Gene editing strikes oil: High-efficiency Camelina revolutionizes biofuels

Scientists have increased oil production in Camelina sativa by 21.4% by editing the TT8 gene, paving the way for more efficient biofuel crops.

As initiatives to achieve net-zero carbon emissions from transportation fuels gain momentum, the need for oil from non-food crops is increasing. These crops use sunlight to convert carbon dioxide from the atmosphere into oil, which is stored in their seeds. Crop breeders looking to maximize oil production often prefer yellow-seeded plants because they typically yield more oil than brown-seeded varieties in oilseed crops such as canola. This is due to a protein that turns the seeds brown, which is absent in plants with yellow seeds, and which also plays a key role in oil production.

Breakthrough in the development of biofuel crops

Now, plant biochemists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory – who are interested in increasing the synthesis of vegetable oil for the sustainable production of biofuels and other bioproducts – have leveraged this knowledge to create a new, high-yield variety of oilseed crops. In an article just published in The Plant Biotechnology Journal,< they describe how they used modern genetics tools to produce a yellow seed variety Camelina sativaa close relative of canola, which collects 21.4% more oil than regular camelina.

“If breeders can achieve a few percent increase in oil production, they consider that significant, because even a small increase in yield can lead to a large increase in oil production when you plant millions of hectares,” says biochemist John Shanklin of Brookhaven Lab. chair of the Lab’s Biology Department and leader of the vegetable oil research program. “Our increase of almost 22% was unexpected and could potentially result in a dramatic increase in production,” he said.

The Brookhaven Lab research team (from left to right): Jin Chai, Jodie Cui, Shreyas Prakash, Xiao-Hong Yu, John Shanklin, Jorg Schwender, Hai Shi and Sanket Anaokar. They are all members of Brookhaven Lab’s Department of Biology; Prakash and Cui are students at Cornell University and Stony Brook University, respectively, participating in the U.S. Department of Energy-sponsored Science Undergraduate Laboratory Internship program. Credit: Jessica Rotkiewicz/Brookhaven National Laboratory

Simple idea, unusual plant

The idea behind developing this high-yielding camelina variety was simple: mimic what happens in the naturally occurring, high-yielding yellow-seeded canola varieties.

“Breeders had identified plants with more oil, some of which happened to have yellow seeds, and they weren’t really concerned about the mechanism,” Shanklin said. But once scientists discovered the gene responsible for both yellow seed color and increased oil content, they had a way to potentially increase oil yields in other countries. kind.

Gene editing for improved oil production

The gene has the instructions for making a protein known as Transparent Testa8 (TT8), which regulates the production of compounds that, among other things, give seeds their brown color. Importantly, TT8 also inhibits some of the genes involved in oil synthesis.

Xiao-Hong Yu, who led this project, hypothesized that getting rid of TT8 in Camelina should remove the inhibition of oil synthesis – and free up some carbon that can be channeled into oil production.

Getting rid of a single gene in camelina is quite a challenge, because this plant is unusual among living things. Instead of two sets of chromosomes – that is, two copies of each gene – it has six sets.

“This ‘hexaploid’ genome explains why there are no yellow-seed varieties of camelina in nature,” Yu explains. “It would be very unlikely that mutations would occur simultaneously in all six copies of TT8 and completely disrupt its function.”

Gene Editing Strikes Oil

Thanks to the tools of modern genetics, the Brookhaven team was able to eliminate all six examples of the TT8. They used gene editing technology known as CRISPR/Cas9 to target the specific sequences of DNA within the TT8 genes. They used the technology to splice the DNA at these locations and then create mutations that deactivated the genes. Yu and the team then performed a series of biochemical and genetic analyzes to monitor the effects of their targeted gene editing.

“The yellow seed phenotype we were looking for was a great visual guide to our search,” said Yu. “This helped us find the seeds we were looking for by screening fewer than 100 plants – among which we identified three independently occurring lineages in which all six genes were disrupted.”

The results: The color of the seed coat changed from brown to yellow only in plants in which all six copies of the TT8 gene were disrupted. The yellow seeds had lower levels of “flavonoid” compounds and “mucilage” – both normally produced by biochemical pathways controlled by TT8 – than brown seeds from camelina strains with unedited genomes.

In addition, many genes involved in oil synthesis and the production of fatty acids, the building blocks of oil, were highly expressed in seeds of the CRISPR/Cas9-edited plants. This resulted in the dramatic increase in oil accumulation. The modified seeds contained another positive surprise, namely that the levels of protein and starch remained unchanged.

The targeted mutations in TT8 were carried over into subsequent generations of camelina plants, indicating that the improvements would be stable and long-lasting.

“Our results demonstrate the potential for creating new camelina lines through gene editing, in this case by manipulating TT8 to improve oil biosynthesis. Understanding further details of how TT8 and other factors control biochemical pathways may provide additional gene targets for increasing oil yields,” Shanklin said.

Reference: “Creating Yellow Seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa8” by Yuanheng Cai, Yuanxue Liang, Hai Shi, Jodie Cui, Shreyas Prakash, Jianhui Zhang, Sanket Anaokar, Jin Chai, Jorg Schwender, Chaofu Lu, Xiao-Hong Yu and John Shanklin, June 10, 2024, Journal of Plant Biotechnology.
DOI: 10.1111/pbi.14403

This research was funded by the DOE Office of Science – in part through a project known as “Enhancing Camelina Oilseed Production with Minimal Nitrogen Fertilization in Sustainable Cropping Systems” led by Montana State University; the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a DOE-funded bioenergy research center led by the University of Illinois Urbana-Champaign; and Brookhaven Lab’s Physical Biosciences program. Students supported by the Office of Science also contributed to this research. In addition, the scientists used a confocal microscope at the Center for Functional Nanomaterials (CFN), which functions as a DOE Office of Science user facility at Brookhaven Lab.