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

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Abstract

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.

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  • 03 May 2013

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

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Acknowledgements

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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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). https://doi.org/10.1038/nbt.2509

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