (trpP.O) during continuous and fed-batch cultivation with a limited supply of glucose. The expression of ifnα1 could be improved after insertion of the catabolite activator region (cap) upstream to trpP.O during cultivation of the modified E. coli TG1(pHRW500cap) in glucose-controlled continuous and fed-batch cultures. The cap-mediated stimulatory effect on the expression of cap-trpP.O-ifnα1 increased with decreasing dilution rate. These results are in line with the increase in the level of cAMP with declining dilution rate and the well-known positive effects of cAMP-catabolite gene activator protein (CAP) at the transcriptional level. In addition, expression of the galactokinase gene (trpP.O-galK) in E. coli TG1(pDR720) could be improved in the same way with cap-trpP.O-galK in E. coli TG1(pDR720cap). Determinations of plasmid copy numbers, cellular amounts of galactokinase-mRNA, activity of galactokinase (AGalK) and the concentration of galactokinase at various dilution rates (D) strengthen the conclusion that the increase in AGalK with decreasing D was indeed due to the cap-mediated enhancement of transcription of the galK gene. We suggest that expression of other recombinant genes directed by various promoters that allow permanent transcription during growth with limited glucose supply in chemostat and fed-batch fermentors can be improved by appropriate insertion of the cap region.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aiba H, Adhya S, DeCrombrugghe B (1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256:11 905–11 910
Bailey JE, DaSilva NA, Perethi SW, Seo J-H, Srienc F (1986) Studies of host-plasmid interactions in recombinant microorganisms. Ann N Y Acad Sci 469:194–211
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Breitling R, Gerlach D, Hartmann M, Behnke D (1989) Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals. Mol Gen Genet 217:384–391
Buettner MJ, Spitz E, Rickenberg HV (1973) Cyclic adenosine 3′,5′-monophosphate in Escherichia coli. J Bacteriol 114:1068–1073
Calcott PH (1982) Cyclic AMP and cyclic GMP control of synthesis of constitutive enzymes in Escherichia coli. J Gen Microbiol 128:705–712
Carter P, Bedouelle H, Winter G (1985) Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13:4431–4443
Ebright RH, Ebright YW, Gunasekera A (1989) Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site. Nucleic Acids Res 17:10295–10305
Engberg B, Nordström K (1975) Replication of R-factor R1 in Escherichia coli K-12 at different growth rates. J Bacteriol 123:179–186
Farabaugh PJ (1978) Sequence of the lacI gene. Nature 274:765–769
Fineberg AF, Vogelstein B (1985) Addendum to “A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity”. Anal Biochem 137:266–267
Günther HH, Bergter F (1971) Bestimmung der Trockenmasse von Zellsuspensionen durch Extinktionsmessungen. Z Allg Mikrobiol 11:191–197
Hartmann M, Reichardt W, Walter F, Birch-Hirschfeld E, Tonew M (1986) Verfahren zur Herstellung von Expressionsplasmiden für reifes humanes Interferon alpha 1 in Escherichia coli. GDR patent no. 250335
Knorre WA (1989) Herstellung von rDNA-Produkten, Hochproduktivitätsfermentationen mit E. coli. BTF-Biotechforum 6:20–24
Knorre WA, Deckwer WD, Korz D, Pohl HD, Riesenberg D, Ross A, Sanders E, Schulz V (1990) Hochzelldichte-Fermentationen von E. coli for rDNA-Produkte. BioEngineering 5:28–33
Koizumi JI, Monden Y, Aiba S (1985) Effects of temperature and dilution rate on the copy number of recombinant plasmid in continuous culture of Bacillus stearothermophilus (pLP11). Biotechnol Bioeng 27:721–728
Mandecki W, Caruthers MH (1984) Mutants of the lac promoter with large insertions and deletions between the CAP-binding site and the — 35 region. Gene 31:263–267
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–565
McKenney K, Shimatake H, Court D, Schmeisser K, Brady D, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In: Chirikjian JC, Papas TS (eds) Gene amplification and analysis, vol II. Analysis of nucleic acids by enzymatic methods. Elsevier North Holland, Amsterdam, pp 383–415
Pastan I, Adhya S (1976) Cyclic adenosine 3′,5′-monophosphate in Escherichia coli. Bacteriol Rev 40:527–551
Peterkofsky A, Gazdar C (1971) Glucose and the metabolism of adenosine 3′,5′-cyclic monophosphate in Escherichia coli. Proc Natl Acad Sci USA 68:2794–2798
Reinikainen P, Vikajärvi I (1989) Escherichia coli growth and plasmid copy numbers in continuous cultivations. Biotechnol Lett 11:225–230
Riesenberg D, Menzel K, Schulz V, Schumann K, Veith G, Zuber G, Knorre WA (1990) High-cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1. Appl Microbiol Biotechnol 34:77–82
Riesenberg D, Schulz V, Knorre WA, Pohl HD, Korz D, Sanders EA, Ross A, Deckwer WD (1991) High cell density cultivation of Escherichia coli at controlled specific growth rate. J Biotechnol 20:17–28
Rosenthal A, Schwertner S, Hahn V, Hunger HD (1985) Solidphase methods for sequencing of nucleic acids. I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper. Nucleic Acids Res 13:1173–1184
Saier MJ Jr, Feucht BU, McCaman MT (1975) Regulation of intracellular adenosine cyclic 3′,5′-monophosphate levels in Escherichia coli and Salmonella typhimurium. Evidence for energy-dependent excretion of the cyclic nucleotide. J Biol Chem 250:7593–7601
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Cold Spring Harbor, N. Y.
Seo J-H, Bailey JE (1986) Continuous cultivation of recombinant Escherichia coli: existence of an optimum dilution rate for maximum plasmid and gene product concentration. Biotechnol Bioeng 28:1590–1594
Siegel R, Ryu DDY (1985) Kinetic study of instability of recombinant plasmid pPLc23trpA1 in E. coli using two-stage continuous culture system. Biotechnol Bioeng 27:28–33
Ullmann A, Danchin A (1983) Role of cyclic AMP in bacteria. Adv Cyclic Nucleotide Res 15:1–53
Weiss R, Birch-Hirschfeld E, Reinert H (1986) Synthesis of medium sized gene fragments on a polystyrene grafted PTFE support. In: Bruzik KS, Stec WS (eds) Biophosphates and their analogues — synthesis, structure, metabolism and activity. Elsevier Science Publishers, Amsterdam
Wollweber L, Wisniewski HG, Herrmann C, Hoffmann S, Siller K, Tonew M (1988) Verfahren zur immunchemischen Bestimmung von humanem Interferon alpha. German patent no DD 281 660 AP
Wright LF, Milne DP, Knowless CJ (1979) The regulatory effects of growth rate and cyclic AMP levels on carbon catabolism and respiration in Escherichia coli-K 12. Biochim Biophys Acta 583:73–80
Author information
Authors and Affiliations
Additional information
Offprint requests to: D. Riesenberg
Rights and permissions
About this article
Cite this article
Schroeckh, V., Hartmann, M., Birch-Hirschfeld, E. et al. Improvement of recombinant gene expression in Escherichia coli for glucose-controlled continuous and fed-batch cultures. Appl Microbiol Biotechnol 36, 487–492 (1992). https://doi.org/10.1007/BF00170189
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00170189