Regiobiochemical analysis reveals the role of castor LPAT2 in the accumulation of hydroxy fatty acids in transgenic lesquerella seeds

Authors: Chen, Grace; Lin, Jiann; VAN ERP, HARRIE - University Of Hertfordshire; Johnson, Kumiko; LU, CHAOFU - Montana State University

Submitted to: Biocatalysis and Biotransformation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2020
Publication Date: 4/21/2020
Citation: Chen, G.Q., Lin, J.T., Van Erp, H., Johnson, K., Lu, C. 2020. Regiobiochemical analysis reveals the role of castor LPAT2 in the accumulation of hydroxy fatty acids in transgenic lesquerella seeds. Biocatalysis and Biotransformation. 25:10167. https://doi.org/10.1016/j.bcab.2020.101617.
DOI: https://doi.org/10.1016/j.bcab.2020.101617

Interpretive Summary: The conventional source of hydroxy fatty acids (HFA) is from castor (Ricinus communis), which contains 90% of ricinoleic acid in its seed oil (TAG). HFA are used as a raw material for manufacturing a range of products, such as lubricants, plasticizers and surfactants. The production of castor oil, however, is hampered by the presence of the toxin ricin and hyperallergic 2S albumins in seeds. Lesquerella (Physaria fendleri) does not have such biologically toxic compounds and also contains a major HFA, lesquerolic acid at 55-60% of seed oil. Therefore, lesquerella is being developed as an alternative industrial oilseed crop in the US. We have demonstrated castor RcLPAT2 gene can be used to ricinoleis acid at the sn-2 position of TAG. We further characterized the molecular species of TAG and their positional isomers (regioisomers) in those transgenic lesquerella oils using high-performance liquid chromatography and Electrospray Ionization Mass Spectrometry. These analyses, which are able to reveal regioisomeric structures of TAG with high resolution and accuracy, provide detailed information that are important to understand the mechanisms of HFA synthesis in seeds.

Technical Abstract: Castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) increases hydroxy fatty acids (HFA) content at the sn-2 position of seed triacylglycerols (TAG). Determining TAG species and their regioisomers from transgenic lesquerella seeds expressing RcLPAT2 provides information for understanding the mechanisms of RcLPAT2 in HFA-containing TAG biosynthesis. Using high-performance liquid chromatography and electrospray ionization mass spectrometry, four molecular species of 3-HFA-TAG (tri-HFA-TAG), five of 2-HFA-TAG, and nine of 1-HFA-TAG were revealed, and the ratios of the regioisomers of these molecular species were quantified. HFA were identified as lesquerolic acid (Ls, OH20:1), ricinoleic acid (R, OH18:1), auricolic acid (Au, OH20:2), and densipolic acid (De, OH18:2). Ls was predominately at the sn-1,3 positions in 1-, 2- and 3-HFA-TAG. RcLPAT2 increased significantly all four kinds of HFA at the sn-2 position of the 3-HFA-TAG, and the content of regioisomer with R at the sn-2 in LsLsR alone counted about 91–94% of the total R in the transgenics. RcPLAT2 slightly decreased each 2-HFA-TAG species. There was no Ls, R and De, but trace amount of Au found at the sn-2 position of 2-HFA-TAG. RcLPAT2 also elevated each 1-HFA-TAG species with Ls found to be the only HFA, and likely at the sn-3 position.