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  • Review Article
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Recombinant Protein Expression in High Cell Density Fed-Batch Cultures of Escherichia Coli

Bio/Technology volume 10, pages 1550–1556 (1992)Cite this article

Abstract

Whereas cell concentrations of 5–10 grams dry cell weight per liter (gDCW/1) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/1. Feeding strategies for fed-batch cultures include feedback control as well as pre-determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration. Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli. The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described.

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References

  1. Yamane, T. and Shimizu, S. 1984. Fed-batch techniques in microbial processes. Adv. Biochem. Eng. 30: 147–194.

    CAS  Google Scholar 

  2. Thompson, B.G., Kole, M. and Gerson, D.F. 1985. Control of ammonium concentration in Escherichia coli fermentations. Biotechnol. Bioeng. 27: 818–824.

    Article  CAS  Google Scholar 

  3. Doelle, H.W., Ewings, K.N. and Hollywood, N.W. 1982. Regulation of glucose metabolism in bacterial systems. Adv. Biochem. Eng/Biotech. 23: 1–35.

    CAS  Google Scholar 

  4. Shiloach, J. and Bauer, S. 1975. High-yield growth of E. coli at different temperatures in a bench scale fermentor. Biotechnol. Bioeng. 17: 227–239.

    Article  CAS  Google Scholar 

  5. Bauer, S. and Shiloach, J. 1974. Maximal exponential growth rate and yield of E. coli obtainable in a bench-scale fermentor. Biotechnol. Bioeng. 16: 933–941.

    Article  CAS  Google Scholar 

  6. Bauer, S. and White, M.D. 1976. Pilot scale exponential growth of Escherichia coli W to high cell concentration with temperature variation. Biotechnol. Bioeng. 18: 839–846.

    Article  CAS  Google Scholar 

  7. Bauer, S. and Ziv, E. 1976. Dense growth of aerobic bacteria in a bench-scale fermentor. Biotechnol. Bioeng. 18: 81–94.

    Article  CAS  Google Scholar 

  8. Mori, H., Yano, T., Kobayashi, T. and Shimizu, S. 1979. High density cultivation of biomass in fed-batch system with DO-Stat. J. Chem. Eng. Japan 12: 313–319.

    Article  CAS  Google Scholar 

  9. Cutayar, J.M. and Poillon, D. 1989. High cell density culture of E. coli in a fed-batch system with dissolved oxygen as substrate feed indicator. Biotechnol. Lett. 11: 155–160.

    Article  CAS  Google Scholar 

  10. Fieschko, J. and Ritch, T. 1986. Production of human alpha consensus interferon in recombinant Escherichia coli. Chem. Eng Commun. 45: 229–240.

    Article  CAS  Google Scholar 

  11. Mizutani, S., Mori, H., Shimizu, S., Sakaguchi, S. and Kobayashi, T. 1986. Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid. Biotechnol. Bioeng. 28: 204–209.

    Article  CAS  Google Scholar 

  12. Strandberg, L. and Enfors, S.-O. 1991. Batch and fed-batch cultivations for the temperature induced production of a recombinant protein in Escherichia coli. Biotechnol. Lett. 13: 609–614.

    Article  CAS  Google Scholar 

  13. Riesenberg, D., Menzel, K., Schulz, V., Schumann, K., Veith, G., Zuber, G. and Knorre, W.A. 1990. High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1. Appl. Microbiol. Biotechnol. 34: 77–82.

    Article  CAS  Google Scholar 

  14. Dalboge, H., Bech Jensen, E., Tottrup, H., Grubb, A., Abrahamson, M., Olafsson, I. and Carlsen, S. 1989. High-level expression of active human cystatin C in Escherichia coli, Gene 79: 325–332.

    Article  CAS  Google Scholar 

  15. Jung, G., Denefle, P., Becquart, J. and Mayaux, J.-F. 1988. High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1β. Ann. Inst. Pasteur/Microbiol. 139: 129–146.

    Article  CAS  Google Scholar 

  16. Crabtree, H.G. 1929. Observations on the carbohydrate metabolism of tumours. Biochem. J. 23: 536–545.

    Article  CAS  Google Scholar 

  17. Mandelstam, J. 1960. The intracellular turnover of protein and nucleic acids and its role in biochemical differentiation. Bact. Rev. 24: 289–308.

    CAS  PubMed  Google Scholar 

  18. Bech Jensen, E. and Carlsen, S. 1990. Production of recombinant human growth hormone in Escherichia coli: Expression of different precursors and physiological effects of glucose, acetate, and salts. Biotechnol. Bioeng. 36: 1–11.

    Article  Google Scholar 

  19. Zabriskie, D.W., Wareheim, D.A. and Polansky, M.J. 1987. Effects of fermentation feeding strategies prior to induction of expression of a recombinant malaria antigen in Escherichia coli. J. Ind. Microbiol. 2: 87–95.

    Article  CAS  Google Scholar 

  20. Curless, C., Fu, K., Swank, R., Menjares, A., Fieschko, J. and Tsai, L. 1991. Design and evaluation of a two-stage, cyclic, recombinant fermentation process. Biotechnol. Bioeng. 38: 1082–1090.

    Article  CAS  Google Scholar 

  21. Luli, G.W. and Strohl, W.R. 1990. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl. Environ. Microbiol. 56: 1004–1011.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Landwall, P. and Holme, T. 1977. Removal of inhibitors of bacterial growth by dialysis culture. J. Gen. Microbiol. 103: 345–352.

    Article  CAS  Google Scholar 

  23. Pan, J.G., Rhee, J.S. and LeBeault, J.M. 1987. Physiological contraints in increasing biomass concentration of Escherichia coli B in fed-batch culture. Biotechnol. Lett. 9: 89–94.

    Article  CAS  Google Scholar 

  24. Robbins, J.W. and Taylor, K.B. 1989. Optimization of Escherichia coligrowth by controlled addition of glucose. Biotechnol. Bioeng. 34: 1289–1294.

    Article  CAS  Google Scholar 

  25. Reiling, H.E., Laurila, H. and Fiechter, A. 1985. Mass culture of Escherichia coli: Medium development for low and high density cultivation of Escherichia coli B/r in minimal and complex media. J. Biotechnol. 2: 191–206.

    Article  CAS  Google Scholar 

  26. Smirnova, G.V. and Oktyabr'skii, O.N. 1985. Influence of acetate on the growth of Escherichia coli under aerobic and anaerobic conditions, mikrobiologiya 54: 252–256. (English translation: Microbiology (USSR), 54: 205–209.)

  27. Meyer, H.-R., Leist, C. and Fiechter, A. 1984. Acetate formation in continuous culture of Escherichia coli K12 D1 on defined and complex media. J. Biotechnol. 1: 355–358.

    Article  CAS  Google Scholar 

  28. Paalme, T., Tiisma, K., Kahru, A., Vanatalu, K. and Vilu, R. 1990. Glucose-limited fed-batch cultivation of Escherichia coliwith computer-controlled fixed growth rate. Biotechnol. Bioeng. 35: 312–319.

    Article  CAS  Google Scholar 

  29. Meyer, H.-P., Kuhn, H.J., Brown, S.W. and Fiechter, A. Production of human leucocyte interferon by E. coli. Proceed. of 3rd Eureopean Congress on Biotechnol., Munich, Germany, Sept. 10-14, 1984, Verlag Che-mie GmbH, Weinheim, Fed. Rep. of Germany 1: 499–505.

  30. El-Mansi, E.M.T. and Holms, W.H. 1989. Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures. J. Gen. Microbiol. 135: 2875–2883.

    CAS  PubMed  Google Scholar 

  31. Brown, S.W., Meyer, H.-P. and Fiechter, A. 1985. Continuous production of human leukocyte interferon with Escherichia coli and continuous cell lysis in a two stage chemostat. Appl. Microbiol. Biotechnol. 23: 5–9.

    Article  CAS  Google Scholar 

  32. Siegel, R. and Ryu, D.Y. 1985. Kinetic study of instability of recombinant plasmid pPLc23trpAI in E. coli using two-stage continuous culture system. Biotechnol. Bioeng. 27: 28–33.

    Article  CAS  Google Scholar 

  33. Seo, J.-H and Bailey, J. E. 1985. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol. Bioeng. 27: 1668–1674.

    Article  CAS  Google Scholar 

  34. Lee, J.-H., Choi, Y.-H., Kang, S.-K., Park, H.-H. and Kwon, I.-B. 1989. Production of human leukocyte interferon in Escherichia coli by control of growth rate in fed-batch fermentation. Biotechnol. Lett. 2: 695–698.

    Google Scholar 

  35. Curless, C., Pope, J. and Tsai, L. 1990. Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli. Biotechnol. Prog. 6: 149–152.

    Article  CAS  Google Scholar 

  36. Konstantinov, K., Kishimoto, M., Seki, T. and Yoshida, T. 1990. A balanced DO-Stat and its application to the control of acetic acid excretion by recombinant Escherichia coli. Biotechnol.Bioeng. 36: 750–758.

    Article  CAS  Google Scholar 

  37. Mizutani, S., Iijima, S., Morikawa, M., Shimkizu, K., Matsubara, M., Ogawa, Y., Izumi, R., Matsumoto, K. and Kobayashi, T. 1987. On-line control of glucose concentration using an automatic glucose analyzer. J. Ferment. Technol. 65: 325–331.

    Article  CAS  Google Scholar 

  38. Luli, G.W., Schlasner, S.M., Ordaz, D.E., Mason, M. and Strohl, W.R. 1987. An automatic, on-line glucose analyzer for feed-back control of fed-batch growth of Escherichia coli. Biotechnol. Tech. 1: 225–230.

    Article  CAS  Google Scholar 

  39. Iijima, S., Yamashita, S., Matsunaga, K., Miura, H., Morikawa, M., Shimizu, K., Matsubara, M. and Kobayshi, T. 1987. Use of a novel turbi-dimeter to monitor microbial growth and control glucose concentration. J. Chem. Tech. Biotechnol. 40: 203–213.

    CAS  Google Scholar 

  40. Zabriskie, D.W. and Humphrey, A.E. 1978. Estimation of fermentation biomass concentration by measuring culture fluorescence. Appl. Environ. Microbiol. 35: 337–343.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Randolph, T.W., Marison, I.W., Martens, D.E. and Stockar, U.von. 1990. Calorimetric control of fed-batch fermentations. Biotechnol. Bioeng. 36: 678–684.

    Article  CAS  Google Scholar 

  42. Wang, H.Y., Cooney, C.L. and Wang, D.I.C. 1979. Computer control of Bakers' yeast production. Biotechnol. Bioeng. 21: 975–995.

    Article  CAS  Google Scholar 

  43. Lee, Y.L. and Chang, H.N. 1990. High cell density culture of a recombinant Escherichia coliproducing penicillin acylase in a membrane cell recycle fermentor. Biotechnol. Bioeng. 36: 330–337.

    Article  CAS  Google Scholar 

  44. Shimizu, N., Fukuzono, S., Fujimori, K., Nishimura, N. and Odawara, Y. 1988. Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring. J. Ferment. Technol. 66: 187–191.

    Article  CAS  Google Scholar 

  45. Shimizu, N., Fukuzono, S., Harade, Y., Fujimori, K., Gotoh, K. and Yamazaki, Y. 1991. Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli. Biotechnol. Bioeng. 38: 37–42.

    Article  CAS  Google Scholar 

  46. Allen, B.R. and Luli, G.W. 1987. A gradient-feed process for E.coli fermentations. BioPharm. 1: 38–41.

    Google Scholar 

  47. Tsai, L.B., Mann, M., Morris, F., Rotger, C. and Fenton, D. 1987. The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell density Escherichia coli fermentations. J. Ind. Microbiol. 2: 181–187.

    Article  CAS  Google Scholar 

  48. Shimizu, N., Fukuzono, S., Nishimura, N., Odawara, Y. and Fujiwara, K. 1987. Cultivation of Escherichia coli harbouring hybrid plasmids. J. Ferment. Technol. 65: 7–10.

    Article  CAS  Google Scholar 

  49. Horn, U., Krug, M. and Sawistowski, J. 1990. Effect of high density cultivation on plasmid copy number in recombinant Escherichia coli cells. Biotechnol. Lett. 12: 191–196.

    Article  CAS  Google Scholar 

  50. Jones, I.M., Primrose, S.B., Robinson, A. and Ellwood, D.C. 1980. Maintenance of some ColEl-type plasmids in chemostat culture. Molec. Gen. Genet. 180: 579–584.

    Article  CAS  Google Scholar 

  51. Jones, S.A. and Melling, J. 1984. Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures. FEMS Microbiol. Lett. 22: 239–243.

    Article  CAS  Google Scholar 

  52. Epstein, W. and Schultz, S.G. 1965. Cation transport in Escherichia coli, V. Regulation of cation content. J. Gen. Physiol. 49: 221–234.

    Article  CAS  Google Scholar 

  53. Tempest, D.W. 1969. Quantitative relationships between inorganic cations and anionic polymers in growing bacteria, p. 87–111. In: Microbial Growth: 19th Symp. Gen. Microbiol, London. University Press, Cambridge.

    Google Scholar 

  54. Bailey, F.J., Blankenship, J., Condra, J.H., Maigetter, R.Z. and Ellis, R.W. 1987. High-cell-density fermentation studies of a recombinant E coli that expresses atrial natriuretic factor. J. Ind. Microbiol. 2: 47–52.

    Article  CAS  Google Scholar 

  55. Seo, J.-H., Loffler, A.I. and Bailey, J. E. 1988. A parametric study of cloned fusion protein expression in Escherichia coli. Biotechnol. Bioeng. 32: 725–730.

    Article  CAS  Google Scholar 

  56. Tsai, L.B., Hsieng, S.L., Kenney, W.C., Curless, C.C., Klein, M.L., Lai, P.-H., Fenton, D.M., Altrock, B.W. and Mann, M.B. 1988. Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli. Biochem. Biophys. Res. Comm. 156: 733–739.

    Article  CAS  Google Scholar 

  57. Mizukami, T., Komatsu, Y., Hosoi, N., Itoh, S. and Oka, T. 1986. Production of active human interferon-β in E. coli, II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid. Biotechnol. Lett. 8: 611–614.

    Article  CAS  Google Scholar 

  58. Wood, T.K. and Peretti, S.W. 1991. Effect of chemically-induced, cloned-gene expression on protein synthesis in E. coli.. Biotechnol. Bioeng. 38: 397–412.

    Article  CAS  Google Scholar 

  59. Rosenfeld, S.A., Brandis, J.W., Ditullio, D.F., Lee, J.F. and Armiger, W.B. 1991. High-cell-density fermentations based on culture fluorescence, p. 23–33. In: ACS Sym. Series: Expression systems and processes for rDNA products. Hatch, R.T Goochee, C., Moreira,A. and Y. (Eds.). 477, ACSWashington, D.C.

    Chapter  Google Scholar 

  60. Lee, L. and Blanch, H.W. To appear. 1992. Recombinant trypsin production in high cell density fed-batch cultures of E. coli. Biotechnol. Bioeng.

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  1. H. W. Blanch: Corresponding author.

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  1. Department of Chemical Engineering, University of California at Berkeley, Berkeley, California, 94702

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Yee, L., Blanch, H. Recombinant Protein Expression in High Cell Density Fed-Batch Cultures of Escherichia Coli. Nat Biotechnol 10, 1550–1556 (1992). https://doi.org/10.1038/nbt1292-1550

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