2012 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.
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.
Production of antibodies against full-length tung DGAT enzymes. Detailed characterization of important oil synthesizing enzymes, like tung DGAT1 and DGAT2, has been slowed by an inability to study the cells they are produced in, their targeting within cells, the proteins they interact with, and the ways in which they are produced, maintained, and then broken down. The most important tools needed to address these questions are high-titer antibodies against the DGAT1 and DGAT2 enzymes. Last year, ARS researchers at the Southern Regional Research Center in New Orleans, LA, achieved the first production of soluble forms of these two otherwise insoluble enzymes, and have now produced antibodies against them. These antibodies are now undergoing a battery of tests to assess their properties and purity. These antibodies will be a powerful tool for diagnostic analysis of the level of expression and efficiency of natural and mutant forms of DGAT enzymes, which are key components for production of high-performing transgenic industrial oilseed crops.
Identification of a potential third type of the PAP enzyme. In oilseed crops, the flow of carbon into its ultimate storage location, oil droplets, is controlled to different degrees by many enzymes, some of which are known and some of which are not. One potentially important enzyme is phosphatidic acid phosphohydrolase (PAP). PAP plays several roles in living cells, one of which is to synthesize the diacylglycerol (DAG) necessary to produce storage oils. Currently two types of PAP are known, but neither displays properties consistent with a role in storage oil synthesis. ARS researchers at the Southern Regional Research Center in New Orleans, LA, have been analyzing seed extracts from oil-producing seeds of bottle gourd (a close relative to one of the plants used heavily in the current project) and have shown a potential third type of PAP enzyme. The primary biochemical properties of this protein are substantially different than the previous two classes, and may represent the PAP activity that fuels storage oil synthesis. Peptide sequences that may represent parts of the protein responsible are in hand, which will allow for isolation of the full-length genes from bottle gourd, tung tree, and other important oilseed crops. These genes could become an important component of our metabolic engineering strategies and be put to use in transgenic crops and microbes to help produce higher levels of novel value-added oils.
Shockey, J., Chapital, D., Gidda, S., Mason, C., Davis, G., Klasson, K., 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.
Cao, H. 2011. Bioengineering recombinant diacylglycerol acyltransferases. In: Carpi, A., editor. Progress in molecular and environmental bioengineering-from analysis and modeling to technology applications. Rijeka, Croatia: InTech Open Access Publisher. p. 467-482.
Cao, Heping. 2011. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Research Notes. 4:249 (24 pages).