Abstract
Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.
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References
Barg H, Malten M, Jahn M, Jahn D (2005) Protein and vitamin production in Bacillus megaterium. In: Barredo JL (ed) Microbial processes and products. Humana, Totowa, pp 165–184
Biedendieck R, Beine R, Gamer G, Jordan E, Buchholz K, Seibel J, Dijkhuizen L, Malten M, Jahn D (2007a) Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium. Appl Microbiol Biotechnol 74:1062–1073
Biedendieck R, Gamer M, Jaensch L, Meyer S, Rohde M, Deckwer WD, Jahn D (2007b) A sucrose-inducible promoter system for the intra- and extracellular protein production in Bacillus megaterium. J Biotechnol 132:426–430
Biedendieck R, Yang Y, Deckwer WD, Malten M, Jahn D (2007c) Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium. Biotechnol Bioeng 96:525–537
Brunschwig E, Darzins A (1992) A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. Gene 111:35–41
Conrad B, Savchenko RS, Breves R, Hofemeister J (1996) A T7 promoter-specific, inducible protein expression system for Bacillus subtilis. Mol Gen Genet 250:230–236
Darmon E, Noone D, Masson A, Bron S, Kuipers OP, Devine KM, van Dijl JM (2002) A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis. J Bacteriol 184:5661–5671
Davanloo P, Rosenberg AH, Dunn JJ, Studier FW (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 81:2035–2039
Elroy-Stein O, Moss B (1990) Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. Proc Natl Acad Sci U S A 87:6743–6747
Englard S, Seifter S (1990) Precipitation techniques. In: Deutscher MP (ed) Methods in enzymology. Academic, San Diego, pp 285–301
Homann A, Biedendieck R, Gotze S, Jahn D, Seibel J (2007) Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochem J 407:189–198
Hyyrylainen HL, Bolhuis A, Darmon E, Muukkonen L, Koski P, Vitikainen M, Sarvas M, Pragai Z, Bron S, van Dijl JM, Kontinen VP (2001) A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol Microbiol 41:1159–1172
Jordan E, Hust M, Roth A, Biedendieck R, Jahn D, Schirrmann T, Dübel S (2007) Production of recombinant antibody fragments in Bacillus megaterium. Microb Cell Fact 15:2
Kieselburg MK, Weickert M, Vary PS (1984) Analysis of resident and transformant plasmids in Bacillus megaterium. Bio/Technology 2:254–259
Kunnimalaiyaan M, Stevenson DM, Zhou Y, Vary PS (2001) Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551. Mol Microbiol 39:1010–1021
Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218
Malten M, Biedendieck R, Gamer M, Drews AC, Stammen S, Buchholz K, Dijkhuizen L, Jahn D (2006) A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium. Appl Environ Microbiol 72:1677–1679
Ozimek LK, van Hijum SA, van Koningsveld GA, van Der Maarel MJ, van Geel-Schutten GH, Dijkhuizen L (2004) Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121. FEBS Lett 560:131–133
Rosenberg AH, Goldman E, Dunn JJ, Studier FW, Zubay G (1993) Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bacteriol 175:716–722
Rygus T, Hillen W (1991) Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol 35:594–599
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Sumer JB, Howell SF (1935) A method of determination of invertase activity. J Biol Chem 108:51–54
Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A 82:1074–1078
Vary PS (1994) Prime time for Bacillus megaterium. Microbiology 140:1001–1013
Vary PS, Biedendieck R, Fuerch T, Meinhardt F, Rohde M, Deckwer WD, Jahn D (2007) Bacillus megaterium—from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76:957–967
Vellanoweth RL (1993) Translation and its regulation. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other Gram-positive Bacteria. ASM, Washington, pp 699–711
Wittchen KD, Meinhard F (1995) Inactivation of the major protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:871–877
Yang Y, Malten M, Grote A, Jahn D, Deckwer WD (2007) Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium. Biotechnol Bioeng 96:780–794
Acknowledgements
We gratefully thank Dr. Patricia Vary for contributing the plasmid pYZ5, and we acknowledge financial support granted by the “Deutsche Forschungsgemeinschaft (SFB 578)”.
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Gamer, M., Fröde, D., Biedendieck, R. et al. A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium . Appl Microbiol Biotechnol 82, 1195–1203 (2009). https://doi.org/10.1007/s00253-009-1952-5
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DOI: https://doi.org/10.1007/s00253-009-1952-5