Abstract
Sterols attract increasing attention due to their important bioactivities. The oleaginous yeast Yarrowia lipolytica has large lipid droplets, which provide storage for the accumulated steroid compounds. In this study, we have successfully constructed a campesterol biosynthetic pathway by modifying the synthetic pathway of ergosterol in Y. lipolytica with different capacity of lipid synthesis. The results showed that the maximal campesterol production was produced in the engineered strain YL-D+M−E−, as the optimal lipid content. Furthermore, we found that campesterol mainly exists in the lipid droplets. The campesterol production was further accumulated through the overexpression of two copies of dhcr7. Finally, the maximal campesterol production of 837 mg/L was obtained using a 5-L bioreactor in the engineered YL-D+D+M−E−, exhibiting a 3.7-fold increase compared with the initial strain YL-D+E−. Our results demonstrate that the proper promotion of lipid content plays an important role in campesterol biosynthesis in Y. lipolytica, and what we found provides an effective strategy for the production of hydrophobic compounds.
Key Points
• Campesterol was biosynthesized by deleting erg5 and introducing heterologous dhcr7.
• Campesterol production elevated via promotion of lipid content.
• Campesterol was mainly found in lipid droplets.
• Promotion of lipid content is an effective strategy to produce hydrophobic compounds.
Similar content being viewed by others
References
Abdel-Mawgoud AM, Markham KA, Palmer CM, Liu N, Stephanopoulos G, Alper HS (2018) Metabolic engineering in the host Yarrowia lipolytica. Metab Eng 50:192–208. https://doi.org/10.1016/j.ymben.2018.07.016
Caspeta L, Chen Y, Ghiaci P, Feizi A, Buskov S, Hallstrom BM, Nielsen J (2014) Altered sterol composition renders yeast thermotolerant. Sci. 346(6205):75–78
Demonty I, Ras RT, van der Knaap HC, Duchateau GS, Meijer L, Zock PL, Trautwein EA (2008) Continuous dose-response relationship of the LDL-cholesterol-lowering effect of phytosterol intake. J Nutr 139(2):271–284
Du HX, Xiao W-H, Wang Y, Zhou X, Zhang Y, Liu D, Chardot T (2016) Engineering Yarrowia lipolytica for campesterol overproduction. PLoS One 11(1):e0146773
Duport C, Spagnoli R, Degryse E, Pompon D (1998) Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast. Nat Biotechnol 16(2):186–189
Fernandes P, Cabral JMS (2007) Phytosterols: applications and recovery methods. Bioresour Technol 98(12):2335–2350
Fickers, P., Dall, M. T. L., Gaillardin, C., Thonart, P., Nicaud, J. M. (2003). New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiolo Meth, 55(3), 727–737. doi:org/https://doi.org/10.1016/j.mimet.2003.07.003
Gao S, Tong Y, Wen Z, Zhu L, Ge M, Chen D, Yang S (2016) Multiplex gene editing of theYarrowia lipolytica genome using the CRISPR-Cas9 system. J Ind Microbiol Biotechnol 43(8):1085–1093
Jenner AM, Brown SH (2017) Sterol analysis by quantitative mass spectrometry. Methods Mol Biol 1583:221–239. https://doi.org/10.1007/978-1-4939-6875-6-17
Lees ND, Bard M, Kirsch DR (2000) Biochemistry and molecular biology of sterol synthesis in Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 34(1):33
Li AP, Ye LD, Bian Q, Yong Q (2017) Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae. Enzym Microb Technol 100:28–36. https://doi.org/10.1016/j.enzmictec.2017.02.006
Liu SY, Lu H, Guo X, Sun LJ, Ge SY (2012) Extraction of phytosterols from Jatropha seed oil by the saponification and acid hydrolysis method in chemical engineering. Adv Mater Res 577:77–80
Livak K j, Schmittgen TD (2000) Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△Ct method. Meth. 25(4):402–408. https://doi.org/10.1006/meth.2001
Ma BX, Ke X, Tang XL, Zheng RC, Zheng YG (2018) Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3beta-ol accumulation by metabolic engineering. World J Microbiol Biotechnol 34(4):55
Matthaus F, Ketelhot M, Gatter M, Barth G (2014) Production of lycopene in the non-carotenoid-producing yeast Yarrowia lipolytica. Appl Environ Microbiol 80(5):1660–1669. https://doi.org/10.1128/AEM.03167-13
Mlickova K, Roux E, Athenstaedt K, Andrea S, Daum G, Chardot T (2004) Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica. Appl Environ Microbiol 70(7):3918–3924
Piironen V, Lindsay DG, Miettinen TA, Toivo J, Lampi AM (2000) Plant sterols: biosynthesis, biological function and their importance to human nutrition. J Sci Food Agric 80(7):939–966
Qiao, K., Abidi, S. H. I., Liu, H., Zhang, H., Chakraborty, S., Watson, N., Stephanopoulos, G. (2015). Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng, 29, 56-65. Doi: org/https://doi.org/10.1016/j.ymben.2015.02.005
Szczebara FM, Chandelier C, Villeret C, Masurel A, Bourot S, Duport C, Costaglioli P (2003) Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol 21(2):143–149
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. https://doi.org/10.1016/j.ymben.2012.08.007
Tan S, Niu Y, Liu L, Su A, Hu C, Meng Y (2019) Development of a GC-MS/SIM method for the determination of phytosteryl esters. Food Chem 281:236–241. https://doi.org/10.1016/j.foodchem.2018.12.092
Veen M, Lang C (2005) Interactions of the ergosterol biosynthetic pathway with other lipid pathways. Biochem Soc Trans 33(5):1178–1181
Wang G, Xiong X, Rishikesh G, Wang P, Meng Y, Chen S (2016) Exploring fatty alcohol-producing capability of Yarrowia lipolytica. Biotechnol Biofu 9(1):1–10. https://doi.org/10.1186/s13068-016-0512-3
Wriessnegger T, Leitner E, Belegratis MR (2008) Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris. Chem Phys Lipids 149:S47. https://doi.org/10.1016/j.chemphyslip.2007.06.107
Yan FX, Dong GR, Qiang S, Niu YJ, Hu CY, Meng YH (2020) Overexpression of Δ12, Δ15-desaturases for enhanced lipids synthesis in Yarrowia lipolytica. Front Microbiol 11(289). https://doi.org/10.3389/fmicb.2020.00289
Madzak C, Tréton B, Blanchinroland S (2000) Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol 2:207–216
Data availability statement
All datasets obtained for this study are included in the manuscript/Supplementary material.
Funding
This research was supported by the National Natural Science Foundation (31972089) and National Key Research and Development Program of China (2019YFD1002402, 2019YFD100240205).
Author information
Authors and Affiliations
Contributions
ST, GD, and YM designed the study. GD, ST, and YN carried out the experiment. YQ, GD, ST, YN, CH, and YM analyzed the data and wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical statement
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.
Electronic supplementary material
The Supplementary material for this article can be found on the AMB website. Table S1 shows the list of primers used in this study. Table S2 shows the primers and sequences used during qPCR. Table S3 shows the nucleotide sequence of the codon-optimized dhcr7 gene. Figure S1 shows the fermentation characteristics of the initial strain YL-D+E−.
ESM 1
(PDF 75 kb)
Rights and permissions
About this article
Cite this article
Qian, Y.D., Tan, S.Y., Dong, G.R. et al. Increased campesterol synthesis by improving lipid content in engineered Yarrowia lipolytica. Appl Microbiol Biotechnol 104, 7165–7175 (2020). https://doi.org/10.1007/s00253-020-10743-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10743-4