Periodic Reporting for period 1 - NaturalCOLOURS (NatureCOLOURS: Natural colours delivered by yeast fermentation)
Periodo di rendicontazione: 2023-07-01 al 2024-12-31
Colour is an essential feature of food and beverages. Food colour affects consumer perception of flavour, freshness and product quality. Natural and synthetic food dyes are therefore frequently added to processed foods to enhance or correct food colour to meet consumer expectations. Concerns raised by consumers and food authorities alike about synthetic food additives have increased the demand for natural food colourants. The global market for natural food colours was estimated at US$1.5–1.75 billion in 2022 and is expected to grow. Most natural food colors are currently produced by extraction from plant sources, but the process is notoriously inefficient and resource-intensive, given the pigments' low natural concentration, impurities that get co-extracted, and the seasonality of plant harvesting.
A biotechnological process where colors can be produced by fermentation, similar to how enzymes and bioethanol are made, presents an attractive alternative. Natural colors can be made cheaper, with less environmental impact, and on demand.
In this project, we tackled the challenge of producing betalain-type natural food colours. Betalains are radiant water-soluble colours in the yellow-to-red spectrum. They have strong antioxidant properties and are health-beneficial. While over 75 different betalains have been found in nature, only one betalain pigment is extracted commercially - betanin from red beet, marketed in the EU as the food additive ‘beetroot red’ (E162). Beetroot contains only 0.2% of betanin on wet weight basis, hence, the production generates a lot of beetroot pulp as a side product. We have engineered oleaginous yeast Yarrowia lipolytica to produce several betalain-type pigments at high titers from glucose or glycerol as the sole carbon sources. Engineered Y. lipolytica strains are being used in industry for the production of omega-3-fatty acids (DuPont), stevia glucosides and carotenoids (DSM), lipids (Novogy), Lepidoptera pheromones (BioPhero), and other products. It is a yeast species that commonly occurs in food such as cheese or sausages. Our technology can deliver a pallet of betalain colours to the food industry and consumers, with variations of hue and stability properties, at lower cost and with less environmental footprint.
A biotechnological process where colors can be produced by fermentation, similar to how enzymes and bioethanol are made, presents an attractive alternative. Natural colors can be made cheaper, with less environmental impact, and on demand.
In this project, we tackled the challenge of producing betalain-type natural food colours. Betalains are radiant water-soluble colours in the yellow-to-red spectrum. They have strong antioxidant properties and are health-beneficial. While over 75 different betalains have been found in nature, only one betalain pigment is extracted commercially - betanin from red beet, marketed in the EU as the food additive ‘beetroot red’ (E162). Beetroot contains only 0.2% of betanin on wet weight basis, hence, the production generates a lot of beetroot pulp as a side product. We have engineered oleaginous yeast Yarrowia lipolytica to produce several betalain-type pigments at high titers from glucose or glycerol as the sole carbon sources. Engineered Y. lipolytica strains are being used in industry for the production of omega-3-fatty acids (DuPont), stevia glucosides and carotenoids (DSM), lipids (Novogy), Lepidoptera pheromones (BioPhero), and other products. It is a yeast species that commonly occurs in food such as cheese or sausages. Our technology can deliver a pallet of betalain colours to the food industry and consumers, with variations of hue and stability properties, at lower cost and with less environmental footprint.
We have successfully achieved the project objectives. We have developed engineered strains, fermentation, and downstream processes for the production of four betalain-type food colors.
The results on the production of betanin have been published in Thomsen et al (2023) “Beet red food colorant can be more sustainably produced with engineered Yarrowia lipolytica”. Nature Microbiology, 8: 2290–2303 [https://www.nature.com/articles/s41564-023-01517-5] and in Babaei et al (2023) “Combinatorial engineering of betalain biosynthesis pathway in yeast Saccharomyces cerevisiae”. Biotechnol Biofuels Bioprod, 16:128. [https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02374-4].
We also published a review on the topic of natural food colors: Thomsen PT, Nielsen SR, Borodina I (2024). “Recent advances in engineering microorganisms for the production of natural food colorants”. Current Opinion in Chemical Biology, 81:102477 [https://www.sciencedirect.com/science/article/pii/S136759312400053X]
We discovered biosynthesis pathways towards two additional red betalains - amaranthin and phyllocactin. We reported the results on betalain biosynthetic enzymes in Glitz, C., Dyekjær, J.D. Vaitkus, D. et al. "Screening of Plant UDP-Glycosyltransferases for Betanin Production in Yeast." Appl Biochem Biotechnol (2025), [https://doi.org/10.1007/s12010-024-05100-4] and in Glitz, Christiane; Dyekjær, Jane; Mattitsch, Sophia; Babaei, Mahsa; Borodina, Irina “BAHD acyltransferase from dragon fruit enables production of phyllocactin in engineered yeast”. FEMS Yeast Research, in press.
Patents have been filed to protect the innovation. [published patent: WO2022253815A1 "Methods for producing betalains in yeast."]
Several lay audience articles describing the work can be found here:
o DTU Biosustain - Nature's Palette Reinvented: New fermentation breakthrough in sustainable food colouring. https://www.biosustain.dtu.dk/about/stories/nyhed?id=75ee0e99-b24f-4b36-8a75-c0337d667379(si apre in una nuova finestra)
o Food color overhaul: Scientists unlock innovative fermentation process to formulate betanin from yeast. https://www.foodingredientsfirst.com/news/food-color-overhaul-scientists-unlock-innovative-fermentation-process-to-formulate-betanin-from-yeast.html(si apre in una nuova finestra)
o Beet red reimagined: how engineered yeast is revolutionising natural colourings. https://sciencenews.dk/en/beet-red-reimagined-how-engineered-yeast-is-revolutionising-natural-colourings(si apre in una nuova finestra)
o Eurekalert - Nature's Palette Reinvented: New fermentation breakthrough in sustainable food coloring. https://www.eurekalert.org/news-releases/1009664(si apre in una nuova finestra)
The results on the production of betanin have been published in Thomsen et al (2023) “Beet red food colorant can be more sustainably produced with engineered Yarrowia lipolytica”. Nature Microbiology, 8: 2290–2303 [https://www.nature.com/articles/s41564-023-01517-5] and in Babaei et al (2023) “Combinatorial engineering of betalain biosynthesis pathway in yeast Saccharomyces cerevisiae”. Biotechnol Biofuels Bioprod, 16:128. [https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02374-4].
We also published a review on the topic of natural food colors: Thomsen PT, Nielsen SR, Borodina I (2024). “Recent advances in engineering microorganisms for the production of natural food colorants”. Current Opinion in Chemical Biology, 81:102477 [https://www.sciencedirect.com/science/article/pii/S136759312400053X]
We discovered biosynthesis pathways towards two additional red betalains - amaranthin and phyllocactin. We reported the results on betalain biosynthetic enzymes in Glitz, C., Dyekjær, J.D. Vaitkus, D. et al. "Screening of Plant UDP-Glycosyltransferases for Betanin Production in Yeast." Appl Biochem Biotechnol (2025), [https://doi.org/10.1007/s12010-024-05100-4] and in Glitz, Christiane; Dyekjær, Jane; Mattitsch, Sophia; Babaei, Mahsa; Borodina, Irina “BAHD acyltransferase from dragon fruit enables production of phyllocactin in engineered yeast”. FEMS Yeast Research, in press.
Patents have been filed to protect the innovation. [published patent: WO2022253815A1 "Methods for producing betalains in yeast."]
Several lay audience articles describing the work can be found here:
o DTU Biosustain - Nature's Palette Reinvented: New fermentation breakthrough in sustainable food colouring. https://www.biosustain.dtu.dk/about/stories/nyhed?id=75ee0e99-b24f-4b36-8a75-c0337d667379(si apre in una nuova finestra)
o Food color overhaul: Scientists unlock innovative fermentation process to formulate betanin from yeast. https://www.foodingredientsfirst.com/news/food-color-overhaul-scientists-unlock-innovative-fermentation-process-to-formulate-betanin-from-yeast.html(si apre in una nuova finestra)
o Beet red reimagined: how engineered yeast is revolutionising natural colourings. https://sciencenews.dk/en/beet-red-reimagined-how-engineered-yeast-is-revolutionising-natural-colourings(si apre in una nuova finestra)
o Eurekalert - Nature's Palette Reinvented: New fermentation breakthrough in sustainable food coloring. https://www.eurekalert.org/news-releases/1009664(si apre in una nuova finestra)
The project aimed to enable sustainable and low-cost production of multiple natural betalain colours for food, cosmetics, and other applications. In contrast to synthetic colours, betalains do not have any adverse health effects and are even health-promoting due to their antioxidant nature.
If the technology is commercialized and betalain colours make it to the market, it may have the following key impacts for various stakeholders:
Improved Health and Food Quality for Consumers. Replacement of synthetic colours with antioxidant betalains will have multiple health benefits. Furthermore, new betalain variants with different hues and improved heat or pH stability will expand the potential applications of betalains.
New Industry & Job Creation. The technology could be commercialized by creating a new industrial biotech company or by licensing to an existing company. This would create high-tech workplaces in Europe for manufacturing, sales, and further R&D. The company would contribute to creating a strong European biotechnology industry cluster.
More Sustainable Production. Our sustainability assessment shows that producing betanin by fermentation has 3-5 less environmental impact than extraction from red beets. Betalain colours could be produced with better purity and less footprint.
If the technology is commercialized and betalain colours make it to the market, it may have the following key impacts for various stakeholders:
Improved Health and Food Quality for Consumers. Replacement of synthetic colours with antioxidant betalains will have multiple health benefits. Furthermore, new betalain variants with different hues and improved heat or pH stability will expand the potential applications of betalains.
New Industry & Job Creation. The technology could be commercialized by creating a new industrial biotech company or by licensing to an existing company. This would create high-tech workplaces in Europe for manufacturing, sales, and further R&D. The company would contribute to creating a strong European biotechnology industry cluster.
More Sustainable Production. Our sustainability assessment shows that producing betanin by fermentation has 3-5 less environmental impact than extraction from red beets. Betalain colours could be produced with better purity and less footprint.