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

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 in transgenic Arabidopsis is much less than the level found in castor seeds which produce 90% HFA in seed oil.

We designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleate [Formula: see text] 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 HFA counting for 25% in seed oil. 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, seedling growth and seed mucilage content of pCam5-atfae1 lines were not affected by the genetic modification.

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.