Small protein with great potential

Hard work in the laboratories of BRAIN AG and with academic partners: In order to find out how the exchange of a single amino acid affects the stability and sensory profile of the sweet-tasting protein brazzein, biologists and biochemists at BRAIN created a gene sequence library of around 2,000 brazzein variants and subsequently expressed this library in the yeast Pichia pastoris.

Why go to all this trouble? Brazzein has long been known as a natural sweetener and is found naturally in berries of the West African plant Pentadiplandra brazzeana. Since the taste researchers at BRAIN are researching alternative sweeteners and sweetness enhancers, one of their tasks is to investigate the molecular structures of sweet-tasting proteins.

Back to the gene sequence library: Scientists at BRAIN therefore introduced a gene sequence variant into each of the yeasts of the species Pichia pastoris mentioned above via a "plasmid taxi" and synthesized the corresponding protein variant in the fermenter. In this way they produced over 2,000 protein variants, and subsequently characterized them in cooperation with the French INRA (merged into INRAE in January 2020, France's new National Research Institute for Agriculture, Nutrition and the Environment).

From the 2,000 expressed variants, they selected the ones with the highest yield, and then produced them on a larger scale, purified and tested them for their thermal stability, sensory profile and sweetness intensity. Using a proprietary cell-based assay system, they also examined the protein variants for their ability to activate the human sweet taste receptor (TAS1R2/TAS1R3).

What remains of the hard work?

The results of these tests will enable BRAIN scientists to identify a wide range of new brazzein variants, those that are either sweeter, just as sweet or less sweet compared to wild-type brazzein. However, whether the sweetness sets in sooner or later, whether it disappears quickly or lingers longer – this evaluation of the temporal sensation and also off-tastes such as bitter or metallic is performed by a "human sensor": the human tongue of testers. In addition, the hard work has also led to the finding that the expression level in yeasts and the sensory profile of some brazzein variants can be much better in comparison to wild-type brazzein if only one single amino acid is exchanged.

The "Taste Team" at BRAIN recently presented its results at the (virtual) scientific conference ISOT 2020 (International Symposium on Olfaction and Taste).

Funding: The work described here is part of the Natural Life Excellence Network 2020 (NatLifE 2020) strategic alliance funded by the German Federal Ministry of Education and Research (BMBF, Funding reference numbers 031A206B / 031B0089A). Alliance partners have been working since 2013 to develop sustainable production processes and new assay systems for innovative bioactive ingredients, proteins and small molecules for the food and cosmetics industries.