Bacteria have been engineered to produce a sweetener

Many substances beneficial to humans, such as insulin, have long been made with bacteria. However, the production of many types of proteins using bacteria has so far failed, as producing proteins that contain disulfide bond can be  problematic. CyDisCoTM (cytoplasmic disulfide bond formation in E. coli) is a method developed at the University of Oulu, which allows successful disulfide bond formation. The modified microbes are able to produce, for instance, the sweet tasting protein brazzein, which is being developed to become an alternative to existing low-calorie sweeteners.

Bacteria are great manufacturers, they are cheap and divide quickly. Bacteria are commonly used in the production of a wide variety of proteins including enzymes. The pharmaceutical industry is moving rapidly from small chemical drugs to the use of therapeutic biological products. The problem however is that bacteria are bad at producing complex disulfide bond containing proteins.

Researchers Lloyd Ruddock, Anna Gaciarz, Yuko Uchida and Mirva Saaranen have a reason to celebrate. On the right is Maarit Jokela, Innovation Manager from University of Oulu, who has helped the researchers with further development of the invention (Image: Juha Sarkkinen).

Disulfide bonds are chemical structures found in many proteins that are formed by the oxidation of cysteine amino acids. They are essential in order for the proteins to be functional.

Professor Lloyd Ruddock from the Faculty of Biochemistry and Molecular Medicine has for more than two decades studied how proteins fold inside the cell.

"Many proteins require disulfide bonds in order to fold. If the disulfide bonds are missing the protein does not fold correctly, but forms aggregates inside the cell.  The correct three-dimensional fold of a protein is essential for them to work in their assigned roles, for example, as enzymes or as hormones”, professor Ruddock says.

Professor Ruddock, together with his research group, has developed a method in which mammalian proteins are introduced into E. coli bacterial cells. The modified bacteria can produce functional disulfide bond containing proteins.

“The system has been named CyDisCoTM. The system has been patented and the method has been licensed to companies that use it to produce their own proteins, for example, pharmaceutical proteins and enzymes required in food and detergents.”

Developing a new, natural sweetener for the market

CyDisCoTM technology has been tested with about 300 different proteins. Some of the proteins are academically interesting and others are industrially relevant.

“I had been looking for proteins that have not previously been commercially viable. I came across brazzein, and we started experimenting with its production in CyDisCoTM -modified bacterial cells,” says Professor Ruddock.

Brazzein is a sweet tasting protein extracted from the fruit of the West African plant Pentadiplandra brazzeana. It was first isolated in 1994. Brazzein is calorie-free and up to 2000 times sweeter than sugar, which immediately aroused great interest in the sweetener market.

Producing brazzein by extracting it from the fruit is so expensive that it does not support the commercialisation of the sweetener," Ruddock explains.

Sufficient amounts of the sweet protein were however isolated for taste tests. The flavour profile of brazzein is very similar to sugar and it does not have a bitter aftertaste. Brazzein can also withstand heating very well, unlike some commonly used artificial sweeteners.

“The production of brazzein in bacteria and yeast was already experimented with in the 1990's. However, it was not economically viable because brazzein has cysteine amino acids, which form disulfide bonds when the protein folds inside the cell.”

Now brazzein production is being tested using the CyDisCoTM modified bacteria. Its potential for commercialisation is currently being studied in a TUTLI (Research for New Business) project funded by Business Finland. If the production is deemed commercially viable then the market for a new sweetener is open. Natural, tasty brazzein that is suitable for use in baking and hot drinks will certainly attract interest in the food industry, as well as with consumers.

Artificial sweeteners such as aspartame, saccharin and acesulfame K have been used for decades and they are easy and inexpensive to produce. However, their taste does not appeal to everyone, and there is constantly new information about the disadvantages of sweeteners, the latest being that some artificial sweeteners destroy beneficial intestinal bacteria.

A decade ago, Stevia was heralded as revolutionary is the sweetener market. Stevia is extracted from the stevia plant, so it is a natural sweetener. Its liquorice taste however isn’t for everyone.

“If brazzein can be commercialised it will certainly interest consumers," Lloyd Ruddock says.

"In the commercialisation project, we are investigating under what conditions brazzein should be produced and how to safely purify it from the bacteria's own proteins. All the stages are documented as required by the regulations so that the product is ready to be commercialised at the end of the project. We are negotiating with food companies and before the end of the project they will get brazzein for testing in their own products.”

Help is available for commercialising inventions

Innovation Manager Maarit Jokela from the Innovation Services at Oulu University assists natural and life sciences researchers with inventions and patents.

“I am here to listen as soon as an idea for an invention has formed”, Jokela says. “Together we can work out how the invention can be used and if it can be patented and commercialised. We also have to think about how further development and testing of the invention would be funded.”

The patenting process is often long, for example, patenting the CyDisCoTM invention was started in 2008 and the first patent was obtained in 2016. “This doesn’t however mean that the invention should be kept a secret all this time. Details of the invention can be published as soon as the patent application has been filed with the patent authority”, Jokela points out.

Patent applications have an 18-month delay before they are added to international patent databases. "After that the information in them is available to all and can be freely used. It may be possible to solve a problem described in a patent in a better way, which can lead to a new patentable invention.”

Since 2015, the University has awarded Proof of Concept (PoC) funding for the initial testing of inventions, which can be up to 20,000 euros.

"Professor Ruddock's research group was able to demonstrate with the PoC funding that the CyDisCoTM method produces brazzein. After that we applied for the TUTLI funding from Business Finland, which can be granted for 12 to 18 months for preparation for commercialisation,” Jokela explains.

"The TUTLI projects identify who the invention is for, map the needs and wishes of potential customers, and consider the business model. Commercialisation can take place either through a new start-up company or by licensing a method to existing companies.”

Innovation Manager Maarit Jokela assists researchers with questions relating to inventions and patents. Professor Lloyd Ruddock’s research team’s invention received a patent in 2016. Investigating the commercialisation of the method is in progress (Image: Juha Sarkkinen).

Since 2012, the University of Oulu has received TUTLI funding from Business Finland for 34 projects. They have generated ten start-up companies.

Text: Satu Räsänen

Last updated: 11.12.2018