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. CyDisCo (cytoplasmic disulfide bond formation in E. coli) is a method developed at the University of Oulu (UniOulu), 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.

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. He explained that 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, 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. It 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.

CyDisCoTM technology has been tested with about 300 different proteins. Some of the proteins are academically interesting and others are industrially relevant. Prof Ruddock mentioned that he had been looking for proteins that have not previously been commercially viable. He came across brazzein, and researchers started experimenting with its production in CyDisCoTM -modified bacterial cells.

Brazzein is a sweet tasting protein extracted from the fruit of the West African plant Pentadiplandra brazzeana. It is calorie-free and up to 2000 times sweeter than sugar, which immediately aroused great interest in the sweetener market. However, the producing brazzein by extracting it from the fruit is so expensive that it does not support the commercialisation of the sweetener. 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 1990s. 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. Prof Ruddock emphasized if brazzein can be commercialised it will certainly interest consumers.