Perspective | Published:

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

Nature Chemical Biology volume 11, pages 649659 (2015) | Download Citation

  • An Erratum to this article was published on 20 October 2015

This article has been updated

Abstract

Natural products continue to play a pivotal role in drug-discovery efforts and in the understanding of human health. The ability to extend nature's chemistry through combinatorial biosynthesis—altering functional groups, regiochemistry and scaffold backbones through the manipulation of biosynthetic enzymes—offers unique opportunities to create natural product analogs. Incorporating emerging synthetic biology techniques has the potential to further accelerate the refinement of combinatorial biosynthesis as a robust platform for the diversification of natural chemical drug leads. Two decades after the field originated, we discuss the current limitations, the realities and the state of the art of combinatorial biosynthesis, including the engineering of substrate specificity of biosynthetic enzymes and the development of heterologous expression systems for biosynthetic pathways. We also propose a new perspective for the combinatorial biosynthesis of natural products that could reinvigorate drug discovery by using synthetic biology in combination with synthetic chemistry.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 23 September 2015

    In the version of this article initially published, there were four typographical errors in the abstract and main text. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. 1.

    et al. Production of 'hybrid' antibiotics by genetic engineering. Nature 314, 642–644 (1985).

  2. 2.

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

  3. 3.

    & Chemo- and site-selective derivatizations of natural products enabling biological studies. Nat. Prod. Rep. 31, 318–334 (2014).

  4. 4.

    & Drug discovery and natural products: end of an era or an endless frontier? Science 325, 161–165 (2009).

  5. 5.

    , , , & Recent advances in the biochemistry of spinosyns. Appl. Microbiol. Biotechnol. 82, 13–23 (2009).

  6. 6.

    The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Edn Engl. 48, 4688–4716 (2009).

  7. 7.

    , & Nonribosomal peptides: from genes to products. Nat. Prod. Rep. 20, 275–287 (2003).

  8. 8.

    et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products. Proc. Natl. Acad. Sci. USA 96, 1846–1851 (1999).

  9. 9.

    , , & Engineered biosynthesis of novel polyketides. Science 262, 1546–1550 (1993).

  10. 10.

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

  11. 11.

    Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways. ACS Synth. Biol. 3, 748–758 (2014).

  12. 12.

    , , & Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line. Angew. Chem. Int. Edn Engl. 53, 1560–1564 (2014).

  13. 13.

    , , , & Heterologous expression and manipulation of three tetracycline biosynthetic pathways. Angew. Chem. Int. Edn Engl. 51, 11136–11140 (2012).

  14. 14.

    & Beyond ethylmalonyl-CoA: the functional role of crotonyl-CoA carboxylase/reductase homologs in expanding polyketide diversity. Nat. Prod. Rep. 29, 72–86 (2012).

  15. 15.

    & Ribosome-independent biosynthesis of biologically active peptides: Application of synthetic biology to generate structural diversity. FEBS Lett. 586, 2065–2075 (2012).

  16. 16.

    , & Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of combinatorial biosynthesis. Nat. Prod. Rep. 27, 571–616 (2010).

  17. 17.

    et al. Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine. Proc. Natl. Acad. Sci. USA 106, 12295–12300 (2009).

  18. 18.

    et al. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J. Am. Chem. Soc. 133, 976–985 (2011).

  19. 19.

    , & De novo biosynthesis of terminal alkyne–labeled natural products. Nat. Chem. Biol. 11, 115–120 (2015).

  20. 20.

    et al. Multiplexing of combinatorial chemistry in antimycin biosynthesis: expansion of molecular diversity and utility. Angew. Chem. Int. Edn Engl. 52, 12308–12312 (2013).

  21. 21.

    et al. Enzyme-directed mutasynthesis: a combined experimental and theoretical approach to substrate recognition of a polyketide synthase. ACS Chem. Biol. 8, 443–450 (2013).

  22. 22.

    et al. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases. ACS Chem. Biol. 8, 200–208 (2013).

  23. 23.

    et al. Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity. Angew. Chem. Int. Edn Engl. 51, 7181–7184 (2012).

  24. 24.

    et al. Reprogramming nonribosomal peptide synthetases for “clickable” amino acids. Angew. Chem. Int. Edn Engl. 53, 10105–10108 (2014).

  25. 25.

    , , , & Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo. Chem. Biol. 18, 601–607 (2011).

  26. 26.

    Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 27, 996–1047 (2010).

  27. 27.

    et al. Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways. Science 341, 1089–1094 (2013).

  28. 28.

    & Engineering the acyltransferase substrate specificity of assembly line polyketide synthases. J. R. Soc. Interface 10, 20130297 (2013).

  29. 29.

    , , , & Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Proc. Natl. Acad. Sci. USA 104, 11951–11956 (2007).

  30. 30.

    , , & Crystal structure of the termination module of a nonribosomal peptide synthetase. Science 321, 659–663 (2008).

  31. 31.

    et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014).

  32. 32.

    et al. Structural rearrangements of a polyketide synthase module during its catalytic cycle. Nature 510, 560–564 (2014).

  33. 33.

    , & Probing the phosphopantetheine arm conformations of acyl carrier proteins using vibrational spectroscopy. J. Am. Chem. Soc. 136, 11240–11243 (2014).

  34. 34.

    et al. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria. J. Am. Chem. Soc. 135, 1155–1162 (2013).

  35. 35.

    et al. Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation. Proc. Natl. Acad. Sci. USA 109, 4110–4115 (2012).

  36. 36.

    , , & Dissecting and exploiting intermodular communication in polyketide synthases. Science 284, 482–485 (1999).

  37. 37.

    et al. The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 723–731 (2003).

  38. 38.

    & Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proc. Natl. Acad. Sci. USA 101, 15585–15590 (2004).

  39. 39.

    et al. Cyanobacterial polyketide synthase docking domains: a tool for engineering natural product biosynthesis. Chem. Biol. 20, 1340–1351 (2013).

  40. 40.

    , , & Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat. Prod. Rep. 30, 1121–1138 (2013).

  41. 41.

    , & Metagenomic small molecule discovery methods. Curr. Opin. Microbiol. 19, 70–75 (2014).

  42. 42.

    , & Microbial genome mining for accelerated natural products discovery: is a renaissance in the making? J. Ind. Microbiol. Biotechnol. 41, 175–184 (2014).

  43. 43.

    & Opportunities for synthetic biology in antibiotics: expanding glycopeptide chemical diversity. ACS Synth. Biol. 4, 195–206 (2015).

  44. 44.

    , & DNA assembly techniques for next-generation combinatorial biosynthesis of natural products. J. Ind. Microbiol. Biotechnol. 41, 469–477 (2014).

  45. 45.

    Programming biological operating systems: genome design, assembly and activation. Nat. Methods 11, 521–526 (2014).

  46. 46.

    , , & DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

  47. 47.

    et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440–446 (2012).

  48. 48.

    & TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat. Rev. Genet. 7, 805–812 (2006).

  49. 49.

    , & Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster. J. Am. Chem. Soc. 132, 11902–11903 (2010).

  50. 50.

    , & Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites. Proc. Natl. Acad. Sci. USA 108, 12629–12634 (2011).

  51. 51.

    et al. Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. USA 111, 1957–1962 (2014).

  52. 52.

    , , , & PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 100, 1541–1546 (2003).

  53. 53.

    , & Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler. Mol. Biosyst. 7, 1056–1059 (2011).

  54. 54.

    et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  55. 55.

    & SLIC: a method for sequence- and ligation-independent cloning. Methods Mol. Biol. 852, 51–59 (2012).

  56. 56.

    et al. Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angew. Chem. Int. Edn Engl. 51, 10664–10669 (2012).

  57. 57.

    et al. Microbial biosynthesis of medicinally important plant secondary metabolites. Nat. Prod. Rep. 31, 1497–1509 (2014).

  58. 58.

    & Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb. Biotechnol. 4, 207–215 (2011).

  59. 59.

    , , , & Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl. Acad. Sci. USA 107, 2646–2651 (2010).

  60. 60.

    et al. Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor. FEMS Microbiol. Lett. 333, 169–179 (2012).

  61. 61.

    et al. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites. ACS Synth. Biol. 2, 384–396 (2013).

  62. 62.

    et al. Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains. J. Ind. Microbiol. Biotechnol. 30, 480–488 (2003).

  63. 63.

    , , & High titer production of tetracenomycins by heterologous expression of the pathway in a Streptomyces cinnamonensis industrial monensin producer strain. Metab. Eng. 11, 319–327 (2009).

  64. 64.

    , , , & Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001).

  65. 65.

    , , , & Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host. Chem. Biol. 17, 1232–1240 (2010).

  66. 66.

    et al. Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli. Nat. Chem. Biol. 2, 423–428 (2006).

  67. 67.

    et al. Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. ChemBioChem 13, 1946–1952 (2012).

  68. 68.

    , , & Targeted capture and heterologous expression of the Pseudoalteromonas alterochromide gene cluster in Escherichia coli represents a promising natural product exploratory platform. ACS Synth. Biol. 4, 414–420 (2015).

  69. 69.

    , , & Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli. ACS Synth. Biol. 4, 559–565 (2015).

  70. 70.

    et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

  71. 71.

    , , & De novo production of the plant-derived alkaloid strictosidine in yeast. Proc. Natl. Acad. Sci. USA 112, 3205–3210 (2015).

  72. 72.

    et al. Complete reconstitution of a highly reducing iterative polyketide synthase. Science 326, 589–592 (2009).

  73. 73.

    et al. An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans. J. Am. Chem. Soc. 135, 7720–7731 (2013).

  74. 74.

    , , & Heterologous production of epothilone C and D in Escherichia coli. Biochemistry 45, 1321–1330 (2006).

  75. 75.

    et al. Modular construction of a functional artificial epothilone polyketide pathway. ACS Synth. Biol. 3, 759–772 (2014).

  76. 76.

    et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).

  77. 77.

    , , & Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470–474 (2011).

  78. 78.

    et al. DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. 40, 1879–1889 (2012).

  79. 79.

    et al. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res. 40, 8773–8781 (2012).

  80. 80.

    , & Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

  81. 81.

    , , & Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metab. Eng. 19, 98–106 (2013).

  82. 82.

    et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  83. 83.

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

  84. 84.

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

  85. 85.

    et al. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth. Biol. 4, 585–594 (2014).

  86. 86.

    , , , & RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

  87. 87.

    , & High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2014).

  88. 88.

    et al. Synthetic RNA silencing of actinorhodin biosynthesis in Streptomyces coelicolor A3(2). PLoS ONE 8, e67509 (2013).

  89. 89.

    et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat. Biotechnol. 31, 170–174 (2013).

  90. 90.

    , , & Gene expression enabling synthetic diversification of natural products: chemogenetic generation of pacidamycin analogs. J. Am. Chem. Soc. 132, 12243–12245 (2010).

  91. 91.

    et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

  92. 92.

    , , , & 2-Deoxystreptamine-containing aminoglycoside antibiotics: recent advances in the characterization and manipulation of their biosynthetic pathways. Nat. Prod. Rep. 30, 11–20 (2013).

  93. 93.

    & Metabolic engineering of microorganisms for isoprenoid production. Nat. Prod. Rep. 25, 656–661 (2008).

  94. 94.

    , & Biosynthesis of fungal indole alkaloids. Nat. Prod. Rep. 31, 1474–1487 (2014).

  95. 95.

    et al. Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci. USA 102, 7315–7320 (2005).

  96. 96.

    , & Assessing the combinatorial potential of the RiPP cyanobactin tru pathway. ACS Synth. Biol. 4, 482–492 (2015).

  97. 97.

    , , & Computational tools for the synthetic design of biochemical pathways. Nat. Rev. Microbiol. 10, 191–202 (2012).

  98. 98.

    et al. Bioretrosynthetic construction of a didanosine biosynthetic pathway. Nat. Chem. Biol. 10, 392–399 (2014).

Download references

Acknowledgements

We thank K. Rathwell for critically reading this manuscript. Research in Y.J.Y.'s laboratory has been supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MISP) (2013R1A2A1A01014230), the Intelligent Synthetic Biology Center of the Global Frontier Project funded by MISP (20110031961), the High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and the National Research Council of Science and Technology through the Degree & Research Center program (DRC-14-3-KBSI). Combinatorial biosynthetic work in B.S.M.'s laboratory is supported by US National Institutes of Health grants R01-CA127622 and R01-GM085770.

Author information

Affiliations

  1. Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Republic of Korea.

    • Eunji Kim
    •  & Yeo Joon Yoon
  2. Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California, USA.

    • Bradley S Moore
  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California, USA.

    • Bradley S Moore

Authors

  1. Search for Eunji Kim in:

  2. Search for Bradley S Moore in:

  3. Search for Yeo Joon Yoon in:

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bradley S Moore or Yeo Joon Yoon.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.1893

Further reading