Abstract
As synthetic regulatory programs expand in sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often ‘part mined’ from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. Fourteen repressor variants were evolved with unique regulatory profiles across five operators and three ligands. The logic of individual repressors can be modularly programmed by creating heterodimeric fusions, resulting in single proteins that display logic functions, such as ‘NAND’. Despite the evolutionarily limited regulatory role of TrpR, vast functional spaces exist around this highly conserved protein scaffold and can be harnessed to create synthetic regulatory programs.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
López-Maury, L., Marguerat, S. & Bähler, J. Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation. Nat. Rev. Genet. 9, 583–593 (2008).
Kiel, C., Yus, E. & Serrano, L. Engineering signal transduction pathways. Cell 140, 33–47 (2010).
Motlagh, H. N., Wrabl, J. O., Li, J. & Hilser, V. J. The ensemble nature of allostery. Nature 508, 331–339 (2014).
Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–1134 (2005).
Chen, Y., Kim, J. K., Hirning, A. J., Josić, K. & Bennett, M. R. Emergent genetic oscillations in a synthetic microbial consortium. Science 349, 986–989 (2015).
Raman, S., Rogers, J. K., Taylor, N. D. & Church, G. M. Evolution-guided optimization of biosynthetic pathways. Proc. Natl. Acad. Sci. USA 111, 17803–17808 (2014).
Tabor, J. J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).
Moon, T. S., Lou, C., Tamsir, A., Stanton, B. C. & Voigt, C. A. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012).
Stanton, B. C. et al. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat. Chem. Biol. 10, 99–105 (2014).
Bennett, G. N. & Yanofsky, C. Sequence analysis of operator constitutive mutants of the tryptophan operon of Escherichia coli. J. Mol. Biol. 121, 179–192 (1978).
Gunsalus, R. P. & Yanofsky, C. Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc. Natl. Acad. Sci. USA 77, 7117–7121 (1980).
Squires, C. L., Lee, F. D. & Yanofsky, C. Interaction of the trp repressor and RNA polymerase with the trp operon. J. Mol. Biol. 92, 93–111 (1975).
Zhang, R. G. et al. The crystal structure of trp aporepressor at 1.8 A shows how binding tryptophan enhances DNA affinity. Nature 327, 591–597 (1987).
Otwinowski, Z. et al. Crystal structure of trp repressor/operator complex at atomic resolution. Nature 335, 321–329 (1988).
Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. The three-dimensional structure of trp repressor. Nature 317, 782–786 (1985).
Yanofsky, C., Kelley, R. L. & Horn, V. Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J. Bacteriol. 158, 1018–1024 (1984).
Yanofsky, C. Attenuation in the control of expression of bacterial operons. Nature 289, 751–758 (1981).
Luisi, B. F. & Sigler, P. B. The stereochemistry and biochemistry of the trp repressor-operator complex. Biochim. Biophys. Acta 1048, 113–126 (1990).
Arvidson, D. N., Bruce, C. & Gunsalus, R. P. Interaction of the Escherichia coli trp aporepressor with its ligand, l-tryptophan. J. Biol. Chem. 261, 238–243 (1986).
Xie, G., Keyhani, N. O., Bonner, C. A. & Jensen, R. A. Ancient origin of the tryptophan operon and the dynamics of evolutionary change. Microbiol. Mol. Biol. Rev. 67, 303–342 (2003).
Madan Babu, M. & Teichmann, S. A. Evolution of transcription factors and the gene regulatory network in Escherichia coli. Nucleic Acids Res. 31, 1234–1244 (2003).
Arvidson, D. N. et al. The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae. Nucleic Acids Res. 22, 1821–1829 (1994).
Manson, M. D. & Yanofsky, C. Tryptophan operon regulation in interspecific hybrids of enteric bacteria. J. Bacteriol. 126, 679–689 (1976).
Ellefson, J. W. et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat. Biotechnol. 32, 97–101 (2014).
Maranhao, A. C. & Ellington, A. D. Evolving orthogonal suppressor tRNAs to incorporate modified amino acids. ACS Synth. Biol. 6, 108–119 (2017).
Yanofsky, C. et al. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 9, 6647–6668 (1981).
Hurlburt, B. K. & Yanofsky, C. Enhanced operator binding by trp superrepressors of Escherichia coli. J. Biol. Chem. 265, 7853–7858 (1990).
Zhang, H. et al. The solution structures of the trp repressor-operator DNA complex. J. Mol. Biol. 238, 592–614 (1994).
Marmorstein, R. Q. & Sigler, P. B. Stereochemical effects of l-tryptophan and its analogues on trp repressor’s affinity for operator-DNA. J. Biol. Chem. 264, 9149–9154 (1989).
Rogers, J. K. et al. Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res. 43, 7648–7660 (2015).
Hurlburt, B. K. & Yanofsky, C. trp repressor/trp operator interaction. Equilibrium and kinetic analysis of complex formation and stability. J. Biol. Chem. 267, 16783–16789 (1992).
Yang, J. et al. In vivo and in vitro studies of TrpR-DNA interactions. J. Mol. Biol. 258, 37–52 (1996).
Carey, J. Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. Proc. Natl. Acad. Sci. USA 85, 975–979 (1988).
Bass, S., Sorrells, V. & Youderian, P. Mutant Trp repressors with new DNA-binding specificities. Science 242, 240–245 (1988).
Czernik, P. J., Shin, D. S. & Hurlburt, B. K. Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli. J. Biol. Chem. 269, 27869–27875 (1994).
Shao, X., Hensley, P. & Matthews, C. R. Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein. Biochemistry 36, 9941–9949 (1997).
De Croos, P. Z. et al. Hemoglobin S antigelation agents based on 5-bromotryptophan with potential for sickle cell anemia. J. Med. Chem. 33, 3138–3142 (1990).
Bush, J. A., Long, B. H., Catino, J. J., Bradner, W. T. & Tomita, K. Production and biological activity of rebeccamycin, a novel antitumor agent. J. Antibiot. (Tokyo) 40, 668–678 (1987).
Craig, A. G. et al. A novel post-translational modification involving bromination of tryptophan. Identification of the residue, l-6-bromotryptophan, in peptides from Conus imperialis and Conus radiatus venom. J. Biol. Chem. 272, 4689–4698 (1997).
Menon, B. R. K. et al. Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes. Org. Biomol. Chem. 14, 9354–9361 (2016).
Dietrich, J. A., McKee, A. E. & Keasling, J. D. High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu. Rev. Biochem. 79, 563–590 (2010).
Burg, J. M. et al. Large-scale bioprocess competitiveness: the potential of dynamic metabolic control in two-stage fermentations. Curr. Opin. Chem. Eng. 14, 121–136 (2016).
Anesiadis, N., Cluett, W. R. & Mahadevan, R. Dynamic metabolic engineering for increasing bioprocess productivity. Metab. Eng. 10, 255–266 (2008).
Brophy, J. A. N. & Voigt, C. A. Principles of genetic circuit design. Nat. Methods 11, 508–520 (2014).
Nevozhay, D., Adams, R. M., Van Itallie, E., Bennett, M. R. & Balázsi, G. Mapping the environmental fitness landscape of a synthetic gene circuit. PLOS Comput. Biol. 8, e1002480 (2012).
Lawrence, J. G. & Ochman, H. Molecular archaeology of the Escherichia coli genome. Proc. Natl. Acad. Sci. USA 95, 9413–9417 (1998).
Schell, M. A. Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47, 597–626 (1993).
Maddocks, S. E. & Oyston, P. C. F. Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154, 3609–3623 (2008).
Top, E. M. & Springael, D. The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds. Curr. Opin. Biotechnol. 14, 262–269 (2003).
Pribnow, D. Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J. Mol. Biol. 99, 419–443 (1975).
Kelley, R. L. & Yanofsky, C. Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. Proc. Natl. Acad. Sci. USA 82, 483–487 (1985).
Bass, S., Sugiono, P., Arvidson, D. N., Gunsalus, R. P. & Youderian, P. DNA specificity determinants of Escherichia coli tryptophan repressor binding. Genes Dev. 1, 565–572 (1987).
Pabo, C. O. & Sauer, R. T. Protein-DNA recognition. Annu. Rev. Biochem. 53, 293–321 (1984).
Günes, C. & Müller-Hill, B. Mutants in position 69 of the Trp repressor of Escherichia coli K12 with altered DNA-binding specificity. Mol. Gen. Genet. 251, 338–346 (1996).
Lawson, C. L. & Carey, J. Tandem binding in crystals of a trp repressor/operator half-site complex. Nature 366, 178–182 (1993).
Acknowledgements
We would like to thank funding from the Air Force Office of Scientific Research (FA9550-14-1-0089) and the Welch Foundation (F-1654). M.P.L. was supported by a National Science Foundation Graduate Research Fellowship (Grant No. NSF/DGE-1346837).
Author information
Authors and Affiliations
Contributions
J.W.E. conceived the project and performed all experiments with assistance from M.P.L. A.D.E., M.P.L., and J.W.E. wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9, Supplementary Tables 1 and 2, and Supplementary Note
Rights and permissions
About this article
Cite this article
Ellefson, J.W., Ledbetter, M.P. & Ellington, A.D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nat Chem Biol 14, 361–367 (2018). https://doi.org/10.1038/s41589-018-0006-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41589-018-0006-7
This article is cited by
-
Latent generative landscapes as maps of functional diversity in protein sequence space
Nature Communications (2023)
-
Customizing cellular signal processing by synthetic multi-level regulatory circuits
Nature Communications (2023)
-
Robust and flexible platform for directed evolution of yeast genetic switches
Nature Communications (2021)
-
L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering: progress and prospects
Amino Acids (2021)
-
Engineered systems of inducible anti-repressors for the next generation of biological programming
Nature Communications (2020)