Using fermentation, University of Alberta researchers have come up with a way to produce higher amounts of a healthy fatty acid found mainly in pomegranates.
Working with baker's yeast that initially contained none, they developed a new strain containing high levels of punicic acid, offering a sustainable way to produce both the value-added fatty acid and yeast biomass — a supplemental protein source used in food and animal feed industries — with additional health benefits.
"This means we could produce this high-value lipid much more quickly and economically in the future, without needing to use arable land, and it also shows how we can develop and nutritionally enhance sustainable sources of specialty oil," says study co-author Guanqun (Gavin) Chen, associate professor in the Faculty of Agricultural, Life & Environmental Sciences and Canada Research Chair in Plant Lipid Biotechnology.
Derived from the seed oil of the exotic fruit, punicic acid offers several health benefits, with cholesterol-lowering, anti-inflammatory and anti-carcinogenic properties.
But compared with other oilseed crops such as canola, the pomegranate has very low seed-to-fruit ratio and oil yield, which limits supply and makes it a more costly commodity, Chen notes.
Using a gene-editing approach called CRISPR-based gene shuffling, Chen and study co-author Juli Wang randomly integrated certain genes potentially involved in punicic acid synthesis and accumulation directly into the genome — the complete set of DNA — of baker's yeast.
Their experiments mark the first time CRISPR-based gene shuffling has been used in engineering yeast to produce plant-derived, unusual fatty acids, and the work has resulted in a faster, more efficient way to find out which of the genes work well together.
Instead of the more standard and labour-intensive practice of having to test gene combinations one by one for their influence, "the gene shuffling process allowed us to randomly add genes to the yeast strains to make a library, then screen that library to identify the best ones," says Wang, who conducted the experiments to earn a PhD in plant science.
"We get the screening out of the best strain first and then figure out what genes are transformed," he adds. "This guarantees better performance in our results, because it tells us which genes work better with one another."
The experiments boosted the punicic acid content by 80-fold, to 26.7 per cent, the highest level achieved and reported by scientists in engineered micro-organisms or plants so far — and a number "that is high enough to show great potential for commercial-scale production," Chen notes.
The yeast strain they produced also has stable punicic acid content, which is another promising development for eventual large-scale use, Wang adds.
"For bioindustrial production, it means that the genes that get added into the yeast don't get lost from one batch of fermentation to the next."
The discoveries, which have resulted in a provisional patent application, build on an earlier study by the researchers, initially identifying the dynamics of increasing punicic acid content in yeast via gene-stacking. They now plan to grow their high-yield strain in lab-scale fermenters, a step toward scaling up for potential commercial production.
In addition, their CRISPR-based gene shuffling approach is versatile enough that it could be used in engineering baker's yeast to produce other valuable unusual fatty acids, such as from castor oil, and along with that, "exciting potential for developing other bioproducts," Chen notes.
The study was funded by Natural Sciences and Engineering Research Council of Canada Discovery and Alliance grants, the Canada Research Chairs Program, Alberta Innovates, Canadian Poultry Research Council, Cargill/Diamond V, Results Driven Agriculture Research, Canada Foundation for Innovation-John R. Evans Leaders Fund and Research Capacity Program of Alberta. Wang was supported in his work by an Alberta Innovates Graduate Student Scholarship.
