Biotechnology and Bioprocess Engineering

, Volume 17, Issue 1, pp 1–7 | Cite as

Synthetic regulatory tools for microbial engineering

Review Paper


Microbial engineering requires accurate information about cellular metabolic networks and a set of molecular tools that can be predictably applied to the efficient redesign of such networks. Recent advances in the field of metabolic engineering and synthetic biology, particularly the development of molecular tools for synthetic regulation in the static and dynamic control of gene expression, have increased our ability to efficiently balance the expression of genes in various biological systems. It would accelerate the creation of synthetic pathways and genetic programs capable of adapting to environmental changes in real time to perform the programmed cellular behavior. In this paper, we review current developments in the field of synthetic regulatory tools for static and dynamic control of microbial gene expression.


metabolic engineering synthetic biology expression control synthetic regulatory tools 


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  1. 1.
    Nandagopal, N. and M. B. Elowitz (2011) Synthetic biology: Integrated gene circuits. Science 333: 1244–1248.CrossRefGoogle Scholar
  2. 2.
    Mitchell, W. (2011) Natural products from synthetic biology. Curr. Opin. Chem. Biol. 15: 505–515.CrossRefGoogle Scholar
  3. 3.
    Feng, Z. -H., Y. -S. Wang, and Y. -G. Zheng (2011) A new microtiter plate-based screening method for microorganisms producing Alpha-amylase inhibitors. Biotechnol. Bioprocess Eng. 16: 894–900.CrossRefGoogle Scholar
  4. 4.
    Meng, H., Y. Wang, Q. Hua, S. Zhang, and X. Wang (2011) In silico analysis and experimental improvement of taxadiene heterologous biosynthesis in Escherichia coli. Biotechnol. Bioprocess Eng. 16: 205–215.CrossRefGoogle Scholar
  5. 5.
    Keasling, J. D. (2008) Synthetic biology for synthetic chemistry. ACS. Chem. Biol. 3: 64–76.CrossRefGoogle Scholar
  6. 6.
    Meng, H., Z. Lu, Y. Wang, X. Wang, and S. Zhang (2011) In silico improvement of heterologous biosynthesis of erythromycin precursor 6-deoxyerythronolide B in Escherichia coli. Biotechnol. Bioprocess Eng. 16: 445–456.CrossRefGoogle Scholar
  7. 7.
    Won, W., C. Park, S. Lee, K. Lee, and J. Lee (2011) Parameter estimation and dynamic control analysis of central carbon metabolism in Escherichia coli. Biotechnol. Bioprocess Eng. 16: 216–228.CrossRefGoogle Scholar
  8. 8.
    Ajikumar, P. K., W. H. Xiao, K. E. Tyo, Y. Wang, F. Simeon, E. Leonard, O. Mucha, T. H. Phon, B. Pfeifer, and G. Stephanopoulos (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330: 70–74.CrossRefGoogle Scholar
  9. 9.
    Anderson, J. C., C. A. Voigt, and A. P. Arkin (2007) Environmental signal integration by a modular AND gate. Mol. Syst. Biol. 3: 133.CrossRefGoogle Scholar
  10. 10.
    Lee, S., E. Jeon, H. Yun, and J. Lee (2011) Improvement of fatty acid biosynthesis by engineered recombinant Escherichia coli. Biotechnol. Bioproc. Eng. 16: 706–713.CrossRefGoogle Scholar
  11. 11.
    Braatsch, S., S. Helmark, H. Kranz, B. Koebmann, and P. R. Jensen (2008) Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning. Biotechniques 45: 335–337.CrossRefGoogle Scholar
  12. 12.
    Miksch, G., F. Bettenworth, K. Friehs, E. Flaschel, A. Saalbach, T. Twellmann, and T. W. Nattkemper (2005) Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli. J. Biotechnol. 120: 25–37.CrossRefGoogle Scholar
  13. 13.
    De Mey, M., J. Maertens, G. J. Lequeux, W. K. Soetaert, and E. J. Vandamme (2007) Construction and model-based analysis of a promoter library for E. coli: An indispensable tool for metabolic engineering. BMC. Biotechnol. 7: 34.CrossRefGoogle Scholar
  14. 14.
    Rud, I., P. R. Jensen, K. Naterstad, and L. Axelsson (2006) A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiol. 152: 1011–1019.CrossRefGoogle Scholar
  15. 15.
    Jensen, P. R. and K. Hammer (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol. 64: 82–87.Google Scholar
  16. 16.
    Solem, C. and P. R. Jensen (2002) Modulation of gene expression made easy. Appl. Environ. Microbiol. 68: 2397–2403.CrossRefGoogle Scholar
  17. 17.
    Nevoigt, E., J. Kohnke, C. R. Fischer, H. Alper, U. Stahl, and G. Stephanopoulos (2006) Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 72: 5266–5273.CrossRefGoogle Scholar
  18. 18.
    Alper, H., C. Fischer, E. Nevoigt, and G. Stephanopoulos (2005) Tuning genetic control through promoter engineering. Proc. Natl. Acad. Sci. U S A 102: 12678–12683.CrossRefGoogle Scholar
  19. 19.
    Qin, X., J. Qian, G. Yao, Y. Zhuang, S. Zhang, and J. Chu (2011) GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl. Environ. Microbiol. 77: 3600–3608.CrossRefGoogle Scholar
  20. 20.
    Blazeck, J., L. Liu, H. Redden, and H. Alper (2011) Tuning gene expression in Yarrowia lipolytica using a hybrid promoter approach. Appl. Environ. Microbiol. 22: 7905–7014.CrossRefGoogle Scholar
  21. 21.
    Davis, J. H., A. J. Rubin, and R. T. Sauer (2011) Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39: 1131–1141.CrossRefGoogle Scholar
  22. 22.
    Kelly, J. R., A. J. Rubin, J. H. Davis, C. M. Ajo-Franklin, J. Cumbers, M. J. Czar, K. de Mora, A. L. Glieberman, D. D. Monie, and D. Endy (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard. J. Biol. Eng. 3: 4.CrossRefGoogle Scholar
  23. 23.
    Seo, S. W., J. Yang, and G. Y. Jung (2009) Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli. Biotechnol. Bioeng. 104: 611–616.CrossRefGoogle Scholar
  24. 24.
    Park, Y. S., S. W. Seo, S. Hwang, H. S. Chu, J. H. Ahn, T. W. Kim, D. M. Kim, and G. Y. Jung (2007) Design of 5′-untranslated region variants for tunable expression in Escherichia coli. Biochem. Biophys. Res. Commun. 356: 136–141.CrossRefGoogle Scholar
  25. 25.
    de Smit, M. H. and J. van Duin (2003) Translational standby sites: How ribosomes may deal with the rapid folding kinetics of mRNA. J. Mol. Biol. 331: 737–743.CrossRefGoogle Scholar
  26. 26.
    de Smit, M. H. and J. van Duin (1994) Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data. J. Mol. Biol. 244: 144–150.CrossRefGoogle Scholar
  27. 27.
    de Smit, M. H. and J. van Duin (1994) Translational initiation on structured messengers. Another role for the Shine-Dalgarno interaction. J. Mol. Biol. 235: 173–184.Google Scholar
  28. 28.
    de Smit, M. H. and J. van Duin (1990) Secondary structure of the ribosome binding site determines translational efficiency: A quantitative analysis. Proc. Natl. Acad. Sci. U S A 87: 7668–7672.CrossRefGoogle Scholar
  29. 29.
    Pfleger, B. F., D. J. Pitera, C. D. Smolke, and J. D. Keasling (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24: 1027–1032.CrossRefGoogle Scholar
  30. 30.
    Ochi, K. (2007) From microbial differentiation to ribosome engineering. Biosci. Biotechnol. Biochem. 71: 1373–1386.CrossRefGoogle Scholar
  31. 31.
    Ochi, K., S. Okamoto, Y. Tozawa, T. Inaoka, T. Hosaka, J. Xu, and K. Kurosawa (2004) Ribosome engineering and secondary metabolite production. Adv. Appl. Microbiol. 56: 155–184.CrossRefGoogle Scholar
  32. 32.
    Chin, J. W. (2006) Programming and engineering biological networks. Curr. Opin. Struct. Biol. 16: 551–556.CrossRefGoogle Scholar
  33. 33.
    Rackham, O. and J. W. Chin (2005) A network of orthogonal ribosome x mRNA pairs. Nat. Chem. Biol. 1: 159–166.CrossRefGoogle Scholar
  34. 34.
    Xia, X. X., Z. G. Qian, C. S. Ki, Y. H. Park, D. L. Kaplan, and S. Y. Lee (2010) Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. U S A 107: 14059–14063.CrossRefGoogle Scholar
  35. 35.
    Salis, H. M., E. A. Mirsky, and C. A. Voigt (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27: 946–950.CrossRefGoogle Scholar
  36. 36.
    Lim, H. N., Y. Lee, and R. Hussein (2011) Fundamental relationship between operon organization and gene expression. Proc. Natl. Acad. Sci. U S A 108: 10626–10631.CrossRefGoogle Scholar
  37. 37.
    Dueber, J. E., G. C. Wu, G. R. Malmirchegini, T. S. Moon, C. J. Petzold, A. V. Ullal, K. L. Prather, and J. D. Keasling (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27: 753–759.CrossRefGoogle Scholar
  38. 38.
    Tsuji, S. Y., D. E. Cane, and C. Khosla (2001) Selective proteinprotein interactions direct channeling of intermediates between polyketide synthase modules. Biochem. 40: 2326–2331.CrossRefGoogle Scholar
  39. 39.
    Roughan, P. G. (1997) Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: Evidence for substrate channelling within the chloroplast fatty acid synthase. Biochem. J. 327: 267–273.Google Scholar
  40. 40.
    Clancy, K. and C. A. Voigt (2010) Programming cells: Towards an automated ‘Genetic Compiler’ Curr. Opin. Biotechnol. 21: 572–581.CrossRefGoogle Scholar
  41. 41.
    Yoon, S. H., S. H. Lee, A. Das, H. K. Ryu, H. J. Jang, J. Y. Kim, D. K. Oh, J. D. Keasling, and S. W. Kim (2009) Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. J. Biotechnol. 140: 218–226.CrossRefGoogle Scholar
  42. 42.
    Ro, D. K., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Chang, S. T. Withers, Y. Shiba, R. Sarpong, and J. D. Keasling (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943.CrossRefGoogle Scholar
  43. 43.
    Tamsir, A., J. J. Tabor, and C. A. Voigt (2011) Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’. Nature 469: 212–215.CrossRefGoogle Scholar
  44. 44.
    Cheung, J. and W. A. Hendrickson (2010) Sensor domains of two-component regulatory systems. Curr. Opin. Microbiol. 13: 116–123.CrossRefGoogle Scholar
  45. 45.
    Clarke, E. J. and C. A. Voigt (2011) Characterization of combinatorial patterns generated by multiple two-component sensors in E. coli that respond to many stimuli. Biotechnol. Bioeng. 108: 666–675.CrossRefGoogle Scholar
  46. 46.
    Hong, H. J., M. I. Hutchings, and M. J. Buttner (2008) Vancomycin resistance VanS/VanR two-component systems. Adv. Exp. Med. Biol. 631: 200–213.CrossRefGoogle Scholar
  47. 47.
    Prohinar, P., S. A. Forst, D. Reed, I. Mandic-Mulec, and J. Weiss (2002) OmpR-dependent and OmpR-independent responses of Escherichia coli to sublethal attack by the neutrophil bactericidal/permeability increasing protein. Mol. Microbiol. 43: 1493–1504.CrossRefGoogle Scholar
  48. 48.
    Tabor, J. J., H. M. Salis, Z. B. Simpson, A. A. Chevalier, A. Levskaya, E. M. Marcotte, C. A. Voigt, and A. D. Ellington (2009) A synthetic genetic edge detection program. Cell 137: 1272–1281.CrossRefGoogle Scholar
  49. 49.
    Sinha, J., S. J. Reyes, and J. P. Gallivan (2010) Reprogramming bacteria to seek and destroy an herbicide. Nat. Chem. Biol. 6: 464–470.CrossRefGoogle Scholar
  50. 50.
    Elowitz, M. B. and S. Leibler (2000) A synthetic oscillatory network of transcriptional regulators. Nature 403: 335–338.CrossRefGoogle Scholar
  51. 51.
    Gardner, T. S., C. R. Cantor, and J. J. Collins (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.CrossRefGoogle Scholar
  52. 52.
    Lee, S. K., H. H. Chou, B. F. Pfleger, J. D. Newman, Y. Yoshikuni, and J. D. Keasling (2007) Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters. Appl. Environ. Microbiol. 73: 5711–5715.CrossRefGoogle Scholar
  53. 53.
    Cox, R. S., M. G. Surette, and M. B. Elowitz (2007) Programming gene expression with combinatorial promoters. Mol. Syst. Biol. 3: 145.Google Scholar
  54. 54.
    Lutz, R. and H. Bujard (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25: 1203–1210.CrossRefGoogle Scholar
  55. 55.
    Stock, A. M., V. L. Robinson, and P. N. Goudreau (2000) Twocomponent signal transduction. Annu. Rev. Biochem. 69: 183–215.CrossRefGoogle Scholar
  56. 56.
    Utsumi, R., R. E. Brissette, A. Rampersaud, S. A. Forst, K. Oosawa, and M. Inouye (1989) Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate. Science 245: 1246–1249.CrossRefGoogle Scholar
  57. 57.
    Farmer, W. R. and J. C. Liao (2000) Improving lycopene production in Escherichia coli by engineering metabolic control. Nat. Biotechnol. 18: 533–537.CrossRefGoogle Scholar
  58. 58.
    Nystrom, T. (1995) Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis. J. Bacteriol. 177: 5707–5710.Google Scholar
  59. 59.
    Bulter, T., S. G. Lee, W. W. Wong, E. Fung, M. R. Connor, and J. C. Liao (2004) Design of artificial cell-cell communication using gene and metabolic networks. Proc. Natl. Acad. Sci. U S A 101: 2299–2304.CrossRefGoogle Scholar
  60. 60.
    Fung, E., W. W. Wong, J. K. Suen, T. Bulter, S. G. Lee, and J. C. Liao (2005) A synthetic gene-metabolic oscillator. Nature 435: 118–122.CrossRefGoogle Scholar
  61. 61.
    Liang, J. C., R. J. Bloom, and C. D. Smolke (2011) Engineering biological systems with synthetic RNA molecules. Mol. Cell. 43: 915–926.CrossRefGoogle Scholar
  62. 62.
    Mironov, A. S., I. Gusarov, R. Rafikov, L. E. Lopez, K. Shatalin, R. A. Kreneva, D. A. Perumov, and E. Nudler (2002) Sensing small molecules by nascent RNA: A mechanism to control transcription in bacteria. Cell 111: 747–756.CrossRefGoogle Scholar
  63. 63.
    Winkler, W., A. Nahvi, and R. R. Breaker (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: 952–956.CrossRefGoogle Scholar
  64. 64.
    Winkler, W. C., A. Nahvi, A. Roth, J. A. Collins, and R. R. Breaker (2004) Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428: 281–286.CrossRefGoogle Scholar
  65. 65.
    Batey, R. T., S. D. Gilbert, and R. K. Montange (2004) Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432: 411–415.CrossRefGoogle Scholar
  66. 66.
    Suess, B., B. Fink, C. Berens, R. Stentz, and W. Hillen (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res. 32: 1610–1614.CrossRefGoogle Scholar
  67. 67.
    Winkler, W. C., S. Cohen-Chalamish, and R. R. Breaker (2002) An mRNA structure that controls gene expression by binding FMN. Proc. Natl. Acad. Sci. U S A 99: 15908–15913.CrossRefGoogle Scholar
  68. 68.
    Muranaka, N., K. Abe, and Y. Yokobayashi (2009) Mechanismguided library design and dual genetic selection of synthetic OFF riboswitches. Chembiochem. 10: 2375–2381.CrossRefGoogle Scholar
  69. 69.
    Sharma, V., Y. Nomura, and Y. Yokobayashi (2008) Engineering complex riboswitch regulation by dual genetic selection. J. Am. Chem. Soc. 130: 16310–16315.CrossRefGoogle Scholar
  70. 70.
    Nomura, Y. and Y. Yokobayashi (2007) Reengineering a natural riboswitch by dual genetic selection. J. Am. Chem. Soc. 129: 13814–13815.CrossRefGoogle Scholar
  71. 71.
    Kim, D. S., V. Gusti, S. G. Pillai, and R. K. Gaur (2005) An artificial riboswitch for controlling pre-mRNA splicing. RNA 11: 1667–1677.CrossRefGoogle Scholar
  72. 72.
    Culler, S. J., K. G. Hoff, and C. D. Smolke (2010) Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science 330: 1251–1255.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Sang Woo Seo
    • 1
  • Seong Cheol Kim
    • 2
  • Gyoo Yeol Jung
    • 1
    • 2
  1. 1.Department of Chemical EngineeringPohang University of Science and TechnologyPohangKorea
  2. 2.School of Interdisciplinary Bioscience and BioengineeringPohang University of Science and TechnologyPohangKorea

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