Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium

Journal of Biological Engineering

Peter Lindblad¹  Thorsten Heidorn²  Daniel Camsund¹ 

[1] Microbial Chemistry, Department of Chemistry–Ångström Laboratory, Science for Life Laboratory, Uppsala University, P.O. Box 523, SE-75120 Uppsala, Sweden;Bioforsk, Frederik A Dahls vei 20, 1432 Ås Oslo, Norway

关键词: Transcriptional regulation;    Metabolic engineering tools;    LacI;    Synechocystis;    Ptrc;    Transcriptional systems;    Synthetic biology;    Lac repressor;    Cyanobacteria;   

Others  :  804839
DOI  :  10.1186/1754-1611-8-4

 received in 2013-08-16, accepted in 2013-12-26,  发布年份 2014

【 摘 要 】

Background

Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library).

Results

We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis.

Conclusions

The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.

【 授权许可】

   
2014 Camsund et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Ducat DC, Way JC, Silver PA: Engineering cyanobacteria to generate high-value products. Trends Biotechnol 2011, 29:95-103.
• [2]Lindblad P, Lindberg P, Oliveira P, Stensjö K, Heidorn T: Design, engineering, and construction of photosynthetic microbial cell factories for renewable solar fuel production. Ambio 2012, 41:163-168.
• [3]Keasling JD: Synthetic biology and the development of tools for metabolic engineering. Metab Eng 2012, 14:189-195.
• [4]Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjö K, Lindblad P: Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods Enzymol 2011, 497:539-579.
• [5]Huang HH, Lindblad P: Wide-dynamic-range promoters engineered for cyanobacteria. J Biol Eng 2013, 7:10. BioMed Central Full Text
• [6]Cardinale S, Arkin AP: Contextualizing context for synthetic biology - identifying causes of failure of synthetic biological systems. Biotechnol J 2012, 7:856-866.
• [7]Rao CV: Expanding the synthetic biology toolbox: engineering orthogonal regulators of gene expression. Curr Opin Biotechnol 2012, 23:689-694.
• [8]Wang B, Wang J, Zhang W, Meldrum DR: Application of synthetic biology in cyanobacteria and algae. Front Microbiol 2012, 3:344.
• [9]Gilbert W, Müller-Hill B: Isolation of the lac repressor. Proc Natl Acad Sci USA 1966, 56:1891-1898.
• [10]Lewis M: The lac repressor. C R Biol 2005, 328:521-548.
• [11]Barkley MD, Riggs AD, Jobe A, Burgeois S: Interaction of effecting ligands with lac repressor and repressor-operator complex. Biochemistry (Mosc) 1975, 14:1700-1712.
• [12]Sadler JR, Sasmor H, Betz JL: A perfectly symmetric lac operator binds the lac repressor very tightly. Proc Natl Acad Sci U S A 1983, 80:6785-6789.
• [13]Oehler S, Amouyal M, Kolkhof P, Von Wilcken-Bergmann B, Muller-Hill B: Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J 1994, 13:3348-3355.
• [14]Mossing MC, Record MT Jr: Upstream operators enhance repression of the lac promoter. Science 1986, 233:889-892.
• [15]Krämer H, Niemoller M, Amouyal M, Revet B, Von Wilcken-Bergmann B, Muller-Hill B: lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J 1987, 6:1481-1491.
• [16]Bellomy GR, Mossing MC, Record MT: Physical properties of DNA in vivo as probed by the length pependence of the lac operator looping process. Biochemistry (Mosc) 1988, 27:3900-3906.
• [17]Oehler S, Eismann ER, Kramer H, Muller-Hill B: The three operators of the lac operon cooperate in repression. EMBO J 1990, 9:973-979.
• [18]Law SM, Bellomy GR, Schlax PJ, Record MT Jr: In vivo thermodynamic analysis of repression with and without looping in lac constructs: estimates of free and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid DNA in vivo. J Mol Biol 1993, 230:161-173.
• [19]Garcia HG, Sanchez A, Boedicker JQ, Osborne M, Gelles J, Kondev J, Phillips R: Operator sequence alters gene expression independently of transcription factor occupancy in bacteria. Cell Reports 2012, 2:150-161.
• [20]Bond LM, Peters JP, Becker NA, Kahn JD, Maher LJ: Gene repression by minimal lac loops in vivo. Nucleic Acids Res 2010, 38:8072-8082.
• [21]Lee DH, Schleif RF: In vivo DNA loops in araCBAD: size limits and helical repeat. Proc Natl Acad Sci USA 1989, 86:476-480.
• [22]Zhang Y, McEwen AE, Crothers DM, Levene SD: Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping. PLoS One 2006, 1:e136.
• [23]Müller J, Oehler S, Müller-Hill B: Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. J Mol Biol 1996, 257:21-29.
• [24]Brosius J, Erfle M, Storella J: Spacing of the−10 and−35 regions in the tac promoter–effect on its in vivo activity. J Biol Chem 1985, 260:3539-3541.
• [25]Huang HH, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a synthetic biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38:2577-2593.
• [26]Imamura S, Asayama M: Sigma factors for cyanobacterial transcription. Gene Regul Syst Biol 2009, 3:65-87.
• [27]Geerts D, Bovy A, De Vrieze G, Borrias M, Weisbeek P: Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942. Microbiology 1995, 141(Pt 4):831-841.
• [28]Guerrero F, Carbonell V, Cossu M, Correddu D, Jones PR: Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS One 2012, 7:e50470.
• [29]de Boer HA, Comstock LJ, Vasser M: The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 1983, 80:21-25.
• [30]Elhai J: Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol Lett 1993, 114:179-184.
• [31]Schneider GJ, Haselkorn R: RNA-polymerase subunit homology among cyanobacteria, other eubacteria, and archaebacteria. J Bacteriol 1988, 170:4136-4140.
• [32]Xie WQ, Jager K, Potts M: Cyanobacterial RNA-Polymerase Genes rpoc1 and rpoc2 Correspond to rpoc of Escherichia coli. J Bacteriol 1989, 171:1967-1973.
• [33]Asayama M: Regulatory system for light-responsive gene expression in photosynthesizing bacteria: cis-elements and trans-acting factors in transcription and post-transcription. Biosci Biotechnol Biochem 2006, 70:565-573.
• [34]The iGEM registry of standard biological parts. http://parts.igem.org/ webcite
• [35]Griese M, Lange C, Soppa J: Ploidy in cyanobacteria. FEMS Microbiol Lett 2011, 323:124-131.
• [36]Lutz R, Bujard H: Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements. Nucleic Acids Res 1997, 25:1203-1210.
• [37]Mitschke J, Georg J, Scholz I, Sharma CM, Dienst D, Bantscheff J, Voss B, Steglich C, Wilde A, Vogel J, Hess WR: An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp PCC6803. Proc Natl Acad Sci U S A 2011, 108:2124-2129.
• [38]Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, De Mora K, Glieberman AL, Monie DD, Endy D: Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 2009, 3:4. BioMed Central Full Text
• [39]Dong GG, Kim YI, Golden SS: Simplicity and complexity in the cyanobacterial circadian clock mechanism. Curr Opin Genet Dev 2010, 20:619-625.
• [40]Temme K, Hill R, Segall-Shapiro TH, Moser F, Voigt CA: Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res 2012, 40:8773-8781.
• [41]Shis DL, Bennett MR: Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. Proc Natl Acad Sci USA 2013, 110:5028-5033.
• [42]De Smit MH, Vanduin J: Secondary structure of the ribosome binding-site determines translational efficiency–a quantitative-analysis. Proc Natl Acad Sci USA 1990, 87:7668-7672.
• [43]Welch M, Villalobos A, Gustafsson C, Minshull J: You’re one in a googol: optimizing genes for protein expression. J R Soc Interface 2009, 6(4):S467-S476.
• [44]Saecker RM, Record MT Jr, Dehaseth PL: Mechanism of bacterial transcription initiation: RNA polymerase–promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J Mol Biol 2011, 412:754-771.
• [45]Sclavi B, Zaychikov E, Rogozina A, Walther F, Buckle M, Heumann H: Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter. Proc Natl Acad Sci USA 2005, 102:4706-4711.
• [46]Chakraborty A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R, et al.: Opening and closing of the bacterial RNA polymerase clamp. Science 2012, 337:591-595.
• [47]Schyns G, Jia L, Coursin T, Tandeau de Marsac N, Houmard J: Promoter recognition by a cyanobacterial RNA polymerase: in vitro studies with the Calothrix sp. PCC 7601 transcriptional factors RcaA and RcaD. Plant Mol Biol 1998, 36:649-659.
• [48]Imashimizu M, Fujiwara S, Tanigawa R, Tanaka K, Hirokawa T, Nakajima Y, Higo J, Tsuzuki M: Thymine at−5 is crucial for cpc promoter activity of Synechocystis sp. strain PCC 6714. J Bacteriol 2003, 185:6477-6480.
• [49]Imashimizu M, Tanaka K, Shimamoto N: Comparative study of cyanobacterial and E. coli RNA Polymerases: misincorporation, abortive transcription, and dependence on divalent cations. Genet Res Int 2011, 572689.
• [50]Leveau JH, Lindow SE: Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria. J Bacteriol 2001, 183:6752-6762.
• [51]Becker NA, Kahn JD, Maher LJ III: Bacterial repression loops require enhanced DNA flexibility. J Mol Biol 2005, 349:716-730.
• [52]Becker NA, Kahn JD, Maher LJ III: Eukaryotic HMGB proteins as replacements for HU in E. coli repression loop formation. Nucleic Acids Res 2008, 36:4009-4021.
• [53]CyanoBase. http://genome.microbedb.jp/CyanoBase webcite
• [54]Goodson KA, Wang Z, Haeusler AR, Kahn JD, English DS: LacI-DNA-IPTG loops: equilibria among conformations by single-molecule FRET. J Phys Chem B 2013, 117:4713-4722.
• [55]Becker NA, Peters JP, Maher LJ III, Lionberger TA: Mechanism of promoter repression by Lac repressor-DNA loops. Nucleic Acids Res 2012, 41:156-166.
• [56]Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D: Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods 2013, 10:353-354.
• [57]Müller-Hill B, Crapo L, Gilbert W: Mutants that make more lac repressor. Proc Natl Acad Sci USA 1968, 59:1259-1264.
• [58]Stanier RY, Kunisawa R, Mandel M, Cohenbaz G: Purification and properties of unicellular blue-green algae (Order Chroococcales). Bacteriol Rev 1971, 35:171.
• [59]Shetty RP, Endy D, Knight TF Jr: Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2008, 2:5. BioMed Central Full Text
• [60]Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP: Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 1997, 179:1998-2005.
• [61]Milkowski C, Quinones A, Hagemann M: A DNA fragment from the cyanobacterium Synechocystis sp. PCC 6803 mediates gene expression inducible by osmotic stress in E. coli. Curr Microbiol 1998, 37:108-116.
• [62]Kunert A, Hagemann M, Erdmann N: Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J Microbiol Methods 2000, 41:185-194.
• [63]Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:U341-U343.
• [64]Hillson NJ, Rosengarten RD, Keasling JD: j5 DNA assembly design automation software. ACS Synthetic Biol 2012, 1:14-21.
• [65]Kremers GJ, Goedhart J, van den Heuvel DJ, Gerritsen HC, Gadella TWJ: Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry (Mosc) 2007, 46:3775-3783.