Researchers have engineered yeast to make fruity, milky flavours in a one-step process. Making these lovely smelling flavours involves bat genes and can make production easier and cheaper.
When you think of fruity, milky or ’coconutty’ aromas, your mouth may begin to water. Now, researchers from The Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark have proven that these delicious food flavours can be made in an ‘oil-loving’ yeast cell factory in a one-step process. The research is published in Metabolic Engineering.
Many food flavours used for example in peachy soft drinks and strawberry fruit teas have traditionally been produced by chemical synthesis, which relies on fossil fuels.
Furthermore, in 2008, the EU changed its legislation, which means that chemically produced flavours must be labeled as chemicals. To a lot of consumers, chemicals and food don’t mix for obvious reasons. Therefore, flavor manufacturers are looking for natural, bio-based alternative production methods.
Natural extraction is not an option
In plants, lactones are only found in tiny amounts. Hence, extraction is not an option.
For these reasons, manufacturers are using microbes to do the job. For example, hydroxy fatty acid from castor oil can be converted by yeast to lactone. But this oil contains ricin – a toxin that is even more lethal than cyanide. Thus, the ricin must be removed very carefully before making food ingredients, adding to the price of these natural flavours, which belong to the lactone family of molecules.
A two-step process has been the only alternative for bio-production of lactones. The first step involved the hydroxylation of fatty acids found in, for instance, sweet potatoes. Afterward, the fats are converted to the wanted lactones by a yeast cell factory.
First of all, hydroxy fatty acids in sweet potato and other sources are not abundant, so a lot of energy needed to obtain it. Second, the yeast that converts the fat to lactones is not so efficient. So, a one-step process from more abundant oil-types with more efficient yeast would be desirable.
“We used oleic acid and linoleic acid we can get from used cooking oil and animal waste and optimized the yeast strain to use these oils – and it worked,” says Roy Marella, first author of this study and PhD student in the Yeast Metabolic Engineering group at The Novo Nordisk Foundation Center for Biosustainability.
The researchers started engineering the oleaginous yeast Yarrowia lipolytica. This yeast naturally take up and accumulate large amounts of fat, just like adipose tissue.
To make lactones, a microbe has to be engineered to carry out fatty acid hydroxylation and a controlled chain shortening.
Naturally, cells degrade hydroxylated acids to energy and biomass production, rendering the molecules useless for lactone formation. Therefore, the scientists had to delete several oxidase genes, meaning that the hydroxylated fatty acids did not get chopped to pieces. They also had to insert a hydroxylating enzyme to achieve hydroxylation, as well as long-chain oxidases to end up with fatty acid of 10-12 carbon molecules. Therefore, the team tried out many different combinations of enzymes from moth, pumpkin, bat, and yeast because the literature showed that they might have the right enzymatic activity.
It turned out that the combination of a bat and yeast gene worked the best to achieve 10-12 carbon lactones. After the fermentation, the oily layer traps the produced lactones, so no product-capture equipment is needed. Since lactones are volatile, trapping them in an oily layer is necessary, so they do not disappear up into the air.
Further optimisation needed
The main lactones produced by this optimised Y. lipolytica strain was gamma-dodecalactone (γ-dodecalactone), which smells like peach, as well as delta-decalactone (δ-decalactone), which has a rich coconutty, fruity scent and taste. In the best fed-batch fermentation, the scientists produced 282 mg/L. This yield is still too low, and Roy Marella expects that the yield should be around 100 times as high (approximately 20 g/L) before this strain becomes commercially interesting.
“This is quite a straightforward engineering, so I think it is possible, but we need to make more strain engineering, high throughput screening of enzymes and test whether it can be scaled up.”
Another advantage of using Y. lipolytica as a production host is that some of the strains have been classified as a GRAS or ‘Generally Recognized As Safe’. For example, GRAS status has been approved for Y. lipolytica strains that produce omega-3 fatty acids by DuPont and stevia sweetener by DSM.
Besides optimising the pathways for increased yield, the researchers also think that finding essential bottlenecks, select more efficient enzymes and fine-tuning expression could be a way to go about further optimisation.