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Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol

  • Bioenergy and biofuels
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Abstract

Saccharomyces cerevisiae naturally produces small amounts of isobutanol and 3-methyl-1-butanol via Ehrlich pathway from the catabolism of valine and leucine, respectively. In this study, we engineered CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, for the production of isobutanol and 3-methyl-1-butanol. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. We also increased transcription of endogenous genes in the valine and leucine biosynthetic pathways by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production (ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4 D578Y, a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol.

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References

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

    Article  PubMed  CAS  Google Scholar 

  • Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC (2010) Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 85:651–657

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Baichwal VR, Cunningham TS, Gatzek PR, Kohlhaw GB (1983) Leucine biosynthesis in yeast: identification of two genes (LEU4, LEU5) that affect alpha-Isopropylmalate synthase activity and evidence that LEU1 and LEU2 gene expression is controlled by alpha-Isopropylmalate and the product of a regulatory gene. Curr Genet 7:369–377

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  • Boer VM, Daran JM, Almering MJ, de Winde JH, Pronk JT (2005) Contribution of the Saccharomyces cerevisiae transcriptional regulator Leu3p to physiology and gene expression in nitrogen- and carbon-limited chemostat cultures. FEMS Yeast Res 5:885–897

    Article  PubMed  CAS  Google Scholar 

  • Boles E, de Jong-Gubbels P, Pronk JT (1998) Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. J Bacteriol 180:2875–2882

    PubMed  CAS  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Buijs NA, Siewers V, Nielsen J (2013) Advanced biofuel production by the yeast Saccharomyces cerevisiae. Curr Opin Chem Biol 17:480–488

    Article  PubMed  CAS  Google Scholar 

  • Chen X, Nielsen KF, Borodina I, Kielland-Brandt MC, Karhumaa K (2011) Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism. Biotechnol Biofuels 4:21

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Connor MR, Liao JC (2008) Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 74:5769–5775

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Connor MR, Liao JC (2009) Microbial production of advanced transportation fuels in non-natural hosts. Curr Opin Biotechnol 20:307–315

    Article  PubMed  CAS  Google Scholar 

  • Dickinson JR, Harrison SJ, Hewlins MJ (1998) An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae. J Biol Chem 273:25751–25756

    Article  PubMed  CAS  Google Scholar 

  • Eden A, Simchen G, Benvenisty N (1996) Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branched-chain amino acid aminotransferases. J Biol Chem 271:20242–20245

  • Friden P, Schimmel P (1987) LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes. Mol Cell Biol 7:2708–2717

    PubMed  CAS  PubMed Central  Google Scholar 

  • Friden P, Schimmel P (1988) LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol 8:2690–2697

    PubMed  CAS  PubMed Central  Google Scholar 

  • Friden P, Reynolds C, Schimmel P (1989) A large internal deletion converts yeast LEU3 to a constitutive transcriptional activator. Mol Cell Biol 9:4056–4060

    PubMed  CAS  PubMed Central  Google Scholar 

  • Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30:e23

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hazelwood LA, Daran JM, van Maris AJ, Pronk JT, Dickinson JR (2008) The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl Environ Microbiol 74:2259–2266

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hohmann S (2002) Osmotic adaptation in yeast–control of the yeast osmolyte system. Int Rev Cytol 215:149–187

    Article  PubMed  CAS  Google Scholar 

  • Horton CE, Huang KX, Bennett GN, Rudolph FB (2003) Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase genes in Clostridium acetobutylicum and Escherichia coli for the production of isoamyl acetate. J Ind Microbiol Biotechnol 30:427–432

    Article  PubMed  CAS  Google Scholar 

  • Ihrig J, Hausmann A, Hain A, Richter N, Hamza I, Lill R, Muhlenhoff U (2010) Iron regulation through the back door: iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in Saccharomyces cerevisiae. Eukaryot Cell 9:460–471

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Kispal G, Steiner H, Court DA, Rolinski B, Lill R (1996) Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein. J Biol Chem 271:24458–24464

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Kondo T, Tezuka H, Ishii J, Matsuda F, Ogino C, Kondo A (2012) 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

    Article  PubMed  CAS  Google Scholar 

  • Lee WH, Seo SO, Bae YH, Nan H, Jin YS, Seo JH (2012) Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes. Bioprocess Biosyst Eng 35:1467–1475

    Article  PubMed  CAS  Google Scholar 

  • Lilly M, Bauer FF, Styger G, Lambrechts MG, Pretorius IS (2006) The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates. FEMS Yeast Res 6:726–743

    Article  PubMed  CAS  Google Scholar 

  • Matsuda F, Ishii J, Kondo T, Ida K, Tezuka H, Kondo A (2013) Increased isobutanol production in Saccharomyces cerevisiae by eliminating competing pathways and resolving cofactor imbalance. Microbiol Cell Fact 12:119

    Article  Google Scholar 

  • Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122

    Article  PubMed  CAS  Google Scholar 

  • Nevoigt E (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 72:379–412

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Oba T, Nomiyama S, Hirakawa H, Tashiro K, Kuhara S (2005) Asp578 in LEU4p is one of the key residues for leucine feedback inhibition release in sake yeast. Biosci Biotechnol Biochem 69:1270–1273

    Article  PubMed  CAS  Google Scholar 

  • Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, Mattanovich D (2011) Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems? Appl Microbiol Biotechnol 89:939–948

    Article  PubMed  CAS  Google Scholar 

  • Romagnoli G, Luttik MA, Kotter P, Pronk JT, Daran JM (2012) Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae. Appl Environ Microbiol 78:7538–7548

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Ryan ED, Kohlhaw GB (1974) Subcellular localization of isoleucine-valine biosynthetic enzymes in yeast. J Bacteriol 120:631–637

    PubMed  CAS  PubMed Central  Google Scholar 

  • Schoondermark-Stolk SA, Tabernero M, Chapman J, Ter Schure EG, Verrips CT, Verkleij AJ, Boonstra J (2005) Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol. FEMS Yeast Res 5:757–766

    Article  PubMed  CAS  Google Scholar 

  • Todd RB, Andrianopoulos A (1997) Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif. Fungal Genet Biol 21:388–405

    Article  PubMed  CAS  Google Scholar 

  • Vadali RV, Horton CE, Rudolph FB, Bennett GN, San KY (2004) Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2. Appl Microbiol Biotechnol 63:698–704

  • Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E (2010) Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol 87:1303–1315

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto S, Suda M, Niimi S, Inui M, Yukawa H (2013) Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation. Biotechnol Bioeng 110:2938–2948

    Article  PubMed  CAS  Google Scholar 

  • Zhou K, Brisco PR, Hinkkanen AE, Kohlhaw GB (1987) Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control. Nucleic Acids Res 15:5261–5273

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Zhou KM, Bai YL, Kohlhaw GB (1990) Yeast regulatory protein LEU3: a structure-function analysis. Nucleic Acids Res 18:291–298

    Article  PubMed  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Research Foundation (NRF) of Korea Grant funded by the Korean Government (2012-R1A1A-3011963).

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  1. School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-744, Republic of Korea

    Seong-Hee Park, Sujin Kim & Ji-Sook Hahn

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  1. Seong-Hee Park
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  2. Sujin Kim
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Correspondence to Ji-Sook Hahn.

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Park, SH., Kim, S. & Hahn, JS. Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol. Appl Microbiol Biotechnol 98, 9139–9147 (2014). https://doi.org/10.1007/s00253-014-6081-0

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