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  • Review Article
  • Published:

Synthetic biology to access and expand nature's chemical diversity

Nature Reviews Microbiology volume 14, pages 135–149 (2016)Cite this article

Subjects

Key Points

  • This Review covers the recent advances in synthetic biology and how these advances will affect the field of natural products.

  • There has been an emphasis on creating genetic parts, such as promoters, that generate precise levels of gene expression. The generation of large libraries of well-characterized parts and the development of biophysical and bioinformatic models to predict the behaviour of genetic parts in different organisms will aid in the transfer of biosynthetic gene clusters between hosts.

  • The capacity of DNA synthesis has exploded over the past decade and it is routine to synthesize the 20–100 kb required for a large gene cluster. In addition, new DNA assembly methods enable the rapid construction of different genetic part permutations or to substitute many genetic parts in a single step.

  • With regard to synthetic regulation, genetic circuits have been constructed that function as logic gates, timers, switches and oscillators. Sensors have also been developed that respond to many inducible inputs as well as metabolite levels. These could be incorporated into natural product pathways to control the timing of expression of different genes or to implement feedback in response to a toxic intermediate.

  • It is often desirable to make many simultaneous genomic changes. Methods such as CRISPR–Cas9 can target essentially any region of the genome and have been shown to function in many species, including several host species that are well suited for the industrial-scale production of small molecules.

Abstract

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products.

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Figure 1: Natural product biosynthetic gene clusters.
Figure 2: Genetic refactoring and genetic parts for controlling levels of gene expression.
Figure 3: Advanced regulation relevant to natural product biosynthesis.
Figure 4: Using refactored systems for genetic optimization and host transfer.
Figure 5: Multiplexed genome editing with CRISPR–Cas9.

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References

  1. Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 75, 311–335 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Demain, A. L. Importance of microbial natural products and the need to revitalize their discovery. J. Ind. Microbiol. Biotechnol. 41, 185–201 (2014).

    CAS  PubMed  Google Scholar 

  3. Davies, J. How to discover new antibiotics: harvesting the parvome. Curr. Opin. Chem. Biol. 15, 5–10 (2011).

    CAS  PubMed  Google Scholar 

  4. Röttig, M. et al. NRPSpredictor2 — a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res. 39, W362–W367 (2011).

    PubMed  PubMed Central  Google Scholar 

  5. Kim, J. & Yi, G.-S. PKMiner: a database for exploring type II polyketide synthases. BMC Microbiol. 12, 169 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Cimermancic, P. et al. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158, 412–421 (2014). This study provides a comparative analysis of 33,000 putative BGCs present in more than 1,000 sequenced bacterial and archaeal genomes. A large family of aryl polyene gene clusters was characterized as a result.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. van Heel, A. J., de Jong, A., Montalbán-López, M., Kok, J. & Kuipers, O. P. BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides. Nucleic Acids Res. 41, W448–W453 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Blin, K. et al. antiSMASH 2.0 — a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204–W212 (2013).

    PubMed  PubMed Central  Google Scholar 

  9. Medema, M. H. et al. AntiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39, W339–W346 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Weber, T. et al. antiSMASH 3.0 — a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, W237–W243 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Watve, M. G., Tickoo, R., Jog, M. M. & Bhole, B. D. How many antibiotics are produced by the genus Streptomyces? Arch. Microbiol. 176, 386–390 (2001).

    CAS  PubMed  Google Scholar 

  12. Doroghazi, J. R. et al. A roadmap for natural product discovery based on large-scale genomics and metabolomics. Nat. Chem. Biol. 10, 963–968 (2014). This study describes a multi-parameter distance metric for comparing BGCs and applies this metric to organize more than 11,000 actinobacterial BGCs into families of gene clusters.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Galm, U. et al. In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway. J. Biol. Chem. 283, 28236–28245 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Piel, J. A polyketide synthase–peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles. Proc. Natl Acad. Sci. USA 99, 14002–14007 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Galm, U. & Shen, B. Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis. Expert Opin. Drug Discov. 1, 409–437 (2006).

    CAS  PubMed  Google Scholar 

  16. Williams, G. J. Engineering polyketide synthases and nonribosomal peptide synthetases. Curr. Opin. Struct. Biol. 23, 603–612 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Pickens, L. B., Tang, Y. & Chooi, Y. H. Metabolic engineering for the production of natural products. Annu. Rev. Chem. Biomolecular Engineer. 2, 211–236 (2011).

    CAS  Google Scholar 

  18. Demain, A. L. & Adrio, J. L. Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation. Prog. Drug Res. 65, 252–289 (2008).

    Google Scholar 

  19. Voigt, C. A. Synthetic biology. ACS Synth. Biol. 1, 1–2 (2012).

    CAS  PubMed  Google Scholar 

  20. Way, J. C., Collins, J. J. & Keasling, J. D. & Silver, P. A. Integrating biological redesign: where synthetic biology came from and where it needs to go. Cell 157, 151–161 (2014).

    CAS  PubMed  Google Scholar 

  21. Medema, M. H., Breitling, R., Bovenberg, R. & Takano, E. Exploiting plug-and-play synthetic biology for drug discovery and production in microorganisms. Nat. Rev. Microbiol. 9, 131–137 (2011).

    CAS  PubMed  Google Scholar 

  22. Luo, Y., Cobb, R. E. & Zhao, H. Recent advances in natural product discovery. Curr. Opin. Biotechnol. 30, 230–237 (2014).

    CAS  PubMed  Google Scholar 

  23. Luo, Y. et al. Engineered biosynthesis of natural products in heterologous hosts. Chem. Soc. Rev. 44, 5265–5290 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Rutledge, P. J. & Challis, G. L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat. Rev. Microbiol. 13, 509–523 (2015).

    CAS  PubMed  Google Scholar 

  25. Keasling, J. D. Synthetic biology and the development of tools for metabolic engineering. Metab. Eng. 14, 189–195 (2012).

    CAS  PubMed  Google Scholar 

  26. Fischbach, M. & Voigt, C. A. Prokaryotic gene clusters: a rich toolbox for synthetic biology. Biotechnol. J. 5, 1277–1296 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Endy, D. Foundations for engineering biology. Nature 438, 449–453 (2005).

    CAS  PubMed  Google Scholar 

  28. Temme, K., Zhao, D. & Voigt, C. A. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc. Natl Acad. Sci. USA 109, 7085–7090 (2012).

    PubMed  PubMed Central  Google Scholar 

  29. Chan, L. Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005).

    PubMed  PubMed Central  Google Scholar 

  30. Shao, Z. et al. Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold. ACS Synth. Biol. 2, 662–669 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Oßwald, C. et al. Modular construction of a functional artificial epothilone polyketide pathway. ACS Synth. Biol. 3, 759–772 (2012).

    PubMed  Google Scholar 

  32. Luo, Y. et al. Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster. Nat. Commun. 4, 2894 (2013).

    PubMed  Google Scholar 

  33. Ajikumar, P. K. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330, 70–74 (2010). This study showcases the approach of multivariate modular metabolic engineering for optimizing the biosynthesis of a Taxol precursor to 1 gram per litre in E. coli.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).

    CAS  PubMed  Google Scholar 

  35. Mutalik, V. K. et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat. Methods 10, 347–353 (2013).

    CAS  PubMed  Google Scholar 

  36. Chen, Y.-J. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Methods 10, 659–664 (2013).

    CAS  PubMed  Google Scholar 

  37. Seghezzi, N., Amar, P., Koebmann, B., Jensen, P. R. & Virolle, M. J. The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters. Appl. Microbiol. Biotechnol. 90, 615–623 (2011).

    CAS  PubMed  Google Scholar 

  38. Alper, H., Fischer, C., Nevoigt, E. & Stephanopoulos, G. Tuning genetic control through promoter engineering. Proc. Natl Acad. Sci. USA 102, 12678–12683 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Farasat, I. et al. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Mol. Syst. Biol. 10, 731 (2014).

    PubMed  PubMed Central  Google Scholar 

  41. Nielsen, A. A. K., Segall-Shapiro, T. H. & Voigt, C. A. Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression. Curr. Opin. Chem. Biol. 17, 878–892 (2013).

    CAS  PubMed  Google Scholar 

  42. Kosuri, S. et al. Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proc. Natl Acad. Sci. USA 110, 14024–14029 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354–360 (2013).

    CAS  PubMed  Google Scholar 

  44. Cardinale, S. & Arkin, A. P. Contextualizing context for synthetic biology - identifying causes of failure of synthetic biological systems. Biotechnol. J. 7, 856–866 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Lou, C., Stanton, B., Chen, Y.-J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137–1142 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Bai, C. et al. Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces. Proc. Natl Acad. Sci. USA 112, 12181–12186 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Davis, J. H., Rubin, A. J. & Sauer, R. T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).

    CAS  PubMed  Google Scholar 

  48. Kalir, S. et al. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292, 2080–2083 (2001).

    CAS  PubMed  Google Scholar 

  49. Temme, K. et al. Induction and relaxation dynamics of the regulatory network controlling the type III secretion system encoded within Salmonella pathogenicity Island 1. J. Mol. Biol. 377, 47–61 (2008).

    CAS  PubMed  Google Scholar 

  50. Brophy, J. A. N. & Voigt, C. A. Principles of genetic circuit design. Nat. Methods 11, 508–520 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Liu, G., Chater, K. F., Chandra, G., Niu, G. & Tan, H. Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol. Mol. Biol. Rev. 77, 112–143 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Chatterjee, A. et al. Convergent transcription in the butyrolactone regulon in Streptomyces coelicolor confers a bistable genetic switch for antibiotic biosynthesis. PLoS ONE 6, e21974 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Sherwood, E. J. & Bibb, M. J. The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba. Proc. Natl Acad. Sci. USA 110, E2500–E2509 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Chen, Y., Smanski, M. J. & Shen, B. Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation. Appl. Microbiol. Biotechnol. 86, 19–25 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Smanski, M. J., Peterson, R. M., Rajski, S. R. & Shen, B. Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin. Antimicrob. Agents Chemother. 53, 1299–1304 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Tahlan, K. et al. Initiation of actinorhodin export in Streptomyces coelicolor. Mol. Microbiol. 63, 951–961 (2007).

    CAS  PubMed  Google Scholar 

  57. Stevens, J. T. & Carothers, J. M. Designing RNA-based genetic control systems for efficient production from engineered metabolic pathways. ACS Synth. Biol. 4, 107–115 (2015).

    CAS  PubMed  Google Scholar 

  58. Zhang, F., Carothers, J. M. & Keasling, J. D. Design of a dynamic sensor–regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol. 30, 354–359 (2012).

    CAS  PubMed  Google Scholar 

  59. Kushwaha, M. & Salis, H. M. A portable expression resource for engineering cross-species genetic circuits and pathways. Nat. Commun. 6, 7832 (2015).

    CAS  PubMed  Google Scholar 

  60. Solomon, K. V., Sanders, T. M. & Prather, K. L. J. A dynamic metabolite valve for the control of central carbon metabolism. Metab. Eng. 14, 661–671 (2012).

    CAS  PubMed  Google Scholar 

  61. Brockman, I. M. & Prather, K. L. J. Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites. Metab. Eng. 28, 104–113 (2015).

    CAS  PubMed  Google Scholar 

  62. Nieselt, K. et al. The dynamic architecture of the metabolic switch in Streptomyces coelicolor. BMC Genomics 11, 10 (2010).

    PubMed  PubMed Central  Google Scholar 

  63. Ellis, T., Wang, X. & Collins, J. J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat. Biotechnol. 27, 465–471 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    CAS  PubMed  Google Scholar 

  65. Hasty, J., McMillen, D. & Collins, J. J. Engineered gene circuits. Nature 420, 224–230 (2002).

    CAS  PubMed  Google Scholar 

  66. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013). This study describes CRISPRi, which utilizes a catalytically inactive dCas9 protein to block gene expression by RNA polymerase.

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Gilbert, L. A. et al. XCRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013). This study demonstrates that the CRISPR–dCas9 system can be used to activate gene expression by fusing transcriptional activator domains to dCas9.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Tong, Y., Charusanti, P., Zhang, L., Weber, T. & Lee, S. Y. CRISPR–Cas9 based engineering of actinomycetal genomes. ACS Synth. Biol. 4, 1020–1029 (2015).

    CAS  PubMed  Google Scholar 

  69. Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578–589 (2015).

    CAS  PubMed  Google Scholar 

  70. Lv, L., Ren, Y., Chen, J., Wu, Q. & Chen, G. Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: controllable P (3HB-co-4HB) biosynthesis. Metab. Eng. 29, 160–168 (2015).

    CAS  PubMed  Google Scholar 

  71. Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2014).

    PubMed  PubMed Central  Google Scholar 

  72. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    CAS  PubMed  Google Scholar 

  73. Sánchez, C. et al. Combinatorial biosynthesis of antitumor indolocarbazole compounds. Proc. Natl Acad. Sci. USA 102, 461–466 (2005).

    PubMed  Google Scholar 

  74. Menzella, H. G. et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat. Biotechnol. 23, 1171–1176 (2005).

    CAS  PubMed  Google Scholar 

  75. Kakule, T. B., Lin, Z. & Schmidt, E. W. Combinatorialization of fungal polyketide synthase–peptide synthetase hybrid proteins J. Am. Chem. Soc. 136, 17882–17890 (2014).

    CAS  PubMed  Google Scholar 

  76. Nguyen, K. T. et al. Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc. Natl Acad. Sci. USA 103, 17462–17467 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Ru, D. E., Schmidt, E. W. & Heemstra, J. R. Assessing the combinatorial potential of the RiPP cyanobactin tru pathway. ACS Synth. Biol. 4, 482–492 (2015). The authors use high-throughput mutagenesis and analytical chemistry to create more than 300 structural variants of the cyanobacterial RiPP, trunkamide. The data were analysed to establish rules for amino acid preference at various positions along the core scaffold.

    Google Scholar 

  79. Mitchell, D. A. et al. Structural and functional dissection of the heterocyclic peptide cytotoxin streptolysin S. J. Biol. Chem. 284, 13004–13012 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat. Biotechnol. 32, 1241–1249 (2014).

    CAS  PubMed  Google Scholar 

  81. Appleton, E., Tao, J., Haddock, T. & Densmore, D. Interactive assembly algorithms for molecular cloning. Nat. Methods 11, 657–662 (2014).

    CAS  PubMed  Google Scholar 

  82. Freestone, T. S. & Zhao, H. Combinatorial pathway engineering for optimized production of the anti-malarial FR900098. Biotechnol. Bioeng. 113, 384–392 (2015).

    PubMed  Google Scholar 

  83. Biggs, B. W., De Paepe, B., Santos, C. N. S., De Mey, M. & Ajikumar, P. K. Multivariate modular metabolic engineering for pathway and strain optimization. Curr. Opin. Biotechnol. 29, 156–162 (2014).

    CAS  PubMed  Google Scholar 

  84. Thodey, K., Galanie, S. & Smolke, C. D. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat. Chem. Biol. 10, 837–844 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Li, L. et al. A stepwise increase in pristinamycin II biosynthesis by Streptomyces pristinaespiralis through combinatorial metabolic engineering. Metab. Eng. 29, 12–25 (2015).

    PubMed  Google Scholar 

  86. Zhao, S. et al. Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metab. Eng. 28, 43–53 (2015).

    CAS  PubMed  Google Scholar 

  87. Du, Y.-L. & Ryan, K. S. Expansion of bisindole biosynthetic pathways by combinatorial construction. ACS Synth. Biol. 4, 682–688 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Chemler, J. a. et al. Evolution of efficient modular polyketide synthases by homeologous recombination. J. Am. Chem. Soc. 137, 10603–10609 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Smanski, M. J. et al. Expression of the platencin biosynthetic gene cluster in heterologous hosts yielding new platencin congeners. J. Nat. Prod. 75, 2158–2167 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Wang, B., Kitney, R. I., Joly, N. & Buck, M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Commun. 2, 508 (2011).

    PubMed  Google Scholar 

  91. Hajimorad, M., Gray, P. R. & Keasling, J. D. A framework and model system to investigate linear system behavior in Escherichia coli. J. Biol. Eng. 5, 3 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Moser, F. et al. Genetic circuit performance under conditions relevant for industrial bioreactors. ACS Synth. Biol. 1, 555–564 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Siegl, T., Tokovenko, B., Myronovskyi, M. & Luzhetskyy, A. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metab. Eng. 19, 98–106 (2013).

    CAS  PubMed  Google Scholar 

  94. Sarrion-Perdigones, A. et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Zhang, Y., Perry, K., Vinci, V. & Powell, K. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 5–7 (2002).

    CAS  Google Scholar 

  96. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    CAS  PubMed  Google Scholar 

  97. Warner, J. R., Reeder, P. J., Karimpour-Fard, A., Woodruff, L. B. A. & Gill, R. T. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat. Biotechnol. 28, 856–862 (2010).

    CAS  PubMed  Google Scholar 

  98. Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591–593 (2012).

    PubMed  PubMed Central  Google Scholar 

  100. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Wang, H. H. & Church, G. M. Multiplexed genome engineering and genotyping methods: applications for synthetic biology and metabolic engineering. Methods Enzymol. 498, 409–426 (2011).

    CAS  PubMed  Google Scholar 

  102. Wang, H. H. et al. Multiplexed in vivo his-tagging of enzyme pathways for in vitro single-pot multienzyme catalysis. ACS Synth. Biol. 1, 43–52 (2012).

    PubMed  PubMed Central  Google Scholar 

  103. Bonde, M. T. et al. Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides. ACS Synth. Biol. 4, 17–22 (2015). In this study, the authors develop a workflow for the integration of microarray-based DNA synthesis with MAGE and apply their approach to introduce T7 promoters upstream of 2,500 operons in the E. coli genome.

    CAS  PubMed  Google Scholar 

  104. Binder, S., Siedler, S., Marienhagen, J., Bott, M. & Eggeling, L. Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res. 41, 6360–6369 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Dicarlo, J. E. et al. Yeast oligo-mediated genome engineering (YOGE). ACS Synth. Biol. 2, 741–749 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Sandoval, N. R. et al. Strategy for directing combinatorial genome engineering in Escherichia coli. Proc. Natl Acad. Sci. USA 109, 10540–10545 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Belhaj, K., Chaparro-Garcia, A., Kamoun, S. & Nekrasov, V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9, 39 (2013).

    PubMed  PubMed Central  Google Scholar 

  108. Cobb, R. E., Wang, Y. & Zhao, H. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2014). This is the first study to demonstrate CRISPR–Cas9 genome editing in Streptomyces spp.; the authors achieve up to 100% editing efficiency for the deletion of single genes or whole gene clusters.

    PubMed  PubMed Central  Google Scholar 

  109. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Jakočiūnas, T. et al. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metab. Eng. 28, 213–222 (2015).

    PubMed  Google Scholar 

  112. Huang, H., Zheng, G., Jiang, W., Hu, H. & Lu, Y. One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim. Biophys. Sin. (Shanghai). 47, 231–243 (2015).

    CAS  PubMed  Google Scholar 

  113. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    CAS  PubMed  Google Scholar 

  114. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1–9 (2014).

    Google Scholar 

  115. Bao, Z., Cobb, R. E. & Zhao, H. Accelerated genome engineering through multiplexing. Wiley Interdiscip. Rev. Syst. Biol. Med. 8, 5–21 (2015).

    PubMed  PubMed Central  Google Scholar 

  116. Chen, C., Fenk, L. a. & De Bono, M. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination. Nucleic Acids Res. 41, e193 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. Michener, J. K., Thodey, K., Liang, J. C. & Smolke, C. D. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. Metab. Eng. 14, 212–222 (2012).

    CAS  PubMed  Google Scholar 

  118. An, G. H., Bielich, J., Auerbach, R. & Johnson, E. A. Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting. Biotechnology 9, 70–73 (1991).

    CAS  PubMed  Google Scholar 

  119. Cho, E. J., Lee, J.-W. & Ellington, A. D. Applications of aptamers as sensors. Annu. Rev. Anal. Chem. (Palo Alto. Calif.). 2, 241–264 (2009).

    CAS  Google Scholar 

  120. Wachsmuth, M., Findeiss, S., Weissheimer, N., Stadler, P. F. & Morl, M. De novo design of a synthetic riboswitch that regulates transcription termination. Nucleic Acids Res. 41, 2541–2551 (2012).

    PubMed  PubMed Central  Google Scholar 

  121. Weigand, J. E. & Suess, B. Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast. Nucleic Acids Res. 35, 4179–4185 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Michener, J. K. & Smolke, C. D. High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch. Metab. Eng. 14, 306–316 (2012).

    CAS  PubMed  Google Scholar 

  123. Weigand, J. E. et al. Screening for engineered neomycin riboswitches that control translation initiation. RNA 14, 89–97 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Schoukroun-Barnes, L. R., Wagan, S. & White, R. J. Enhancing the analytical performance of electrochemical RNA aptamer-based sensors for sensitive detection of aminoglycoside antibiotics. Anal. Chem. 86, 1131–1137 (2014).

    CAS  PubMed  Google Scholar 

  125. Farjami, E. et al. RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine. Anal. Chem. 85, 121–128 (2013).

    CAS  PubMed  Google Scholar 

  126. Chen, J., Fang, Z., Liu, J. & Zeng, L. A simple and rapid biosensor for ochratoxin A based on a structure-switching signaling aptamer. Food Control 25, 555–560 (2012).

    CAS  Google Scholar 

  127. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Grkovic, S., Hardie, K. M., Brown, M. H. & Skurray, R. A. Interactions of the QacR multidrug-binding protein with structurally diverse ligands: implications for the evolution of the binding pocket. Biochemistry 42, 15226–15236 (2003).

    CAS  PubMed  Google Scholar 

  129. Ramos, J. L. et al. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 69, 326–356 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Mohn, W. W., Garmendia, J., Galvao, T. C. & De Lorenzo, V. Surveying biotransformations with à la carte genetic traps: translating dehydrochlorination of lindane (γ-hexachlorocyclohexane) into lacZ-based phenotypes. Environ. Microbiol. 8, 546–555 (2006).

    CAS  PubMed  Google Scholar 

  131. Mustafi, N., Grünberger, A., Kohlheyer, D., Bott, M. & Frunzke, J. The development and application of a single-cell biosensor for the detection of L-methionine and branched-chain amino acids. Metab. Eng. 14, 449–457 (2012).

    CAS  PubMed  Google Scholar 

  132. Tang, S. Y. & Cirino, P. C. Design and application of a mevalonate-responsive regulatory protein. Angew. Chem. Int. Ed. Engl. 50, 1084–1086 (2011).

    CAS  PubMed  Google Scholar 

  133. Tang, S. Y. et al. Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J. Am. Chem. Soc. 135, 10099–10103 (2013).

    CAS  PubMed  Google Scholar 

  134. Buskirk, A. R., Ong, Y.-C., Gartner, Z. J. & Liu, D. R. Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc. Natl Acad. Sci. USA 101, 10505–10510 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Peck, S. H., Chen, I. & Liu, D. R. Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells. Chem. Biol. 18, 619–630 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. DeLoache, W. C. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 11, 465–471 (2015).

    CAS  PubMed  Google Scholar 

  137. Gandía-Herrero, F. & García-Carmona, F. Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci. 18, 334–343 (2013).

    PubMed  Google Scholar 

  138. Meyer, A. et al. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat. Chem. 7, 673–678 (2015).

    CAS  PubMed  Google Scholar 

  139. Pfleger, B. F., Pitera, D. J., Newman, J. D., Martin, V. J. J. & Keasling, J. D. Microbial sensors for small molecules: development of a mevalonate biosensor. Metab. Eng. 9, 30–38 (2007).

    CAS  PubMed  Google Scholar 

  140. Bertels, F., Merker, H. & Kost, C. Design and characterization of auxotrophy-based amino acid biosensors. PLoS ONE 7, e41349 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Bugni, T. S. et al. Marine natural product libraries for high-throughput screening and rapid drug discovery. J. Nat. Prod. 71, 1095–1098 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Kotula, J. W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl Acad. Sci. USA 111, 4838–4843 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Claesen, J. & Fischbach, M. A. Synthetic microbes as drug delivery systems. ACS Synth. Biol. 4, 358–364 (2015).

    CAS  PubMed  Google Scholar 

  144. Fischbach, M. A., Bluestone, J. A. & Lim, W. A. Cell-based therapeutics: the next pillar of medicine. Sci. Transl. Med. 5, 179ps7 (2013).

    PubMed  PubMed Central  Google Scholar 

  145. Qian, P.-Y., Xu, Y. & Fusetani, N. Natural products as antifouling compounds: recent progress and future perspectives. Biofouling 26, 223–234 (2010).

    CAS  PubMed  Google Scholar 

  146. Yoshino, T. & Matsunaga, T. Development of efficient expression system for protein display on bacterial magnetic particles. Biochem. Biophys. Res. Commun. 338, 1678–1681 (2005).

    CAS  PubMed  Google Scholar 

  147. Dayan, F. E., Cantrell, C. L. & Duke, S. O. Natural products in crop protection. Bioorg. Med. Chem. 17, 4022–4034 (2009).

    CAS  PubMed  Google Scholar 

  148. Zimmermann, M. & Fischbach, M. A. A family of pyrazinone natural products from a conserved nonribosomal peptide synthetase in Staphylococcus aureus. Chem. Biol. 17, 925–930 (2010).

    CAS  PubMed  Google Scholar 

  149. Smanski, M. J. et al. Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis. Proc. Natl Acad. Sci. USA 108, 13498–13503 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Sudek, S. et al. Identification of the putative bryostatin polyketide synthase gene cluster from 'Candidatus Endobugula sertula', the uncultivated microbial symbiont of the marine bryozoan Bugula neritina. J. Nat. Prod. 70, 67–74 (2007).

    CAS  PubMed  Google Scholar 

  151. Wong, F. T. & Khosla, C. Combinatorial biosynthesis of polyketides — a perspective. Curr. Opin. Chem. Biol. 16, 117–123 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  152. Medema, M. H. Minimum information about a biosynthetic gene cluster. Nat. Chem. Biol. 11, 625–631 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  153. Carlson, R. Time for new DNA synthesis and sequencing cost curves. Synthesis [online], (2014).

    Google Scholar 

  154. Gustafsson, C., Govindarajan, S. & Minshull, J. Codon bias and heterologous protein expression. Trends Biotechnol. 22, 346–353 (2004).

    CAS  PubMed  Google Scholar 

  155. Rodríguez-García, A., Combes, P., Pérez-Redondo, R., Smith, M. C. A. & Smith, M. C. M. Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces. Nucleic Acids Res. 33, e87 (2005).

    PubMed  PubMed Central  Google Scholar 

  156. Stanton, B. C. et al. Systematic transfer of prokaryotic sensors and circuits to mammalian cells. ACS Synth. Biol. 19, 880–891 (2014).

    Google Scholar 

  157. Selle, K. & Barrangou, R. Harnessing CRISPR–Cas systems for bacterial genome editing. Trends Microbiol. 23, 225–232 (2015).

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology, and Biophysics and the BioTechnology Institute, University of Minnesota Twin Cities, Saint Paul, 55108, Minnesota, USA

    Michael J. Smanski

  2. Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Michael J. Smanski, Hui Zhou & Christopher A. Voigt

  3. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, 94158, California, USA

    Jan Claesen & Michael A. Fischbach

  4. Departments of Chemistry and Molecular Therapeutics, The Scripps Research Institute, Jupiter, 33458, Florida, USA

    Ben Shen

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  1. Michael J. Smanski
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  4. Ben Shen
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  5. Michael A. Fischbach
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Correspondence to Christopher A. Voigt.

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PowerPoint slides

Glossary

Polyketide

One of a family of natural products that share a common biosynthetic pathway through the decarboxylative condensation of substituted malonyl-CoA-derived extender units and acyl-CoA starter units on polyketide synthase enzymes.

Non-ribosomal peptide

One of a family of natural products that share a common biosynthetic pathway through the condensation of proteogenic or non-proteogenic amino acids on modular non-ribosomal peptide synthetase enzymes.

Terpene

One of a family of natural products that share a common biosynthetic pathway through the polymerization of branched five-carbon isoprene units and cyclization by terpene synthases.

Ribosomally synthesized and post-translationally modified peptide

(RiPP). One of a family of natural products, including the lanthipeptides, bacteriocins, and thiazole-modified or oxazole-modified microcins, that share a common biosynthetic pathway through the translation of an mRNA-encoded core peptide and subsequent modification.

Random chemical mutagenesis

A process by which cells or organisms are exposed to chemical mutagens to introduce mutations at random locations in the genome.

5′ untranslated region

(5′ UTR). The untranslated region of an mRNA transcript that is upstream of the start codon. The sequence of the 5′ UTR can influence translation initiation and mRNA stability.

Bicistrons

Transcripts that contain two coding DNA sequences (CDSs). For translational control, the first CDS encodes a short, non-functional peptide and is located immediately upstream of the ribosome binding site for the second CDS.

Actinorhodin

A benzoisochromanequinone polyketide pigment produced by Streptomyces coelicolor.

para-aminostyrene

An industrially relevant vinyl aromatic monomer with applications in materials and biomedicine.

Undecylprodigiosin

A tripyrrole polyketide pigment produced by Streptomyces coelicolor.

Gibson assembly

A restriction-enzyme-independent method for the joining of several DNA fragments in a single isothermal reaction.

Pristinamycin II

One of two structurally unrelated chemical components of the clinical antibiotic pristinamycin. Pristinamycin II is a depsipeptide antibiotic produced by Streptomyces pristinaespiralis.

Catechin

A plant flavonoid with antioxidant properties.

Indolocarbazoles

A family of five-ring heterocyclic aromatic compounds that share a common biosynthetic pathway from two tryptophan molecules.

Platencin

An antibiotic produced by Streptomyces platensis containing an amino-dihydroxybenzoic acid moiety fused to a modified diterpene core.

Shunt metabolites

Chemically modified intermediates of a biosynthetic pathway that can no longer proceed through the biosynthetic pathway.

Tylosin

A glycosylated macrolide antibiotic produced by Streptomyces venezuelae.

Okazaki fragments

Short, newly synthesized single-stranded DNA oligomers that are formed on the lagging template strand during DNA replication.

Lycopene

A symmetrical tetraterpene pigment formed by the tail-to-tail condensation of two molecules of geranylgeranyl diphosphate.

T7 RNA polymerase promoters

Short DNA sequences that are recognized by T7 RNA polymerase to initiate transcription.

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Smanski, M., Zhou, H., Claesen, J. et al. Synthetic biology to access and expand nature's chemical diversity. Nat Rev Microbiol 14, 135–149 (2016). https://doi.org/10.1038/nrmicro.2015.24

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