Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3β-ol accumulation by metabolic engineering


Ergosterol is the predominant nature sterol constituent of plasma membrane in Saccharomyces cerevisiae. Herein, the biosynthetic pathway of ergosterol was proposed to be metabolically engineered for the efficient production of ergosta-5,7-dien-3β-ol, which is the precursor of vitamin D4. By target disruption of erg5, involved in the end-steps of post-squalene formation, predominantly accumulated ergosta-5,7-dien-3β-ol (4.12 mg/g dry cell weight). Moreover, the rate-limiting enzymes of ergosta-5,7-dien-3β-ol biosynthesis were characterized. Overexpression of Hmg1p led to a significant accumulation of squalene, and induction of Erg1p/Erg11p expression raised the yield of both total sterols and ergosta-5,7-dien-3β-ol with no obvious changes in growth behavior. Furthermore, the transcription factor allele upc2-1 was overexpressed to explore the effect of combined induction of rate-limiting enzymes. Compared with an obviously enhanced yield of ergosterol in the wild-type strain, decreases of both the ergosta-5,7-dienol levels and the total sterol yield were found in Δerg5-upc2-1, probably due to the unbalanced NADH/NAD+ ratio observed in the erg5 knockouts, suggesting the whole-cell redox homeostasis was also vital for end-product biosynthesis. The data obtained in this study can be used as reference values for the production of sterol-related intermediates involved in the post-squalene biosynthetic pathway in food-grade S. cerevisiae strains.

Graphical Abstract

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Adams BG, Parks LW (1968) Isolation from yeast of a metabolically active water-soluble form of ergosterol. J Lipid Res 9:8–11

  2. Aguilar PS et al (2010) Structure of sterol aliphatic chains affects yeast cell shape and cell fusion during mating. Proc Natl Acad Sci USA 107:4170–4175. https://doi.org/10.1073/pnas.0914094107

  3. Bard M et al (1996) Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase. Proc Natl Acad Sci USA 93:186–190

  4. Bloch K (1981) Sterol structure and membrane. Curr Top Cell Regul 18:289–299

  5. Borkowski O, Bricio Garberi C, Murgiano M, Stan G-B, Ellis T (2017) Cell-free prediction of protein expression costs for growing cells. bioRxiv. https://doi.org/10.1101/172627

  6. Caspeta L et al (2014) Biofuels. Altered sterol composition renders yeast thermotolerant. Science 346:75–78. https://doi.org/10.1126/science.1258137

  7. Chen X, Li S, Liu L (2014) Engineering redox balance through cofactor systems. Trends Biotechnol 32:337–343. https://doi.org/10.1016/j.tibtech.2014.04.003

  8. Cheng KK, Wang GY, Zeng J, Zhang JA (2013) Improved succinate production by metabolic engineering. BioMed Res Int 2013:538790. https://doi.org/10.1155/2013/538790

  9. Crowley JH, Leak FW Jr, Shianna KV, Tove S, Parks LW (1998) A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae. J Bacteriol 180:4177–4183

  10. Dai Z, Liu Y, Zhang X, Shi M, Wang B, Wang D, Huang L (2013) Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng 20:146–156. https://doi.org/10.1016/j.ymben.2013.10.004

  11. Gaber RF, Copple DM, Kennedy BK, Vidal M, Bard M (1989) The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol Cell Biol 9:3447–3456

  12. Gachotte D, Barbuch R, Gaylor J, Nickel E, Bard M (1998) Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis. Proc Natl Acad Sci USA 95:13794–13799

  13. Gachotte D, Sen SE, Eckstein J, Barbuch R, Krieger M, Ray BD, Bard M (1999) Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation. Proc Natl Acad Sci USA 96:12655–12660

  14. Ghodasara A, Voigt CA (2017) Balancing gene expression without library construction via a reusable sRNA pool. Nucleic Acids Res 45:8116–8127. https://doi.org/10.1093/nar/gkx530

  15. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360. https://doi.org/10.1002/yea.320110408

  16. Holick MF (2002) Vitamin D. Clin Rev Bone Miner Metab 1(3–4):181–207

  17. Hughes TR et al (2000) Functional discovery via a compendium of expression profiles. Cell 102:109–126

  18. Ignea C, Pontini M, Maffei ME, Makris AM, Kampranis SC (2014) Engineering monoterpene production in Yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synth Biol 3:298–306. https://doi.org/10.1021/sb400115e

  19. Kodedova M, Sychrova H (2015) Changes in the sterol composition of the plasma membrane affect membrane potential, salt tolerance and the activity of multidrug resistance pumps in Saccharomyces cerevisiae. PLoS ONE 10:e0139306. https://doi.org/10.1371/journal.pone.0139306

  20. Korn ED, Von BT, Tobie EJ (1969) The sterols of Trypanosoma cruzi and Crithidia fasciculata. Comp Biochem Physiol 30:601

  21. Krings U, Berger RG (2014) Dynamics of sterols and fatty acids during UV-B treatment of oyster mushroom. Food Chem 149:10–14. https://doi.org/10.1016/j.foodchem.2013.10.064

  22. Leidig-Bruckner G, Bruckner T, Raue F, Frank-Raue K (2016) Long-term follow-up and treatment of postoperative permanent hypoparathyroidism in patients with medullary thyroid carcinoma: differences in complete and partial disease. Horm Metab Res 48:806–813. https://doi.org/10.1055/s-0042-118181

  23. MacPherson S, Akache B, Weber S, De Deken X, Raymond M, Turcotte B (2005) Candida albicans zinc cluster protein Upc2p. confers resistance to antifungal drugs and is an activator of ergosterol biosynthetic genes. Antimicrob Agents Chemother 49:1745–1752. https://doi.org/10.1128/Aac.49.5.1745-1752

  24. Mo CQ, Bard M (2005) Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex. J Lipid Res 46:1991–1998. https://doi.org/10.1194/jlr.M500153-JLR200

  25. Mysiakina IS, Sergeeva YE, Ivashechkin AA, Feofilova EP (2012) Impact of morphogenetic effectors on the growth pattern and the lipid composition of the mycelium and the yeastlike cells of the fungus Mucor hiemalis. Microbiology 81:676–683. https://doi.org/10.1134/S0026261712060100

  26. Nielsen JJMC (2007) The role of metabolomics in systems biology. Metabolomics 18:1–10

  27. Parks LW, Smith SJ, Crowley JH (1995) Biochemical and physiological effects of sterol alterations in yeast: a review. Lipids 30:227–230

  28. Phillips KM, Horst RL, Koszewski NJ, Simon. RR (2012) Vitamin D4 in mushrooms. PLoS ONE 7:e40702. https://doi.org/10.1371/journal.pone.0040702.g001

  29. Pierson CA, Eckstein J, Barbuch R, Bard M (2004) Ergosterol gene expression in wild-type and ergosterol-deficient mutants of Candida albicans. Med Mycol 42:385–389

  30. Polakowski T, Stahl U, Lang C (1998) Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Appl Microbiol Biotechnol 49:66–71

  31. Scallen TJ (1965) Chemical synthesis of cholesta-5,7,24-trien-3-beta-ol and demonstration of its conversion to cholesterol in the rat. Biochem Biophys Res Commun 21:149–155

  32. Sharma SC (2006) Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae. FEMS Yeast Res 6:1047–1051. https://doi.org/10.1111/j.1567-1364.2006.00149.x

  33. Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N (1998) Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 64:2676–2680

  34. Skaggs BA et al (1996) Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. Gene 169:105–109

  35. Su W, Xiao WH, Wang Y, Liu D, Zhou X, Yuan YJ (2015) Alleviating redox imbalance enhances 7-dehydrocholesterol production in engineered Saccharomyces cerevisiae. PLoS ONE 10:e0130840. https://doi.org/10.1371/journal.pone.0130840

  36. Sun X et al (2013) Sterol C-22 desaturase ERG5 mediates the sensitivity to antifungal azoles in Neurospora crassa and Fusarium verticillioides. Front Microbiol 4:127. https://doi.org/10.3389/fmicb.2013.00127

  37. Tiedje C, Holland DG, Just U, Hofken T (2007) Proteins involved in sterol synthesis interact with Ste20 and regulate cell polarity. J Cell Sci 120:3613–3624. https://doi.org/10.1242/jcs.009860

  38. Tokuhiro K et al (2009) Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. Appl Environ Microbiol 75:5536–5543. https://doi.org/10.1128/AEM.00277-09

  39. Tsuji M, Tachibana Y, Yokoyama S, Ikekawa N (1992) 1α,25-dihydroxyvitamin D4 compounds, ergosta-5,7-diene compounds and processes for the preparation thereof. US5157135

  40. Veen M, Stahl U, Lang C (2003) Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Res 4:87–95. https://doi.org/10.1016/s1567-1356(03)00126-0

  41. Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J (2007) Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104:2402–2407. https://doi.org/10.1073/pnas.0607469104

  42. Yang H, Tong J, Lee CW, Ha S, Eom SH, Im YJ (2015) Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2. Nat Commun 6:6129. https://doi.org/10.1038/ncomms7129

  43. Yun Y, Yin D, Dawood DH, Liu X, Chen Y, Ma Z (2014) Functional characterization of FgERG3 and FgERG5 associated with ergosterol biosynthesis, vegetative differentiation and virulence of Fusarium graminearum. Fungal Genet Biol 68:60–70. https://doi.org/10.1016/j.fgb.2014.04.010

  44. Zhou P et al (2017) Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae. Enzyme Microb Technol 100:28–36. https://doi.org/10.1016/j.enzmictec.2017.02.006

Download references


This work was supported by the National Natural Science Foundation of China (No. 31400978) and the Natural Science Foundation of Zhejiang Province (No. LY18B020019).

Author information

Correspondence to Yu-Guo Zheng.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 41 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ma, B., Ke, X., Tang, X. et al. Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3β-ol accumulation by metabolic engineering. World J Microbiol Biotechnol 34, 55 (2018). https://doi.org/10.1007/s11274-018-2440-9

Download citation


  • Ergosta-5,7-dien-3β-ol
  • Ergosterol synthesis pathway
  • Rate-limiting steps
  • Saccharomyces cerevisiae
  • Upc2-1