The project intends to promote a more extensive use of natural carotenoids as safe food additives in Europe. Carotenoids, which have important beneficial effects on human health, are produced by chemical or biological means. The fungi Blakeslea and Xanthophyllomyces are used by European industry for the production of beta-carotene and astaxanthin. Biological production is favoured by current preferences for natural products, but to compete with chemical industries, fungal carotenoid yields must be improved. The major goal of this project is to increase the knowledge on (a) the genes and the enzymes of the carotenoid pathway; (b) the relationships between gene expression, enzyme levels, and carotenoid production; and (c) the mechanisms of accumulation or sequestering of carotenoids in the cell. This information will allow the scientific partners to get through genetic engineering fungal strains with improved carotenoid biosynthesis; to be used by industrial partners for the biotechnological production of carotenes and the development of new carotene-rich food products.
The objectives are grouped in five main topics:
- Genes and enzymes of the carotenoid pathway. Cloning of new crt and car genes in Xanthophyllomyces and Blakeslea. Expression of available and newly isolated genes in E. coli, characterisation of the enzymes, and production of specific antibodies.
- Cellular mechanisms for carotenoid storage in Xanthophyllomyces and Blakeslea. Relation between lipid biosynthesis and carotenoid accumulation. Subcellular localisation of carotenoid biosynthesis. Proteins and genes responsible for carotenoid storage in lipid globules.
- New improved strains of Xanthophyllomyces. Metabolic pathway engineering. Manipulation of regulatory signals and expression of engineered homologous and heterologous genes.
- New improved strains of Blakeslea. Development of a transformation method.
- Applied use of improved fungal strains. Improvement of fermentative conditions. Formulation of carotenoid products. Development of new carotene-rich food and beverages.
The set of available genes will be completed with new isolations, including the first carotene hydroxylase and ketolase genes from fungi. The genes will be expressed in E. coli and their protein products will be characterised and purified for the preparation of specific antibodies. A single biofunctional gene, responsible for phytoene svnthase and lycopene cyclase, will be investigated at the biochemical level and their respective domains will be separated in independent peptides. The relationship between carotenoid accumulation and lipid composition in Xanthophyllomyces and Blakeslea will be established. The compartments where carotenoids are accumulated will be identified and characterised. The identification of the proteins and lipids involved in carotenoid storage in Blakeslea will allow the cloning of the corresponding genes. Overexpression of these genes may remove the upper limits for the production of carotenoids. The regulatory mechanisms governing the expression of the carotenoid genes of Blakeslea and Xanthophyllomyces will be investigated. Cloned genes will allow the determination of transcription levels and antibodies that of enzyme levels. Checking the effect of targeted deletions on gene expression will identify key sequences for promoter activity.
Novel strains of Xanthophyllomyces with improved quantitative and qualitative production of carotenoids will be obtained. Structural genes will be overexpressed to overcome bottlenecks in the biosynthetic pathway. Gene shuffling in E. coli will improve the activity of heterologous gene products. New genes inducing or repressing carotenoid biosynthesis will be identified.
A transformation method will be developed for Blakeslea and novel strains with improved beta-carotene and lycopene production will be created. The hydroxylase and ketolase genes of Xanthophyllomyces will be expressed in Blakeslea to obtain new strains producing xanthophylls.
The industrial partners will verify the practical value of the new strains of Xanthophyllomyces and Blakeslea provided by the scientific partners. The fermentation conditions at pilot and industrial scale will be improved for these new strains. New raw materials will be tested to reduce fermentation costs. The new carotene fermentation products will be formulated to produce new carotene-rich food and beverages.