Abstract
Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG.
Highlights
Levels of ricinoleic acid (RA) than RcFAH-expressing lines[14]; the expression of CpFAH and the resulting RA production was shown to markedly suppress cell growth in fission yeast[12]
We report that a CpFAH-expressing transgenic C. gracilis produced RA in photoautotrophic conditions, without any negative effects on cell growth, and that increased RA levels were achieved by co-expressing a palmitic acid (16:0)-specific fatty acid elongase, Mortierella alpina long chain fatty acid elongase[1] (MALCE1)
Most of the synthesised RA accumulated as monoestolide triacylglycerols (ME TAGs), in which the RA hydroxyl group was masked by other fatty acids, which might explain its reduced cellular toxicity
Summary
Levels of RA than RcFAH-expressing lines[14]; the expression of CpFAH and the resulting RA production was shown to markedly suppress cell growth in fission yeast[12] This cellular toxicity was probably caused by the incorporation of RA into phospholipid fractions, which could affect membrane properties[12]. Holic et al.[12] improved RA production and prevented its cellular toxicity, and Yazawa et al.[5] reported that the co-expression of CpFAH and a phospholipase gene suppressed RA toxicity as well These authors demonstrated that phospholipase-expressing fission yeast lines secreted RA into the culture medium[15,16]. Most of the synthesised RA accumulated as monoestolide triacylglycerols (ME TAGs), in which the RA hydroxyl group was masked by other fatty acids, which might explain its reduced cellular toxicity
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