Skip to main content
SpringerLink
Log in

Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP+-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19 % more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95 % producing up to 186 g l-1 KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Aurich A, Stottmeister U (2006) Verbundvorhaben: Biotechnologische Gewinnung von Carbonsäuren, Teilvorhaben 1: Biotechnologische Gewinnung von Oxocarbonsäuren als Synthesebausteine. Präambel, Endbericht. Projekt der Fachagentur für nachwachsende Rohstoffe, FZ: 220010701. http://www.fnr-server.de/ftp/pdf/berichte/220010701.pdf

  • Aurich A, Specht R, Müller RA, Stottmeister U, Yovkova V, Otto C, Holz M, Barth G, Heretsch P, Thomas F, Sicker D, Giannis A (2012) Microbiologically produced carboxylic acids used as buildin blocks in organic synthesis. Subcell Biochem 64:391–423

    Article  CAS  PubMed  Google Scholar 

  • Barth G, Gaillardin C (1996) Yarrowia lipolytica. In: Wolf K (ed) Nonconventional yeasts in biotechnology. Springer-Verlag, Berlin, pp 313–388

    Chapter  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chernyavskaya OG, Shishkanova NV, Il'chenko AP, Finogenova TV (2000) Synthesis of alpha-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol. Appl Microbiol Biotechnol 53:152–158

    Article  CAS  PubMed  Google Scholar 

  • Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241:779–786

    Article  CAS  PubMed  Google Scholar 

  • Dujon B, Sherman D, Fischer G, Durrens P, Cassaregola S, Lafontaine I, De Montigny J, Marck C, Neuvéglise C, Talla E, Goffard N, Frageul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisramé A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud J-M, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML, Suleau A, Swennen D, Tekaia F, Wesolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet JL (2004) Genome evolution in yeasts. Nature 430:35–44

    Article  PubMed  Google Scholar 

  • Finogenova TV, Morgunov IG, Kamzolova SV, Chernyavskaya OG (2005) Organic acid production by the yeast Yarrowia lipolytica: a review of prospects. Appl Biochem Microbiol 41:418–425

    Article  CAS  Google Scholar 

  • Flores CL, Gancedo C (2005) Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype. Eukaryot Cell 4(2):356–364

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Förster A, Köllner K, Mauersberger S, Barth G (2006) Verbundvorhaben: Biotechnologische Gewinnung von Carbonsäuren, Teilvorhaben 3: Genetic engineering oxocarbonsäurebildender Hefen. Endbericht. Projekt der Fachagentur für nachwachsende Rohstoffe, FZ: 22002505. http://www.fnr-server.de/ftp/pdf/berichte/220010701.pdf

  • Haselbeck RJ, McAlister-Henn L (1991) Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J Biol Chem 266:2339–2345

    CAS  PubMed  Google Scholar 

  • Haselbeck RJ, McAlister-Henn L (1993) Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases. J Biol Chem 268:12116–12122

    CAS  PubMed  Google Scholar 

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272

    Article  CAS  PubMed  Google Scholar 

  • Holz M, Otto C, Kretzschmar A, Yovkova V, Aurich A, Pötter M, Marx A, Barth G (2011) Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids. Appl Microbiol Biotechnol 89:1519–1526

    Article  CAS  PubMed  Google Scholar 

  • Huang HJ, Liu LM, Li Y, Du GC, Chen J (2006) Redirecting carbon flux in Torulopsis glabrata from pyruvate to alpha-ketoglutaric acid by changing metabolic co-factors. Biotechnol Lett 28:95–98

    Article  CAS  PubMed  Google Scholar 

  • Il'chenko AP, Chernyavskaya OG, Shishkanova NV, Finogenova TV (2002) Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of α-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes (Russ). Microbiology (Moscow) 71(3):269–274; Mikrobiologiya 71(3):315–322

    Google Scholar 

  • Kamawoto S, Tanaka A, Yamamura M, Teranishi Y, Fukui S, Osumi M (1977) Microbody of n-alkane grown yeast: Enzyme localization in the isolated microbody. Arch Microbiol 67:1–8

    Article  Google Scholar 

  • Kamzolova SV, Morgunov IG (2013) α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast. Appl Microbiol Biotechnol 97:5517–5525

    Article  CAS  PubMed  Google Scholar 

  • Kamzolova SV, Chiglintseva MN, Lunina JN, Morgunov IG (2012) α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation. Appl Microbiol Biotechnol 96:783–791

    Article  CAS  PubMed  Google Scholar 

  • Kretzschmar A (2010) Die Beeinflussung der Succinatproduktion durch die veränderte Aktivität der Succinyl-CoA Synthetase und der Pyruvat-Carboxylase in Yarrowia lipolytica. Dissertation, Technische Universität Dresden

  • Maldonado P, Desmarquest JP, Gaillardin C, Binet D, Malmaison R (1973) Process for getting diploid Candida lipolytica strains for alpha-ketoglutarate fermentation. Institut Français du Petrole, Patent FR 2207989

  • Marchal R, Vandecasteele JP, Metche M (1977) Regulation of central metabolism in relation to citric acid production in Saccharomycopsis lipolytica. Arch Microbiol 133:99–104

    Article  Google Scholar 

  • Mauersberger S, Kruse K, Barth G (2003) Chapter 63. Induction of citric acid/isocitric acid and α-ketoglutaric acid production in the yeast Yarrowia lipolytica. In: Wolf K, Breunig K, Barth G (eds) Non-conventional yeasts in genetics, biochemistry and biotechnology. Practical protocols. Springer, Berlin, pp 393–400

    Chapter  Google Scholar 

  • Otto C (2010) Gezielte Beeinflussung der Ausbeute und des Nebenproduktspektrums der α-Ketoglutarsäure Ausbeute in Yarrowia lipolytica mittels “metabolic engineering”. Dissertation, Technische Universität Dresden

  • Otto C, Yovkova V, Barth G (2011) Overproduction and secretion of α-ketoglutaric acid by microorganisms. Appl Microbiol Biotechnol 92:689–695

    Article  CAS  PubMed  Google Scholar 

  • Otto C, Yovkova V, Aurich A, Mauersberger S, Barth G (2012) Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica. Appl Microbiol Biotechnol 95:905–917

    Article  CAS  PubMed  Google Scholar 

  • Pignède G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud J-M (2000) Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 66:3283–3289

    Article  PubMed Central  PubMed  Google Scholar 

  • Stottmeister U, Weissbrodt E (1991) Product formation by Yarrowia lipolytica—some generalizing aspects. In: Finogenova TV, Sharyshev AA (eds) Alkane metabolism and oversynthesis of metabolites by microorganisms. Center of Biological Research USSR Academy of Science, Pushchino, Russia, pp 147–158

    Google Scholar 

  • Stottmeister U, Aurich A, Wilde H, Andersch J, Schmidt S, Sicker D (2005) White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses. J Ind Microbiol Biotechnol 32:651–664

    Article  CAS  PubMed  Google Scholar 

  • van Urk H, Schipper D, Breedveld GJ, Mak PR, Scheffers WA, van Dijken JP (1989) Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim Biophys Acta 992(1):78–86

    Article  PubMed  Google Scholar 

  • Verseck S, Karau A, Weber M (2007/2009) Fermentative production of alpha-ketoglutaric acid. Evonik Degussa, Patents DE102007051451.6 (2007) and WO2009053489 (2009)

  • Walker ME, Val DL, Rohde M, Devenish RJ, Wallace JC (1991) Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes. Biochem Biophys Res Commun 176:1210–1217

    Article  CAS  PubMed  Google Scholar 

  • Weißbrodt E, Gey M, Barth G, Weber H, Stottmeister U, Düresch R, Richter H-P (1989) Verfahren zur Herstellung von 2-Oxoglutarsäure durch Hefen. Patent DD 267999

  • Yin X, Madzak C, Du G, Zhou J, Chen J (2012) Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway. Appl Microbiol Biotechnol 96(6):1527–1537

    Article  CAS  PubMed  Google Scholar 

  • Yovkova V (2011) Gentechnische Optimierung der Hefe Yarrowia lipolytica für die biotechnologische Gewinnung von α-Ketoglutarsäure. Dissertation, Technische Universität Dresden

  • Zhang D, Liang N, Shi Z, Liu L, Chen J, Du G (2009) Enhancement of α-ketoglutarate production in Torulopsis glabrata: Redistribution of carbon flux from pyruvate to α-ketoglutarate. Biotechnol Bioprocess Eng 14:134–139

    Article  CAS  Google Scholar 

  • Zhao WN, McAlister-Henn L (1996) Expression and gene disruption analysis of the isocitrate dehydrogenase family in yeast. Biochemistry 35:7873–7878

    Article  CAS  PubMed  Google Scholar 

  • Zhou J, Zhou H, Du G, Liu L, Chen J (2010) Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction. Lett Appl Microbiol 51:264–271

    Article  CAS  PubMed  Google Scholar 

  • Zhou J, Yin X, Madzak C, Du G, Chen J (2012) Enhanced α-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism. J Biotechnol 161:257–264

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was partially co-financed by the European Union, the Land of North-Rhine Westphalia of Germany and by the Evonik Degussa. We thank Dr. Jean-Marc Nicaud (INRA-CNRS Paris-Grignon, France) for supplying the vector JMP6.

Author information

Authors and Affiliations

  1. Institut für Mikrobiologie, Technische Universität Dresden, 01062, Dresden, Germany

    Venelina Yovkova, Christina Otto, Stephan Mauersberger & Gerold Barth

  2. Umwelt- und Biotechnologisches Zentrum (UBZ), Helmholtz-Zentrum für Umweltforschung - UFZ, 04318, Leipzig, Germany

    Andreas Aurich

Authors
  1. Venelina Yovkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christina Otto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andreas Aurich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephan Mauersberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gerold Barth
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Venelina Yovkova.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 82 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yovkova, V., Otto, C., Aurich, A. et al. Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica . Appl Microbiol Biotechnol 98, 2003–2013 (2014). https://doi.org/10.1007/s00253-013-5369-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-5369-9

Keywords

Search

Navigation