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.
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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.
Alonso, D. M., Bond, J. Q., & Dumesic, J. A. (2010). Catalytic conversion of biomass to biofuels. Green Chemistry, 12(9), 1493–1513.
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.
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.
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.
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.
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.
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.
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.
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.
Byme, B. (2015). Pichia pastoris as an expression host for membrane protein structural biology. Current Opinion in Structural Biology, 32, 9–17.
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.
Joshi, H. J., & Gupta, R. (2015). Eukaryotic glycosylation: online methods for site prediction on protein sequences. Methods in Molecular Biology, 1273, 127–137.
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.
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.
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.
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.
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.
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.
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.
Funding
This study was financially supported by the National Natural Science Foundation of China (No. 21576253, No. 21877110 and No. 31470432).
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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.
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Feng, Y., Zhang, Y., Ding, W. et al. Expanding of Phospholipid:Diacylglycerol AcylTransferase (PDAT) from Saccharomyces cerevisiae as Multifunctional Biocatalyst with Broad Acyl Donor/Acceptor Selectivity. Appl Biochem Biotechnol 188, 824–835 (2019). https://doi.org/10.1007/s12010-019-02954-x
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DOI: https://doi.org/10.1007/s12010-019-02954-x