Advertisement

Applied Microbiology and Biotechnology

, Volume 100, Issue 3, pp 1297–1305 | Cite as

Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides

  • Lina Zhao
  • José T. Cánovas-Márquez
  • Xin Tang
  • Haiqin Chen
  • Yong Q. Chen
  • Wei Chen
  • Victoriano GarreEmail author
  • Yuanda SongEmail author
  • Colin Ratledge
Applied genetics and molecular biotechnology

Abstract

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Keywords

Mucor circinelloides Malate transporter Lipid content Malate concentration Citrate/malate/pyruvate shuttle 

Notes

Acknowledgments

This study was funded by the National Natural Science Foundation of China (31271812, 81071685, 21276108), the National High Technology Research and Development Program of China (2012AA022105C), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249), the Program for New Century Excellent Talents (NCET-13-0831), and the Strategic Merieux Research Grant. The authors declare that they have no conflict of interest.

Compliance with ethical standards

Ethical statement

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

Supplementary material

253_2015_7079_MOESM1_ESM.pdf (188 kb)
ESM 1 (PDF 187 kb)

References

  1. Bartnicki-García S, Nickerson WJ (1962) Nutrition, growth and morphogenesis of Mucor rouxii. J Bacteriol 84:841–858PubMedPubMedCentralGoogle Scholar
  2. Benito EP, Campuzano V, López-Matas MA, De Vicente JI, Eslava AP (1995) Isolation, characterization and transformation, by autonomous replication, of Mucor circinelloides OMPdecase-deficient mutans. Mol Gen Genet 248:126–135. doi: 10.1007/BF02190793 PubMedCrossRefGoogle Scholar
  3. Chaney AL, Marbach EP (1962) Modified reagents for determination of urea and ammonia. Clin Chem 8:130–132PubMedGoogle Scholar
  4. Evans CT, Scragg AH, Ratledge C (1983) A comparative study of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts. Eur J Biochem 130:195–204. doi: 10.1111/j.1432-1033.1983.tb07136.x PubMedCrossRefGoogle Scholar
  5. Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  6. Fontanille P, Kumar V, Christophe G, Nouaille R, Larroche C (2012) Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour Technol 114:443–439. doi: 10.1016/j.biortech.2012.02.091 PubMedCrossRefGoogle Scholar
  7. Gong Z, Wang Q, Shen H, Hu C, Jin G, Zhao ZK (2012) Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production. Bioresour Technol 117:20–24. doi: 10.1016/j.biortech.2012.04.063 PubMedCrossRefGoogle Scholar
  8. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–563. doi: 10.1016/S0022-2836(83)80284-8 PubMedCrossRefGoogle Scholar
  9. Illman AM, Scragg AH, Shales SW (2000) Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzym Microb Technol 27:631–635. doi: 10.1016/S0141-0229(00)00266-0 CrossRefGoogle Scholar
  10. Kendrick A, Ratledge C (1992) Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. Eur J Biochem 209:667–673. doi: 10.1111/j.1432-1033.1992.tb17334.x PubMedCrossRefGoogle Scholar
  11. Khozin-Goldberg I, Cohen Z (2006) The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67:696–701. doi: 10.1016/j.phytochem.2006.01.010 PubMedCrossRefGoogle Scholar
  12. Lin H, Wang Q, Shen Q, Zhan J, Zhao Y (2013) Genetic engineering of microorganisms for biodiesel production. Bioengineered 4:292–304. doi: 10.4161/bioe.23114 PubMedPubMedCentralCrossRefGoogle Scholar
  13. Nicolás FE, de Haro JP, Torres-Martínez S, Ruiz-Vázquez RM (2007) Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genet Biol 44:504–516. doi: 10.1016/j.fgb.2006.09.003 PubMedCrossRefGoogle Scholar
  14. Ratledge C (2014) The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems. Biotechnol Lett 36:1557–1568. doi: 10.1007/s10529-014-1532-3 PubMedCrossRefGoogle Scholar
  15. Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51. doi: 10.1016/S0065-2164(02)51000-5 PubMedCrossRefGoogle Scholar
  16. Rodríguez-Frómeta RA, Gutiérrez A, Torres-Martínez S, Garre V (2013) Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides. Appl Microbiol Biotechnol 97:3063–3072. doi: 10.1007/s00253-012-4432-2 PubMedCrossRefGoogle Scholar
  17. Roncero MIG, Jepsen LP, Strøman P, van Heeswijck R (1989) Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84:335–343. doi: 10.1016/0378-1119(89)90508-8 PubMedCrossRefGoogle Scholar
  18. Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid derived biofuels and chemicals. Metab Eng 21:103–113. doi: 10.1016/j.ymben.2013.07.003 PubMedCrossRefGoogle Scholar
  19. Solovchenko AE, Khozin-Goldberg I, Didi-Cohen S, Cohen Z, Merzlyak MN (2008) Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa. J Appl Phycol 20:245–251. doi: 10.1007/s10811-007-9233-0 CrossRefGoogle Scholar
  20. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. doi: 10.1016/j.ymben.2012.08.007 PubMedCrossRefGoogle Scholar
  21. Tang X, Zhang H, Chen H, Chen YQ, Chen W, Song Y (2014) Effects of 20 standard amino acids on the growth, total fatty acids production and γ-linolenic acid yield in Mucor circinelloides. Curr Microbiol 69:899–908. doi: 10.1007/s00284-014-0671-z PubMedCrossRefGoogle Scholar
  22. Torres-Martínez S, Ruiz-Vázquez RM, Garre V, López-García S, Navarro E, Vila A (2012) Molecular tools for carotenogenesis analysis in the zygomycete Mucor circinelloides. Methods Mol Biol 898:85–107. doi: 10.1007/978-1-61779-918-1-5 PubMedCrossRefGoogle Scholar
  23. Vongsangnak W, Ruenwai R, Tang X, Hu X, Zhang H, Shen B, Song Y, Laoteng K (2013) Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi. Gene 521:180–190. doi: 10.1016/j.gene.2013.03.012 PubMedCrossRefGoogle Scholar
  24. Wu S, Zhao X, Shen H, Wang Q, Zhao ZK (2011) Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions. Bioresour Technol 102:1803–1807. doi: 10.1016/j.biortech.2010.09.033 PubMedCrossRefGoogle Scholar
  25. Wynn JP, Ratledge C (1997) Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans. Microbiology 143:253–257. doi: 10.1099/00221287-143-1-253 CrossRefGoogle Scholar
  26. Wynn JP, bin Abdul HA, Ratledge C (1999) The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology 145:1911–1917. doi: 10.1099/13500872-145-8-1911 PubMedCrossRefGoogle Scholar
  27. Wynn JP, Hamid AA, Li Y, Ratledge C (2001) Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology 147:2857–2864PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Lina Zhao
    • 1
  • José T. Cánovas-Márquez
    • 2
  • Xin Tang
    • 1
  • Haiqin Chen
    • 1
    • 3
  • Yong Q. Chen
    • 1
    • 3
  • Wei Chen
    • 1
    • 3
  • Victoriano Garre
    • 2
    Email author
  • Yuanda Song
    • 1
    Email author
  • Colin Ratledge
    • 4
  1. 1.State Key Laboratory of Food Science and Technology, School of Food Science and TechnologyJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.Departamento de Genética y Microbiología (Unidad asociada al IQFR-CSIC), Facultad de BiologíaUniversidad de MurciaMurciaSpain
  3. 3.Synergistic Innovation Center for Food Safety and NutritionWuxiPeople’s Republic of China
  4. 4.Department of Biological SciencesUniversity of HullHullUK

Personalised recommendations