Research Accomplishment Reports 2007

Epoxy Fatty Acid Accumulation in Seed Oil

D.Hildebrand
Department of Plant and Soil Sciences

 

Project Description

The overall goal of this project is to develop soybeans with seed oil accumulating high levels of oxygenated fatty acids (e.g. epoxy fatty acids) for providing additional renewable resources that are industrially valuable.

Previously, we developed a system to synthesize labeled and unlabeled fatty acids and derivatives for in vitro biochemical studies on the enzymes responsible for TAG synthesis. Our previous data showed that microsomal DGATs from Vernonia and Stokesia exhibited high enzymatic activities and selective incorporation of vernolic acids (an epoxy fatty acid) into TAG (Yu et al., 2006). In order to identify which enzymes are responsible for the selective accumulation of vernolic acids in these plant seeds, we isolated two DGAT1 cDNA clones (VgDGAT1a and 1b) and one DGAT2 clone (VgDGAT2) from Vernonia.  Two DGAT1 cDNAs were also cloned respectively from Euphorbia (ElDGAT1a and 1b) and soybean (GmDGAT1a and 1b).  Soybean is our target oilseed plant for engineering to produce epoxy fatty acids.  Understanding of the enzymatic properties related to TAG synthesis in soybean is also helpful to achieve our goal.

In this period, we further investigated the biochemical functions of these cDNA clones using our yeast expression and in vitro assay system. VgDGAT1a and 1b showed very high enzymatic activities, and no substrate specificities with vernolic acid containing substrates. VgDGAT2 expression in yeast were further evaluated after incorporation of a Kozak translation initiation sequence along with GmDGAT1a, GmDGAT1b and ElDGAT1a.  No enzymatic activity and therefore vernolic acid substrate preference of VgDGAT2 and ElDGAT1a have yet to be assessed by our yeast in vitro experiments. We recently tested the impact of VgDGAT1a and VgDGAT2 in a new rapid plant gene test system involving Agro-infiltration of petunia leaves and find VgDGAT1a increases the accumulation of the epoxy fatty acid, vernolic acid (Va), moderately and VgDGAT2 increases dramatically. 

At the linear range assay conditions, GmDGAT1a and GmDGAT1b exhibited much higher activity relative to the vector control. No difference was found between GmDGAT1a and GmDGAT1b in the assays. Specifically, oleoyl-CoA (18:1-CoA)/dioleoyl-diacylglycerol (sn-DODAG) has the highest activity and vernoloyl-CoA/ divernoyl-diacylglycerol (sn-DVDAG) has the lowest activity. The two substrate combinations of vernoloyl-CoA/ sn-DODAG and 18:1-CoA/ sn-DVDAG have high activity in the middle levels. Within these middle levels, the substrate combination of 18:1-CoA/ sn-DVDAG has higher DGAT activities than those of substrate combination of vernoloyl-CoA/ sn-DODAG. Such results suggest that GmDGAT1s are not very helpful for vernolic acid accumulation into TAG although high DGAT activities indicate their important roles in TAG synthesis.

VgDGAT1a and 1b as well as VgDGAT2 are the main target enzymes for further analysis because their expression patterns are consistent with the epoxy fatty acid accumulation in Vernonia seed development and Vernonia is the highest accumulator of epoxy fatty acids among these plants. Our current yeast expression and in vitro assay above do not exclude their functions in vivo although no vernolic acid substrate preference was found for VgDGAT1a or1b. Additional function studies on VgDGAT1s and VgDGAT2 are in process using other test systems such as in vivo yeast expression assay and transgenic expression in soybean somatic embryos and Arabidopsis seeds.  As mentioned above we recently tested the impact of VgDGAT1a and VgDGAT2 in a new rapid plant gene test system involving Agro-infiltration of petunia leaves and find VgDGAT1a increases the accumulation of the epoxy fatty acid, vernolic acid (Va), moderately and VgDGAT2 increases dramatically.

Somatic embryos of transgenic lines expressing Stokesia epoxygenase plants have been produced. However, the seed set for these plants is very limited probably due to the long culture periods in the somatic embryos (SE) stages. Further soybean transformations were performed using Stokesia epoxygenase (STE-pCambia1201) and VgDGAT2 (both a linear fragment in a phaseolin seed-specific expression cassette and a whole plasmid in pCambia 1301). Close to 20 hygromycin positive lines have been recovered using the  STE-pC1201 and the VgDGAT2-phaseolin linear fragment.  Some of the SEs have been matured and preliminary data indicates a large increase in Va accumulation when Stokesia epoxygenase is co-expressed with VgDGAT2.

Soybean transformation with VgDGAT1a-pC1201 was also performed and more than a dozen hygromycin positive lines have been recovered. VgDGAT1a-pC1301 was also constructed for further soybean transformation.  Some of these soybean SEs have also been matured and preliminary data indicates a moderate increase in Va accumulation when Stokesia epoxygenase is co-expressed with VgDGAT1a.

Impact

Engineering oilseeds for high epoxy fatty acid accumulation in triglyceride remains the specific goal of this research.  A considerable market currently exists for epoxy fatty acids particularly for epoxy coatings and plasticizers and currently most of these are derived from petroleum.  There is no known way to produce a commercial oilseed that accumulates epoxy fatty acids by conventional breeding and genetics.  Certain genotypes of several plant species, however, accumulate high levels of epoxy fatty acids in the seed oil.  The best examples of this are Vernonia galamensis, Stokesia laevis and Euphorbia lagascae. There are two major project objectives:  1) Further investigation of the mechanism of high-oxygenated fatty acid accumulation in triacylglycerol.  2) Evaluate genes responsible for high epoxy fatty acid accumulation in source plants and test these genes in soybean embryos. 

V. galamensis, S. laevis or E. lagascae cannot readily be grown to produce seed on an industrial scale limiting their current potential to replace much fossil fuel use as sources of epoxy compounds.  Epoxygenase genes have been cloned from several accumulators of vernolic acid and high expression in developing Arabidopsis or soybean embryos can result in up to ~10% epoxy fatty acid in the seed oil but this is insufficient for commercial production of epoxides in oilseeds. Enzymes that synthesize the final triacylglycerol of the oil such as diacylglycerol acyltransferase (DGAT) and/or phospholipid: diacylglycerol acyltransferase (PDAT) are of major importance to the accumulation of epoxy fatty acids in seed oil triacylglycerols.  As mentioned in our previous report our cumulative experiments and related research indicates that DGAT is the main enzyme responsible for epoxy oil synthesis with DGAT2 apparently being most important at least in the case of Vernonia and Stokesia.

Vernonia DGAT1a and Vernonia DGAT1b are highly active when expressed in yeast. However, they do not have substrate preference toward vernolic acid bearing substrates. Instead, oleoyl-CoA is a preferred substrate over vernoloyl-CoA and sn-1,2-dioleoylglycerol is a preferred substrate over sn-1,2-divernoloylglycerol. Such results with DGAT1s again implicate DGAT2 in the synthesis of epoxy TAG in Vernonia and Euphorbia. A full-length Vernonia DGAT2 was cloned and sequenced.Thus, the Vernonia DGAT2 was placed in a seed-specific expression vector and co-expressed with Stokesia epoxygenase in soybeans for high vernolic acid accumulation. Close to 20 hygromycin positive lines have been recovered from these soybean transformations.

Soybean DGAT1a and soybean DGAT1b have also been expressed in yeast. They have also exhibited much higher activity relative to the vector control. No difference was found between GmDGAT1a and GmDGAT1b in the assays. Specifically, 18:1-CoA/ sn-DODAG has the highest activity and vernoloyl-CoA/ sn-DVDAG has the lowest activity.  Such results suggest that GmDGAT1s are not very helpful for vernolic acid accumulation into TAG. This is consistent with the low accumulation of Va in soybeans transformed with Stokesia epoxygenase alone.  Thus, a DGAT that prefers vernolic acid bearing substrates is being co-expressed with an epoxygenase for high vernolic acid accumulation in transgenic soybeans.

Plants from transgenic soybean lines expressing only Stokesia epoxygenase have been produced. Further soybean transformations are being performed with the above mentioned Stokesia epoxygenase and Vernonia DGAT2. Soybean transformation with Vernonia DGAT1a has also been performed to evaluation this gene’s effect on soybean oil accumulation. These efforts are leading to achieving high (> 50%) epoxy fatty acid accumulation in seed oil of a commercial crop.  Preliminary data indicate a moderate increase in Va accumulation when Vernonia DGAT1a is co-expressed with Stokesia epoxygenase and a large increase when Vernonia DGAT2 is co-expressed with Stokesia epoxygenase.

Publications

Yu, K., R. Li, T. Hatanaka and D. Hildebrand. (2008). Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis, Phytochemistry (in press).

Hilker, B.L., H. Fukushige, C. Hou and D. Hildebrand. (2008). Comparison of Bacillus monooxygenase genes for unique fatty acid production.  Progress in Lipid Research 47:1-14.

Hildebrand. D., R. Li and T. Hatanaka. (2008). Genomics of soybean oil traits. In Soybean Genomics, G. Stacey, ed. (in press).

Yu, K., C. T. McCracken, Jr., and D. Hildebrand. (2007). Synthesis of sn-1,2-diacyl[U-14C]glycerol with high specific activity. In: Current Advances in the Biochemistry and Cell Biology of Plant Lipids (eds. Benning, C and J. Ohlrogge). Proceedings for the 17th International Symposium on Plant Lipids, pp 6-10. Aardvark Global Publishing Company, LLC, Salt Lake City, UT.

Kachroo, A, J. Shanklin, E. Whittle, L. Lapchyk, D. Hildebrand and P. Kachroo. (2007). The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis. Plant Molec. Biol. 63: 257-271.

Hilker, B.L., H. Fukushige, C. Hou and D. Hildebrand (2008) Some properties of a self-sufficient cytochrome P-450 from Bacillus megaterium strain ALA2. In “Biocatalysis and Bioenergy”, Hou, C.T. and J.F. Shaw eds., John Wiley & Sons (In press).

Mendu, V. and David F. Hildebrand. (2007). Plant Hydroperoxide Lyases and Related Enzymes. Chapt. 24 In: Biocatalysis and Biotechnology for Functional Foods and Industrial Products, Ed., Ching Hou, Taylor & Francis, N.Y., pp 399-417.

Hildebrand. D. (2007) Biotechnology and Crop Improvement in Agriculture.  In Teaching Innovations in Lipid Science, R. Weselake, ed. Taylor and Francis Group, LLC.