1887

Abstract

A synthetic promoter library (SPL) for has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in and . A wide range of promoter strengths was obtained with the approach, covering 3–4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive -glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10–15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in and , which indicates the potential of the SPL for other species. The SPL enables fine-tuning of stable gene expression for various applications in .

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2006-04-01
2019-10-14
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References

  1. Ahrné, S., Nobaek, S., Jeppsson, B., Adlerberth, I., Wold, A. E. & Molin, G. ( 1998; ). The normal Lactobacillus flora of healthy human rectal and oral mucosa. J Appl Microbiol 85, 88–94.[CrossRef]
    [Google Scholar]
  2. Alander, M., De Smet, I., Nollet, L., Verstraete, W., von Wright, A. & Mattila-Sandholm, T. ( 1999; ). The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Int J Food Microbiol 46, 71–79.[CrossRef]
    [Google Scholar]
  3. Aukrust, T. & Blom, H. ( 1992; ). Transformation of Lactobacillus strains used in meat and vegetable fermentations. Food Res Int 25, 253–261.[CrossRef]
    [Google Scholar]
  4. Axelsson, L. ( 2004; ). Lactic acid bacteria: classification and physiology. In Lactic Acid Bacteria: Microbiological and Functional Aspects, 3rd edn, pp. 1–66. Edited by S. Salminen, A. von Wright & A. Ouwehand. New York: Marcel Dekker.
  5. Axelsson, L. & Ahrné, S. ( 2000; ). Lactic acid bacteria. In Applied Microbial Systematics, pp. 365–386. Edited by F. G. Priest & M. Goodfellow. The Netherlands: Kluwer Academic Publishers.
  6. Axelsson, L., Lindstad, G. & Naterstad, K. ( 2003; ). Development of an inducible gene expression system for Lactobacillus sakei. Lett Appl Microbiol 37, 115–120.[CrossRef]
    [Google Scholar]
  7. Chaves, A. C., Fernandez, M., Lerayer, A. L., Mierau, I., Kleerebezem, M. & Hugenholtz, J. ( 2002; ). Metabolic engineering of acetaldehyde production by Streptococcus thermophilus. Appl Environ Microbiol 68, 5656–5662.[CrossRef]
    [Google Scholar]
  8. Chen, Y. S. & Steele, J. L. ( 2005; ). Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria. J Appl Microbiol 98, 64–72.[CrossRef]
    [Google Scholar]
  9. de Vos, W. & Simons, G. ( 1994; ). Gene cloning and expression systems in lactococci. In Genetics and Biotechnology of Lactic Acid Bacteria, pp. 52–105. Edited by M. Gasson & W. de Vos. Oxford: Chapman and Hall.
  10. Exterkate, F. A. ( 1984; ). Location of peptidases outside and inside the membrane of Streptococcus cremoris. Appl Environ Microbiol 47, 177–183.
    [Google Scholar]
  11. Havenith, C. E. G., Seegers, J. F. M. L. & Pouwels, P. H. ( 2002; ). Gut-associated lactobacilli for oral immunisation. Food Res Int 35, 151–163.[CrossRef]
    [Google Scholar]
  12. Hugenholtz, J., Kleerebezem, M., Starrenburg, M., Delcour, J., de Vos, W. & Hols, P. ( 2000; ). Lactococcus lactis as a cell factory for high-level diacetyl production. Appl Environ Microbiol 66, 4112–4114.[CrossRef]
    [Google Scholar]
  13. Jensen, P. R. & Hammer, K. ( 1998a; ). Artificial promoters for metabolic optimization. Biotechnol Bioeng 58, 191–195.[CrossRef]
    [Google Scholar]
  14. Jensen, P. R. & Hammer, K. ( 1998b; ). The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64, 82–87.
    [Google Scholar]
  15. Jensen, P. R., Westerhoff, H. V. & Michelsen, O. ( 1993; ). The use of lac-type promoters in control analysis. Eur J Biochem 211, 181–191.[CrossRef]
    [Google Scholar]
  16. Kahala, M. & Palva, A. ( 1999; ). The expression signals of the Lactobacillus brevis slpA gene direct efficient heterologous protein production in lactic acid bacteria. Appl Microbiol Biotechnol 51, 71–78.[CrossRef]
    [Google Scholar]
  17. Koebmann, B. J., Andersen, H. W., Solem, C. & Jensen, P. R. ( 2002a; ). Experimental determination of control of glycolysis in Lactococcus lactis. Antonie van Leeuwenhoek 82, 237–248.[CrossRef]
    [Google Scholar]
  18. Koebmann, B. J., Solem, C., Pedersen, M. B., Nilsson, D. & Jensen, P. R. ( 2002b; ). Expression of genes encoding F1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis. Appl Environ Microbiol 68, 4274–4282.[CrossRef]
    [Google Scholar]
  19. Krüger, C., Hu, Y., Pan, Q. & 10 other authors ( 2002; ). In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotechnol 20, 702–706.[CrossRef]
    [Google Scholar]
  20. Mathiesen, G., Sørvig, E., Blatny, J., Naterstad, K., Axelsson, L. & Eijsink, V. G. H. ( 2004; ). High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter. Lett Appl Microbiol 39, 137–143.[CrossRef]
    [Google Scholar]
  21. McCracken, A., Turner, M. S., Giffard, P., Hafner, L. M. & Timms, P. ( 2000; ). Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species. Arch Microbiol 173, 383–389.[CrossRef]
    [Google Scholar]
  22. Mercenier, A., Pouwels, P. & Chassy, B. ( 1994; ). Genetic engineering of lactobacilli, leuconostocs and Streptococcus thermophilus. In Genetics and Biotechnology of Lactic Acid Bacteria, pp. 252–293. Edited by M. Gasson & W. de Vos. Oxford: Chapman and Hall.
  23. Mercenier, A., Pavan, S. & Pot, B. ( 2003; ). Probiotics as biotherapeutic agents: present knowledge and future prospects. Curr Pharm Des 9, 175–191.[CrossRef]
    [Google Scholar]
  24. Miller, J. M. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  25. Møretrø, T., Hagen, B. F. & Axelsson, L. ( 1998; ). A new, completely defined medium for meat lactobacilli. J Appl Microbiol 85, 715–722.[CrossRef]
    [Google Scholar]
  26. Møretrø, T., Naterstad, K., Wang, E., Aasen, I. M., Chaillou, S., Zagorec, M. & Axelsson, L. ( 2005; ). Sakacin P non-producing Lactobacillus sakei strains contain homologues of the sakacin P gene cluster. Res Microbiol 156, 949–960.[CrossRef]
    [Google Scholar]
  27. Neu, T. & Henrich, B. ( 2003; ). New thermosensitive delivery vector and its use to enable nisin-controlled gene expression in Lactobacillus gasseri. Appl Environ Microbiol 69, 1377–1382.[CrossRef]
    [Google Scholar]
  28. Pavan, S., Hols, P., Delcour, J., Geoffroy, M. C., Grangette, C., Kleerebezem, M. & Mercenier, A. ( 2000; ). Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects. Appl Environ Microbiol 66, 4427–4432.[CrossRef]
    [Google Scholar]
  29. Pouwels, P. H. & Leer, R. J. ( 1993; ). Genetics of lactobacilli: plasmids and gene expression. Antonie van Leeuwenhoek 64, 85–107.
    [Google Scholar]
  30. Pouwels, P. H., Leer, R. J. & Boersma, W. J. ( 1996; ). The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens. J Biotechnol 44, 183–192.[CrossRef]
    [Google Scholar]
  31. Pouwels, P. H., Leer, R. J., Shaw, M., Heijne den Bak-Glashouwer, M. J., Tielen, F. D., Smit, E., Martinez, B., Jore, J. & Conway, P. L. ( 1998; ). Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes. Int J Food Microbiol 41, 155–167.[CrossRef]
    [Google Scholar]
  32. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  33. Schillinger, U. & Lücke, F.-K. ( 1989; ). Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 55, 1901–1906.
    [Google Scholar]
  34. Schultz, M., Veltkamp, C., Dieleman, L. A., Grenther, W. B., Wyrick, P. B., Tonkonogy, S. L. & Sartor, R. B. ( 2002; ). Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 8, 71–80.[CrossRef]
    [Google Scholar]
  35. Solem, C. & Jensen, P. R. ( 2002; ). Modulation of gene expression made easy. Appl Environ Microbiol 68, 2397–2403.[CrossRef]
    [Google Scholar]
  36. Solem, C., Koebmann, B. J. & Jensen, P. R. ( 2003; ). Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. J Bacteriol 185, 1564–1571.[CrossRef]
    [Google Scholar]
  37. Sybesma, W., Starrenburg, M., Kleerebezem, M., Mierau, I., de Vos, W. M. & Hugenholtz, J. ( 2003; ). Increased production of folate by metabolic engineering of Lactococcus lactis. Appl Environ Microbiol 69, 3069–3076.[CrossRef]
    [Google Scholar]
  38. Sørvig, E., Grönqvist, S., Naterstad, K., Mathiesen, G., Eijsink, V. G. & Axelsson, L. ( 2003; ). Construction of vectors for inducible gene expression in Lactobacillus sakei and L. plantarum. FEMS Microbiol Lett 229, 119–126.[CrossRef]
    [Google Scholar]
  39. Sørvig, E., Mathiesen, G., Naterstad, K., Eijsink, V. G. & Axelsson, L. ( 2005; ). High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology 151, 2439–2449.[CrossRef]
    [Google Scholar]
  40. van de Guchte, M., Kok, J. & Venema, G. ( 1991; ). Distance-dependent translational coupling and interference in Lactococcus lactis. Mol Gen Genet 227, 65–71.[CrossRef]
    [Google Scholar]
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