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Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy

  • Applied genetics and molecular biotechnology
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Abstract

Yarrowia lipolytica is a promising platform for single cell oil production. It is well-known for its metabolism oriented toward utilization of hydrophobic substrates and accumulation of storage lipids. Multiple copies of DGA2 under constitutive promoter were introduced into the Q4 strain, a quadruple mutant deleted for the four acyltransferases (Δdga1, Δdga2, Δlro1, and Δare1) to improve lipid accumulation. The Q4-DGA2 x3 strain contains three copies of DGA2. Further increase in accumulation was accomplished by blocking the β-oxidation pathway through MFE1 gene deletion yielding Q4-Δmfe DGA2 x3. In order to use molasses as a substrate for single cell oil production, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene yielding Q4-SUC2 DGA2 x3 and Q4-Δmfe SUC2 DGA2 x3. During cultivation on sucrose medium with a carbon to nitrogen ratio of 80, both strains accumulated more than 40 % of lipids, which was a 2-fold increase in lipid storage. Q4-Δmfe SUC2 DGA2 x3 accumulated more lipids than Q4-SUC2 DGA2 x3 (49 vs. 43 %) but yielded less biomass (13.7 vs. 15 g/L). When grown on 8 % (v/v) molasses, both strains accumulated more than 30 % of lipids after 3 days, while biomass yield was higher in Q4-SUC2 DGA2 x3 (16.4 vs. 14.4 g/L). Further addition of molasses at 72 h resulted in higher biomass yield, 26.6 g/L for Q4-SUC2 DGA2 x3, without modification of lipid content. This work presents genetically modified strains of Y. lipolytica as suitable tools for direct conversion of industrial molasses into value added products based on single cell oils.

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References

  • Arshad M, Ahmed S, Zia MA, Rajoka MI (2014) Kinetics and thermodynamics of ethanol production by Saccharomyces cerevisiae MLD10 using molasses. Appl Biochem Biotechnol 172:2455–2464. doi:10.1007/s12010-013-0689-x

    Article  CAS  PubMed  Google Scholar 

  • Bankar AV, Kumar AR, Zinjarde SS (2009) Environmental and industrial applications of Yarrowia lipolityca. Appl Microbiol Biotechnol 84:847–865. doi:10.1007/s00253-009-2156-8

    Article  CAS  PubMed  Google Scholar 

  • Barth G, Gaillardin C (1996) The dimorphic fungus Yarrowia lipolytica. In: Wolf K (ed) Genetics, biochemistry, and molecular biology of non conventional yeasts. Springer, Berlin, pp 313–388

    Google Scholar 

  • Beopoulos A, Mrozova Z, Thevenieau F, Le Dall MT, Hapala I, Papanikolaou S, Chardot T, Nicaud JM (2008) Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl Environ Microbiol 74:7779–7789. doi:10.1128/AEM.01412-08

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 48:375–387. doi:10.1016/j.plipres.2009.08.005

    Article  CAS  PubMed  Google Scholar 

  • Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud JM (2012) Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol 93:1523–1537. doi:10.1007/s00253-011-3506-x

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Čertík M, Shimidzu S (2000) Kinetic analysis of biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4. Appl Microbiol Biotechnol 54:224–230

    Article  PubMed  Google Scholar 

  • Chuang LT, Chen DC, Nicaud JM, Madzak C, Chen YH, Huang YS (2010) Co-expression of heterologous desaturase genes in Yarrowia lipolytica. N Biotechnol 27:277–282. doi:10.1016/j.nbt.2010.02.006

    Article  CAS  PubMed  Google Scholar 

  • Dulermo T, Nicaud JM (2011) Involvement of G3P shuttle and β-oxidation pathway into the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab Eng 13:482–491. doi:10.1016/j.ymben.2011.05.002

    Article  CAS  PubMed  Google Scholar 

  • Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55:727–737

    Article  CAS  PubMed  Google Scholar 

  • Fickers P, Marty A, Nicaud JM (2011) The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications. Biotechnol Adv 29:632–644. doi:10.1016/j.biotechadv.2011.04.005

    Article  CAS  PubMed  Google Scholar 

  • Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509

    CAS  PubMed  Google Scholar 

  • Gaillardin C, Ribet AM (1987) LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica. Curr Genet 11:369–375

    Article  CAS  PubMed  Google Scholar 

  • Gasmi N, Lassoued R, Ayed A, Tréton B, Chevret D, Nicaud JM, Kallel H (2012) Production and characterization of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 96:89–101. doi:10.1007/s00253-012-4141-x

    Article  CAS  PubMed  Google Scholar 

  • Grenfell-Lee D, Zeller S, Cardoso R, Pucaj K (2014) The safety of β-carotene from Yarrowia lipolytica. Food Chem Toxicol 65:1–11. doi:10.1016/j.fct.2013.12.010

    Article  CAS  PubMed  Google Scholar 

  • Groenewald M, Boekhout T, Neuvéglise C, Gaillardin C, van Dijck PW, Wyss M (2014) Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential. Crit Rev Microbiol 40:187–206. doi:10.3109/1040841X.2013.770386

    Article  CAS  PubMed  Google Scholar 

  • Haddouche R, Poirier Y, Delessert S, Sabirova J, Pagot Y, Neuvéglise C, Nicaud JM (2011) Engineering polyhydroxyalkanoate content and monomer composition in the oleaginous yeast Yarrowia lipolytica by modifying the ß-oxidation multifunctional protein. Appl Microbiol Biotechnol 91:1327–1340. doi:10.1007/s00253-011-3331-2

    Article  CAS  PubMed  Google Scholar 

  • He J, Wu AM, Chen D, Yu B, Mao X, Zheng P, Yu J, Tian G (2014) Cost-effective lignocellulolytic enzyme production by Trichoderma reesei on a cane molasses medium. Biotechnol Biofuels 7:43. doi:10.1186/1754-6834-7-43

    Article  PubMed Central  PubMed  Google Scholar 

  • Kabran P, Rossignol T, Gaillardin C, Nicaud JM, Neuvéglise C (2012) Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica. DNA Res 19:231–244. doi:10.1093/dnares/dss007

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Karatay SE, Dönmez G (2010) Improving the lipid accumulation properties of the yeast cells for biodiesel production using molasses. Bioresour Technol 101:7988–7990. doi:10.1016/j.biortech.2010.05.054

    Article  CAS  PubMed  Google Scholar 

  • Lazar Z, Walczak E, Robak M (2011) Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC + transformants. Bioresour Technol 102:6982–6989. doi:10.1016/j.biortech.2011.04.032

    Article  CAS  PubMed  Google Scholar 

  • Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M (2013) Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications. J Ind Microbiol Biotechnol 40:1273–1283. doi:10.1007/s10295-013-1323-1

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Le Dall MT, Nicaud JM, Gaillardin C (1994) Multi-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44

    Article  PubMed  Google Scholar 

  • Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–81

    Article  CAS  PubMed  Google Scholar 

  • Makri A, Fakas S, Aggelis G (2010) Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour Technol 101:2351–2358. doi:10.1016/j.biortech.2009.11.024

    Article  CAS  PubMed  Google Scholar 

  • Nicaud J-M, Fabre E, Gaillardin C (1989) Expression of invertase activity in Yarrowia lipolytica and its use as a selective marker. Curr Genet 16:253–260

    Article  CAS  PubMed  Google Scholar 

  • Olbrich H (2006) The molasses. Biotechnologie-Kempe GmbH, Berlin

    Google Scholar 

  • Ortiz ME, Fornaguera MJ, Raya RR, Mozzi F (2012) Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source. Appl Microbiol Biotechnol 95:991–999. doi:10.1007/s00253-012-3945-z

    Article  CAS  PubMed  Google Scholar 

  • Papanikolaou S, Aggelis G (2002) Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture. Bioresour Technol 82:43–49

    Article  CAS  PubMed  Google Scholar 

  • Papanikolaou S, Aggelis G (2010) Yarrowia lipolytica: a model microorganism used for the production of tailor-made lipids. Eur J Lipid Sci Technol 112:639–654. doi:10.1002/ejlt.200900197

    Article  CAS  Google Scholar 

  • Papanikolaou S, Beopoulos A, Koletti A, Thevenieau F, Koutinasa A, Nicaud JM, Aggelis G (2013) Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica. J Biotechnol 168:303–314. doi:10.1016/j.jbiotec.2013.10.025

    Article  CAS  PubMed  Google Scholar 

  • Pignède G, Wang H, Fudalej F, Gaillardin C, Seman M, Nicaud J-M (2000) Autocloning vectors for gene expression and amplification for the yeast Y. lipolytica. Appl Environ Microbiol 66:3283–3289

    Article  PubMed Central  PubMed  Google Scholar 

  • Querol A, Barrio E, Huerta T, Ramon D (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 58:2948–2953

    PubMed Central  CAS  PubMed  Google Scholar 

  • Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86:807–815

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Tsigie YA, Wang CY, Kasim NS, Diem QD, Huynh LH, Ho QP, Truong CT, Ju YH (2012) Oil production from Yarrowia lipolytica Po1g using rice bran hydrolysate. J Biomed Biotechnol 2012:378384. doi:10.1155/2012/378384

    Article  PubMed Central  PubMed  Google Scholar 

  • Xia J, Xu Z, Xu H, Liang J, Li S, Feng X (2014) Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1. Bioresour Technol 164:241–247. doi:10.1016/j.biortech.2014.04.078

    Article  CAS  PubMed  Google Scholar 

  • Zhu Z, Zhang S, Liu H, Shen H, Lin X, Yang F, Zhou YJ, Jin G, Ye M, Zou H, Zhao ZK (2012) A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat Commun 3:1112. doi:10.1038/ncomms2112

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The work was supported by grant VEGA 1/0574/15 from the Grant Agency of the Ministry of Education, Slovak Republic and by grant APVV-0662-11 from the Slovak Research and Development Agency, Slovak Republic. This work was funded by the French National Institute for Agricultural Research (Institut National de la Recherche Agronomique).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia

    Peter Gajdoš & Milan Čertík

  2. INRA, UMR1319 Micalis, F-78352, Jouy-en-Josas, France

    Jean-Marc Nicaud & Tristan Rossignol

  3. AgroParisTech, UMR Micalis, F-78352, Jouy-en-Josas, France

    Jean-Marc Nicaud & Tristan Rossignol

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  1. Peter Gajdoš
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  4. Milan Čertík
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Correspondence to Milan Čertík.

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Gajdoš, P., Nicaud, JM., Rossignol, T. et al. Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy. Appl Microbiol Biotechnol 99, 8065–8074 (2015). https://doi.org/10.1007/s00253-015-6733-8

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  • DOI: https://doi.org/10.1007/s00253-015-6733-8

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