Molecular cloning and characterization of a KCS gene from Cardamine graeca and its heterologous expression in Brassica oilseeds to engineer high nervonic acid oils for potential medical and industrial use

Abstract

Database: The C. gracea KCS sequence information has been submitted to the EMBL ⁄ GenBank under accession number: bankit1110928.Keywords: 3-keto-acyl-CoA synthase, Cardamine graeca, Nervonic acid, Brassica carinata, Brassica napus, Arabidopsis thaliana, Health and Industry. SummaryNervonic acid 24:1 D15 (cis-tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid.There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells.We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T 3 generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic ⁄ highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical ⁄ nutraceutical applications to improve human and animal health.

Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas

“…KCS is a component of the elongation complex responsible for the synthesis of very long chains of monounsaturated fatty acids (VLCMFA) in the seeds of plants [16]. The KCS gene showed an expression pattern of Category III, which was constitutively expressed throughout endosperm development (Figure 5H).…”

“…Taylor et al (2009) produced transgenic Arabidopsis and Brassica Carinata plants that expressed Cardamine KCS gene. The seed-specific expression of the Cardamine KCS gene led to 55-fold and 15-fold increase in nervonic acid proportions in Arabidopsis and B. carinata seed oil, respectively [16]. …”

A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds

“…A number of reports have described the ability to increase total oil content of seeds by transgenic expression of DGAT1 and DGAT2 enzymes of plant and fungal origin (Jako et al ., 2001; Lardizabal et al ., 2008; Oakes et al ., 2011). Seed-specific overexpression of Arabidopsis DGAT1, for example, increased oil content by up to 15 and 46% in seeds of transgenic Brassica napus and Arabidopsis , respectively (Jako et al ., 2001; Sharma et al ., 2008; Taylor et al ., 2009). Small increases in oil content of maize and soybean seeds were conferred by expression of the fungus Umbelopsis ramanniana DGAT2 (Lardizabal et al ., 2008; Oakes et al ., 2011).…”

Renewable feedstocks for lubricant production

“…Suitable plants for such manipulation include Brassica species such as B. napus, B. juncea and B. rapa, Crambe species such as C. abyssinica and C. hispanica, and Limnanthes species such as L. alba alba and L. douglasii (meadowfoam). seed-specifi c reduction in Δ15 desaturase activity.In another recent development Taylor et al[238] have reported the signifi cant accumulation of the unusual VLCFA nervonic acid (C24:1Δ15) in Brassica oilseeds by metabolic engineering. Annual honesty (Lunaria annua…”