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John Shanklin

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Duckweed. Photo from BNL
Scientists drive oil accumulation in rapidly growing aquatic plants

Scientists at the U.S. Department of Energy’s Brookhaven National Laboratory and collaborators at Cold Spring Harbor Laboratory (CSHL) have engineered duckweed to produce high yields of oil. The team added genes to one of nature’s fastest growing aquatic plants to “push” the synthesis of fatty acids, “pull” those fatty acids into oils, and “protect” the oil from degradation. As the scientists explain in a paper published in Plant Biotechnology Journal, such oil-rich duckweed could be easily harvested to produce biofuels or other bioproducts.

John Shanklin. Photo from BNL

The paper describes how the scientists engineered a strain of duckweed, Lemna japonica, to accumulate oil at close to 10 percent of its dry weight biomass. That’s a dramatic, 100-fold increase over such plants growing in the wild—with yields more than seven times higher than soybeans, today’s largest source of biodiesel.

“Duckweed grows fast,” said Brookhaven Lab biochemist John Shanklin [https://www.bnl.gov/staff/shanklin], who led the team. “It has only tiny stems and roots—so most of its biomass is in leaf-like fronds that grow on the surface of ponds worldwide. Our engineering creates high oil content in all that biomass.

“Growing and harvesting this engineered duckweed in batches and extracting its oil could be an efficient pathway to renewable and sustainable oil production,” he said.

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.

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 [https://www.bnl.gov/staff/schwend] 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 alone did not significantly increase fatty acid levels in Lemna japonica fronds. But plants engineered with all three modifications accumulated up to 16 percent of their dry weight as fatty acids and 8.7 percent as oil when results were averaged across several different transgenic lines. The best plants accumulated up to 10 percent TAG—more than 100 times the level of oil that accumulates in unmodified wild type plants.

Some combinations of two modifications (WD and DO) increased fatty acid content and TAG accumulation dramatically relative to their individual effects. These results are called synergistic, where the combined effect of two genes increased production more than the sum of the two separate modifications.

These results were also revealed in images of lipid droplets in the plants’ fronds, produced using a confocal microscope at the Center for Functional Nanomaterials [https://www.bnl.gov/cfn/] (CFN), a DOE Office of Science user facility at Brookhaven Lab. When the duckweed fronds were stained with a chemical that binds to oil, the images showed that plants with each two-gene combination (OD, OW, WD) had enhanced accumulation of lipid droplets relative to plants where these genes were expressed singly—and also when compared to control plants with no genetic modification. Plants from the OD and OWD lines both had large oil droplets, but the OWD line had more of them, producing 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.”

That scale-up work has several main thrusts: 1) designing the types of large-scale culture vessels for growing the modified plants, 2) optimizing large-scale growth conditions, and 3) developing methods to efficiently extract oil at high levels.

This work was funded by the DOE Office of Science (BER). CFN is also supported by the Office of Science (BES).

Brookhaven National Laboratory is supported by the Office of Science of the U.S. Department of Energy. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, visit science.energy.gov. [https://www.energy.gov/science/]

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In left photo, Zhiyang Zhai, on the right, with John Shanklin and Jantana Keereetaweep; in right photo, Zhiyang Zhai with Hui Liu. Photos courtesy of BNL

By Daniel Dunaief

In a highly competitive national award process, the Department of Energy provides $2.5 million to promising researchers through Early Career Research Funding.

Recently, the DOE announced that Zhiyang Zhai, an associate biologist at Brookhaven National Laboratory, was one of 83 scientists from around the country to receive this funding.

“Supporting talented researchers early in their career is key to fostering scientific creativity and ingenuity within the national research community,” DOE Office of Science Director Asmeret Asefaw Berhe, said in a statement.

Zhai, who has worked at BNL for 11 years, is studying a signaling protein called Target Of Rapamycin (TOR) kinase, which is important in the plant and animal kingdom.

He hopes to develop a basic understanding of the way this kinase reacts to different conditions, such as the presence of carbon, to trigger reactions in a plant, including producing oils through photosynthesis or making seeds.

Zhiyang Zhai. Photo from BNL

“Ancient systems like this evolve in different lineages (like plants and animals) to work differently and [Zhai] wants to find out the details of how it works in plants,” John Shanklin, chair of BNL’s Biology Department, explained in an email. 

Zhai is trying to define which upstream signals interact with TOR and what the effects of those interactions are on TOR to learn how the kinase works.

He is hoping to get a clear idea of how different nodes interact and how signaling through carbon, nutrients and sunlight affects TOR kinase levels and its configuration.

Researchers may eventually use the knowledge of upstream regulators to reprogram responses by introducing enzymes that would cause the synthesis, or degradation, of upstream regulatory metabolites, Shanklin suggested.

This could be a way to “tune” the sensor kinase activity to increase the synthesis of storage compounds like oil and starch.

In the bigger picture, this type of research could have implications and applications in basic science that could enhance the production of renewable resources that are part of a net-zero carbon fuel strategy.

The DOE sponsors “basic science programs to discover how plants and other organisms convert and store carbon that will enable a transition towards a net zero carbon economy to reduce the use of fossil fuels,” Shanklin said.

In applying for the award, Zhai paid “tremendous attention” to what the DOE’s mission is in this area, Shanklin said. Zhai picked out a project that, if successful, will directly contribute to some of the goals of the DOE.

Through an understanding of the way TOR kinase works, Zhai hopes to provide more details about metabolism.

Structure and function

Jen Sheen, Professor in the Department of Genetics at the Harvard Medical School, conducted pioneering work on how TOR kinase regulates cell growth in plants in 2013. Since then, TOR has attracted attention from an increasing number of biologists and has become “a hot and rapidly-developing research direction in plant biology,” Zhai explained.

He hopes to study the structure of TOR using BNL’s Laboratory of Biomolecular Structure at the National Synchrotron Lightsource II.

Zhai, who hopes to purify the plant version of TOR, plans to study how upstream signaling molecules interact with and modify the structure of the enzyme.

He will also use the cryo-electron microscope to get a structure. He is looking at molecular changes in TOR in the presence or absence of molecules or compounds that biochemically bind to it.

Through this funded research, Zhai hopes to explain how signals such as carbon supply, nutrients and sunlight regulates cell growth.

Once he’s conducted his studies on TOR, Zhai plans to make mutants of TOR and test them experimentally to see if a new version, which Zhai described as “TOR 2.0,” has the anticipated effects.

Zhai is building on his experience with another regulatory kinase, called SnRK1, which is involved in energy signaling.

“His expertise in defining SnRK1’s mechanism ideally positions him to perform this work,” Shanklin said.

At this point, Zhai is focused on basic science. Other researchers will apply what he learns to the development of plants for commercial use.

A seminal moment and a call home

Zhai described the award as “very significant” for him. He plans to continue with his passionate research to explore the unknown.

He will use the funds to hire new postdoctoral researchers to build up his research team. He also hopes this award gives him increased visibility and an opportunity to add collaborators at BNL and elsewhere.

The funding will support part of Zhai’s salary as well as that of his staff. He will also purchase some new lab instruments and tap into the award to attend conferences and publish papers.

When he learned he had won the award, Zhai called his mother Ruiming, who lives in his native China. “She is so proud of me and immediately spread the good news to my other relatives in China,” Zhai recalled.

When Shanklin spoke with Zhai after the two had learned of the award, he said he had “never seen Zhai look happier.”Shanklin suggested that this is a “seminal moment” in a career that he expects will have other such milestones in the future.

A resident of Mt. Sinai, Zhai lives with his wife Hui Liu, who is a Research Associate in Shanklin’s group specializing in plant transformation, fatty acids and lipidomics analysis.The couple has two sons, nine-year-old Terence and three-year-old Steven.

As for his work, Zhai hopes it has broader implications.

“The knowledge of TOR signaling will provide us [with] tools to achieve hyperaccumulation of lipids in plant vegetative tissues, which is a promising source for renewable energy,” he said.

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F. William Studier

By Daniel Dunaief

People around the world are lining up, and in some cases traveling great distances, to get vaccinations to COVID-19 that will provide them with immune protection from the virus.

An important step in the vaccinations from Pfizer-BioNTech and Moderna, the two messenger RNA vaccinations, originated with basic research at Brookhaven National Laboratory in the 1980’s, close to 40 years before the pandemic infected millions and killed close to three million people.

At the national laboratory, scientists including F. William Studier, Alan Rosenberg, and the late John Dunn, among others, worked on another virus, called the T7 bacteriophage, which infects bacteria. T7 effectively corrupts a bacteria’s genetic machinery, turning it into a machine that makes copies of itself.

From top graphic, the T7 virus uses RNA polymerase and a promoter to start a process inside a bacteria that makes copies of itself; researchers use copies of the promoter and the polymerase to insert genes that code for a specific protein; the mRNAs are injected into our arms where human ribosomes make COVID-19 spike proteins. Those spike proteins train the attack dog cells of our immune system to recognize the virus if it attempts to invade.

Back in the 1980’s, Studier and Dunn in BNL’s Biology Department were trying to do something no one else had accomplished: they wanted to clone the T7 RNA polymerase. The use of this genetic region, along with a promoter that starts the process of transcription, enabled scientists to mimic the effect of the virus, directing a cell to make copies of genetic sequences or proteins.

The BNL researchers perfected that process amid a time when numerous labs were trying to accomplish the same molecular biological feat.

“Although there were several labs that were trying to clone the T7 RNA polymerase, we understood what made its cloning difficult,” said Alan Rosenberg, who retired as a senior scientist at BNL in 1996. The patented technology “became the general tool that molecular biologists use to produce the RNA and proteins they want to study.”

The scientists who worked on the process, as well as researchers who currently work at BNL, are pleased that this type of effort, which involves a desire for general knowledge and understanding before policy makers and funders are aware of all the implications and benefits, led to such life-saving vaccinations.

“This is an excellent example of the value of basic science in that the practical applications were quite unanticipated,” John Shanklin, Chair of BNL’s Biology Department, wrote in an email. 

“The goal of the work Studier and his team did was to understand fundamental biological principles using a virus that infects bacteria. Once discovered, those principles led to a transformation of how biochemists and biomedical researchers around the world produce and analyze proteins in addition to providing a foundational technology that allowed the rapid development of mRNA vaccines,” he wrote.

Shanklin described Studier, who recruited him to join BNL 30 years ago from Michigan, as a mentor to numerous researchers, including himself. Shanklin credits Studier for helping him develop his career and is pleased that Studier is getting credit for this seminal work.

“I am tremendously proud that the basic research done in the Biology Department has been instrumental in accelerating the production of a vaccine with the potential to save millions of lives worldwide,” Shanklin wrote. “I couldn’t be happier for [Studier] and his team being recognized for their tremendous basic science efforts.”

Steve Binkley, Acting Director of the Department of Energy’s Office of Science, acknowledged the importance of the earlier work.

“The fact that scientific knowledge and tools developed decades ago are now being used to produce today’s lifesaving mRNA vaccines for COVID-19 is a great example of how the Department of Energy’s long-term investments in fundamental research at our National Laboratories can improve American lives today and into the future,” Binkley said in a statement.

Studier explained that his interests were more modest when he started studying this particular virus, which infects the bacteria E. coli.

“T7 was not a well-studied bacteriophage when I came to Brookhaven in 1964,” Studier, who is a senior biophysicist Emeritus, said in a statement. “I was using it to study properties of DNA and decided also to study its molecular genetics and physiology. My goal, of course, was to understand as much as possible about T7 and how it works.”

In an email, Studier said he did not realize the connection between his work and the vaccinations until Venki Ramakrishnan, a Nobel-Prize winning structural biologists from the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, told him.

“I am pleased that our work with T7 is relevant for fighting this world-wide pandemic,” Studier wrote. “History shows that some of the most useful discoveries come from basic research that could not have been predicted.”

While BNL is one of 17 Department of Energy facilities, it has conducted scientific research in numerous fields.

Several translational achievements originated at BNL, Shanklin wrote, including the thalium stress test for evaluating heart function, the development of Fluoro Deoxy Glucose for Positron Emission Tomography and the first chemical synthesis for human insulin, which allowed human insulin to replace animal insulin.

As for the effort that led to the T7 discoveries, Studier worked with Parichehre Davanloo, who was a postdoctoral fellow, Rosenberg, Dunn and Barbara Moffatt, who was a graduate student.

Rosenberg appreciated the multi-national background of the researchers who came together to conduct this research, as Moffatt is Canadian and Davanloo is Iranian.

Rosenberg added that while the group had “an inkling” of the potential usefulness of the processes they were perfecting, they couldn’t anticipate its value over the next 40 years and, in particular, its current contribution.

“Nobody really understood or thought just how widely spread its use would be,” Rosenberg said. “We certainly had no idea it would be an important element in the technology” that would lead to the Pfizer and Moderna vaccinations.

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