Advertisement

Retrosynthetic Design of Heterologous Pathways

  • Pablo Carbonell
  • Anne-Gaëlle Planson
  • Jean-Loup Faulon
Part of the Methods in Molecular Biology book series (MIMB, volume 985)

Abstract

Tools from metabolic engineering and synthetic biology are synergistically used in order to develop high-performance cell factories. However, the number of successful applications has been limited due to the complexity of exploring efficiently the metabolic space for the discovery of candidate heterologous pathways. To address this challenge, retrosynthetic biology provides an integrated framework to formalize and rationalize the problem of importing biosynthetic pathways into a chassis organism using methods at the interface from bottom-up and top-down strategies. Here, we describe step by step the process of implementing a retrosynthetic framework for the design of heterologous biosynthetic pathways in a chassis organism. The method consists of the following steps: choosing the chassis and the target, selection of an in silico model for the chassis, definition of the metabolic space, pathway enumeration, gene selection, estimation of yields, toxicity prediction of pathway metabolites, definition of an objective function to select the best pathway candidates, and pathway implementation and verification.

Key words

Synthetic biology Metabolic engineering Retrosynthesis Metabolic pathway 

Notes

Acknowledgements

This work was funded by Genopole® (ATIGE grant) and Agence Nationale de la Recherche (ANR Chaire d’excellence).

References

  1. 1.
    Oberhardt MA, Palsson BO, Papin JA (2009) Applications of genome-scale metabolic reconstructions. Mol Syst Biol 5:320CrossRefGoogle Scholar
  2. 2.
    Yadav VG, De Mey M, Lim CG et al (2012) The future of metabolic engineering and synthetic biology: towards a systematic practice. Metab Eng 14:233–241CrossRefGoogle Scholar
  3. 3.
    Curran KA, Crook NC, Alper HS (2012) Using flux balance analysis to guide microbial metabolic engineering. Methods Mol Biol 834:197–216CrossRefGoogle Scholar
  4. 4.
    Planson AG, Carbonell P, Grigoras I et al (2012) A retrosynthetic biology approach to therapeutics: from conception to delivery. Curr Opin Biotechnol. doi: 10.1016/j.copbio.2012.03.009
  5. 5.
    Caspi R, Altman T, Dreher K et al (2012) The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res 40:D742–D753CrossRefGoogle Scholar
  6. 6.
    Kanehisa M, Goto S, Sato Y et al (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40:D109–D114CrossRefGoogle Scholar
  7. 7.
    Chang A, Scheer M, Grote A et al (2009) BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res 37:D588–D592CrossRefGoogle Scholar
  8. 8.
    Schellenberger J, Park J, Conrad T et al (2010) BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions. BMC Bioinformatics 11:213CrossRefGoogle Scholar
  9. 9.
    Li C, Donizelli M, Rodriguez N et al (2010) BioModels database: an enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol 4:92CrossRefGoogle Scholar
  10. 10.
    Carbonell P, Fichera D, Pandit SB et al (2012) Enumerating metabolic pathways for the production of heterologous target chemicals in chassis organisms. BMC Syst Biol 6:10CrossRefGoogle Scholar
  11. 11.
    Schellenberger J, Que R, Fleming RMT et al (2011) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat Protoc 6:1290–1307CrossRefGoogle Scholar
  12. 12.
    Rocha I, Maia P, Evangelista P et al (2010) OptFlux: an open-source software platform for in silico metabolic engineering. BMC Syst Biol 4:45CrossRefGoogle Scholar
  13. 13.
    Hoops S, Sahle S, Gauges R et al (2006) COPASI—a COmplex PAthway SImulator. Bioinformatics 22:3067–3074CrossRefGoogle Scholar
  14. 14.
    Planson AG, Carbonell P, Paillard E et al (2012) Compound toxicity screening and structure-activity relationship modeling in Escherichia coli. Biotechnol Bioeng 109:846–850CrossRefGoogle Scholar
  15. 15.
    Carbonell P, Planson AG, Fichera D et al (2011) A retrosynthetic biology approach to metabolic pathway design for therapeutic production. BMC Syst Biol 5:122CrossRefGoogle Scholar
  16. 16.
    Feist AM, Herrgard MJ, Thiele I et al (2008) Reconstruction of biochemical networks in microorganisms. Nat Rev Microbiol 7:129–143Google Scholar
  17. 17.
    Menzella H, Reeves C (2007) Combinatorial biosynthesis for drug development. Curr Opin Microbiol 10:238–245CrossRefGoogle Scholar
  18. 18.
    Ajikumar PK, Xiao WH, Tyo KEJ et al (2010) Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 330:70–74CrossRefGoogle Scholar
  19. 19.
    Orth JD, Conrad TM, Na J et al (2011) A comprehensive genome-scale reconstruction of Escherichia coli metabolism-2011. Mol Syst Biol 7:535CrossRefGoogle Scholar
  20. 20.
    Krivoruchko A, Siewers V, Nielsen J (2011) Opportunities for yeast metabolic engineering: lessons from synthetic biology. Biotechnol J 6:262–276CrossRefGoogle Scholar
  21. 21.
    Boghigian BA, Seth G, Kiss R et al (2010) Metabolic flux analysis and pharmaceutical production. Metab Eng 12:81–95CrossRefGoogle Scholar
  22. 22.
    Halls C, Yu O (2008) Potential for metabolic engineering of resveratrol biosynthesis. Trends Biotechnol 26:77–81CrossRefGoogle Scholar
  23. 23.
    Beekwilder J, Wolswinkel R, Jonker H et al (2006) Production of resveratrol in recombinant microorganisms. Appl Environ Microbiol 72:5670–5672CrossRefGoogle Scholar
  24. 24.
    Lim CGG, Fowler ZL, Hueller T et al (2011) High-yield resveratrol production in engineered Escherichia coli. Appl Environ Microbiol 77:3451–3460CrossRefGoogle Scholar
  25. 25.
    Brunk E, Neri M, Tavernelli I et al (2012) Integrating computational methods to retrofit enzymes to synthetic pathways. Biotechnol Bioeng 109:572–582CrossRefGoogle Scholar
  26. 26.
    Cho A, Yun H, Park JHH et al (2010) Prediction of novel synthetic pathways for the production of desired chemicals. BMC Syst Biol 4:35CrossRefGoogle Scholar
  27. 27.
    Limem I, Guedon E, Hehn A et al (2008) Production of phenylpropanoid compounds by recombinant microorganisms expressing plant-specific biosynthesis genes. Process Biochem 43:463–479CrossRefGoogle Scholar
  28. 28.
    Kamp A, Schuster S (2006) Metatool 5.0: fast and flexible elementary modes analysis. Bioinformatics 22:1930–1931CrossRefGoogle Scholar
  29. 29.
    Gerlee P, Lizana L, Sneppen K (2009) Pathway identification by network pruning in the metabolic network of Escherichia coli. Bioinformatics 25:3282–3288CrossRefGoogle Scholar
  30. 30.
    Welch M, Villalobos A, Gustafsson C et al (2011) Designing genes for successful protein expression. Methods Enzymol 498:43–66CrossRefGoogle Scholar
  31. 31.
    Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  32. 32.
    Kingsford C, Salzberg SL (2008) What are decision trees? Nat Biotechnol 26:1011–1013CrossRefGoogle Scholar
  33. 33.
    Yamaguchi T, Kurosaki F, Suh D et al (1999) Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli. FEBS Lett 460:457–461CrossRefGoogle Scholar
  34. 34.
    Patil K, Rocha I, Forster J et al (2005) Evolutionary programming as a platform for in silico metabolic engineering. BMC Bioinformatics 6:308CrossRefGoogle Scholar
  35. 35.
    Ma SM, Garcia DE, Redding-Johanson AM et al (2011) Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. Metab Eng 13:588–597CrossRefGoogle Scholar
  36. 36.
    Pitera DJ, Paddon CJ, Newman JD et al (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9:193–207CrossRefGoogle Scholar
  37. 37.
    Tropsha A, Golbraikh A (2010) Predictive quantitative structure-activity relationship modeling. Development and validation of QSAR models. In: Faulon JL, Benders A (eds) Handbook of chemoinformatics algorithms. Chapman and Hall/CRC, London, pp 211–232CrossRefGoogle Scholar
  38. 38.
    Mevik BH, Wehrens R (2007) The pls package: principal component and partial least squares regression in R. J Stat Softw 18:1CrossRefGoogle Scholar
  39. 39.
    Hughes RA, Miklos AE, Ellington AD (2011) Gene synthesis: methods and applications. Methods Enzymol 498:277–309CrossRefGoogle Scholar
  40. 40.
    Tolia NH, Joshua-Tor L (2006) Strategies for protein coexpression in Escherichia coli. Nat Methods 3:55–64CrossRefGoogle Scholar
  41. 41.
    Scheich C, Kummel D, Soumailakakis D et al (2007) Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res 35:e43CrossRefGoogle Scholar
  42. 42.
    Tsvetanova B, Peng L, Liang X et al (2011) Genetic assembly tools for synthetic biology. Methods Enzymol 498:327–348CrossRefGoogle Scholar
  43. 43.
    Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361CrossRefGoogle Scholar
  44. 44.
    Santos CNS, Koffas M, Stephanopoulos G (2011) Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab Eng 13:392–400CrossRefGoogle Scholar
  45. 45.
    Makino T, Skretas G, Georgiou G (2011) Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 10:32CrossRefGoogle Scholar
  46. 46.
    Roux A, Lison D, Junot C et al (2011) Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: a review. Clin Biochem 44:119–135CrossRefGoogle Scholar
  47. 47.
    Garcia DE, Baidoo EE, Benke PI et al (2008) Separation and mass spectrometry in microbial metabolomics. Curr Opin Microbiol 11:233–239CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Pablo Carbonell
    • 1
  • Anne-Gaëlle Planson
    • 1
  • Jean-Loup Faulon
    • 1
  1. 1.Institute of Systems & Synthetic Biology (ISSB)EvryFrance

Personalised recommendations