To fulfill biofuels’ promise, scientists boost plant oils with gene editing

A researcher from the ROGUE project examines plant oil that can be converted into biodiesel and jet biofuel. Credit: Claire Benjamin/University of Illinois

If you’ve ever squeezed raw sunflower seeds or peanuts, you know if you press hard enough, you can collect oil from them. The same is true of seeds from the flowering plant Camelina sativa—although chances are, you’ve probably not squeezed these seeds firsthand.

Plant scientist Heike Sederoff of North Carolina State University, however, is very familiar with the oils that camelina offers. The highly concentrated fatty acids produced by the flaxlike plant are sought for their ability to be converted into biofuel, which Sederoff studies. Camelina seeds, in fact, have already been used to manufacture commercial jet biofuel.

Despite how environmentally friendly it sounds to use plants like camelina to make biofuels for transportation needs, how to do it in a cost-effective and sustainable way has been an issue.

Some studies suggest that the use of biofuels could reduce greenhouse gas emissions by 60 to 94% relative to fossil fuels (Agriculture 2017, DOI: 10.3390/agriculture7040032). But some scientists doubt the sustainability of biofuels, pointing to the land, water, and other resources required to produce them.

One thing everyone can agree on, however, is that innovation is needed for biofuels to fulfill their promise. Ideally, this innovation will be backed by industry rather than just enforced by government policy and incentives.

The idea is that by being produced and sold on the market, biofuel technologies could ride on a “virtuous cycle of innovation” and become more economically viable, says Gregory Graff, a professor of agricultural and resource economics at Colorado State University. Economic viability in turn would enable “future breakthroughs that could become game changers in terms of energy provision.”

One avenue of innovation being pursued by Sederoff and others is to genetically tinker with plants like camelina to either boost their oil production or reduce the chain length of the fatty acids they generate, diminishing the energy that refineries have to use to convert those compounds into usable fuels. These actions, the scientists hope, would make biofuels more economically viable.

OBSTACLES TO OVERCOME

Biofuels’ potential to lower greenhouse gas emissions and reduce countries’ dependence on foreign oil continues to drive government investment in research. Even with funding in hand, however, scientists who are genetically engineering crops to increase biofuels’ profitability could face a big roadblock. Skepticism over gene-edited crops has led to stringent regulations in the European Union: A July 25 ruling will make it difficult for scientists to conduct field trials in EU countries and to bring gene-edited products to the European market.

In the U.S., though, the Department of Agriculture says it is not going to regulate gene-edited crops that could otherwise be produced by conventional breeding methods, as long as the crop isn’t edited to include a plant-pest-derived DNA sequence. According to Neil Hoffman, a scientific adviser at USDA’s Animal & Plant Health Inspection Service (APHIS), USDA would be more likely to regulate a gene-edited crop if it were considered a plant pest or weed and would harm other plants if the gene-edited version spread into the environment.

For example, if someone engineered switchgrass, a weedy plant that can be converted to biofuel, to have improved drought tolerance, would a serious weed problem occur?

“We don’t believe that genetically engineered organisms need to be regulated” just because they are genetically engineered, Hoffman says. Researchers and companies planning to release a gene-edited crop into the environment can go through a process called “Am I Regulated?” to identify whether their products need to apply for a permit from USDA.

A recent gene-edited plant that received APHIS’s nonregulated status is a camelina plant from Yield10 Bioscience, a biotech company that hopes to improve plant oil production and oil quality.

Oliver Peoples, Yield10’s CEO, tells C&EN the company is planning on field trials for this camelina line in 2019. Having the plant identified as nonregulated by USDA definitely accelerated the process for the firm to pursue development in a cost-effective way, Peoples says.

To Peoples, the future looks promising. A 20% increase in the yield of conventional oil crops like canola and soybean could boost the economic value of these crops by $10 billion, he says. If the current techniques work well in camelina, the company will attempt similar gene edits in canola and soybeans.

Although Yield10’s primary goal is to sell its camelina oil for human health products and as an ingredient for agricultural animal feed rather than as a feedstock for biofuels, Peoples says the gene-editing discovery made by Yield10 could be beneficial for that type of application too.

Yield10 won’t disclose the exact edits its scientists made to improve camelina’s oil production, citing proprietary information. But presentation slides shared online on Nov. 4 for investors indicate that edits to genes that the firm calls C3008a, C3008b, and C3009 work to increase oil production by simultaneously increasing fatty acid biosynthesis and blocking the metabolism of the synthesized lipid.

At the same time, Yield10’s Canadian subsidiary, Metabolix Oilseeds, is working to edit a gene the company has called C3007 in camelina and canola. Gene C3007 encodes a protein that negatively regulates a key enzyme in fatty acid biosynthesis, acetyl-CoA carboxylase (Plant Cell 2016, DOI: 10.1105/tpc.16.00317). By decreasing the activity of this negative regulator, the firm aims to boost the enzyme’s activity and make more plant oil.

North Carolina State’s Sederoff is one of the scientists collaborating with Yield10. In a study from 2015, she and her colleagues successfully increased the seed yield from camelina by 57 to 73% (Biotechnol. Biofuels DOI: 10.1186/s13068-015-0357-1). The researchers accomplished this by engineering camelina to produce enzymes that reduce photorespiration, a process that diverts energy away from synthesizing carbon compounds.

Sederoff’s group is looking at other options to further improve seed yield. “If a farmer doesn’t get enough money out of it, he’s not going to grow it,” she says.

Recognizing the need to streamline processing steps and reduce cost in biofuel manufacturing, Sederoff is also working to engineer camelina to produce oil that requires less energy to convert it into biodiesel.

When plants take in carbon dioxide and then “fix” the molecule’s carbon during photosynthesis, the carbon can end up in a number of places. It might end up in sugar, cellulose, lignin, starch, proteins, or lipids. One of the main components that biofuel proponents care about is a group of lipid compounds called triacylglycerides (TAGs). One role for TAGs in plants is energy storage. Biofuel manufacturers can convert TAGs into biodiesel via transesterification or into jet biofuel via hydrotreatment.

The TAGs produced naturally by camelina plants are fatty acids that contain unsaturated, 16-to-18-carbon chains. Genetic engineering might not only reduce the length of those chains but also take them from unsaturated to saturated (PLOS One 2017, DOI: 10.1371/journal.pone.0172296). Such TAGs wouldn’t have to go through as much energy-intensive hydrogenation and cracking to convert them into usable fuel, Sederoff explains.

Credit: Agriculture 2017, DOI: 10.3390/agriculture7040032; Ind. Crops Prod. 2008, DOI: 10.1016/j.indcrop.2008.06.001; Stephen Long; USDA

BEYOND CAMELINA

Increasing oil production in multiple plants is essential to meet the world’s biofuel needs, says Nanyang Technological University plant biologist Wei Ma.

In 2012, researchers predicted that the global demand for plant oil would double by 2030 (J. Biol. Chem., DOI:10.1074/jbc.R111.290072). “The only way we can meet this huge demand is to increase the oil production. The question is how,” Ma says.

Ma’s research has its roots in the discovery, made in 2004, of a master gene regulator of plant oil production (Plant J. 2004, DOI: 10.1111/j.1365-313X.2004.02235.x). The transcription factor, aptly named WRINKLED1 because its mutation leads to wrinkled seeds that produce little oil, got the scientific world’s attention when Christoph Benning and Alex Cernac at Michigan State University discovered that overexpressing its gene could increase fatty acid biosynthesis in Arabidopsis, a model plant used in many labs.

Ma later did research in Benning’s lab at Michigan State and now continues to work on WRINKLED1 in his own lab at NTU.

Since that seminal study in 2004, scientists have pinpointed the equivalent version of Arabidopsis’s gene for WRINKLED1 in many other plant species, including avocado, oil palm, camelina, and maize plants (Plant Sci. 2018, DOI: 10.1016/j.plantsci.2018.04.013). The prevalence of the gene suggests that fundamental research on plants like Arabidopsis could lead to better oil yields in a broad range of crops.

Ma is searching for the biomolecules that interact with WRINKLED1. In collaboration with Benning, Ma found phosphopeptide-binding proteins called 14-3-3, which help stabilize WRINKLED1, presenting a new direction for boosting oil production in plants (Plant J. 2016, DOI: 10.1111/tpj.13244).

It’s not easy to elucidate all of WRINKLED1’s interactions. For example, the gene that encodes it is also expressed in plant roots and has been linked to maintaining homeostasis of a plant growth hormone called auxin (J. Exp. Bot. 2017, DOI: 10.1093/jxb/erx275). Overexpressing the gene without considering other factors could lead to alterations in multiple physiological processes that might affect the growth of some plant oil crops.

Ma says that’s why his lab is also collaborating with others to see if they can reengineer the gene for WRINKLED1 to increase its binding specificity for the right partners and reduce side effects.

Although Ma is focusing on fundamental lab research at the moment, he acknowledges the need to bring products to the market. “It’s definitely our responsibility to better use plant oil regulators to maximize efficiency,” he says.

A genetically engineered tobacco plant (right) accumulates more lipids (collected in droplets) in its leaves and has a higher oil content compared with a typical tobacco plant (left). Red is higher oil content, and purple is lower oil content. Credit: Metab. Eng. 2017, DOI: 10.1016/j.ymben.2016.12.007

USING MORE PARTS OF THE PLANT

TAGs are typically stored in the fruits, nuts, and seeds of plants and are present in only small amounts in vegetative tissues such as leaves and stems. To increase oil production, some scientists are looking to engineer plants to bulk up the fatty acids synthesized in their leafy, green bits.

One study performed on tobacco plants, a model organism typically used in plant biotechnology research, demonstrated that tobacco’s leaves could reach a level of TAG accumulation similar to what’s found in soybean seeds. The induced genetic changes—which boosted TAG production and accumulation—didn’t otherwise affect the crop’s development or seed viability (Plant Biotechnol. J. 2013, DOI: 10.1111/pbi.12131).

James Petrie of the Commonwealth Scientific & Industrial Research Organisation, the lead author on that study, has helped launch the company Folear on the basis of the study’s gene-editing findings. Folear’s primary purpose is to commercialize the technology, ultimately moving it into high-biomass plants and bringing down the cost of producing plant oil to less than $500 per metric ton, compared with soybean oil’s $800 per metric ton.

“We currently use about 180 million metric tons of plant oil each year. It is mostly used for food, and about 20 million is used as chemical feedstock and fuel,” Petrie says. Many of the potential applications, especially fuel, are limited by plant oil’s production cost and the need to use plants for food rather than fuel.

“By switching on plant oil production in the leaves and stems of plants, rather than the seed alone, we have the opportunity to produce massive amounts of oil per acre,” Petrie says. He is currently collaborating with a team at the University of Kentucky to run a field trial on a gene-edited tobacco variety to test its oil production ability. So far the crop is doing “really well,” he says.

Another plant that scientists recently engineered to accumulate oil in its leaves and stems is sugarcane. Sugarcane is attractive to biofuel proponents because of its high biomass. Typically, sugarcane would be harvested by biofuel manufacturers for its sugars, and they would convert the saccharides into ethanol.

But researchers have calculated that increasing TAGs to 5% of sugarcane’s stem weight could, because of the plant’s high biomass, produce about four times as much oil per hectare of land as soybeans, plus more than 10,000 L of ethanol (GCB Bioenergy 2017, DOI: 10.1111/gcbb.12478).

For this reason, researchers from the University of Illinois, Brookhaven National Laboratory, the University of Florida, and Mississippi State University have banded together to study gene editing in sugarcane. The project, dubbed ROGUE for Renewable Oil Generated with Ultra-productive Energycane, received a five-year, $10.6 million grant from the U.S. Department of Energy in February.

Previously, the team managed to raise TAG levels in the leaves of sugarcane to 8% by weight. Ultimately, the researchers aim to produce plants capable of accumulating 20% oil by weight in their leaves and stems, which could generate more than 42 bbl of oil per hectare. Soybeans produce less than 3 barrels per hectare, according to Illinois’s Stephen Long, lead researcher of the ROGUE project.

The intended market for this sugarcane variety, which the team calls energycane, would be jet biofuel. Long is confident that, although the project might take at least 20 years to scale up, fuel from energycane would become increasingly competitive against projected fossil-fuel prices.

Thomas Foust, a mechanical engineer at the U.S. National Renewable Energy Laboratory and Colorado State University, cautions that in the past, the predominant driver for developing biofuels was to head off an oil crisis. “That’s not the driver today,” he says, pointing to how fracking and horizontal drilling have enabled a much bigger world supply of fossil fuels.

Given the current situation, Foust says, biofuels and their blends with fossil-fuel counterparts really need to enable higher efficiency compared with petrochemicals while also contributing to other markets, such as biodegradable chemicals and plastics. Those are really the drivers for developing biofuels today, he says.