Skip to main content

Advertisement

SpringerLink
Log in

Isoprene Production Via the Mevalonic Acid Pathway in Escherichia coli (Bacteria)

  • Published:
BioEnergy Research Aims and scope Submit manuscript

Abstract

There is a need to develop renewable fuels and chemicals that will help meet global demands for energy and synthetic chemistry feedstock, without contributing to climate change or environmental degradation. Isoprene (C5H8) is one such key chemical ingredient, required for the production of synthetic rubber or plastic products, and a potential biofuel. Enabling a sustainable microbial fermentation for the production of isoprene is an attractive alternative to a petroleum origin. This work demonstrates transgenic expression of the Pueraria montana (kudzu vine) isoprene synthase gene (kIspS) and heterologous isoprene production in Escherichia coli. Enhancements in the amount of E. coli isoprene production were achieved upon over-expression of the native 2-C-methyl-d-erythritol-4-phosphate (MEP) biosynthetic pathway and, independently, upon heterologous over-expression of the entire mevalonic acid (MVA) pathway. A direct comparison of the efficiency of cellular organic carbon flux through the MEP and MVA pathways is provided, under conditions when these are expressed in the same host using the same plasmid, and same ribosome-binding sites (RBS). These alternative isoprenoid biosynthetic pathways were assembled in and expressed through a superoperon, suitable for transformation of E. coli. Introduction of specific RBS and nucleotide spacers between individual genes in the superoperon structure enabled maximal expression in E. coli batch cultures and translated to an improved production from 0.4 mg isoprene per liter of culture (control) to 5 mg isoprene per liter of culture (MEP superoperon transformants) and up to 320 mg isoprene per liter of culture (MVA superoperon transformants). This 800-fold increase in isoprene concentration from the MVA transformants and the attendant isoprene-to-biomass 0.78:1 carbon partitioning ratio suggested that the engineered MVA pathway introduces a bypass in the flux of endogenous substrate in E. coli to isopentenyl-diphosphate and dimethylallyl-diphosphate, thus overcoming flux limitations imposed upon the regulation of the native MEP pathway by the cell.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Abbreviations

IPP:

Isopentenyl-diphosphate

DMAPP:

Dimethylallyl-diphosphate

DCW:

Dry cell weight

RBS:

Ribosome-binding site

TIR:

Translation initiation region

References

  1. Guenther A, Hewitt CN, Erickson D, Fall R, Geron C, Graedel T et al (1995) A global model of natural volatile organic compound emissions. J Geophys Res 100:8873–8892

    Article  CAS  Google Scholar 

  2. Sharkey TD, Singsass EL (1995) Why plants emit isoprene? Nature 374:769–769

    Article  CAS  Google Scholar 

  3. Paulot F, Crounse JD, Kjaergaard HG, Kürten A, St Clair JM, Seinfeld JH et al (2009) Unexpected epoxide formation in the gasphase photooxidation of isoprene. Science 325:730–733

    Article  PubMed  CAS  Google Scholar 

  4. Behnke K, Ehlting B, Teuber M, Bauerfeind M, Louis S, Hänsch R et al (2007) Transgenic, nonisoprene emitting poplars don’t like it hot. Plant J 51:485–499

    Article  PubMed  CAS  Google Scholar 

  5. Singsaas EL, Lerdau M, Winter K, Sharkey TD (1997) Isoprene increases thermotolerance of isoprene-emitting species. Plant Physiol 115:1413–1420

    PubMed  CAS  Google Scholar 

  6. Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127:1781–1787

    Article  PubMed  CAS  Google Scholar 

  7. Vickers CE, Possell M, Cojocariu CI, Velikova VB, Laothawornkitkul J, Ryan A et al (2009) Isoprene synthesis protects transgenic tobacco plants from oxidative stress. Plant Cell Environ 32:520–531

    Article  PubMed  CAS  Google Scholar 

  8. Loivamäki M, Mumm R, Dicke M, Schnitzler JP (2008) Isoprene interferes with the attraction of bodyguards by herbaceous plants. Proc Natl Acad Sci U S A 105:17430–17435

    Article  PubMed  Google Scholar 

  9. Laothawornkitkul J, Paul ND, Vickers CE, Possell M, Taylor JE, Mullineaux PM et al (2008) Isoprene emissions influence herbivore feeding decisions. Plant Cell Environ 31:1410–1415

    Article  PubMed  CAS  Google Scholar 

  10. Miller B, Oschinski C, Zimmer W (2001) First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli. Planta 213:483–487

    Article  PubMed  CAS  Google Scholar 

  11. Silver GM, Fall R (1995) Characterization of aspen isoprene synthase, an enzyme responsible for leaf isoprene emission to the atmosphere. J Biol Chem 270:13010–13016

    Article  PubMed  CAS  Google Scholar 

  12. Fortunati A, Barta C, Brilli F, Centritto M, Zimmer I, Schnitzler JP et al (2008) Isoprene emission is not temperature-dependent during and after severe drought-stress: a physiological and biochemical analysis. Plant J 55:687–697

    Article  PubMed  CAS  Google Scholar 

  13. Sasaki K, Ohara K, Yazaki K (2005) Gene expression and characterization of isoprene synthase from Populus alba. FEBS Lett 579:2514–2518

    Article  PubMed  CAS  Google Scholar 

  14. Sharkey TD, Yeh S, Wiberley AE, Falbel TG, Gong D, Fernandez DE (2005) Evolution of the isoprene biosynthetic pathway in kudzu. Plant Physiol 137:700–712

    Article  PubMed  CAS  Google Scholar 

  15. Köksal M, Zimmer I, Schnitzler JP, Christianson DW (2010) Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission. J Mol Biol 402(2):363–373

    Article  PubMed  Google Scholar 

  16. Lindberg P, Park S, Melis A (2010) Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab Eng 12:70–79

    Article  PubMed  CAS  Google Scholar 

  17. Bentley FK, Melis A (2012) Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms. Biotech Bioeng 109:100–109

    Article  CAS  Google Scholar 

  18. Whited GM, Feher FJ, Benko DA, Cervin MA, Chotani GK, McAuliffe JC et al (2010) Development of a gas-phase bioprocess for isoprene-monomer production using metabolic pathway engineering. Ind Biotechnol 6(3):152–163

    Article  CAS  Google Scholar 

  19. Zhao Y, Yang J, Qin B, Li Y, Sun Y, Su S et al (2011) Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway. Appl Microbiol Biotechnol 90(6):1915–1922

    Article  PubMed  CAS  Google Scholar 

  20. Leonard E, Ajikumar PK, Thayer K, Xiao WH, Mo JD, Tidor B et al (2010) Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc Natl Acad Sci U S A 107(31):13654–13659

    Article  PubMed  CAS  Google Scholar 

  21. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21(7):796–802

    Article  PubMed  CAS  Google Scholar 

  22. Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci U S A 97(24):13172–13177

    Article  PubMed  CAS  Google Scholar 

  23. Lee PC, Schmidt-Dannert C (2002) Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl Microbiol Biotechnol 60(1–2):1–11

    PubMed  CAS  Google Scholar 

  24. Lichtenthaler HK (2000) Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors. Biochem Soc Trans 28:785–789

    Article  PubMed  CAS  Google Scholar 

  25. Rohmer M (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat Prod Rep 16(5):565–574

    Article  PubMed  CAS  Google Scholar 

  26. Rohmer M, Seemann M, Horbach S, Bringer-Meyer S, Sahm H (1996) Glyceraldehyde3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118:2564–2566

    Article  CAS  Google Scholar 

  27. Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol Prog 17(1):57–61

    Article  PubMed  CAS  Google Scholar 

  28. Kajiwara S, Fraser PD, Kondo K, Misawa N (1997) Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J 324:421–426

    PubMed  CAS  Google Scholar 

  29. Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng 72(4):408–415

    Article  PubMed  CAS  Google Scholar 

  30. Vadali RV, Fu Y, Bennett GN, San KY (2005) Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli. Biotechnol Prog 21(5):1558–1561

    Article  PubMed  CAS  Google Scholar 

  31. Yoon SH, Lee SH, Das A, Ryu HK, Jang HJ, Kim JY et al (2009) Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. J Biotechnol 140(3–4):218–226

    Article  PubMed  CAS  Google Scholar 

  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 11–730

    Google Scholar 

  33. Russell RG (2007) Bisphosphonates: mode of action and pharmacology. Pediatrics 119(Suppl 2):S150–S162

    Article  PubMed  Google Scholar 

  34. Melis A (2011) Short chain volatile hydrocarbon production using genetically engineered microalgae, cyanobacteria or bacteria. United States Patent 7,947,478

  35. Cunningham FX Jr, Sun Z, Chamovitz D, Hirschberg J, Gantt E (1994) Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Plant Cell 6(8):1107–1121

    PubMed  CAS  Google Scholar 

  36. Williams DC, McGarvey DJ, Katahira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37(35):12213–12220

    Article  PubMed  CAS  Google Scholar 

  37. Ringquist S, Shinedling S, Barrick D, Green L, Binkley J, Stormo GD et al (1992) Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol 6(9):1219–1229

    Article  PubMed  CAS  Google Scholar 

  38. Lehning A, Zimmer I, Steinbrecher R, Brüggemann N, Schnitzler J-P (1999) Isoprene synthase activity and its relation to isoprene emission in Quercus robur L leaves. Plant Cell Environ 22:495–504

    Article  CAS  Google Scholar 

  39. Schnitzler J-P, Arenz R, Steinbrecher R, Lehning A (1996) Characterization of an isoprene synthase from leaves of Quercus petraea (Mattuschka) Liebl. Bot Acta 109:216–221

    CAS  Google Scholar 

  40. Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18(3):213–219

    Article  PubMed  CAS  Google Scholar 

  41. Dasari MA, Kiatsimkul PP, Sutterlin WR, Suppes GJ (2005) Low-pressure hydrogenolysis of glycerol to propylene glycol. Appl Catal Gen 281:225–231

    Article  CAS  Google Scholar 

  42. Salis H, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950. doi:dx.doi.org

    Article  PubMed  CAS  Google Scholar 

  43. Luria SE (1960) The bacterial protoplasm: composition and organization. In: Gunsales IC, Stanier RY (eds) The bacteria, vol 1. Academic, New York, pp 1–34

    Google Scholar 

Download references

Acknowledgments

A. Z. is the recipient of a Swiss National Science Foundation Postdoctoral Fellowship, grant no. PBBEP3_128360.

Author information

Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720-3102, USA

    Andreas Zurbriggen, Henning Kirst & Anastasios Melis

Authors
  1. Andreas Zurbriggen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henning Kirst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anastasios Melis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anastasios Melis.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 80 kb)

ESM 2

(DOC 40 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zurbriggen, A., Kirst, H. & Melis, A. Isoprene Production Via the Mevalonic Acid Pathway in Escherichia coli (Bacteria). Bioenerg. Res. 5, 814–828 (2012). https://doi.org/10.1007/s12155-012-9192-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12155-012-9192-4

Keywords

Search

Navigation