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Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols

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Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains revealed that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves greater local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion and reducing the loss of intermediates to competing pathways.

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Figure 1: Isobutanol pathways.
Figure 2: Isobutanol production by yeast engineered with mitochondrial and partly cytoplasmic isobutanol pathways.
Figure 3: The effect of cytoplasmic α-ketoisovalerate on isobutanol production.
Figure 4: Production of isopentanol and 2-methyl-1-butanol.
Figure 5: Effects of the CoxIV mitochondrial localization signal on the cellular localization and local concentration of targeted enzymes.

Change history

  • 03 May 2013

    The incorrect version of the supplementary information was posted online and has been replaced as of 3 May 2013.


  1. Attardi, G. & Schatz, G. Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4, 289–333 (1988).

    CAS  PubMed  Google Scholar 

  2. Fukuda, H., Casas, A. & Batlle, A. Aminolevulinic acid: from its unique biological function to its star role in photodynamic therapy. Int. J. Biochem. Cell Biol. 37, 272–276 (2005).

    CAS  PubMed  Google Scholar 

  3. Kohlhaw, G.B. Leucine biosynthesis in fungi: entering metabolism through the back door. Microbiol. Mol. Biol. Rev. 67, 1–15 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumar, A. et al. Subcellular localization of the yeast proteome. Genes Dev. 16, 707–719 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lange, H., Kispal, G. & Lill, R. Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria. J. Biol. Chem. 274, 18989–18996 (1999).

    CAS  PubMed  Google Scholar 

  6. Marquet, A., Bui, B.T. & Florentin, D. Biosynthesis of biotin and lipoic acid. Vitam. Horm. 61, 51–101 (2001).

    CAS  PubMed  Google Scholar 

  7. Neuburger, M., Rebeille, F., Jourdain, A., Nakamura, S. & Douce, R. Mitochondria are a major site for folate and thymidylate synthesis in plants. J. Biol. Chem. 271, 9466–9472 (1996).

    CAS  PubMed  Google Scholar 

  8. Paltauf, F., Kohlwein, S.D. & Henry, S.A. Regulation and compartmentalization of lipid synthesis in yeast. in The Molecular and Cellular Biology of the Yeast Saccharomyces (eds. Jones, E.W., Pringle, J.R. & Broach, J.R.) 415–500 (Cold Spring Harbor Laboratory Press, 1992).

  9. Pierrel, F. et al. Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis. Chem. Biol. 17, 449–459 (2010).

    CAS  PubMed  Google Scholar 

  10. Schonauer, M.S., Kastaniotis, A.J., Kursu, V.A., Hiltunen, J.K. & Dieckmann, C.L. Lipoic acid synthesis and attachment in yeast mitochondria. J. Biol. Chem. 284, 23234–23242 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Shannon, K.W. & Rabinowitz, J.C. Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase. J. Biol. Chem. 263, 7717–7725 (1988).

    CAS  PubMed  Google Scholar 

  12. Sulo, P. & Martin, N.C. Isolation and characterization of LIP5. A lipoate biosynthetic locus of Saccharomyces cerevisiae. J. Biol. Chem. 268, 17634–17639 (1993).

    CAS  PubMed  Google Scholar 

  13. Tran, U.C. & Clarke, C.F. Endogenous synthesis of coenzyme Q in eukaryotes. Mitochondrion 7 (suppl. 7), S62–S71 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Urban-Grimal, D., Volland, C., Garnier, T., Dehoux, P. & Labbe-Bois, R. The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae. Eur. J. Biochem. 156, 511–519 (1986).

    CAS  PubMed  Google Scholar 

  15. Zhang, S., Sanyal, I., Bulboaca, G.H., Rich, A. & Flint, D.H. The gene for biotin synthase from Saccharomyces cerevisiae: cloning, sequencing, and complementation of Escherichia coli strains lacking biotin synthase. Arch. Biochem. Biophys. 309, 29–35 (1994).

    CAS  PubMed  Google Scholar 

  16. Hiltunen, J.K. et al. Mitochondrial fatty acid synthesis type II: more than just fatty acids. J. Biol. Chem. 284, 9011–9015 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Stryer, L. Biochemistry, 4th edn (W.H. Freeman and Company, 1995).

  18. Hu, J., Dong, L. & Outten, C.E. The redox environment in the mitochondrial intermembrane space is maintained separately from the cytosol and matrix. J. Biol. Chem. 283, 29126–29134 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Orij, R., Postmus, J., Ter Beek, A., Brul, S. & Smits, G.J. In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155, 268–278 (2009).

    CAS  PubMed  Google Scholar 

  20. Schnell, N., Krems, B. & Entian, K.D. The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae, a c-jun homologue, is involved in oxygen metabolism. Curr. Genet. 21, 269–273 (1992).

    CAS  PubMed  Google Scholar 

  21. Muhlenhoff, U. & Lill, R. Biogenesis of iron-sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria. Biochim. Biophys. Acta 1459, 370–382 (2000).

    CAS  PubMed  Google Scholar 

  22. Lill, R. & Muhlenhoff, U. Iron-sulfur-protein biogenesis in eukaryotes. Trends Biochem. Sci. 30, 133–141 (2005).

    CAS  PubMed  Google Scholar 

  23. Xu, X.M. & Moller, S.G. Iron-sulfur cluster biogenesis systems and their crosstalk. ChemBioChem 9, 2355–2362 (2008).

    CAS  PubMed  Google Scholar 

  24. Hazelwood, L.A., Daran, J.M., van Maris, A.J., Pronk, J.T. & Dickinson, J.R. The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 74, 2259–2266 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008).

    CAS  PubMed  Google Scholar 

  26. Bastian, S. et al. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab. Eng. 13, 345–352 (2011).

    CAS  PubMed  Google Scholar 

  27. Higashide, W., Li, Y., Yang, Y. & Liao, J.C. Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose. Appl. Environ. Microbiol. 77, 2727–2733 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Jia, X., Li, S., Xie, S. & Wen, J. Engineering a metabolic pathway for isobutanol biosynthesis in Bacillus subtilis. Appl. Biochem. Biotechnol. 168, 1–9 (2012).

    CAS  PubMed  Google Scholar 

  29. Li, S., Wen, J. & Jia, X. Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression. Appl. Microbiol. Biotechnol. 91, 577–589 (2011).

    CAS  PubMed  Google Scholar 

  30. Smith, K.M., Cho, K.M. & Liao, J.C. Engineering Corynebacterium glutamicum for isobutanol production. Appl. Microbiol. Biotechnol. 87, 1045–1055 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, X., Nielsen, K.F., Borodina, I., Kielland-Brandt, M.C. & Karhumaa, K. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism. Biotechnol. Biofuels 4, 21 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kondo, T. et al. Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae. J. Biotechnol. 159, 32–37 (2012).

    CAS  PubMed  Google Scholar 

  33. Lee, W.H. et al. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes. Bioprocess Biosyst. Eng. 35, 1467–1475 (2012).

    CAS  PubMed  Google Scholar 

  34. Hong, K.K. & Nielsen, J. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol. Life Sci. 69, 2671–2690 (2012).

    CAS  PubMed  Google Scholar 

  35. Brat, D., Weber, C., Lorenzen, W., Bode, H.B. & Boles, E. Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast. Saccharomyces cerevisiae. Biotechnol. Biofuels 5, 65 (2012).

    CAS  PubMed  Google Scholar 

  36. Urano, J. et al. US patent. US 2011/0076733 A1 (2011).

  37. Anthony, L.C., Huang, L.L. & Ye, R.W. US patent. US 2010/0129886 A1 (2010).

  38. Buelter, T., Meinhold, P., Smith, C., Aristidou, A., Dundon, C.A. & Urano, J. World patent. WO 2010/075504A2 (2010).

  39. Szczebara, F.M. et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat. Biotechnol. 21, 143–149 (2003).

    CAS  PubMed  Google Scholar 

  40. Farhi, M. et al. Harnessing yeast subcellular compartments for the production of plant terpenoids. Metab. Eng. 13, 474–481 (2011).

    CAS  PubMed  Google Scholar 

  41. Maarse, A.C. et al. Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein. EMBO J. 3, 2831–2837 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Larroy, C., Pares, X. & Biosca, J.A. Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family. Eur. J. Biochem. 269, 5738–5745 (2002).

    CAS  PubMed  Google Scholar 

  43. Dellomonaco, C., Clomburg, J.M., Miller, E.N. & Gonzalez, R. Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–359 (2011).

    CAS  PubMed  Google Scholar 

  44. Feldman, R.M.R. et al. US patent. US 2011/8017375 B2 (2011).

  45. Wu, S. et al. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat. Biotechnol. 24, 1441–1447 (2006).

    CAS  PubMed  Google Scholar 

  46. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H. & Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122 (1992).

    CAS  PubMed  Google Scholar 

  47. Sleight, S.C., Bartley, B.A., Lieviant, J.A. & Sauro, H.M. In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res. 38, 2624–2636 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Blombach, B. & Eikmanns, B.J. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. Bioeng. Bugs 2, 346–350 (2011).

    PubMed  PubMed Central  Google Scholar 

  49. Gregg, C., Kyryakov, P. & Titorenko, V.I. Purification of mitochondria from yeast cells. J. Vis. Exp. 10.3791/1417 (2009).

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We thank K.L. Jones Prather and T.D. Fox for helpful discussions, T.J. Helbig for working on GFP subcellular localization experiments, T. DiCesare for preparing figures, S. Lindquist (Whitehead Institute) for strain Y3929, and members of the Stephanopoulos, Fink and Prather laboratories for discussions and advice. J.L.A. is supported by US National Institutes of Health under Ruth L. Kirchstein National Research Service Award 1F32GM098022-01A1. G.R.F. is supported by National Institutes of Health grant GM040266. This work was supported by Shell Global Solutions (US) Inc.

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Authors and Affiliations



J.L.A., G.R.F. and G.S. conceived the project, designed the experiments, analyzed the results and wrote the manuscript. J.L.A. designed and made the pJLA vectors, constructed all pathways and strains, and executed all the experiments.

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Correspondence to Gerald R Fink or Gregory Stephanopoulos.

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The authors declare no competing financial interests.

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Avalos, J., Fink, G. & Stephanopoulos, G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat Biotechnol 31, 335–341 (2013).

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