Enhanced production of hydroxy fatty acids in arabidopsis seed through modification of multiple gene expression

Authors: PARK, MID EUM - Sejong University; LEE, KYEONG RYEOL - Korean Rural Development Administration; Chen, Grace; KIM, HYUN UK - Sejong University

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2022
Publication Date: 6/18/2022
Citation: Park, M., Lee, K., Chen, G.Q., Kim, H. 2022. Enhanced production of hydroxy fatty acids in arabidopsis seed through modification of multiple gene expression. Biotechnology for Biofuels. 15. Article 66. https://doi.org/10.1186/s13068-022-02167-1.
DOI: https://doi.org/10.1186/s13068-022-02167-1

Interpretive Summary: Castor seeds produce 80–90% of ricinoleic acid, a typical hydroxy fatty acid (HFA) widely used as an industrial raw material for manufacturing high-grade lubricant, paint, coating, plastic, and pharmaceutical products. Castor production is hampered by the presence of deadly toxin ricin and potent allergenic 2S albumins. As such, it is desirable to develop new HFA-producing crops that are safe and suitable for agronomic practices. Arabidopsis has been used as a model to study castor genes involved in HFA synthesis. In this study, we over-expressed five castor genes, and at the same time, deleted one Arabidopsis gene in transgenic Arabidopsis. The resulted lines contain the highest amount of HFA per Arabidopsis seed ever reported. Our strategies provide means for future development of HFA-producing crops through genetic engineering.

Technical Abstract: Background: Castor (Ricinus communis L.) seeds contain unusual fatty acid, hydroxy fatty acid (HFA) used as a chemical feedstock for numerous industrial products. Castor cultivation is limited by the potent toxin ricin in its seeds and other poor agronomic traits, so it is advantageous to develop a suitable HFA-producing crop. Significant research efforts have been made to produce HFA in model Arabidopsis, but the level of HFA produced is far less than the 90% found in castor seeds. Results: We designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleate '12-hydroxylase (FAH12), diacylglycerol (DAG) acyltransferase 2 (DGAT2), phospholipid: DAG acyltransferase 1-2 (PDAT1-2), phosphatidylcholine (PC): DAG cholinephosphotransferase (PDCT) and Lyso-PC acyltransferase (LPCAT). Transgenic Arabidopsis pCam5 lines produced 25% of HFAs in seeds. By knocking out Arabidopsis Fatty acid elongase 1 (AtFAE1) in pCam5 using CRISPR/Cas9 technology, the resulted pCam5-AtFAE1 lines produced over 31% of HFA. Astonishingly, the pCam5-AtFAE1 line increased seed size, weight, and total oil per seed exceeding wild-type by 40%. Seed germination and seedling growth of pCam5-AtFAE1 lines were not affected by the genetic modification. Conclusions: Our results provide not only insights for future research uncovering mechanisms of HFA synthesis in seed, but also metabolic engineering strategies for generating safe HFA-producing crops.