Location: Commodity Utilization Research2012 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 year, increased efforts were initiated, or continued, in the search for additional genes from developing tung seeds that would further increase the amount of value-added lipids that can be produced in transgenic engineered plants or microbes. Several potential target enzymes sit in between the location within cells of developing tung seeds where eleostearic acid (the value-added component of tung oil) is produced and the location where it is ultimately stored in the mature seeds. Finding the enzymes that drive efficient movement between the two sites is a key goal of this project. Ruling out enzymes that do not help can be as useful as identifying those that do. Three additional proteins choline phosphotransferase (CPT), lysophosphatidylcholine acyltransferase (LPCAT), and phospholipid:diacylglycerol acyltransferase (PDAT) were tested. Under the current conditions used, CPT clearly did not seem to have a beneficial effect. LPCAT and PDAT are still being evaluated; recent evidence indicates that accurate answers to the “do they, or don’t they help?” question first requires elimination or reduction of certain types of competing enzymes that are already present in the transgenic plants or microbes. Some lines containing reduced competition are already created and are being analyzed; generation of certain other lines is underway. One of the key steps in the biochemical pathway leading to vegetable oil synthesis is the generation of diacylglycerol (DAG). DAG is a building block used by at least three types of important enzymes that convert DAG to triacylglycerol (TAG), the main form of vegetable oil present in tung seed and all other oil-producing plant seeds. Current evidence suggests that two types of enzymes can produce the necessary pools of DAG. One, LPCAT, has already been cloned from developing tung seeds and is currently being studied. Two different forms of the second enzyme class, phosphatidic acid phosphohydrolase (PAP), have been studied by other groups, but the properties of those enzymes do not seem to be a good biochemical fit with the expected role in TAG production. ARS researchers in the Commodity Utilization Research Unit, in New Orleans, Louisiana, have partially purified an enzyme activity that more closely matches the expected properties of a PAP involved in vegetable oil synthesis. Complete purification of the protein, and isolation of the gene that produces it are actively being pursued. Antibodies against tung DGAT1 and tung DGAT2 have been produced. These antibodies are difficult to produce and had not been available previously. These molecules will now serve as powerful tools in a variety of ways, not the least of which is to provide a new angle in the search for other proteins and enzymes that interact and cooperate with tung DGAT1 and DGAT2. The enzymes identified in this way will be high-priority targets for inclusion in the existing metabolic engineering strategies currently in place.
Shockey, J., Chapital, D., Gidda, S., Mason, C., Davis, G., Klasson, K.T., Cao, H., Mullen, R., Dyer, J. 2011. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 92(6):1207-1217.