Polyoleosins: close sesame

Trans fatty acids are heartbreakers. Decades of epidemiological and laboratory studies have shown that an excess of saturated animal fats in meat and dairy products can significantly increase a person's risk of cardiovascular disease, including heart attack and stroke.

For three decades, researchers have been trying to find a commercially viable way of preventing plant-derived cis polyunsaturated fatty acids - lipids - being transformed into harmful, saturated trans fats in the rumen, the capacious fore-stomach in which ruminant livestock ferment their diet of herbage and grain.

Dr Richard Scott and Dr Nick Roberts, of AgResearch's Grasslands Research Centre in Palmerston North, New Zealand, have conducted a literature search that led them to an ingenious, potentially inexpensive biological solution to the biohydrogenation problem - one 'invented' by plants themselves.

Plants prevent fatty acids in their seeds and pollen coalescing into a single blob by encapsulating them in microspheres, or micelles, made of specialised proteins called polyoleosins.

Forage crops, such as grasses and legumes, could be engineered with polyoleosin transgenes from sesame, causing them to encapsulate triglycerols in self-assembling polyoleosin micelles that seal them off from bacterial activity during transit though the rumen.

The result would be meat and dairy products containing fewer trans fats, and increased levels of health-promoting cis polyunsaturated acids.

Scott says the technology could also have applications in drug delivery, as emulsifiers for the food industry, and as controlled-release systems for the cosmetics industry.

"We looked through the research literature, and found that it was possible to link a whole lot of polyoleosin molecules together to form micelles around lipids," he says.

The proteins initially occur as individual lipophilic molecules, that stabilise lipid monolayers, but as the plant matures, the monomers cross-link to form lipid-filled polyoleosin micelles.

"We can take the plant oil body and mimic this process by cross-linking the oleosin molecules with glutaraldehyde," he says. "But the FDA is not happy about feeding cross-linked proteins to animals."

The AgResearch team, which is collaborating with Professor German Spangenberg's research group at La Trobe University in Melbourne, asked German biotechnology company GeneArt to optimise a selected peptide sequence from a sesame seed oleosin to form a repeating polymer - a pentameric (5x) polyoleosin.

The AgResearch-La Trobe team had spliced a codon into the original gene to facilitate expression in Arabidopsis.

To make it work in the standard plant-gene vector Agrobacterium tumefaciens - a prokaryote - GeneArt geneticists had to remove the intron, eliminate all cryptic splice sites and termination signals, and substitute GC codons for degenerate codons in the repeat sequence.

The highly engineered clone yielded a very stable polyoleosin when expressed in plant cells.

"They achieved in only four weeks what would have taken us two years," Scott says.

Hydrogenation

The plan is to express the polyoleosin transgene in canola and maize, to produce long-chain polyunsaturated fatty acids that will resist rumen hydrogenation.

"We're also interested in co-expressing the polyoleosin transgene in annual ryegrass with the existing diacylglycer acetyle transferase gene, which codes for the last step in the process that produces triglyceride - you can't get polyoleosin accumulating in plants without triglyceride," Scott says.

"We're a forage research group, which is why we're interested in doing it in grass, which has very high levels of the long-chain fatty acids, linoleic and linolenic acid, which you need to protect to get through the rumen."

Why do rumen bacteria hydrogenate long-chain fatty acids to produce trans fats? Because the microbes are susceptible to the highly acidic hydrogen ions in the rumen, and de-fuse them by attaching them to unsaturated sites on the long-chain fatty acids.

The perfume industry could use polyoleosin micelles could be used to release fragrances over several hours. Increasing the number of repeat sequences increases the heat stability of the polyoleosin micelles, allowing polyoleosin repeat numbers, which alter chain lengths have different temperature stabilities, so products could be tailored for timed release, according to whether the individual leads a sedentary or active life.

"The beauty of the system is that polyoleosins can be tailored to requirement - you can also change the size of the micelles, which increases the range of things that can be emulsified, to created a variety of textures and tastes," he says.

AgResearch is commercialising its polyoleosin patents, which it filed 14 months ago, though its own subsidiary, PhytaGro, in partnership with Finisterre Partners in San Diego, which is interested in using it for cosmetics emulsions.

AgResearch is also working with a publicly listed Canadian company, Symbiosis, which already uses oleosin monomers to purify vegetable oils - both the monomers and polymers are lipophilic molecules.

Symbiosis is exploring the possibility of encapsulating insulin in polyoleosin micelles - to do its work of regulating blood sugar levels in diabetics, insulin must be folded correctly.

"Our design also allows use to insert biologicalyl active peptides between the linked repeats," Scott says.

Long-chain fatty acids are energy-rich. Scott says New Zealand's typical ryegrass and white clover pastures could be readily replaced with polyoleosin transgenic varieties, providing high-energy forage for livestock, and a cheap and simple way of producing meat, milk and dairy products enriched in healthful polyunsaturated fats.