Applied Microbiology and Biotechnology

, Volume 101, Issue 1, pp 465–474 | Cite as

Metabolically engineered Saccharomyces cerevisiae for enhanced isoamyl alcohol production

Bioenergy and biofuels
First Online:
Received:
Accepted:

Abstract

Higher chain alcohols have gained much attention as next generation transport fuels because of their higher energy density and low moisture absorption capacity compared to ethanol. In the present study, we attempted to engineer Saccharomyces cerevisiae for the synthesis of isoamyl alcohol via de novo leucine biosynthetic pathway coupled with Ehrlich degradation pathway. To achieve high-level production of isoamyl alcohol, two strategies are used in the current study: (1) reconstruction of a chromosome-based leucine biosynthetic pathway under the control of galactose-inducible promoters; (2) overexpression of the mitochondrial 2-isopropylmalate (α-IPM) transporter to boost the transportation of α-IPM from mitochondria to the cytosol. We found engineered yeast cells with a combinatorially assembled leucine biosynthetic pathway coupled with the Ehrlich degradation pathway resulted in high-level production of isoamyl alcohol; however, there was still a significant amount of isobutanol co-formed during the fermentation process. Further introducing an α-IPM transporter not only boosted the isoamyl alcohol biosynthetic pathway activity but also reduced isobutanol to a much lower level. Taken together, our work represents the first study to construct a chromosome-based leucine biosynthetic pathway for isoamyl alcohol production. Furthermore, the utilization of the mitochondrial compartment coupled with the transporter engineering serves as an effective approach to minimize the by-product formation and to improve the isoamyl alcohol production.

Keywords

Leucine biosynthetic pathway Ehrlich degradation pathway Isoamyl alcohol Pathway assembly 2-isopropylmalate transporter Saccharomyces cerevisiae 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

This work was funded by the National University of Singapore (Start-up Grant: R279 000 364 133).

Authors’ contributions

JY conceived the study, designed the experiments, performed the experiments, analyzed the data, and drafted the manuscript. XC participated in the experiments, analyzed the data, and helped to revise the manuscript. PM participated in the experiments, analyzed the data, and helped to revise the manuscript. CBC supervised the project and revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Ethical approval

This study does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451(7174):86–89CrossRefPubMedGoogle Scholar
  2. Avalos JL, Fink GR, Stephanopoulos G (2013) Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat Biotechnol 31(4):335–341CrossRefPubMedPubMedCentralGoogle Scholar
  3. Blombach B, Eikmanns BJ (2011) Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. Bioeng Bugs 2(6):346–350CrossRefPubMedPubMedCentralGoogle Scholar
  4. Branduardi P, Longo V, Berterame NM, Rossi G, Porro D (2013) A novel pathway to produce butanol and isobutanol in Saccharomyces cerevisiae. Biotechnol Biofuels 6(1):68CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brat D, Weber C, Lorenzen W, Bode HB, Boles E (2012) Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae. Biotechnol Biofuels 5(1):65CrossRefPubMedPubMedCentralGoogle Scholar
  6. Buijs NA, Siewers V, Nielsen J (2013) Advanced biofuel production by the yeast Saccharomyces cerevisiae. Curr Opin Chem Biol 17(3):480–488CrossRefPubMedGoogle Scholar
  7. Cann AF, Liao JC (2008) Production of 2-methyl-1-butanol in engineered Escherichia coli. Appl Microbiol Biotechnol 81(1):89–98CrossRefPubMedGoogle Scholar
  8. 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 4:21CrossRefPubMedPubMedCentralGoogle Scholar
  9. Connor MR, Liao JC (2008) Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 74(18):5769–5775CrossRefPubMedPubMedCentralGoogle Scholar
  10. Connor MR, Cann AF, Liao JC (2010) 3-methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation. Appl Microbiol Biotechnol 86(4):1155–1164CrossRefPubMedPubMedCentralGoogle Scholar
  11. Ferramosca A, Zara V (2013) Biogenesis of mitochondrial carrier proteins: molecular mechanisms of import into mitochondria. Biochim Biophys Acta 1833(3):494–502CrossRefPubMedGoogle Scholar
  12. 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(6):e23CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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(8):2259–2266CrossRefPubMedPubMedCentralGoogle Scholar
  14. Karim AS, Curran KA, Alper HS (2013) Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13(1):107–116CrossRefPubMedGoogle Scholar
  15. 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(1–2):32–37CrossRefPubMedGoogle Scholar
  16. Kuan J, Saier MH Jr (1993) The mitochondrial carrier family of transport proteins: structural, functional, and evolutionary relationships. Crit Rev Biochem Mol Biol 28(3):209–233CrossRefPubMedGoogle Scholar
  17. Kunji ER (2004) The role and structure of mitochondrial carriers. FEBS Lett 564(3):239–244CrossRefPubMedGoogle Scholar
  18. Lopez G, Quezada H, Duhne M, Gonzalez J, Lezama M, El-Hafidi M, Colon M, Martinez de la Escalera X, Flores-Villegas MC, Scazzocchio C, DeLuna A, Gonzalez A (2015) Diversification of paralogous alpha-isopropylmalate synthases by modulation of feedback control and hetero-oligomerization in Saccharomyces cerevisiae. Eukaryot Cell 14(6):564–577CrossRefPubMedPubMedCentralGoogle Scholar
  19. Marobbio CM, Giannuzzi G, Paradies E, Pierri CL, Palmieri F (2008) Alpha-isopropylmalate, a leucine biosynthesis intermediate in yeast, is transported by the mitochondrial oxaloacetate carrier. J Biol Chem 283(42):28445–28453CrossRefPubMedPubMedCentralGoogle Scholar
  20. 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. Microb Cell Factories 12:119CrossRefGoogle Scholar
  21. Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496(7446):528–532CrossRefPubMedGoogle Scholar
  22. Palmieri L, Vozza A, Agrimi G, De Marco V, Runswick MJ, Palmieri F, Walker JE (1999) Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate. J Biol Chem 274(32):22184–22190CrossRefPubMedGoogle Scholar
  23. Park SH, Kim S, Hahn JS (2014) Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol. Appl Microbiol Biotechnol 98(21):9139–9147CrossRefPubMedGoogle Scholar
  24. Peralta-Yahya PP, Keasling JD (2010) Advanced biofuel production in microbes. Biotechnol J 5(2):147–162CrossRefPubMedGoogle Scholar
  25. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440(7086):940–943CrossRefPubMedGoogle Scholar
  27. Sadowski I, Su TC, Parent J (2007) Disintegrator vectors for single-copy yeast chromosomal integration. Yeast 24(5):447–455CrossRefPubMedGoogle Scholar
  28. Savage N (2011) Fuel options: the ideal biofuel. Nature 474(7352):S9–11CrossRefPubMedGoogle Scholar
  29. Smith KM, Liao JC (2011) An evolutionary strategy for isobutanol production strain development in Escherichia coli. Metab Eng 13(6):674–681CrossRefPubMedGoogle Scholar
  30. Smith KM, Cho KM, Liao JC (2010) Engineering Corynebacterium glutamicum for isobutanol production. Appl Microbiol Biotechnol 87(3):1045–1055CrossRefPubMedPubMedCentralGoogle Scholar
  31. Wang BW, Shi AQ, Tu R, Zhang XL, Wang QH, Bai FW (2012) Branched-chain higher alcohols. Adv Biochem Eng Biotechnol 128:101–118PubMedGoogle Scholar
  32. Westfall PJ, Pitera DJ, Lenihan JR, Eng D, Woolard FX, Regentin R, Horning T, Tsuruta H, Melis DJ, Owens A, Fickes S, Diola D, Benjamin KR, Keasling JD, Leavell MD, McPhee DJ, Renninger NS, Newman JD, Paddon CJ (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A 109(3):E111–E118CrossRefPubMedPubMedCentralGoogle Scholar
  33. Yuan J, Ching CB (2014) Combinatorial engineering of mevalonate pathway for improved amorpha-4,11-diene production in budding yeast. Biotechnol Bioeng 111(3):608–617CrossRefPubMedGoogle Scholar
  34. Yuan J, Ching CB (2015a) Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds. ACS Synth Biol 4(1):23–31CrossRefPubMedGoogle Scholar
  35. Yuan J, Ching CB (2015b) Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae. Microb Cell Factories 14(1):38CrossRefGoogle Scholar
  36. Yuan J, Ching CB (2016) Mitochondrial acetyl-CoA utilization pathway for terpenoid productions. Metab Eng. doi:​ 10.1016/j.ymben.2016.07.008

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringNational University of SingaporeSingaporeSingapore
  2. 2.Temasek LaboratoriesNational University of SingaporeSingaporeSingapore
  3. 3.Biotransformation Innovation Platform, Agency for Science, Technology and ResearchSingaporeSingapore
  4. 4.Singapore Institute of TechnologySingaporeSingapore

Personalised recommendations