Skip to main content

Advertisement

SpringerLink
Log in

Enabling Heterologous Synthesis of Lupulones in the Yeast Saccharomyces cerevisiae

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Lupulones, naturally produced by glandular trichomes of hop (Humulus lupulus), are prenylated phloroglucinol derivatives that contribute the bitter flavor of beer and demonstrate antimicrobial and anticancer activities. It is appealing to develop microbial cell factories such that lupulones may be produced via fermentation technology in lieu of extraction from limited plant resources. In this study, the yeast Saccharomyces cerevisiae transformants harboring a synthetic lupulone pathway that consisted of five genes from hop were constructed. The transformants accumulated several precursors but failed to accumulate lupulones. Overexpression of 3-hydroxy-3-methyl glutaryl co-enzyme A reductase, the key enzyme in precursor formation in the mevalonate pathway, also failed to achieve a detectable level of lupulones. To decrease the consumption of the precursors, the ergosterol biosynthesis pathway was chemically downregulated by a small molecule ketoconazole, leading to successful production of lupulones. Our study demonstrated a combination of molecular biology and chemical biology to regulate the metabolism for heterologous production of lupulones. The strategy may be valuable for future engineering microbial process for other prenylated natural products.

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

  1. Almaguer, C., Schönberger, C., Gastl, M., Arendt, E. K., & Becker, T. (2014). Humulus lupulus—a story that begs to be told. A review. Journal of the Institute of Brewing, 120, 289–314.

    CAS  Google Scholar 

  2. Steenackers, B., De Cooman, L., & De Vos, D. (2015). Chemical transformations of characteristic hop secondary metabolites in relation to beer properties and the brewing process: a review. Food Chemistry, 172, 742–756.

    Article  CAS  PubMed  Google Scholar 

  3. Cleemput, M. V., Cattoor, K., Bosscher, K. D., Haegeman, G., Keukeleire, D. D., & Heyerick, A. (2009). Hop (Humulus lupulus)-derived bitter acids as multipotent bioactive compounds. Journal of Natural Products, 72(6), 1220–1230.

    Article  CAS  PubMed  Google Scholar 

  4. Lamy, V., Roussi, S., Chaabi, M., Gosse, F., Schall, N., Lobstein, A., & Raul, F. (2007). Chemopreventive effects of lupulone, a hop β-acid, on human colon cancer-derived metastatic sw620 cells and in a rat model of colon carcinogenesis. Carcinogenesis, 28(7), 1575–1581.

    Article  CAS  PubMed  Google Scholar 

  5. Justé, A., Krause, M. S., Lievens, B., Klingeberg, M., Michiels, C. W., & Willems, K. A. (2007). Protective effect of hop β-acids on microbial degradation of thick juice during storage. Journal of Applied Microbiology, 104, 51–59.

    Google Scholar 

  6. Pollach, G., Hein, W., & Beddie, D. (2002). Application of hop β-acids and rosin acids in the sugar industry. Zuckerindustrie, 127, 921–930.

    CAS  Google Scholar 

  7. Nagel, J., Culley, L. K., Lu, Y., Liu, E., Matthews, P. D., Stevens, J. F., & Page, J. E. (2008). EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol. Plant Cell, 20(1), 186–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Xu, H., Zhang, F., Liu, B., Huhman, D. V., Sumner, L. W., Dixon, R. A., & Wang, G. (2013). Characterization of the formation of branched short-chain fatty acid: CoAs for bitter acid biosynthesis in hop glandular trichomes. Molecular Plant, 6(4), 1301–1317.

    Article  CAS  PubMed  Google Scholar 

  9. Okada, Y., Sano, Y., Kaneko, T., Abe, I., Noguchi, H., & Ito, K. (2004). Enzymatic reactions by five chalcone synthase homologs from hop (Humulus lupulus L.). Bioscience Biotechnology and Biochemistry, 68(5), 1142–1145.

    Article  CAS  Google Scholar 

  10. Li, H., Ban, Z., Qin, H., Ma, L., King, A. J., & Wang, G. (2015). A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway. Plant Physiology, 167(3), 650–659.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhou, Y. J., Gao, W., Rong, Q., Jin, G., Chu, H., Liu, W., Yang, W., Zhu, Z., Li, G., Zhu, G., Huang, L., & Zhao, Z. K. (2012). Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. Journal of the American Chemical Society, 134(6), 3234–3241.

    Article  CAS  PubMed  Google Scholar 

  12. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., & Pease, L. R. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 77(1), 61–68.

    Article  CAS  Google Scholar 

  13. Gietz, R. D., & Schiestl, R. H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2(1), 1–4.

    Article  CAS  PubMed  Google Scholar 

  14. Szkopihska, A., Grabihska, K., Delourme, D., Karst, F., Rytka, J., & Palamarczyk, G. (1997). Polyprenol formation in the yeast Saccharomyces cerevisiae: effect of farnesyl diphosphate synthase overexpression. Journal of Lipid Research, 38, 962–968.

    Google Scholar 

  15. de Macedo-Silva, S. T., Visbal, G., Urbina, J. A., de Souza, W., & Rodrigues, J. C. (2015). Potent in vitro antiproliferative synergism of combinations of ergosterol biosynthesis inhibitors against Leishmania amazonensis. Antimicrobial Agents and Chemotherapy, 59(10), 6402–6418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jachak, G. R., Ramesh, R., Sant, D. G., Jorwekar, S. U., Jadhav, M. R., Tupe, S. G., Deshpande, M. V., & Reddy, D. S. (2015). Silicon incorporated morpholine antifungals: design, synthesis, and biological evaluation. ACS Medicinal Chemistry Letters, 6(11), 1111–1116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Rodrigues, J. C. F., Concepcion, J. L., Rodrigues, C., Caldera, A., Urbina, J. A., & de Souza, W. (2008). In vitro activities of er-119884 and e5700, two potent squalene synthase inhibitors, against Leishmania amazonensis: antiproliferative, biochemical, and ultrastructural effects. Antimicrobial Agents and Chemotherapy, 52(11), 4098–4114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tang, F., Zhou, X., Yang, J., Xu, J., & Li, R. (2009). Study on accumulating lycopene by ergosterol synthesis inhibitor in Rhodothece RY-17. Pharmaceutical Biotechnology, 16, 64–67.

    CAS  Google Scholar 

  19. Urbina, J. A., Concepcion, J. L., Caldera, A., Payares, G., Sanoja, C., Otomo, T., & Hiyoshi, H. (2004). In vitro and in vivo activities of e5700 and er-119884, two novel orally active squalene synthase inhibitors, against Trypanosoma cruzi. Antimicrobial Agents and Chemotherapy, 48(7), 2379–2387.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Peng, B., Plan, M. R., Chrysanthopoulos, P., Hodson, M. P., Nielsen, L. K., & Vickers, C. E. (2017). A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae. Metabolic Engineering, 39, 209–219.

    Article  CAS  PubMed  Google Scholar 

  21. Donald, K., Hampton, R., & Fritz, I. (1997). Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme a reductase on squalene synthesis in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 63, 3341–3344.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Engels, B., Dahm, P., & Jennewein, S. (2008). Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards taxol (paclitaxel) production. Metabolic Engineering, 10(3-4), 201–206.

    Article  CAS  PubMed  Google Scholar 

  23. Grabowska, D., Karst, F., & Szkopinèska, A. (1998). Effect of squalene synthase gene disruption on synthesis of polyprenols in Saccharomyces cerevisiae. FEBS Letters, 434(3), 406–408.

    Article  CAS  PubMed  Google Scholar 

  24. Clark, S. M., Vaitheeswaran, V., Ambrose, S. J., Purves, R. W., & Page, J. E. (2013). Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (humulus lupulus). BMC Plant Biology, 13(1), 12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ignea, C., Pontini, M., Maffei, M. E., Makris, A. M., & Kampranis, S. C. (2014). Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synthetic Biology, 3(5), 298–306.

    Article  CAS  PubMed  Google Scholar 

  26. Bi, H., Bai, Y., Cai, T., Zhuang, Y., Liang, X., Zhang, X., Liu, T., & Ma, Y. (2013). Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives. Applied Microbiology and Biotechnology, 97(24), 10339–10348.

    Article  CAS  PubMed  Google Scholar 

  27. Karkashon, S., Raghupathy, R., Bhatia, H., Dutta, A., Hess, S., Higgs, J., Tifft, C. J., & Little, J. A. (2015). Intermediaries of branched chain amino acid metabolism induce fetal hemoglobin, and repress sox6 and bcl11a, in definitive erythroid cells. Blood Cells, Molecules, and Diseases, 55(2), 161–167.

    Article  CAS  PubMed  Google Scholar 

  28. Dickinson, J. R., Harrison, S. J., & Hewlins, M. J. E. (1998). An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 273(40), 25751–25756.

    Article  CAS  PubMed  Google Scholar 

  29. Dickinson, J. R., Salgado, L. E., & Hewlins, M. J. (2003). The catabolism of amino acids to long chain and complex alcohols in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 278(10), 8028–8034.

    Article  CAS  PubMed  Google Scholar 

  30. Lian, J., Jin, R., & Zhao, H. (2016). Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration. Biotechnology and Bioengineering, 113(11), 2462–2473.

    Article  CAS  PubMed  Google Scholar 

  31. Kim, J. W., Kim, J., Seo, S. O., Kim, K. H., Jin, Y. S., & Seo, J. H. (2016). Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and nadh oxidase activities. Biotechnology for Biofuels, 9(1), 265.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lee, F. W. F., & Silva, D. N. D. (1997). Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 48(3), 339–345.

    Article  CAS  PubMed  Google Scholar 

  33. Xie, W., Ye, L., Lv, X., Xu, H., & Yu, H. (2015). Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metabolic Engineering, 28, 8–18.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Prof. Guodong Wang of Institute of Genetics and Developmental Biology, CAS, for provision of strains and plasmids, and Prof. Fan Yang of Dalian Polytechnic University for provision of hop samples.

Funding

This project is supported by National Natural Science Foundation of China (Nos. 21721004; 51561145014) and Dalian Institute of Chemical Physics, CAS (No. DICP ZZBS201605).

Author information

Authors and Affiliations

  1. Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Xiaojia Guo, Hongwei Shen, Yuxue Liu, Qian Wang, Xueying Wang, Chang Peng, Wujun Liu & Zongbao K. Zhao

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Xiaojia Guo & Yuxue Liu

Authors
  1. Xiaojia Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongwei Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuxue Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qian Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xueying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chang Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wujun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zongbao K. Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zongbao K. Zhao.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

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

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, X., Shen, H., Liu, Y. et al. Enabling Heterologous Synthesis of Lupulones in the Yeast Saccharomyces cerevisiae. Appl Biochem Biotechnol 188, 787–797 (2019). https://doi.org/10.1007/s12010-019-02957-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-019-02957-8

Keywords

Search

Navigation