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Expanding of Phospholipid:Diacylglycerol AcylTransferase (PDAT) from Saccharomyces cerevisiae as Multifunctional Biocatalyst with Broad Acyl Donor/Acceptor Selectivity

  • Yanbin Feng
  • Yunxiu Zhang
  • Wei Ding
  • Peichun Wu
  • Xupeng CaoEmail author
  • Song XueEmail author
Article
  • 45 Downloads

Abstract

Triacylglycerols are considered one of the most promising feedstocks for biofuels. Phospholipid:diacylglycerol acyltransferase (PDAT), responsible for the last step of triacylglycerol synthesis in the acyl-CoA-independent pathway, has attracted much attention by catalyzing membrane lipid transformation. However, due to lack of biochemical and enzymatic studies, PDAT has not carried forward in biocatalyst application. Here, the PDAT from Saccharomyces cerevisiae was expressed in Pichia pastoris. The purified enzymes were studied using different acyl donors and acceptors by thin layer chromatography and gas chromatography. In addition of the preferred acyl donor of PE and PC, the results identified that ScPDAT was capable of using broad acyl donors such as PA, PS, PG, MGDG, DGDG, and acyl-CoA, and ScPDAT was more likely to use unsaturated acyl donors comparing 18:0/18:1 to 18:0/18:0 phospholipids. With regard to acyl acceptors, ScPDAT preferred 1,2 to 1,3-diacylglycerol (DAG), while 12:0/12:0 DAG was identified as the optimal acyl acceptor, followed by 18:1/18:1 and 18:1/16:0 DAG. Additionally, ScPDAT reveals esterification activity that can utilize methanol as acyl acceptor to generate fatty acid methyl esters. The results fully expand the enzymatic selectivity of ScPDAT and provide fundamental knowledge for synthesis of triacylglycerol-derived biofuels.

Keywords

Acyltransferase PDAT Acyl donor Biocatalyst Enzyme Acyl acceptor 

Notes

Author Contributions

SX designed most of the experiments, analyzed the results, and wrote the manuscript. YF conducted most of the experiments, analyzed the results, and wrote the manuscript. XC analyzed the results and provided the useful suggestions for paper. YZ, WD, and PW conducted the experiments.

Funding Information

This study was financially supported by the National Natural Science Foundation of China (No. 21576253, No. 21877110 and No. 31470432).

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no conflict of interest.

References

  1. 1.
    Zheng, L., Shockey, J., Guo, F., Shi, L. M., Li, X. G., Shan, L., Wan, S. B., & Peng, Z. Y. (2017). Discovery of a new mechanism for regulation of plant triacylglycerol metabolism: the peanut diacylglycerol acyltransferase-1 gene family transcriptome is highly enriched in alternative splicing variants. Journal of Plant Physiology, 219, 62–70.CrossRefGoogle Scholar
  2. 2.
    Alonso, D. M., Bond, J. Q., & Dumesic, J. A. (2010). Catalytic conversion of biomass to biofuels. Green Chemistry, 12(9), 1493–1513.CrossRefGoogle Scholar
  3. 3.
    Peng, H., Moghaddam, L., Brinin, A., Williams, B., Mundree, S., & Haritos, V. S. (2018). Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production. Biotechnology and Applied Biochemistry, 65(2), 138–144.CrossRefGoogle Scholar
  4. 4.
    Haslam, R. P., Sayanova, O., Kim, H. J., Cahoon, E. B., & Napier, J. A. (2016). Synthetic redesign of plant lipid metabolism. The Plant Journal, 87(1), 76–86.CrossRefGoogle Scholar
  5. 5.
    Liu, X. Y., Ouyang, L. L., & Zhou, Z. G. (2016). Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in myrmecia incisa during the nitrogen starvation stress. Scientific Reports, 6, 26610.Google Scholar
  6. 6.
    Dahlqvist, A., Stahl, U., Lenman, M., Banas, A., Lee, M., Sandager, L., Ronne, H., & Stymne, H. (2000). Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6487–6492.CrossRefGoogle Scholar
  7. 7.
    van Erp, H., Bates, P. D., Burgal, J., Shockey, J., & Browse, J. (2011). Castor phospholipid: diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiology, 155(2), 683–693.CrossRefGoogle Scholar
  8. 8.
    Yoon, K., Han, D., Li, Y., Sommerfeld, M., & Hu, Q. (2011). Phospholipid:diacylglycerol acyltransferase is involved in lipid synthesis and degradation in Chlamydomonas reinhardtii. Journal of Phycology, 47, S59–S59.CrossRefGoogle Scholar
  9. 9.
    Yoon, K., Han, D. X., Li, Y. T., Sommerfeld, M., & Hu, Q. (2012). Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii. The Plant cell., 24(9), 3708–3724.CrossRefGoogle Scholar
  10. 10.
    Stahl, U., Carlsson, A. S., Lenman, M., Dahlqvist, A., Huang, B., Banas, W., Banas, A., & Stymne, S. (2004). Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant Physiology, 135(3), 1324–1335.CrossRefGoogle Scholar
  11. 11.
    Byme, B. (2015). Pichia pastoris as an expression host for membrane protein structural biology. Current Opinion in Structural Biology, 32, 9–17.CrossRefGoogle Scholar
  12. 12.
    Hua, L., Gao, X., Yang, X. P., Wan, D. Y., He, C. P., Cao, J. Y., & Song, H. F. (2014). Highly efficient production of peptides: N-glycosidase F for N-glycomics analysis. Protein Expression and Purification, 97, 17–22.CrossRefGoogle Scholar
  13. 13.
    Joshi, H. J., & Gupta, R. (2015). Eukaryotic glycosylation: online methods for site prediction on protein sequences. Methods in Molecular Biology, 1273, 127–137.CrossRefGoogle Scholar
  14. 14.
    Ghosal, A., Banas, A., Stahl, U., Dahlqvist, A., Lindqvist, Y., & Stymne, S. (2007). Saccharomyces cerevisiae phospholipid : diacylglycerol acyl transferase (PDAT) devoid of its membrane anchor region is a soluble and active enzyme retaining its substrate specificities. Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids, 1771(12), 1457–1463.CrossRefGoogle Scholar
  15. 15.
    Banas, W., Sanchez Garcia, A., Banas, A., & Stymne, S. (2013). Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds. Planta, 237(6), 1627–1636.CrossRefGoogle Scholar
  16. 16.
    Halim, S. F. A., & Kamaruddin, A. H. (2008). Catalytic studies of lipase on FAME production from waste cooking palm oil in a tert-butanol system. Process Biochemistry, 43(12), 1436–1439.CrossRefGoogle Scholar
  17. 17.
    Rodrigues, J., Perrier, V., Lecomte, J., Dubreucq, E., & Ferreira-Dias, S. (2016). Biodiesel production from crude jatropha oil catalyzed by immobilized lipase/acyltransferase from Candida parapsilosis in aqueous medium. Bioresource Technology, 218, 1224–1229.CrossRefGoogle Scholar
  18. 18.
    Duan, L. W., Zhang, H., Zhao, M. T., Sun, J. X., Chen, W. L., Lin, J. P., & Liu, X. Q. (2017). A non-canonical binding interface in the crystal structure of HIV-1 gp120 core in complex with CD4. Scientific Reports, 7(1), 46733.CrossRefGoogle Scholar
  19. 19.
    Li, S., Cao, X. P., Wang, Y., Zhu, Z., Zhang, H. W., Xue, S., & Tian, J. (2017). A method for microalgae proteomics analysis based on modified filter-aided sample preparation. Applied Biochemistry and Biotechnology, 183(3), 923–930.CrossRefGoogle Scholar
  20. 20.
    Feng, Y. B., Wang, Y. Y., Liu, J., Liu, Y. H., Cao, X. P., & Xue, S. (2017). Structural insight into acyl-ACP thioesterase toward substrate specificity design. ACS Chemical Biology, 12(11), 2830–2836.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalianChina

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