Carotenoid Biosynthesis in Plants and Microorganisms
Overview on carotenoids
Carotenoids are synthesized as functional pigments in microorganisms and plants. Due to their unique property to absorb excitation energy from photosensitized molecules and dissipation as heat, carotenoids are essential components of the oxygenic photosynthetic apparatus as a membrane associated antioxidant. In addition to their antioxidative potential, special sterole-like carotenoids represent structural components within the membranes of extremophilic bacteria.
Humans need a supply of carotenoids in their diet. ß-Carotene is important as provitamin A. The xanthophylls lutein and zeaxanthin have to be constantly integrated into the macula lutea in the central retina of the eye to prevent age-dependent macular degeneration. Concerning general health promoting properties of carotenoids, there is now strong evidence showing that diets rich in carotenoids prevent the onset of certain chronic diseases and certain types of cancer.
The initial steps of carotenoid biosynthesis are quite similar in archea, bacteria, fungi and plants. Nevertheless, there is no direct line of evolution for most of the enzymes involved. The acquirement of novel genes for novel enzymes often results in very diverse reaction mechanisms yielding isomerically modified products. Prominent examples of such unusual isomers are poly-cis desaturation products in plants and the formation of the in the 3R,3'R enantiomer of astaxanthin in Phaffia rhodozyma.
Its focus is on:
Our projects deal with
- elucidation of carotenoid biosynthesis, their relevance for protection of photosynthesis
and their pathway regualtion as stress response,
- functional characterization of genes and enzymes involved in carotenogenesis, enzyme
mechanisms and molecular phylogeny of the pathway,
- genetic engineering of the metabolism to develop cell factories for the production of
high-value industrial carotenoids and to improve the nutritional carotenoid content in
The research approaches involve a combination of molecular, biochemical and physiological techniques and advanced carotenoid analysis involving HPLC combined with diode array spectroscopy and MALDI-TOF mass spectroscopy.
Biosynthesis, genes, enzymes and molecular phylogeny
Analytical identification of unknown carotenoids is the first to elucidate novel carotenoid biosynthetic pathways. This is followed by the cloning of the genes involved, their heterologous expression and enzymological studies with the enzymes produced in this way.
Identical carotenoids can be synthesized via different reaction steps in various organisms. For the identification of the individual catalytic steps, we developed a heterologous complementation system. This involves the co-expression of the gene of an individual enzyme with all genes necessary for the formation of the expected substrate. By analysis of the resulting carotenoid product, the substrate and product specificity of the investigated enzyme and its position in the biosynthetic pathway can be determined. With this approach, we could elucidate the biosynthetic pathways to acyclic carotenoids in purple bacteria and to C50-carotenoids in brevibacteria. By combining functional gene expression with enzyme investigations, we were successful in establishing a novel poly-cis carotenoid biosynthetic pathway for plants and cyanobacteria.
Different reactions for the synthesis of the same carotenoids indicate that diverse genes/enzymes are involved. One of our interests is on their molecular phylogeny and the analysis of their evolutionary roots.
Function in protection and regulation
Chloroplasts of green plants have developed from ancient cyanobacteria. Therefore, they are a very useful model to study the structural requirements of a carotenoid for efficient protection of the photosynthetic apparatus from photooxidation. For such studies, the carotenoid composition of Synechococcus and Synechocystis was genetically modified by disruption of individual genes or by expression of foreign carotenoid genes. Photosynthesis of intact transformants under high-light stress revealed the antioxidative potential of these carotenoids in situ. For in vitro studies on photoprotection, we produce different carotenoids including novel structures by combinatorial biosynthesis in Escherichia coli transforming this bacterium with combinations of carotenogenic genes from various organisms.
Due to carotenoid function as antioxidant under high light stress, carotenoid biosynthesis is up-regulated in a light-dependent manner. In plants, the whole pathway is switched off and on in dark-light cycles. We could show that this regulation is under transcriptional control of the initial enzyme phytoene synthase. This is also the case in cyanobacteria. Depending on species, a second very fast response of hydroxy or keto carotenoid synthesis to high light exists additionally. Both regulatory mechanisms are currently analyzed at the gene level.
Biochemical analysis of fungal carotenoids producers helped to understand the metabolic control at the enzyme level. We exploited these finding and use our genes to over-express rate-limiting enzymes. With this approach, we were successful to increase the desired end product astaxanthin in the yeast Phaffia rhodozyma and to minimize the formation of unwanted side products. Furthermore, we were able to genetically modify Phaffia mutant toward the synthesis of foreign carotenoids.
Since zeaxanthin is a limiting nutrient in our diet, we chose potato as a staple crop to manipulate its biosynthesis. By tuber-specific antisense inactivation of an enzyme within the existing pathway in potato, it was possible to obtain plants with a 130-fold increase of zeaxanthin in the tuber.
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