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United States Department of Agriculture

Agricultural Research Service

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Research Project: Engineering Enzymatic Redirection of Natural Crop Oil Production to Industrial Oil Production

Location: Commodity Utilization Research

2013 Annual Report

1a. Objectives (from AD-416):
The overall objective of this project is to define the minimal sets of genes required for efficient synthesis and accumulation of industrially important fatty acids in transgenic hosts, and to express these genes in microbes and commodity oilseed crops for production of value-added industrial oils. During the project, we will focus on the following objectives: Objective 1: Use model plant systems to identify and refine transgenic expression conditions for critical industrial oil biosynthetic genes. Objective 2: Identify substrate specificity-determining sequences in pertinent genes from tung tree related species. Objective 3: Engineer yeast strains for use in microbial bioconversion system. Objective 4: Transfer knowledge of minimal necessary gene sets from current research (on tung tree genes) to other novel oilseed whose oil represents greater market size or strategic value; i.e., epoxy (from Crepis, Vernonia, and Euphorbia species) or acetylenic fatty acids (also from Crepis). Objective 5: Engineer tung FADX, DGAT2, and other genes from donating organism (tung tree) into commercially important oilseed crop plant such as cotton, soybean, or camelina.

1b. Approach (from AD-416):
Genes encoding the enzymes for tung oil biosynthesis will be identified by homology-based searches and next-generation high volume pyrosequencing technologies. Other necessary enzymes and proteins will be detected via transcriptomic and proteomic analysis of seeds from tung and other species. Comparisons between different species of tung that produce medium or high amounts of eleostearic will also be used to detect evolution of enzymes well-suited to tung oil production. Mutagenesis studies will identify the active sites and critical residues in these enzymes, thus facilitating the design of engineered forms of important proteins. Model laboratory species of plants and microbes will be used to express combinations of multiple tung genes to find the minimal sets necessary to produce useful levels of eleostearic and other novel fatty acids. A microbial expression system tailored for the bioconversion of low-cost oils into tung-like drying oils will be generated by engineering common yeast strains to efficiently use oils as food, convert the common fatty acids to new valuable lipids, and increase the cellular lipid content.

3. Progress Report:
This is the final report for project 6435-41000-106-00D, which has been redirected and combined with existing project 6435-41000-102-00D. Some of the objectives will continue in the new project until a new project plan is written. The biosynthesis of oils and other lipids in plant tissues and organs, including seeds, is an extremely complex process. Substantial progress was made during the three years. Gene discovery studies revealed new forms of enzymes that occupy several of the steps in the biochemical pathway for plant seed oil production. Combining just two of these genes achieved the highest levels of tung-like fatty acids ever observed in transgenic plants. Experience gained from cloning of these genes from tung seeds provided the expertise needed to also identify representative copies of these same important genes from crops, such as cotton, litchi, soybean, bitter gourd, and hawksbeard, that make other types of valuable plant oils. For some of these proteins, new enzyme activity measurement methods were developed that increased the speed of these measurements, while also reducing the cost and reducing the use of hazardous radioactive compounds. We identified three proteins (called Acyl-CoA binding proteins) in the tung tree and determined that at least one of them is important for tung oil production. We made progress determining the profile gene expression in tung oil biosynthesis and looked at 25 genes using two methods in samples of tung seed, leaves, and flowers. The method is important to indentify key factors for tung oil biosynthesis in plants. Novel methods were developed for production and purification of tung oil biosynthetic proteins; the purity of these was such that antibodies could be raised against them. These antibodies will be powerful tools for studying how, when, and where these tung proteins are produced in seed tissues and what their roles are in production of oil and other lipids in tung seeds. Additional studies of one of the key proteins in the tung oil synthesis pathway (type-2 diacylglycerol acyltransferase, or DGAT2) revealed a previously uncharacterized region of the protein that exerts control over the yield of tung-specific fatty acids that accumulate in the oils of transgenic plants expressing this tung protein. We were able to insert the gene responsible for this protein into a bacterium and the first documented protein of this kind. Overall, the impact of this project lies in the contribution to the fundamental understanding of the genetic tool set required for efficient production of oils in plants seeds, and especially the finding that DGAT enzymes are often critical to production of oils containing unusual fatty acids, such as those found in tung oil. The existing project has been primarily dedicated to the study of tung seed genes and proteins. However, the knowledge base developed will likely aid the process of rational experimental design for the study of other plant seed oils, including cottonseed oil. Thus, the findings and impact of the terminated project will still remain relevant, and will quickly contribute to new findings and progress in the cottonseed oil project.

4. Accomplishments
1. Identification of important domain of DGAT2 (diacylglycerol acyltransferase 2) protein. ARS scientists in the Commodity Utilization Research Unit in New Orleans, LA, identified domain of type-2 diacylglycerol acyltransferase (DGAT2) from tung tree that, when altered, can substantially enhance levels of enzyme activity compared to normal enzyme when produced in yeast or plants. While various other previous studies have identified certain amino acids that negatively affect enzyme activity when altered, the current ARS work studying this domain marks one of the first insights into identification of key amino acid residues that positively regulate DGAT2 enzymes. The mutant form of the protein dramatically increases the average level of novel fatty acid product formed in transgenic plants. Determination of the basis for this change may reveal new insights regarding the protein stability, kinetic properties, or substrate selectivity of DGAT2 enzymes. As such, these findings might also guide the production, engineering, and improvement of DGAT2s from other novel oilseed crops.

2. Production of first full-length DGAT2 (diacylglycerol acyltransferase 2) protein purified from a bacterial host. ARS scientists in the Commodity Utilization Research Unit in New Orleans, LA, established first procedure for bacterial expression of full-length recombinant DGAT2 from any species. ARS scientists demonstrated that recombinant DGAT2 is produced as a single unit and as a double unit containing two copies of the protein. Additional analysis showed that DGAT2 is associated with other proteins, lipids, and membranes, and that post-translational modification of recombinant DGAT2 may be required for its enzymatic activity and/or that the bacterial-produced form of the protein is misfolded. This production method may accelerate the identification of enzyme inhibitors for therapeutic treatment of obesity and related diseases (DGATs are key enzymes in oil/fat biosynthesis in plants, animals and humans).

3. Development of non-radioactive colorimetric PAP (Prosphatidic acid phosphatase) assay. ARS scientists in the Commodity Utilization Research Unit in New Orleans, LA, have developed two colorimetric methods to measure phosphatidic acid phosphatase (PAP) activity in developing seeds. This enzyme is considered by lipid biochemists to be one of the key enzymes of Kennedy pathway in oil biosynthesis. Traditional methods of measuring PAP activity relies on incorporating radioactive substrates in the assay mixture which is followed by separating the reactants via silica gel or other cumbersome analytical methods and then finally measuring the radioactivity in phosphatidic acid. As opposed to that, our method relies solely on measuring cleaved phosphates from phosphatidic acid, which is done by simple colorimetric methods, thus increasing speed, reducing the cost of this assay, and increasing safety by reducing the use of hazardous radioactivity compounds.

Review Publications
Ullah, A.H.J., Sethumadhavan, K., Shockey, J. 2012. Measuring phosphatidic acid phosphatase (EC activity using two phosphomolybdate-based colorimetric methods. Advances in Biological Chemistry. 2:416-421.

Ullah, A.H.J., Sethumadhavan, K. 2013. Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of Morordica charantia. Advances in Biological Chemistry. (3):11-17.

Cao, H., Shockey, J.M. 2012. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues. Journal of Agricultural and Food Chemistry. 60(50):12296-12303.

Pastor, S., Sethumadhavan, K., Ullah, A.H.J., Gidda, S., Cao, H., Mason, C., Chapital, D., Scheffler, B., Mullen, R., Dyer, J., Shockey, J. 2013. Molecular properties of the class III subfamily of acyl-coenyzme A binding proteins from tung tree (Vernicia fordii). Plant Science. 203-204:79-88.

Cao, H., Chapital, D.C., Howard Jr, O.D., Deterding, L.J., Mason, C.B., Shockey, J.M., Klasson, K.T. 2012. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2. Applied Microbiology and Biotechnology. 96(3):711-727.

Zhan, Z., Wang, Y., Shockey, J., Chen, Y., Zhou, Z., Yao, X., Ren, H. 2012. Breeding status of tung tree (Vernicia sp.) in China, a multipurpose oilseed crop with industrial uses. Silvae Genetica. 61(6):265-270.

Ullah, A.H.J., Sethumadhavan, K., Grimm, C., Shockey, J. 2012. Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria. Advances in Biological Chemistry. 2(4):403-410.

Last Modified: 05/22/2017
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