Duckweed has been engineered to produce high oil yields by scientists at the US Department of Energy’s Brookhaven National Laboratory and collaborators at Cold Spring Harbor Laboratory (CSHL). The researchers inserted genes into one of nature’s fastest-growing aquatic plants to “push” fatty acid synthesis, “pull” those fatty acids into oils, and “protect” the oil from degradation. According to the scientists, such oil-rich duckweed could be easily harvested to produce biofuels or other bioproducts, as explained in a paper published in Plant Biotechnology Journal.
The researchers engineered a strain of duckweed, Lemna japonica, to accumulate oil at close to 10% of its dry weight biomass. That’s a 100-fold increase over wild plants, with yields more than seven times higher than soybeans, today’s most important source of biodiesel.
“Duckweed grows quickly,” said team leader and Brookhaven Lab biochemist John Shanklin. “It has no stems or roots, so the majority of its biomass is in leaf-like fronds that grow on the surface of ponds all over the world. All of that biomass has a high oil content as a result of our engineering. Growing and harvesting this engineered duckweed in batches and extracting its oil could be a cost-effective way to produce renewable and sustainable oil” He stated.
Two added benefits: As an aquatic plant, oil-producing duckweed wouldn’t compete with food crops for prime agricultural land. It can even grow on runoff from pig and poultry farms.
“That means this engineered plant could potentially clean up agricultural waste streams as it produces oil,” Shanklin said.
Future work will focus on testing push, pull, and protect factors from a variety of different sources, optimizing the levels of expression of the three oil-inducing genes, and refining the timing of their expression. Beyond that we are working on how to scale up production from laboratory to industrial levels.John Shanklin
Leveraging two Long Island research institutions
The current project has roots in Brookhaven Lab research on duckweeds from the 1970s, led by William S. Hillman in the Biology Department. Later, other members of the Biology Department worked with the Martienssen group at Cold Spring Harbor to develop a highly efficient method for expressing genes from other species in duckweeds, along with approaches to suppress expression of duckweeds’ own genes, as desired.
As Brookhaven researchers led by Shanklin and Jorg Schwender over the past two decades identified the key biochemical factors that drive oil production and accumulation in plants, one goal was to leverage that knowledge and the genetic tools to try to modify plant oil production. The latest research, reported here, tested this approach by engineering duckweed with the genes that control these oil-production factors to study their combined effects.
“The current project brings together Brookhaven Lab’s expertise in the biochemistry and regulation of plant oil biosynthesis with Cold Spring Harbor’s cutting-edge genomics and genetics capabilities,” Shanklin said.
One of the oil-production genes identified by the Brookhaven researchers pushes the production of the basic building blocks of oil, known as fatty acids. Another pulls, or assembles, those fatty acids into molecules called triacylglycerols (TAG) — combinations of three fatty acids that link up to form the hydrocarbons we call oils. The third gene produces a protein that coats oil droplets in plant tissues, protecting them from degradation.
From preliminary work, the scientists found that increased fatty acid levels triggered by the “push” gene can have detrimental effects on plant growth. To avoid those effects, Brookhaven Lab postdoctoral researcher Yuanxue Liang paired that gene with a promoter that can be turned on by the addition of a tiny amount of a specific chemical inducer.
“Adding this promoter keeps the push gene turned off until we add the inducer, which allows the plants to grow normally before we turn on fatty acid/oil production,” Shanklin said.
Liang then created a series of gene combinations to express the improved push, pull, and protect factors singly, in pairs, and all together. In the paper these are abbreviated as W, D, and O for their biochemical/genetic names, where W=push, D=pull, and O=protect.
The key findings
Overexpression of each gene modification did not increase fatty acid levels in Lemna japonica fronds significantly. When the results of several different transgenic lines were averaged, plants engineered with all three modifications accumulated up to 16 percent of their dry weight as fatty acids and 8.7 percent as oil. The best plants accumulated up to 10% TAG, which is more than 100 times the amount of oil found in unmodified wild type plants.
Some combinations of two modifications (WD and DO) significantly increased fatty acid content and TAG accumulation compared to their individual effects. Synergistic results occur when the combined effect of two genes increases production more than the sum of the two separate modifications.
These findings were also revealed in images of lipid droplets in plant fronds taken with a confocal microscope at Brookhaven Lab’s Center for Functional Nanomaterials (CFN), a DOE Office of Science user facility. When the duckweed fronds were stained with an oil-binding chemical, the images revealed that plants with each two-gene combination (OD, OW, WD) accumulated more lipid droplets than plants with these genes expressed singly – and also when compared to control plants with no genetic modification. Both the OD and OWD lines produced large oil droplets, but the OWD line produced more of them, resulting in the strongest signals.
“Future work will focus on testing push, pull, and protect factors from a variety of different sources, optimizing the levels of expression of the three oil-inducing genes, and refining the timing of their expression,” Shanklin said. “Beyond that we are working on how to scale up production from laboratory to industrial levels.”