Enabling Heterologous Synthesis of Lupulones in the Yeast Saccharomyces cerevisiae
- 77 Downloads
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.
KeywordsChemical genetics Lupulones Metabolic engineering Mevalonate pathway Saccharomyces cerevisiae
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.
This project is supported by National Natural Science Foundation of China (Nos. 21721004; 51561145014) and Dalian Institute of Chemical Physics, CAS (No. DICP ZZBS201605).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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.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.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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.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.CrossRefGoogle 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.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.CrossRefGoogle Scholar