Skip to main content
SpringerLink
Log in

In silico analysis and experimental improvement of taxadiene heterologous biosynthesis in Escherichia coli

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.

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

Similar content being viewed by others

References

  1. Wani, M. C., H. L. Taylor, M. E. Wall, P. Coggon, and A. T. McPhail (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93: 2325–2327.

    Article  CAS  Google Scholar 

  2. Horwitz, S. B. (1994) How to make taxol from scratch. Nature 367: 593–594.

    Article  CAS  Google Scholar 

  3. Frense, D. (2007) Taxanes: Perspectives for biotechnological production. Appl. Microbiol. Biotechnol. 73: 1233–1240.

    Article  CAS  Google Scholar 

  4. Roberts, S. C. (2007) Production and engineering of terpenoids in plant cell culture. Nature Chem. Biol. 3: 387–395.

    Article  CAS  Google Scholar 

  5. Nicolaou, K. C., Z. Yang, J. J. Liu, H. Ueno, P. G. Nantermet, R. K. Guy, C. F. Claiborne, J. Renaud, E. A. Couladouros, and K. Paulvannan (1994) Total synthesis of taxol. Nature 367: 630–634.

    Article  CAS  Google Scholar 

  6. Kirby, J. and J. D. Keasling (2009) Biosynthesis of plant isoprenoids: Perspectives for microbial engineering. Annu. Rev. Plant Biol. 60: 335–355.

    Article  CAS  Google Scholar 

  7. Ro, D. K., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Chang, S. T. Withers, Y. Shiba, R. Sarpong, and J. D. Keasling (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943.

    Article  CAS  Google Scholar 

  8. Chang, M. C. and J. D. Keasling (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat. Chem. Biol. 2: 674–681.

    Article  CAS  Google Scholar 

  9. Martin, V. J., D. J. Pitera, S. T. Withers, J. D. Newman, and J. D. Keasling (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21: 796–802.

    Article  CAS  Google Scholar 

  10. Tsuruta, H., C. J. Paddon, D. Eng, J. R. Lenihan, T. Horning, L. C. Anthony, R. Regentin, J. D. Keasling, N. S. Renninger, and J. D. Newman (2009) High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One. 4: e4489.

    Article  Google Scholar 

  11. Morrone, D., L. Lowry, M. K. Determan, D. M. Hershey, M. Xu, and R. J. Peters (2010) Increasing diterpene yield with a modular metabolic engineering system in E. coli: Comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85: 1893–1906.

    Article  CAS  Google Scholar 

  12. Huang, Q., C. A. Roessner, R. Croteau, and A. I. Scott (2001) Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol. Bioorg. Med. Chem. 9: 2237–2242.

    Article  CAS  Google Scholar 

  13. Besumbes, O., S. Sauret-Gueto, M. A. Phillips, S. Imperial, M. Rodriguez-Concepcion, and A. Boronat (2004) Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol. Biotechnol. Bioeng. 88: 168–175.

    Article  CAS  Google Scholar 

  14. Dejong, J. M., Y. Liu, A. P. Bollon, R. M. Long, S. Jennewein, D. Williams, and R. B. Croteau (2006) Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol. Bioeng. 93: 212–224.

    Article  CAS  Google Scholar 

  15. Engels, B., P. Dahm, and S. Jennewein (2008) Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab. Eng. 10: 201–206.

    Article  CAS  Google Scholar 

  16. Yuan, L. Z., P. E. Rouviere, R. A. Larossa, and W. Suh (2006) Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab. Eng. 8: 79–90.

    Article  CAS  Google Scholar 

  17. Jin, Y. S. and G. Stephanopoulos (2007) Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli. Metab. Eng. 9: 337–347.

    Article  CAS  Google Scholar 

  18. Alper, H., K. Miyaoku, and G. Stephanopoulos (2005) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat. Biotechnol. 23: 612–616.

    Article  CAS  Google Scholar 

  19. Feist, A. M., C. S. Henry, J. L. Reed, M. Krummenacker, A. R. Joyce, P. D. Karp, L. J. Broadbelt, V. Hatzimanikatis, and B. O. Palsson (2007) A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Mol. Syst. Biol. 3: 121.

    Article  Google Scholar 

  20. Herrgard, M. J., N. Swainston, P. Dobson, W. B. Dunn, K. Y. Arga, M. Arvas, N. Bluthgen, S. Borger, R. Costenoble, M. Heinemann, M. Hucka, N. Le Novere, P. Li, W. Liebermeister, M. L. Mo, A. P. Oliveira, D. Petranovic, S. Pettifer, E. Simeonidis, K. Smallbone, I. Spasic, D. Weichart, R. Brent, D. S. Broomhead, H. V. Westerhoff, B. Kirdar, M. Penttila, E. Klipp, B. O. Palsson, U. Sauer, S. G. Oliver, P. Mendes, J. Nielsen, and D. B. Kell (2008) A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology. Nat. Biotechnol. 26: 1155–1160.

    Article  CAS  Google Scholar 

  21. Oh, Y. K., B. O. Palsson, S. M. Park, C. H. Schilling, and R. Mahadevan (2007) Genome-scale reconstruction of metabolic network in Bacillus subtilis based on high-throughput phenotyping and gene essentiality data. J. Biol. Chem. 282: 28791–28799.

    Article  CAS  Google Scholar 

  22. Becker, S. A., A. M. Feist, M. L. Mo, G. Hannum, B. O. Palsson, and M. J. Herrgard (2007) Quantitative prediction of cellular metabolism with constraint-based models: The COBRA Toolbox. Nat. Protoc. 2: 727–738.

    Article  CAS  Google Scholar 

  23. Varma, A. and B. O. Palsson (1993) Metabolic capabilities of Escherichia coli: I. Synthesis of biosynthetic precursors and cofactors. J. Theor. Biol. 165: 477–502.

    Article  CAS  Google Scholar 

  24. Varmar, A. and B. O. Palsson (1993) Metabolic capabilities of Escherichia-coli: 2. Optimal-growth patterns. J. Theor. Biol. 165: 503–522.

    Article  Google Scholar 

  25. Gonzalez-Lergier, J., L. J. Broadbelt, and V. Hatzimanikatis (2006) Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli. Biotechnol. Bioeng. 95: 638–644.

    Article  CAS  Google Scholar 

  26. Yu, D., H. M. Ellis, E. C. Lee, N. A. Jenkins, N. G. Copeland, and D. L. Court (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA. 97: 5978–5983.

    Article  CAS  Google Scholar 

  27. Datsenko, K. A. and B. L. Wanner (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA. 97: 6640–6645.

    Article  CAS  Google Scholar 

  28. Wang, Y. and B. A. Pfeifer (2008) 6-deoxyerythronolide B production through chromosomal localization of the deoxyerythronolide B synthase genes in E. coli. Metab. Eng. 10: 33–38.

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Sambrook, J. and D. W. Russell (2001) Molecular cloning: A laboratory manual. 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  31. Lau, J., C. Tran, P. Licari, and J. Galazzo (2004) Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli. J. Biotechnol. 110: 95–103.

    Article  CAS  Google Scholar 

  32. Pfeifer, B., Z. Hu, P. Licari, and C. Khosla (2002) Process and metabolic strategies for improved production of Escherichia coliderived 6-deoxyerythronolide B. Appl. Env. Microbiol. 68: 3287–3292.

    Article  CAS  Google Scholar 

  33. Jones, K. L., S. W. Kim, and J. D. Keasling (2000) Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. Metab. Eng. 2: 328–338.

    Article  CAS  Google Scholar 

  34. Edwards, J. S. and B. O. Palsson (2000) Metabolic flux balance analysis and the in silico analysis of Escherichia coli K-12 gene deletions. BMC Bioinformatics 1: 1.

    Article  CAS  Google Scholar 

  35. Segre, D., D. Vitkup, and G. M. Church (2002) Analysis of optimality in natural and perturbed metabolic networks. Proc. Natl. Acad. Sci. USA. 99: 15112–15117.

    Article  CAS  Google Scholar 

  36. Alper, H. and G. Stephanopoulos (2008) Uncovering the gene knockout landscape for improved lycopene production in E. coli. Appl. Microbiol. Biotechnol. 78: 801–810.

    Article  CAS  Google Scholar 

  37. Park, J. H., K. H. Lee, T. Y. Kim, and S. Y. Lee (2007) Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc. Natl. Acad. Sci. USA. 104: 7797–7802.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510-641, China

    Hailin Meng & Xiaoning Wang

  2. Key Laboratory of Synthetic Biology, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200-032, China

    Hailin Meng & Yong Wang

  3. State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology, East China University of Science and Technology, Shanghai, 200-237, China

    Hailin Meng, Qiang Hua & Siliang Zhang

Authors
  1. Hailin Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiang Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Siliang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoning Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yong Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meng, H., Wang, Y., Hua, Q. et al. In silico analysis and experimental improvement of taxadiene heterologous biosynthesis in Escherichia coli. Biotechnol Bioproc E 16, 205–215 (2011). https://doi.org/10.1007/s12257-010-0329-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-010-0329-z

Keywords

Search

Navigation