Advertisement

BioEnergy Research

, Volume 5, Issue 4, pp 814–828 | Cite as

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

  • Andreas Zurbriggen
  • Henning Kirst
  • Anastasios MelisEmail author
Article

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.

Keywords

Isoprene Mevalonic acid pathway Deoxyxylulose-5-phosphate pathway Pueraria montana Terpenoid biosynthesis 

Abbreviations

IPP

Isopentenyl-diphosphate

DMAPP

Dimethylallyl-diphosphate

DCW

Dry cell weight

RBS

Ribosome-binding site

TIR

Translation initiation region

Notes

Acknowledgments

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

Supplementary material

12155_2012_9192_MOESM1_ESM.doc (80 kb)
ESM 1 (DOC 80 kb)
12155_2012_9192_MOESM2_ESM.doc (40 kb)
ESM 2 (DOC 40 kb)

References

  1. 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 100:8873–8892CrossRefGoogle Scholar
  2. 2.
    Sharkey TD, Singsass EL (1995) Why plants emit isoprene? Nature 374:769–769CrossRefGoogle Scholar
  3. 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 325:730–733PubMedCrossRefGoogle Scholar
  4. 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 51:485–499PubMedCrossRefGoogle Scholar
  5. 5.
    Singsaas EL, Lerdau M, Winter K, Sharkey TD (1997) Isoprene increases thermotolerance of isoprene-emitting species. Plant Physiol 115:1413–1420PubMedGoogle Scholar
  6. 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 127:1781–1787PubMedCrossRefGoogle Scholar
  7. 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 32:520–531PubMedCrossRefGoogle Scholar
  8. 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 105:17430–17435PubMedCrossRefGoogle Scholar
  9. 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 31:1410–1415PubMedCrossRefGoogle Scholar
  10. 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 213:483–487PubMedCrossRefGoogle Scholar
  11. 11.
    Silver GM, Fall R (1995) Characterization of aspen isoprene synthase, an enzyme responsible for leaf isoprene emission to the atmosphere. J Biol Chem 270:13010–13016PubMedCrossRefGoogle Scholar
  12. 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 55:687–697PubMedCrossRefGoogle Scholar
  13. 13.
    Sasaki K, Ohara K, Yazaki K (2005) Gene expression and characterization of isoprene synthase from Populus alba. FEBS Lett 579:2514–2518PubMedCrossRefGoogle Scholar
  14. 14.
    Sharkey TD, Yeh S, Wiberley AE, Falbel TG, Gong D, Fernandez DE (2005) Evolution of the isoprene biosynthetic pathway in kudzu. Plant Physiol 137:700–712PubMedCrossRefGoogle Scholar
  15. 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):363–373PubMedCrossRefGoogle Scholar
  16. 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 12:70–79PubMedCrossRefGoogle Scholar
  17. 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 109:100–109CrossRefGoogle Scholar
  18. 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):152–163CrossRefGoogle Scholar
  19. 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):1915–1922PubMedCrossRefGoogle Scholar
  20. 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):13654–13659PubMedCrossRefGoogle Scholar
  21. 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):796–802PubMedCrossRefGoogle Scholar
  22. 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):13172–13177PubMedCrossRefGoogle Scholar
  23. 23.
    Lee PC, Schmidt-Dannert C (2002) Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl Microbiol Biotechnol 60(1–2):1–11PubMedGoogle Scholar
  24. 24.
    Lichtenthaler HK (2000) Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors. Biochem Soc Trans 28:785–789PubMedCrossRefGoogle Scholar
  25. 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):565–574PubMedCrossRefGoogle Scholar
  26. 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 118:2564–2566CrossRefGoogle Scholar
  27. 27.
    Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol Prog 17(1):57–61PubMedCrossRefGoogle Scholar
  28. 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 324:421–426PubMedGoogle Scholar
  29. 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):408–415PubMedCrossRefGoogle Scholar
  30. 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):1558–1561PubMedCrossRefGoogle Scholar
  31. 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):218–226PubMedCrossRefGoogle Scholar
  32. 32.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 11–730Google Scholar
  33. 33.
    Russell RG (2007) Bisphosphonates: mode of action and pharmacology. Pediatrics 119(Suppl 2):S150–S162PubMedCrossRefGoogle Scholar
  34. 34.
    Melis A (2011) Short chain volatile hydrocarbon production using genetically engineered microalgae, cyanobacteria or bacteria. United States Patent 7,947,478Google Scholar
  35. 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):1107–1121PubMedGoogle Scholar
  36. 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):12213–12220PubMedCrossRefGoogle Scholar
  37. 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):1219–1229PubMedCrossRefGoogle Scholar
  38. 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 22:495–504CrossRefGoogle Scholar
  39. 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 109:216–221Google Scholar
  40. 40.
    Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18(3):213–219PubMedCrossRefGoogle Scholar
  41. 41.
    Dasari MA, Kiatsimkul PP, Sutterlin WR, Suppes GJ (2005) Low-pressure hydrogenolysis of glycerol to propylene glycol. Appl Catal Gen 281:225–231CrossRefGoogle Scholar
  42. 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 PubMedCrossRefGoogle Scholar
  43. 43.
    Luria SE (1960) The bacterial protoplasm: composition and organization. In: Gunsales IC, Stanier RY (eds) The bacteria, vol 1. Academic, New York, pp 1–34Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Andreas Zurbriggen
    • 1
  • Henning Kirst
    • 1
  • Anastasios Melis
    • 1
    Email author
  1. 1.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA

Personalised recommendations