Abstract
Crambe is an oil crop suitable for industrial purposes due to the high content of erucic acid (22:1) in the seed oil. The final acylation of diacylglycerols (DAG) with acyl-CoA in the production of triacylglycerols (oil) is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. We identified eight forms of DGATs in crambe and characterized them in microsomal preparations of yeast expressing the enzymes using various acyl-CoAs and both di-6:0-DAG and long-chain DAG species as acyl acceptors. All DGATs accepted 22:1-CoA when using di-6:0-DAG as acyl acceptor. When di-22:1-DAG was the acyl acceptor, the DGAT1 type of enzyme utilized 22:1-CoA at a much-reduced rate compared to assays with sn-1-22:1-sn-2-18:1(oleoyl)-DAG, the most frequently available DAG precursor in crambe seeds. None of the DGAT2 enzymes was able to acylate di-22:1-DAG. Our results indicate that formation of trierucin by crambe DGATs is a limiting step for further increasing the levels of 22:1 in the previously developed transgenic crambe lines due to their poor abilities to acylate di-22:1-DAG. We also show that the acyl-CoA specificities and the enzymatic activities are highly influenced by the fatty acid composition of the DAG acyl acceptor. This finding implies that the use of artificial acyl acceptors (e.g. di-6:0-DAG) may not always reflect the actual acyl-CoA specificities of DGATs in planta. The relevance of the here reported pronounced specificities for specific DAG species exerted by DGAT enzymes is discussed in the context of the findings of DAG pools of distinct catalytic origin in triacylglycerol biosynthesis in the seed oil.
Highlights
Crambe, Crambe hispanica subsp. abyssinica (Hochst. ex R.E.Fr.) Prina has been suggested as a suitable oil crop intended for industrial purposes (Lazzeri et al, 1997; Carlsson et al, 2007)
Eight putative diacylglycerol acyltransferase (DGAT) sequences, four forms of each DGAT1 and DGAT2 isoforms, were isolated from cDNA derived from crambe seeds
In an attempt to boost the 22:1 content in crambe seed oil, genes were introduced to increase the synthesis of di-22:1-DAG and the 22:1-CoA levels, but was unable to increase the erucic acid content above 73% (Li et al, 2012)
Summary
Crambe hispanica subsp. abyssinica (Hochst. ex R.E.Fr.) Prina has been suggested as a suitable oil crop intended for industrial purposes (Lazzeri et al, 1997; Carlsson et al, 2007). Ex R.E.Fr.) Prina has been suggested as a suitable oil crop intended for industrial purposes (Lazzeri et al, 1997; Carlsson et al, 2007). Crambe has a high content (> 55%) of erucic acid (cis-Δ13-22:1, 22:1) in the seed oil. Erucic acid and its derivatives are widely used in industrial applications. Crambe seed oil has appropriate properties as a lubricant and as a quench oil (Lazzeri et al, 1997). Increasing the 22:1 content in seed oil is desirable, due to a drastic reduction of down-stream processing cost when using homogenous or near homogenous starting material in the oleochemical industry (Scarth and Tang, 2006; Li et al, 2012)
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