Applied Microbiology and Biotechnology

, Volume 103, Issue 7, pp 3167–3179 | Cite as

Understanding lipogenesis by dynamically profiling transcriptional activity of lipogenic promoters in Yarrowia lipolytica

  • Huan Liu
  • Monireh Marsafari
  • Li Deng
  • Peng XuEmail author
Applied microbial and cell physiology


Lipogenesis is a complicated process involving global transcriptional reprogramming of lipogenic pathways. It is commonly believed that nitrogen starvation triggers a metabolic shift that reroutes carbon flux from Krebs cycles to lipogenesis. In this study, we systematically surveyed and dynamically profiled the transcriptional activity of 22 lipogenic promoters aiming to delineate a picture how nitrogen starvation regulates lipogenesis in Y. lipolytica. These lipogenic promoters drive the expression of critical pathways that are responsible for the generation of reducing equivalents (NADPH), carbon backbones (acetyl-CoA, malonyl-CoA, DHAP, etc.), synthesis and degradation of fatty acids. Specifically, our investigated promoters span across an array of metabolic pathways, including glycolysis, Krebs cycle, pentose phosphate pathway, mannitol cycle, glutamine–GABA cycle, fatty acid and lipid synthesis, glyoxylate, β-oxidation, and POM (pyruvate–oxaloacetate–malate) cycle. Our work provides evidences that mannitol cycle, glutamine–GABA cycle and amino acid degradation, pyruvate oxidation, and acetate assimilation pathways are lipogenesis-related steps involved in generating cytosolic NADPH and acetyl-CoA precursors. This systematic investigation and dynamic profiling of lipogenic promoters may help us better understand lipogenesis, facilitate the formulation of structure-based kinetic models, as well as develop efficient cell factories for fuels and chemical production in oleaginous species.


Oleaginous yeast Lipogenesis Nitrogen starvations Reducing equivalents Carbon backbones 


Author contributions

PX and HL designed the study. HL performed this study with the promoter cloning work helped by MM and Ms. Lynn Wong. PX and HL wrote the manuscript.


This work was supported by the Cellular and Biochemical Engineering Program of the National Science Foundation under grant no.1805139 and the Department of Chemical, Biochemical and Environmental Engineering at University of Maryland Baltimore County.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

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

Supplementary material

253_2019_9664_MOESM1_ESM.pdf (290 kb)
ESM 1 (PDF 290 kb)


  1. Abghari A, Chen S (2014) Yarrowia lipolytica as an oleaginous cell factory platform for the production of fatty acid-based biofuel and bioproducts. Front Energy 2(21):1–21Google Scholar
  2. Bates PD, Browse J (2012) The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering. In: Front Plant Sci, vol 3Google Scholar
  3. Bellou S, Triantaphyllidou IE, Mizerakis P, Aggelis G (2016) High lipid accumulation in Yarrowia lipolytica cultivated under double limitation of nitrogen and magnesium. J Biotechnol 234:116–126. CrossRefPubMedGoogle Scholar
  4. 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(4):1523–1537. CrossRefPubMedGoogle Scholar
  5. Blazeck J, Liu L, Redden H, Alper H (2011) Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol 77:7905–7914. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Blazeck J, Liu L, Knight R, Alper HS (2013) Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica. J Biotechnol 165(3–4):184–194. CrossRefPubMedGoogle Scholar
  7. Brabender M, Hussain MS, Rodriguez G, Blenner MA (2018) Urea and urine are a viable and cost-effective nitrogen source for Yarrowia lipolytica biomass and lipid accumulation. Appl Microbiol Biotechnol 102(5):2313–2322. CrossRefPubMedGoogle Scholar
  8. Chen D-C, Beckerich J-M, Gaillardin C (1997) One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 48(2):232–235CrossRefGoogle Scholar
  9. Chen Y, Siewers V, Nielsen J (2012) Profiling of cytosolic and peroxisomal acetyl-CoA metabolism in Saccharomyces cerevisiae. PLoS One 7(8): 42475):e42475. CrossRefPubMedPubMedCentralGoogle Scholar
  10. de Jong BW, Shi S, Siewers V, Nielsen J (2014) Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb Cell Factories 13(1):39. CrossRefGoogle Scholar
  11. Dourou M, Mizerakis P, Papanikolaou S, Aggelis G (2017) Storage lipid and polysaccharide metabolism in Yarrowia lipolytica and Umbelopsis isabellina. Appl Microbiol Biotechnol 101(19):7213–7226. CrossRefPubMedGoogle Scholar
  12. Dugar D, Stephanopoulos G (2011) Relative potential of biosynthetic pathways for biofuels and bio-based products. Nat Biotechnol 29:1074–1078. CrossRefPubMedGoogle Scholar
  13. Dulermo T, Lazar Z, Dulermo R, Rakicka M, Haddouche R, Nicaud JM (2015) Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis. Biochim Biophys Acta 1851(9):1107–1117. CrossRefPubMedGoogle Scholar
  14. Fickers P, Benetti PH, Waché Y, Marty A, Mauersberger S, Smit MS, Nicaud JM (2005) Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res 5(6–7):527–543. CrossRefPubMedGoogle Scholar
  15. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA III, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefGoogle Scholar
  16. Grabowska D, Chelstowska A (2003) The ALD6 gene product is indispensable for providing NADPH in yeast cells lacking glucose-6-phosphate dehydrogenase activity. World J Biol Chem 278:13984–13988CrossRefGoogle Scholar
  17. Groenewald M, Boekhout T, Neuvéglise C, Gaillardin C, van Dijck PWM, Wyss M (2014) Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential. Crit Rev Microbiol 40(3):187–206. CrossRefPubMedGoogle Scholar
  18. Haddouche R, Delessert S, Sabirova J, Neuvéglise C, Poirier Y, Nicaud JM (2010) Roles of multiple acyl-CoA oxidases in the routing of carbon flow towards β-oxidation and polyhydroxyalkanoate biosynthesis in Yarrowia lipolytica. FEMS Yeast Res 10(7):917–927. CrossRefPubMedGoogle Scholar
  19. Hiltunen JK, Mursula AM, Rottensteiner H, Wierenga RK, Kastaniotis AJ, Gurvitz A (2003) The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 27(1):35–64CrossRefGoogle Scholar
  20. Hynes MJ, Murray SL (2010) ATP-citrate Lyase is required for production of cytosolic acetyl coenzyme a and development in genus-species Aspergillus nidulans. Eukaryot Cell 9(7):1039–1048CrossRefGoogle Scholar
  21. Jain MR, Zinjarde SS, Deobagkar DD, Deobagkar DN (2004) 2,4,6-Trinitrotoluene transformation by a tropical marine yeast, Yarrowia lipolytica NCIM 3589. Mar Pollut Bull 49(9):783–788. CrossRefPubMedGoogle Scholar
  22. Kavšček M, Bhutada G, Madl T, Natter K (2015) Optimization of lipid production with a genome-scale model of Yarrowia lipolytica. BMC Syst Biol 9(1):72. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kerkhoven EJ, Pomraning KR, Baker SE, Nielsen J (2016) Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica. Npj Syst Biolog Appl 2:16005. CrossRefGoogle Scholar
  24. Kerkhoven EJ, Kim Y-M, Wei S, Nicora CD, Fillmore TL, Purvine SO, Webb-Robertson B-J, Smith RD, Baker SE, Metz TO, Nielsen J (2017) Leucine biosynthesis is involved in regulating high lipid accumulation in Yarrowia lipolytica. mBio 8(3):1–12CrossRefGoogle Scholar
  25. Knothe G (2010) Biodiesel and renewable diesel: a comparison. Prog Energ Combust 36(3):364–373CrossRefGoogle Scholar
  26. Kozak BU, van Rossum HM, Benjamin KR, Wu L, Daran J-MG, Pronk JT, van Maris AJA (2014) Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis. Metab Eng 21:46–59. CrossRefGoogle Scholar
  27. Ledesma-Amaro R, Dulermo R, Niehus X, Nicaud J-M (2016a) Combining metabolic engineering and process optimization to improve production and secretion of fatty acids. Metab Eng 38:38–46. CrossRefPubMedGoogle Scholar
  28. Ledesma-Amaro R, Lazar Z, Rakicka M, Guo Z, Fouchard F, Coq A-MC-L, Nicaud J-M (2016b) Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose. Metab Eng 38:115–124. CrossRefPubMedGoogle Scholar
  29. Li X, Wang P, Ge Y, Wang W, Abbas A, Zhu G (2013) NADP(+)-specific isocitrate dehydrogenase from oleaginous yeast Yarrowia lipolytica CLIB122: biochemical characterization and coenzyme sites evaluation. Appl Biochem Biotechnol 171(2):403–416. CrossRefPubMedGoogle Scholar
  30. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, DeBono A, Durrett TP, Franke RB, Graham IA, Katayama K, Kelly AA, Larson T, Markham JE, Miquel M, Molina I, Nishida I, Rowland O, Samuels L, Schmid KM, Wada H, Welti R, Xu C, Zallot R, Ohlrogge J (2010) Acyl-Lipid Metabolism. The Arabidopsis Book 8:e0133. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Liu L, Markham K, Blazeck J, Zhou N, Leon D, Otoupal P, Alper HS (2015a) Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant. Metab Eng 31:102–111. CrossRefPubMedGoogle Scholar
  32. Liu L, Pan A, Spofford C, Zhou N, Alper HS (2015b) An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica. Metab Eng 29:36–45. CrossRefPubMedGoogle Scholar
  33. Liu N, Qiao K, Stephanopoulos G (2016) 13 C metabolic flux analysis of acetate conversion to lipids by Yarrowia lipolytica. Metab Eng 38:86–97CrossRefGoogle Scholar
  34. Loira N, Dulermo T, Nicaud JM, Sherman DJ (2012) A genome-scale metabolic model of the lipid-accumulating yeast Yarrowia lipolytica. BMC Syst Biol 6:35. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Markham KA, Palmer CM, Chwatko M, Wagner JM, Murray C, Vazquez S, Swaminathan A, Chakravarty I, Lynd NA, Alper HS (2018) Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation. P Natl A Sci 115(9):2096–2101CrossRefGoogle Scholar
  36. Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol R 61(1):17–32Google Scholar
  37. Minard KI, McAlister-Henn L (2005) Sources of NADPH in yeast vary with carbon source. J Biol Chem 280:39890–39896CrossRefGoogle Scholar
  38. Mlícková K, Roux E, Athenstaedt K, d'Andrea S, Daum G, Chardot T, Nicaud JM (2004) Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica. Appl Environ Microbiol 70(7):3918–3924. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Morin N, Cescut J, Beopoulos A, Lelandais G, Le Berre V, Uribelarrea JL, Molina-Jouve C, Nicaud JM (2011) Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica. PLoS One 6(11):e27966. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Pfleger BF, Gossing M, Nielsen J (2015) Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab Eng 29:1–11. CrossRefPubMedGoogle Scholar
  41. Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, Kumaran Ajikumar P, Stephanopoulos G (2015) Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 29:56–65. CrossRefPubMedGoogle Scholar
  42. Qiao K, Wasylenko TM, Zhou K, Xu P, Stephanopoulos G (2017) Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat Biotechnol 35(2):173–177. CrossRefPubMedGoogle Scholar
  43. Ratledge C (2014) The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems. Biotechnol Lett 36(8):1557–1568. CrossRefPubMedGoogle Scholar
  44. Ratledge C, Holdsworth JE (1985) Properties of a pentulose-5-phosphate phosphoketolase from yeast grown on xylose. Appl Microbiol Biotechnol 22:217–221CrossRefGoogle Scholar
  45. Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51. CrossRefPubMedGoogle Scholar
  46. 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(7):3063–3072. CrossRefPubMedGoogle Scholar
  47. Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 21:103–113. CrossRefPubMedGoogle Scholar
  48. Shi S, Chen Y, Siewers V, Nielsen J (2014a) Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1. mBio 5(3):e01130–e01114CrossRefGoogle Scholar
  49. Shi S, Valle-Rodríguez JO, Siewers V, Nielsen J (2014b) Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters. Biotechnol Bioeng 111(9):1740–1747. CrossRefPubMedGoogle Scholar
  50. Shiba Y, Ono C, Fukui F (2000) Effect of ethanol on the production of carboxypeptidase Y using the GAL10 promoter in a Saccharomyces cerevisiae gal80 mutant. J Biosci Bioeng 89:426–430. CrossRefPubMedGoogle Scholar
  51. Shirra MK, Patton-Vogt J, Ulrich A, Liuta-Tehlivets O, Kohlwein SD, Henry SA, Arndt KM (2001) Inhibition of acetyl coenzyme a carboxylase activity restores expression of the INO1 gene in a snf1 mutant strain of Saccharomyces cerevisiae. Mol Cell Biol 21(17):5710–5722CrossRefGoogle Scholar
  52. Silverman AM, Qiao K, Xu P, Stephanopoulos G (2016) Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 100(8):3781–3798. CrossRefPubMedGoogle Scholar
  53. 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. CrossRefPubMedGoogle Scholar
  54. Tai YS, Xiong M, Zhang K (2015) Engineered biosynthesis of medium-chain esters in Escherichia coli. Metab Eng 27:20–28. CrossRefPubMedGoogle Scholar
  55. van Rossum HM, Kozak BU, Pronk JT, van Maris AJA (2016) Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: pathway stoichiometry, free-energy conservation and redox-cofactor balancing. Metab Eng 36:99–115. CrossRefPubMedGoogle Scholar
  56. Wang G, Li D, Miao Z, Zhang S, Liang W, Liu L (2018) Comparative transcriptome analysis reveals multiple functions for Mhy1p in lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. Biochim Biophys Acta (BBA) - Mol Cell Biol L 1863(1):81–90. CrossRefGoogle Scholar
  57. Wasylenko TM, Ahn WS, Stephanopoulos G (2015) The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 30:27–39. CrossRefPubMedGoogle Scholar
  58. Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB (2009) ATP-citrate Lyase links cellular metabolism to histone acetylation. Science 324(5930):1076–1080CrossRefGoogle Scholar
  59. Wenz P, Schwank S, Hoja U, Schüller HJ (2001) A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product. Nucleic Acids Res 29(22):4625–4632CrossRefGoogle Scholar
  60. Wise EM, Ball EG (1964) Malic enzyme and lipogenesis. P Natl A Sci 52(5):1255–1263CrossRefGoogle Scholar
  61. Wong L, Engel J, Jin E, Holdridge B, Xu P (2017) YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metab Eng Commun 5:68–77. CrossRefPubMedPubMedCentralGoogle Scholar
  62. Wynn JP, bin Abdul Hamid A, Ratledge C (1999) The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology 145(Pt 8):1911–1917. CrossRefPubMedGoogle Scholar
  63. Xin Y, Lu Y, Lee Y-Y, Wei L, Jia J, Wang Q, Wang D, Bai F, Hu H, Hu Q, Liu J, Li Y, Xu J (2017) Producing designer oils in industrial microalgae by rational modulation of co-evolving Type-2 diacylglycerol acyltransferases. Mol Plant 10(12):1523–1539. CrossRefPubMedGoogle Scholar
  64. Xing S, Deenen N, Magliano P, Frahm L, Forestier E, Nawrath C, Schaller H, Gronover CS, Prüfer D, Poirier Y (2014) ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways. Plant J 79(2):270–284. CrossRefPubMedGoogle Scholar
  65. Xu P, Gu Q, Wang W, Wong L, Bower AGW, Collins CH, Koffas MAG (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 4:1409. CrossRefPubMedGoogle Scholar
  66. Xu P, Li L, Zhang F, Stephanopoulos G, Koffas M (2014) Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. P Natl A Sci USA 111(31):11299–11304. CrossRefGoogle Scholar
  67. Xu P, Qiao K, Ahn WS, Stephanopoulos G (2016) Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. P Natal A Sci 113(39):10848–10853CrossRefGoogle Scholar
  68. Xu J, Liu N, Qiao K, Vogg S, Stephanopoulos G (2017a) Application of metabolic controls for the maximization of lipid production in semicontinuous fermentation. P Natl A Sci 114(27):E5308–E5316CrossRefGoogle Scholar
  69. Xu P, Qiao K, Stephanopoulos G (2017b) Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica. Biotechnol Bioeng 114(7):1521–1530. CrossRefPubMedGoogle Scholar
  70. Zhang H, Zhang L, Chen H, Chen YQ, Ratledge C, Song Y, Chen W (2013) Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation. Biotechnol Lett 35(12):2091–2098. CrossRefPubMedGoogle Scholar
  71. 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. 3:1112.
  72. Zhu B-H, Zhang R-H, Lv N-N, Yang G-P, Wang Y-S, Pan K-H (2018) The role of malic enzyme on promoting total lipid and fatty acid production in Phaeodactylum tricornutum. Front Plant Sci 9:826–834CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemical, Biochemical and Environmental EngineeringUniversity of MarylandBaltimoreUSA
  2. 2.College of Life Science and TechnologyBeijing University of Chemical TechnologyBeijingChina
  3. 3.Department of Agronomy and Plant BreedingUniversity of GuilanRashtIslamic Republic of Iran

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