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Environment and sustainability

Researchers have finally gotten yeast to produce menthol

For many years, researchers could not get yeast to make enough of the small biological molecules industry uses as fragrances and biofuels. Now researchers have engineered synthetic enzymes into yeast, enabling it to produce the scent of menthol.

For many years, making enough monoterpenoids using microorganisms has been a holy grail for metabolic engineers.

The major classes of terpenoids have been produced using biotechnological methods for many years. However, microorganisms such as yeast could not produce the smaller-size monoterpenoids that are widely used as fragrances in the perfume industry and as flavours in the food industry.

However, this barrier has been removed now that researchers from the University of Copenhagen have successfully engineered a novel biosynthetic reaction chain into common baker’s yeast (Saccharomyces cerevisiae) so that it can produce many substances of enormous interest to industry.

“There are 70,000 terpenoids in nature, and we use them every day for all types of applications. We cannot use natural resources to satisfy our growing needs, so this breakthrough demonstrates how we can make the monoterpenoids biosynthetically,” explains Sotirios Kampranis, Associate Professor, Department of Plant and Environmental Services, University of Copenhagen.

The new study has been published in Nature Communications.

Terpenoids are a huge industry

Terpenoids are a large group of naturally occurring biological molecules. Plants and bacteria use them to communicate with each other and to protect themselves.

Many terpenoids are used as fragrances, such as mint or menthol, but they are also used as biofuels, as colours such as the red in tomatoes, as chemotherapeutic medicines such as paclitaxel and as antimalarial drugs such as artemisinin. Finally, many hormones, such as steroids, are also terpenoids.

Companies harvest the most commercially useful terpenoids from cultivated plants and trees or directly from nature, but neither source is sustainable in the long term. This explains the enormous interest in engineering yeast to make these valuable compounds in large fermentation tanks, avoiding the damage to natural resources.

“Monoterpenoids, like the other terpenoids, are part of a huge industry. Since we cannot use natural resources to supply our entire needs and cannot continue making them by using polluting catalysts and solvents, we need to figure out how we can produce them biosynthetically. We have not yet managed to achieve this, because yeast prefers to make the other terpenoids but not the monoterpenoids,” explains Sotirios Kampranis.

Industry battling yeast for the resources to make monoterpenoids

The problem with yeast is that the building blocks used to produce monoterpenoids are also used to produce sterols, which yeast cells use to build their cell walls.

Thus, although researchers insert the genes into yeast for synthesizing, for example, the scent of menthol, this synthesis competes with the yeast’s propensity to grow and divide.

This affects both the growth of the yeast and the production of the monoterpenoids.

“We therefore needed to discover a whole new way of making monoterpenoids by using yeast,” says Sotirios Kampranis.

A new biosynthetic reaction chain in yeast

The researchers have taken a new approach to solving this very specific problem.

They engineered the yeast so that it could produce an orthogonal substrate: a substrate it cannot use. Yeast produces it as a byproduct as it grows but then has no further use for it. They did this by introducing a new gene that comes from tomatoes into yeast.

Then, the researchers exploited the orthogonal substrate by developing several designer enzymes that could convert it into various monoterpenoids.

The researchers obtained the genes for the designer enzymes from lemon trees, fir trees and sage. However, since the enzymes did not naturally use the orthogonal substrate to make monoterpenoids, they had to be engineered to use this new substrate. The researchers did this by slightly adjusting the genes that make the enzymes so that, instead of their usual substrate, they could cut and paste into the orthogonal substrate.

“The smart thing about using an orthogonal substrate is that yeast has no use for this molecule, and therefore, synthesizing the molecules that produce the monoterpenoids does not compete with yeast growth. We have progressed from trying to create a production chain with something that the yeast needs to grow to creating a production chain with something that the yeast does not need,” says Sotirios Kampranis.

Industry can use the technology soon

The whole idea of inserting the reaction chain into yeast to make it produce both orthogonal substrate and to use it to produce monoterpenes with designer enzymes is quite innovative. However, Sotirios Kampranis is sure that the industry can soon adopt this process in their production.

Companies such as Chr. Hansen and Novo Nordisk can harness the technology to make food colouring and the components of medicine. The perfume industry can also easily implement the technology in developing the next Chanel No5.

“The gene for orthogonal substrate must be inserted as we described in our study, and then genes must be inserted to produce the monoterpenoids needed. This will probably happen within a few years,” says Sotirios Kampranis.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate” has been published in Nature Communications. In 2016, the Novo Nordisk Foundation awarded a grant to Sotirios Kampranis for the project Transforming Yeast Organelles into Micro-factories for the Compartmentalization of Complex Biosynthetic Pathways.

Sotirios Kampranis
Associate Professor
I work in the area of Synthetic Biology/Metabolic Engineering, aiming to devise methods to produce high-value natural products in engineered organisms. My research applies a multidisciplinary approach that begins with the identification of the biosynthetic pathways, continues with the engineering of the enzymes involved, and concludes with the production of the desirable compounds in engineered systems. In recent years, my group has excelled in the identification, characterization and engineering of biosynthetic activities related to plant natural product biosynthesis. We are currently focusing on reproducing the chemodiversity of terpenes found in nature and on synthesizing new natural products and rarely-isolated compounds.