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Reversal of NAD(P)H Cofactor Dependence by Protein Engineering

  • Sabine Bastian
  • Frances H. ArnoldEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 834)

Abstract

There is increasing interest in utilization of engineered microorganisms for the production of renewable chemicals and next-generation biofuels. However, imbalances between the cofactor consumption of the engineered production pathway and the reducing equivalents provided by the cell have been shown to limit yields. This imbalance can be overcome by adjusting the cofactor dependencies of the pathway enzymes to match the available cofactors in the cell. We show how cofactor preference can be reversed by structure-guided directed evolution of the target enzyme.

Key words

Cofactor switch Enzyme engineering Homology modeling Nicotine amide dinucleotide phosphate Nicotine amide dinucleotide Directed evolution 

Notes

Acknowledgments

The authors would like to thank Dr. Christopher Snow for assistance with homology modeling. This work was sponsored by the U.S. Army Research Laboratory and was accomplished under cooperative Agreement number W911NF-09-2-002.

References

  1. 1.
    Rude, M. A., and Schirmer, A. (2009) New microbial fuels: a biotech perspective, Current Opinion in Microbiology 12, 274–281.PubMedCrossRefGoogle Scholar
  2. 2.
    Yan, Y., and Liao, J. (2009) Engineering metabolic systems for production of advanced fuels, Journal of Industrial Microbiology & Biotechnology 36, 471–479.CrossRefGoogle Scholar
  3. 3.
    Weckbecker, A., and Hummel, W. (2004) Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP(+)-dependent alcohol dehydrogenase and NAD(+)-dependent formate dehydrogenase, Biotechnology Letters 26, 1739–1744.PubMedCrossRefGoogle Scholar
  4. 4.
    Nissen, T. L., Anderlund, M., Nielsen, J., Villadsen, J., and Kielland-Brandt, M. C. (2001) Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool, Yeast 18, 19–32.PubMedCrossRefGoogle Scholar
  5. 5.
    Bengtsson, O., Hahn-Hagerdal, B., and Gorwa-Grauslund, M. F. (2009) Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae, Biotechnology for Biofuels 2.Google Scholar
  6. 6.
    Jeppsson, M., Bengtsson, O., Franke, K., Lee, H., Hahn-Hagerdal, R., and Gorwa-Grauslund, M. F. (2006) The expression of a Pichia stipitis xylose reductase mutant with higher Km for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae, Biotechnology and Bioengineering 93, 665–673.PubMedCrossRefGoogle Scholar
  7. 7.
    Bastian, S., Liu, X.; Meyerowitz, J.; Snow, C. D.; Chen, M. M.Y.; Arnold, F. H. (2011) Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic isobutanol production at theoretical yield in Escherichia coli, Metabolic Engineering 13, 345–352.Google Scholar
  8. 8.
    Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., and Branlant, G. (1993) Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase - role of the acidic residue in the fingerprint region of the nucleotide-binding fold, Biochemistry 32, 10178–10184.PubMedCrossRefGoogle Scholar
  9. 9.
    Scrutton, N. S., Berry, A., and Perham, R. N. (1990) Redesign of the coenzyme specificity of a dehydrogenase by protein engineering Nature 343, 38–43.PubMedCrossRefGoogle Scholar
  10. 10.
    Khoury, G. A., Fazelinia, H., Chin, J. W., Pantazes, R. J., Cirino, P. C., and Maranas, C. D. (2009) Computational design of Candida boidinii xylose reductase for altered cofactor specificity, Protein Science 18, 2125–2138.PubMedCrossRefGoogle Scholar
  11. 11.
    Romero, P. A., and Arnold, F. H. (2009) Exploring protein fitness landscapes by directed evolution, Nature Reviews Molecular Cell Biology 10, 866–876.PubMedCrossRefGoogle Scholar
  12. 12.
    Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M., Eramian, D., Shen, M.-y., Pieper, U., Sali, A. (2006) Comparative Protein Structure Modeling Using Modeller, Current Protocols in Bioinformatics 15, 5.6.1–5.6.30.Google Scholar
  13. 13.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymology 154, 367–382.PubMedCrossRefGoogle Scholar
  14. 14.
    Krampitz, L. O. (1957) Preparation and determination of acetoin, diacetyl, and acetolactate, Methods in Enzymology 3, 277–283.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA

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