Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli.

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the -10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.

[1]  E. Ohtsuka,et al.  Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli. , 1996, Protein engineering.

[2]  W. Lotz,et al.  Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. , 1989, Gene.

[3]  V. Ramakrishnan,et al.  Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma S , 1996, Journal of bacteriology.

[4]  G Sawers,et al.  Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR , 1993, Molecular microbiology.

[5]  R. Hengge-aronis,et al.  What makes an Escherichia coli promoter σS dependent? Role of the −13/−14 nucleotide promoter positions and region 2.5 of σS , 2001 .

[6]  Richard Sparling,et al.  Regulation in the rpoS regulon of Escherichia coli , 1998 .

[7]  K. Friehs,et al.  The sequence upstream of the −10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli , 2005, Applied Microbiology and Biotechnology.

[8]  R. Hengge-aronis,et al.  The role of the sigma factor sigma S (KatF) in bacterial global regulation. , 1994, Annual review of microbiology.

[9]  G. Bennett,et al.  Characterization of a pH‐inducible promoter system for high‐level expression of recombinant proteins in Escherichia coli , 1995, Biotechnology and bioengineering.

[10]  S. Gottesman.  Minimizing proteolysis in Escherichia coli: genetic solutions. , 1990, Methods in enzymology.

[11]  Akira Ishihama,et al.  Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase , 2002, Molecular microbiology.

[12]  C. Cagnon,et al.  A new family of sugar-inducible expression vectors for Escherichia coli. , 1991, Protein engineering.

[13]  M. Tuite,et al.  New insights into mRNA decoding--implications for heterologous protein synthesis. , 1993, Trends in biotechnology.

[14]  R. Lobell,et al.  AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein. , 1991, Journal of molecular biology.

[15]  A. Tormo,et al.  Sigma s-dependent promoters in Escherichia coli are located in DNA regions with intrinsic curvature. , 1993, Nucleic acids research.

[16]  I. Charles,et al.  High level heterologous expression in E. coli using the anaerobically-activated nirB promoter. , 1991, Nucleic acids research.

[17]  R. Hengge-aronis,et al.  Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli , 1995, Journal of bacteriology.

[18]  S. C. Lee,et al.  Recent advances in heterologous gene expression in Escherichia coli. , 1993, Current opinion in biotechnology.

[19]  G D Stormo,et al.  High-level translation initiation. , 1990, Methods in enzymology.

[20]  A. Böck,et al.  Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression , 1989, Journal of bacteriology.

[21]  R. Hengge-aronis,et al.  Identification of a central regulator of stationary‐phase gene expression in Escherichia coli , 1991, Molecular microbiology.

[22]  K. Makino,et al.  Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters , 1991, Journal of bacteriology.

[23]  M. Uhlén,et al.  Engineering proteins to facilitate bioprocessing. , 1994, Trends in biotechnology.

[24]  K. Friehs,et al.  The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase , 1997, Archives of Microbiology.

[25]  C. Georgopoulos,et al.  Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[26]  H. Schweizer,et al.  A novel phosphate-regulated expression vector in Escherichia coli. , 1990, Gene.

[27]  R. Burgess,et al.  Promoter recognition and discrimination by EσS RNA polymerase , 2001, Molecular microbiology.

[28]  R. Hengge-aronis,et al.  The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. , 1994, Genes & development.

[29]  E. Bremer,et al.  pOSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli. , 1994, Gene.

[30]  H. Schweizer,et al.  Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli , 1991, Molecular and General Genetics MGG.

[31]  R. Hockney.  Recent developments in heterologous protein production in Escherichia coli. , 1994, Trends in biotechnology.

[32]  W. Boos,et al.  The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor , 1988, Molecular and General Genetics MGG.

[33]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[34]  Kwang-Soo Kim,et al.  Characterization of the nar promoter to use as an inducible promoter , 1996, Biotechnology Letters.

[35]  T. W. Nattkemper,et al.  A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli , 2006, Applied Microbiology and Biotechnology.

[36]  A. Ishihama,et al.  Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38 , 1995, Journal of bacteriology.

[37]  J. Ichikawa,et al.  A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences , 1994, Journal of bacteriology.

[38]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[39]  A. Ishihama,et al.  Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector , 2004, Journal of bacteriology.

[40]  C. Chamizo,et al.  A consensus structure for σs‐dependent promoters , 1996, Molecular microbiology.

[41]  C. J. Collins,et al.  Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells , 1988, Cell.

[42]  N. Otsu.  A threshold selection method from gray level histograms , 1979 .

[43]  J. Bailey,et al.  Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli , 1988, Nature.

[44]  B. Wanner,et al.  Control of bacterial alkaline phosphatase synthesis and variation in an Escherichia coli K-12 phoR mutant by adenyl cyclase, the cyclic AMP receptor protein, and the phoM operon , 1988, Journal of bacteriology.

[45]  K. Tanaka,et al.  Structure of the 5′ upstream region and the regulation of the rpoS gene of Escherichia coli , 1994, Molecular and General Genetics MGG.

[46]  M. Yamasaki,et al.  Synthesis and secretion of human epidermal growth factor by Escherichia coli. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[47]  K. Makino,et al.  Mode of promoter recognition by the Escherichia coli RNA polymerase holoenzyme containing the σs subunit: identification of the recognition sequence of the fic promoter , 1995, Molecular microbiology.

[48]  R. Doi,et al.  Prokaryotic promoters in biotechnology. , 1995, Biotechnology annual review.

[49]  R. Hengge-aronis,et al.  Stationary phase gene regulation: what makes an Escherichia coli promoter sigmaS-selective? , 2002, Current opinion in microbiology.

[50]  D. Webster,et al.  Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. , 1990, Nucleic acids research.

[51]  N. Mackman,et al.  Current status of secretion of foreign proteins by microorganisms , 1986 .

[52]  T. Ferenci,et al.  Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates , 1994, Journal of bacteriology.

[53]  K. Hammer,et al.  The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters , 1998, Applied and Environmental Microbiology.

[54]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[55]  N. Müller,et al.  Plasmid vector for overproduction and export of recombinant protein in Escherichia coli: efficient one-step purification of a recombinant antigen from Echinococcus multilocularis (Cestoda). , 1989, Gene.

[56]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .