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FAIR-CT96-1633
Genetic Engineering of Carotenoid Metabolism: a Novel Route to Vitamins, Colours and Aromas for the European Market |
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Proposal No: | FAIR-CT96-1633 |
| Date Prepared: | July 2001, September 1999 | |
| Source: |
Final Report Progress Report November 1998 |
Source: Final report June 2000
Consortium: The project was co-ordinated by ENEA-CR, Roma (Italy), Royal Holloway college, University of London (UK), CNRS, Strasbourg (France), Institut Biologie II, University Freiburg (Germany), Department of genetics University of Sevilla (Spain), Advanta, Rilland (The Netherlands), Department of Biological Chemistryand Nutrition, University of Hohenheim, Stuttgart (Germany) and the Institute of Plant Science, ETH-Zurich (Switzerland).
Introduction
Carotenoids are synthesised de novo from geranylgeranyl diphosphate by all photosynthetic organisms, where they participate in light harvesting and photoprotection from excess light energy. Many non-photosynthetic bacteria (Erwinia herbicola, Deinococcus radiodurans, Thermus aquaticus) and fungi (Neurospora crassa, Phycomyces blakesleeanus), also synthesise carotenoids. In plants, carotenoids are accumulated, together with chlorophyll, in leaf chloroplasts, and, alone, in the chromoplasts of many fruits, seeds and flowers. While carotenoid composition of chloroplasts is relatively invariant, a wide range of different carotenoids can be found in chromoplasts: lycopene in tomato fruits, beta-carotene (pro-vitamin A) in carrot roots, lutein and zeaxanthin in maize endosperm. The molecular biology of the plant pathway is well elucidated.
Vertebrates do not synthesise carotenoids. They nevertheless depend on dietary carotenoids for making their retinoids, such as retinal (the main visual pigment) retinol (vitamin A), and retinoic acid (a substance controlling morphogenesis). The main precursor of retinoids is beta-carotene, a carotenoid containing two unsubstituted beta-ionone rings at the two ends of the molecule. Beta- carotene is cleaved into retinal by a dioxygenase. A similar enzyme also catalyses the first step in plant ABA biosynthesis.
Avitaminosis A is widespread in the third world, but is also found in poor urban populations of developed countries, among the elderly, heavy drinkers, or smokers. The Recommended Dietary Allowance (RDA) for Vitamin A is 1000 retinol equivalents, equal to 6 mg beta-carotene, per day. Supplementation of the diet with these doses has highly beneficial effects on populations suffering from avitaminosis A.
In a thorough study conducted in Nepal, these doses reduced by 50% the pregnancy-related mortality in women. The World Health Organisation estimates that improving vitamin A nutrition could prevent over 2 million deaths annually, primarily in pre-school children.
Objectives
The objectives were as follows:
Activities
It was proposed to clone plant genes for zeaxanthin biosynthesis and oxidation from Capsicum or Crocus expression libraries. The identity of the enzymes will be confirmed by expression in microbial systems over-producing appropriate carotenoid precursors.
The metabolic re-routing of lycopene, present in tomato fruits, towards industrially valuable xanthophylls will be achieved as follows. First beta-carotene overproducing fruits will be obtained through the use of natural mutants or transformation with appropriate genes (lycopene cyclase, capsanthin-capsorubin synthase). Then, plant and/or bacterial genes for xanthophyll biosynthesis (beta- carotene hydroxylase, ketolase) will be introduced under the control of fruit-specific promoters to produce zeaxanthin and astaxanthin. The same strategy will. be applied to Phycomyces, a beta-carotene accumulating fungus.
To convert the carotenoid precursor GGPP into beta-carotene (pro-vitamin A) in rice endosperm or potato tubers, the following sequential metabolic steps need to be introduced:
GGPP --> phytoene --> lycopene --> beta-carotene.
Genes encoding these metabolic steps will be put under the control of endosperm- or tuber- specific promoters and introduced into transgenic plants. Transgene expression and carotenoid content will be analysed. Plants containing multiple transgene combinations will be produced through multiple transformation of genetic crossing.
After a preliminary molecular and biochemical characterisation of the transgenic lines, high expressing offspring will be used for glasshouse and field trials of the progeny. Nutritional tests will be also performed with selected transgenic crops and fungi with significantly improved beta-carotene content, using a vitamin A- deficient animal model system.
Achievements
Task 1: Isolation of plant genes for xanthophyll biosynthesis and oxidation. The pepper beta- carotene hydroxylase has been cloned. The complete pathway for zeaxanthin oxidation has been cloned from Crocus. Zeaxanthin oxidase, crocetin dialdehyde oxidoreductase, and glucosyl- transferases 1 and 2 have been cloned and antibodies raised against the corresponding enzymes.
The last missing step in plant xanthophyll biosynthesis, neoxanthin synthase, has been cloned from tomato and potato and shown to be a lycopene cyclase paralog. This achievement opens up the possibility for manipulating the levels of ABA in plant tissues.
Task 2: Engineering tomato and Phycomyces into factories for speciality compounds.
Tomato has been transformed using the 35S/tp/crtl transgene. Not only do these tomatoes not accumulate more lycopene, but they also accumulate less total carotenoids. However, up to 50% of these carotenoids (up to 5 mg/100 gm fresh weight) is now beta-carotene, making these tomatoes orange. The character is dominant, and is stable when transmitted to the progeny. These tomatoes are able to deliver the vitamin A RDA with just 120 gms, a dose fully compatible with a normal diet.
Beta-carotene accumulation to similar levels has been also obtained in tomato through the over-expression of the Arabidopsis lycopene beta-cyclase, driven by the fruit-specific Pds promoter. Leaf carotenoids are completely unaltered in these transformants, and the total fruit carotenoids are, if anything, slightly increased.
Transformation of the tomato high-beta genotype, a natural accumulator of beta-carotene, with beta-carotene hydroxylases/ketolases has instead resulted in no change in carotenoids, suggesting that the beta-carotene pool in this mutant is metabolically inactive. A double cyclase/hydroxylase transformant of tomato has instead resulted in significant accumulation of the xanthophylls zeaxanthin and beta-cryptoxanthin in the fruit. This approach (the introduction of multiple metabolic steps) is therefore a viable one for engineering the tomato fruit into a cell factory for speciality carotenoids. The same approach in Phycomyces has failed, mainly due to difficulties in obtaining stable transformants.
Using an antisense approach, high lycopene levels have been engineered in tomato fruits, while the overproduction of astaxanthin in tobacco leaves and nectaries has also been achieved.
Task 3: Engineering potato and rice into dietary beta-carotene sources A major breakthrough has been the engineering of high beta-carotene levels in the endosperm of "golden rice". Rice endosperm accumulates geranylgeranyl diphosphate. Therefore, four novel enzymes are required for beta- carotene synthesis in this tissue: phytoene synthase, phytoene desaturase, beta-carotene desaturase and lycopene beta-cyclase. These enzymes can be reduced to three if one uses crtI, a desaturase from Erwinia able to substitute for the two plant desaturases. In the "golden rice" experiment, a daffodil phytoene synthase and lycopene beta-cyclase, under the control of the endosperm- specific glutelin promoter were used. This was in addition to crtI, fused to a plastidic transit peptide under the control of the CAMV 35S promoter (35S/tp/crtf). It was found that phytoene synthase and crtl alone were sufficient not only to lead to the synthesis of lycopene but also the production of beta-carotene and zeaxanthin.
"Golden rice" contains up to 200g beta-carotene/100 gms, a dose able to deliver 1/10 of the RDA with a daily intake of 300 gms of rice. Although this is far from the RDA, it will have a high dietary impact on populations of Southeast Asia, where avitaminosis A is widespread and rice consumption high. Potato tubers show some low degree of carotenoid formation.
Other experiments were designed to enhance the content by sense transformations. Several cDNAs coding for carotenoid biosynthetic enzymes under patatin promoter control were used for transformation using constructs carrying sequences coding for:
So far none of the transformants obtained displayed a pronounced increase in carotenoid content; maximally a twofold increase was obtained (GGPP-Synthase,. Lycopene Cyclase). However interesting novel carotenoid patterns were observed (capsanthin capsorubin synthase, lycopene cyclase, fibrillin) in some cases accompanied by interference with the tuber's dormancy which are worth being further investigated. Due to greenhouse limitations growth and analysis of transformants represent an ongoing effort that could not be finished by the end of the project.
A surprising result comes from the transformations utilising the cDNA coding for capsanthin capsorubin synthase (CCS). The PCR-based screen for transformants among antibiotic resistant plants revealed the presence of a potato gene that shared a large degree of homology with CCS. The cDNA was isolated and its transient expression in plant protoplasts enabled a functional identity to be assigned, with the protein acting as neoxanthin synthase.
Task 4: Nutritional and agronomic tests Some nutritional tests on high-beta tomatoes have been started. No deliberate releases or open-field agronomic tests have been conducted within Europe, although the "Golden rice" is the object of extensive field testing outside Europe by Zeneca.
Dissemination
A large number of Publications have appeared in high impact factor journals (Science, Nature, Biotechnology, Trends in Plant Science, Plant Journal). Many of these are joint publications, between various project partners. Joint patent applications have been also filed.
Summary
Introduction Carotenoids and their metabolites are pigments widely present in human diet. They occur in many foods and ingredients, ranging from tomatoes to carrots, shrimps, egg yolk, or saffron. Beta-carotene is the main dietary precursor of vitamin A, of which there is a diffuse dietary shortage. This is mainly in the third world, where such deficiency causes acute public health problems. Many dietary xanthophylls and carotenes have been shown to have a beneficial anti-oxidant effect, preventing circulatory diseases. Chemically synthesised carotenoids are widely used as highly safe food, feed and cosmetic colorants; finally, some aromas, such as saffron picrocrocin, derive from carotenoid oxidation. Therefore, genetic engineering of carotenoid metabolism is of importance for both human and animal nutrition.
Objectives The objectives of the project are the following.
Activities Plant genes for zeaxanthin biosynthesis and oxidation will be cloned from Capsicum or Crocus expression libraries. The identity of the enzymes will be confirmed by expression in microbial systems over producing appropriate carotenoid precursors.
The metabolic re-routing of lycopene, present in tomato fruits, towards industrially valuable xanthophylls, will be achieved as follows: first, beta- carotene-over producing fruits will be obtained through the use of natural mutants or transformation with appropriate genes (lycopene cyclase, capsanthin-capsorubin synthase). Then, plant and/or bacterial genes for xanthophyll biosynthesis (beta-carotene hydroxylase, ketolase) will be introduced under the control of fruit-specific promoters to produce zeaxanthin and astaxanthin. The same strategy will be applied to Phycomyces, a beta-carotene accumulating fungus. To convert the carotenoid precursor GGPP into beta-carotene (pro-vitamin A) i n rice endosperm or potato tubers, three sequential metabolic steps need to be introduced:
GGPPÞ phytoene, phytoene Þ lycopene and lycopeneÞ beta- carotene.
Genes encoding these metabolic steps will be put under the control of endosperm- or tuber-specific promoters and introduced into transgenic plants. Transgene expression and carotenoid content will be analysed. Plants containing multiple transgene combinations will be produced through multiple transformation of genetic crossing. After a preliminary, molecular and biochemical characterisation of the transgenic lines, high expressing offspring will be used for glasshouse and field trials of the progeny. Nutritional tests will be also performed with selected transgenic crops and fungi with significantly improved beta-carotene content, using a vitamin A-deficient animal model system.
Results In the second year of the project, the saffron gene for zeaxanthin oxidase has been isolated. All of the initially planned transformations (with the exception of capsanthin-capsorubin synthase introduction in tomato and stable transformation of Phycomyces) have been successfully accomplished and, in most cases, molecularly confirmed. In many cases, the biochemical analysis has been also completed. For potato, over expression of early steps in the pathway does not significantly alter carotenoid composition, while over expression of capsanthin-capsorubin synthase depletes the beta-carotene-derived xanthophylls and gives an early sprouting phenotype. For tomato, the analysis completed only for fruits transformed with the Agrobacterium aurantiacum crtW and- crtZ genes, which do not -bring about a significant change in xanthophyll composition. With Phycomyces and rice, some problems with transgene stability and/or silencing were encountered. For rice, the problem is being solved by resorting to Agrobacterium-mediated transformation. A novel round of transformations with optimised transgenes and double constructs has been almost completed.
Two major break throughs have been accomplished in the second year. These include the isolation of the saffron gene encoding the first step of crocetin/picrocrocin biosynthesis, that has been cloned. Crocetin and picrocrocin are highly priced compounds which constitute, respectively, the major colour and flavour of saffron. This opens up the possibility of metabolic engineering of these compounds into crop plants (molecular farming). The second major discovery concerned the early sprouting phenotype observed in potato plants depleted of beta-carotene-derived xanthophylls (and thus, probably, of ABA). The results confirmed the role of these compounds in tuber dormancy and established a new area for future manipulation.
Future activitiesA novel round of transformations with optimised vectors is under way. Within the third year, production of several GMOs with significantly altered carotenoid content is anticipated.
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