2011 Annual Report
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.
Eleostearic acid is produced in one part of the cells of developing seeds, but packaged into oil at a different site. One limitation to novel fatty acid production is an unknown biochemical blockage between the cellular sites where fatty acids are modified and where they are packaged into oil. One of the challenges to plant oilseed metabolic engineering is to determine which enzymes are necessary to drive efficient movement of the novel fatty acids to the site of oil packaging. Three additional enzymes (or proteins) [choline phosphotransferase (CPT), lysophosphatidylcholine acyltransferase (LPCAT), and phospholipid:diacylglycerol acyltransferase (PDAT)] which are thought to act in between these two sites, are currently being tested.
Most oil synthetic enzymes are targeted to cellular membranes, making them difficult to purify for further study. Tung tree DGAT1 and DGAT2 enzymes were fused to other proteins that are known to increase solubility. When expressed in bacteria, a portion of these fused proteins remained soluble and were partially purified. These studies established the first procedures for expressing full-length DGAT proteins from any species using a bacterial expression system. Recombinant proteins will be further purified for antibody production.
LPAT enzyme activity is known to play an important role in determining the fatty acid composition of several important vegetable oils, such as coconut and canola. Plants contain large families of LPAT genes. To date, six potential LPAT genes have been isolated from tung tree. Paired with the tung DGAT2 gene (which is known to be important to tung oil synthesis), each of the six potential tung LPAT genes has been built into baker’s yeast lines, and will be analyzed for increased production of tung-like drying oils.
Yeasts were modified to produce a self-controlling lipase enzyme (which is an enzyme that breaks down oil so that the yeast can consume it). This lipase is only produced when oils and other complex lipids are present outside the yeast cells. This is an important step in engineering of yeasts, because they are otherwise unable to take up complex lipids from their habitat. Lipase production will assist in conversion of baker’s yeast into a form of “bioreactor” that can grow on low-value waste lipids or commodity vegetable oils to produce various value-added lipids, including tung-like drying oils.
Many proteins and enzymes are chemically modified after they are produced in cells. These modifications help to control the activity of enzymes. Previous studies suggest addition of phosphate may increase enzyme activity. The tung DGAT enzymes were expressed in different types of microbes. Some data suggested that the nature of DGAT enzymes produced in bacteria (which cannot carry out chemical modifications) is different compared to those made in yeast, which can modify proteins.
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