Genetic Engineering of Lesquerella with Increased Ricinoleic Acid Content in Seed Oil.

Grace Q Chen,Kumiko Johnson,Grisel Ponciano,Eva Morales,Edgar B Cahoon,Tara J Nazarenus

doi:10.3390/plants10061093

Abstract

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Highlights

The conventional source of hydroxy fatty acid (HFA) is castor (Ricinus communis), which contains 90% ricinoleic acid (18:1OH) in its seed oil
Line 1 to line 16 were assigned to these transgenics based on descending order in 18:1OH content in their seed oils (Table 1)
The dsRNA fragments in AtFAD3 RNAi and CsFAE1 RNAi contain a 323 bp or 251 bp sequence sharing 91.7% and 82.8% identity with lesquerella PfFAD3-1 (BenBank ID: MF611845) [32] and PfKCS18 (GenBank ID: AF367052) [27], respectively

Summary

Introduction

The conventional source of hydroxy fatty acid (HFA) is castor (Ricinus communis), which contains 90% ricinoleic acid (18:1OH) in its seed oil. The production of castor oil is hampered by the presence of the toxin ricin [3,4] and hyper-allergenic 2S albumins [5,6,7] in its seeds. Lesquerella (Physaria fendleri, Brassicaceae) seed oil contains a major HFA, lesquerolic acid (20:1OH) at 55–60% [8,9,10,11], and seeds of this plant lack any known toxins. Efforts have been made through plant breeding to develop lesquerella as a new oilseed crop that is a safe source of HFA [12,13]. With the success of lesquerella biotechnology [14,15] and the deep knowledge of genes for fatty acid and seed oil biosynthesis [16,17,18], lesquerella oil can be improved through metabolic engineering [15]

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Conclusion