Multiple metabolic engineering of Saccharomyces cerevisiae for the production of lycopene

Abstract

Lycopene is a high-value-added tetraterpenoid, which is widely used in cosmetics, medicine, food, and dietary supplements. The intracellular mevalonate pathway of Saccharomyces cerevisiae provides natural precursors for terpenoid product synthesis, so it is an excellent host for the heterologous production of lycopene. In this study, a recombinant strain named L10 with efficient lycopene production capability was constructed through multiple strategies, such as regulating the gene copy number of key enzymes, increasing nicotinamide adenine dinucleotide phosphate supply, and reducing squalene accumulation. Then, considering that intracellular lycopene accumulation can cause cytotoxicity to S. cerevisiae, we attempted to identify a transporter that can efficiently transport lycopene from intracellular to extracellular space. Molecular docking simulations predicted that the ATP-binding cassette transporter Snq2p may be a potential transporter of lycopene, and its function in promoting lycopene secretion was further determined by overexpression verification. The lycopene secretion titer of the strain L10Z2 overexpressing Snq2p increased to 16.5 times that of the control at the shake-flask level. After optimizing the galactose regulation system, the intracellular and secreted lycopene production of L11Z2 reached 2113.78 and 26.28 mg/L, respectively, after 150 h fed-batch culture in a 3-L bioreactor. This work provides a new research direction for efficient lycopene synthesis in S. cerevisiae cell factory.

Keywords

lycopene / metabolic engineering / Saccharomyces cerevisiae / transporter engineering

References

[1] Asadollahi, M. A., Maury, J., Møller, K., Nielsen, K. F., Schalk, M., Clark, A., & Nielsen, J. (2008). Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnology and Bioengineering, 99(3), 666–677.

[2] Bu, X., Lin, J., Cheng, J., Yang, D., Duan, C., Koffas, M., & Yan, G. (2020). Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of beta-carotene in Saccharomyces cerevisiae. Biotechnology for Biofuels, 13(168), 1–14.

[3] Claus, S., Jezierska, S., & Van Bogaert, I. N. A. (2019). Protein-facilitated transport of hydrophobic molecules across the yeast plasma membrane. FEBS Letters, 593(13), 1508–1527.

[4] Elbourne, L. D. H., Tetu, S. G., Hassan, K. A., & Paulsen, I. T. (2017). TransportDB 2.0: a database for exploring membrane transporters in sequenced genomes from all domains of life. Nucleic Acids Research, 45, D320–D324.

[5] Ford, R. C., & Beis, K. (2019). Learning the ABCs one at a time: structure and mechanism of ABC transporters. Biochemical Society Transactions, 47, 23–36.

[6] Gietz, R. D., & Schiestl, R. H. (2007). Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2(1), 35–37.

[7] Godinho, C. P., Dias, P. J., Ponçot, E., & Sá-Correia, I. (2018). The paralogous genes PDR18 and SNQ2, encoding multidrug resistance ABC transporters, derive from a recent duplication event, PDR18 being specific to the Saccharomyces genus. Frontiers in Genetics, 9, 476.

[8] Guo, W., Ai, L., Hu, D., Chen, Y., Geng, M., Zheng, L., & Bai, L. (2022). [URA3 affects artemisinic acid production by an engineered Saccharomyces cerevisiae in pilot-scale fermentation]. Sheng wu gong cheng xue bao = Chinese Journal of Biotechnology, 38(2), 737–748.

[9] Han, L., Wu, Y., Xu, Y., Zhang, C., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). Engineered Saccharomyces cerevisiae for de novo δ-tocotrienol biosynthesis. Systems Microbiology and Biomanufacturing, 4, 150–164.

[10] Hernández-Almanza, A., Montañez, J., Martínez, G., Aguilar-Jiménez, A., Contreras-Esquivel, J. C., & Aguilar, C. N. (2016). Lycopene: progress in microbial production. Trends in Food Science & Technology, 56, 142–148.

[11] Hong, J., Park, S. H., Kim, S., Kim, S. W., & Hahn, J. S. (2019). Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production. Applied Microbiology and Biotechnology, 103(1), 211–223.

[12] Jing, Y., Wang, Y., Zhou, D., Wang, J., Li, J., Sun, J., Feng, Y., Xin, F., & Zhang, W. (2022). Advances in the synthesis of three typical tetraterpenoids including β-carotene, lycopene and astaxanthin. Biotechnology Advances, 61, 108033.

[13] Li, S., Fu, W., Su, R., Zhao, Y., & Deng, Y. (2023). Producing malonate in Saccharomyces cerevisiae via the β-alanine pathway. Systems Microbiology and Biomanufacturing, 3, 328–338.

[14] Li, X., Wang, Z., Zhang, G., & Yi, L. (2019). Improving lycopene production in Saccharomyces cerevisiae through optimizing pathway and chassis metabolism. Chemical Engineering Science, 193, 364–369.

[15] Liu, J., Wang, X., Jin, K., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). In silico prediction and mining of exporters for secretory bioproduction of terpenoids in Saccharomyces cerevisiae. ACS Synthetic Biology, 12(3), 863–876.

[16] Ma, T., Shi, B., Ye, Z., Li, X., Liu, M., Chen, Y., Xia, J., Nielsen, J., Deng, Z., & Liu, T. (2019). Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metabolic Engineering, 52, 134–142.

[17] Mahé Y., Parle-McDermott, A., Nourani, A., Delahodde, A., Lamprecht, A., & Kuchler, K. (1996). The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: A novel target for the transcription factors Pdr1 and Pdr3. Molecular Microbiology, 20(1), 109–117.

[18] Moon, H. Y., Sim, G. H., Kim, H. J., Kim, K., & Kang, H. A. (2022). Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae. Journal of Microbiology, 60(1), 18–30.

[19] Pais, T. M., Foulquié-Moreno, M. R., Hubmann, G., Duitama, J., Swinnen, S., Goovaerts, A., Yang, Y., Dumortier, F., & Thevelein, J. M. (2013). Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol levels of cell proliferation in yeast. PLoS Genetics, 9(6), e1003548.

[20] Qu, L., Xiu, X., Sun, G., Zhang, C., Yang, H., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2022). Engineered yeast for efficient de novo synthesis of 7-dehydrocholesterol. Biotechnology and Bioengineering, 119(5), 1278–1289.

[21] Reider Apel, A., d’Espaux, L., Wehrs, M., Sachs, D., Li, R. A., Tong, G. J., Garber, M., Nnadi, O., Zhuang, W., Hillson, N. J., Keasling, J. D., & Mukhopadhyay, A. (2017). A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Research, 45(1), 496–508.

[22] Saini, R. K., & Keum, Y. S. (2018). Carotenoid extraction methods: a review of recent developments. Food Chemistry, 240(2), 90–103.

[23] Shi, B., Ma, T., Ye, Z., Li, X., Huang, Y., Zhou, Z., Ding, Y., Deng, Z., & Liu, T. (2019). Systematic metabolic engineering of Saccharomyces cerevisiae for lycopene overproduction. Journal of Agricultural and Food Chemistry, 67(40), 11148–11157.

[24] Srikant, S., & Gaudet, R. (2019). Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters. Nature Structural & Molecular Biology, 26(9), 792–801.

[25] Sterling, T., & Irwin, J. J. (2015). ZINC 15-Ligand discovery for everyone. Journal of Chemical Information and Modeling, 55, 2324–2337.

[26] Su, B., Lai, P., Yang, F., Li, A., Deng, M. R., & Zhu, H. (2022). Engineering a balanced acetyl coenzyme A metabolism in Saccharomyces cerevisiae for lycopene production through rational and evolutionary engineering. Journal of Agricultural and Food Chemistry, 70(13), 4019–4029.

[27] Tunyasuvunakool, K., Adler, J., Wu, Z., Green, T., Zielinski, M., Žídek, A., Bridgland, A., Cowie, A., Meyer, C., Laydon, A., Velankar, S., Kleywegt, G. J., Bateman, A., Evans, R., Pritzel, A., Figurnov, M., Ronneberger, O., Bates, R., Kohl, S. A. A., … Hassabis, D. (2021). Highly accurate protein structure prediction for the human proteome. Nature, 596, 590–596.

[28] Verwaal, R., Wang, J., Meijnen, J. P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350.

[29] Wang, Z., Sun, J., Yang, Q., & Yang, J. (2020). Metabolic engineering Escherichia coli for the production of lycopene. Molecules, 25(14), 3136.

[30] Xie, W., Lv, X., Ye, L., Zhou, P., & Yu, H. (2015). Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metabolic Engineering, 30, 69–78.

[31] Xu, Y., Han, L., Cheng, Y., Zhang, Y., Wu, Y., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). ATP-binding cassette exporter PDR11-mediated terpenoid secretion in engineered yeast. ACS Synthetic Biology, 12(4), 1146–1153.

[32] Yao, Z., Guo, Y., Wang, H., Chen, Y., Wang, Q., Nielsen, J., & Dai, Z. (2023). A highly efficient transcriptome-based biosynthesis of non-ethanol chemicals in Crabtree negative Saccharomyces cerevisiae. Biotechnology for Biofuels and Bioproducts, 16(1), 37.

[33] Zhang, Y., Wang, W., Wei, W., Xia, L., Gao, S., Zeng, W., Liu, S., & Zhou, J. (2023). Regulation of ethanol assimilation for efficient accumulation of squalene in Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 71, 6389–6397.

[34] Zhou, K., Yu, C., Liang, N., Xiao, W., Wang, Y., Yao, M., & Yuan, Y. (2023). Adaptive evolution and metabolic engineering boost lycopene production in Saccharomyces cerevisiae via enhanced precursors supply and utilization. Journal of Agricultural and Food Chemistry, 71, 3821–3831.

RIGHTS & PERMISSIONS

2024 The Author(s). Food Bioengineering published by John Wiley & Sons Australia, Ltd. on behalf of State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.