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Regulation of carotenoid formation of Blakeslea, Phycomyces and Xanthophyllomyces

Several Blakeslea mutants with enhanced ß-carotene production are available. Furthermore, mating of plus and minus strains highly stimulates carotenogenesis. In those mutants and matings up-regulation of enzyme levels and activities were investigated in correlation to the ß-carotene formation. At first, antisera were produced against HMGCoA reductase, the phytoene synthase part of CarRA, the lycopene cyclase part of CarRA (all from Phycomyces) and against IPP isomerase from yeast. After heterologous expression of appropriate segments of these enzymes, the proteins were isolated and used for immunization of rabbits. The partially purified antisera against HMG-CoA reductase and IPP isomerase were not sensitive enough for Blakeslea to detect both proteins either in crude extracts or enriched preparations. Upon subcellular fractioning a protein was detectable in the insoluble fraction of the mated mutants after ultracentrifugation of a size of the lycopene cyclase domain with the lycopene cyclase antiserum. With the phytoene synthase antiserum a corresponding soluble protein was detectable documenting that the bifunctional lycopene cyclase/phytoene synthase protein is post-translationally cleaved to an individual soluble phytoene synthase of 36 kDa and a membrane-bound lycopene cyclase of 30 kDa.

The purified antisera against phytoene synthase allowed the detection and determination of this enzyme under different conditions. With matings, we could establish a kinetic for the formation of phytoene synthase. The levels of this enzyme increased from the mating on to day 4. Then, a steady decline was observed and disappearance of the phytoene synthase band at day 8. In parallel, ß-carotene accumulated over 4 days and afterwards stayed constant. These data are in support of phytoene synthase as a limiting enzyme of carotenogenesis in Blakeslea. Immuno determination of the phytoene synthase in Blakeslea mutants and matings demonstrated that the level of the synthase correlated with the amount of carotene formed. It is quite low in wild type, higher in carotenogenic mutants and increases about 10-fold in matings. In vitro assays of phytoene synthase resulted in considerably higher activity in mutants and matings compared to the wild type. A definite induction of the phytoene synthase / lycopene cyclase mRNA levels in the mated cultures upon mating and a tendency to decrease in the following days was observed.

Transcription of the phytoene synthase / lycopene cyclase and phytoene desaturase genes of Blakeslea was also analyzed by Northern hybridization using RNA samples obtained from pilot-scale feed-batch fermentations for ß-carotene production. Steady-state levels of transcripts were found after 5 days.
In Phycomyces, mRNA levels of the phytoene synthase / lycopene cyclase gene and phytoene desaturase gene were determined in the wild type, four overproducing mutants (carS, carD, carF and carScarF) and four structural mutants (carA, carR, carRA and carB). Cultures were grown for 24 h in the dark, in the presence or absence of two different chemicals (retinol and dimethyl phtalate) activating carotenogenesis, or in the light. After 24 h, transcription of these genes is similar in the wild type, in various mutants and in the wild type exposed to chemical activators. However, light strongly stimulates their mRNA levels in all strains tested, even in a mutant that shows very little increase in carotene synthesis after light exposure. The expression of two genes encoding HMGCoA synthase and reductase, two enzymes of the early terpenoid pathway, was also investigated. No activation of the mRNA levels was found for these genes either under chemical activation of the pathway or mutations in the regulatory genes carS, carF and carD. A minor increase of the mRNA levels was found in the light and after sexual stimulation. With increasing age of the Phycomyces culture, a decrease of the genes for phytoene synthase / lycopene cyclase and phytoene desaturase from day 3 on to day 7 was observed. In contrast, the mRNA levels of the HMGCoA synthase and reductase genes were constant or even slightly increased with age of the culture.

In the course of this project we found physiological conditions that up-regulate carotenogenesis including astaxanthin production in Xanthophyllomyces. Low light intensity as well as oxygen supply increased total carotenoid formation considerably. A combination of both factors, resulted in a 4-fold total carotenoid increase. The astaxanthin yield under those cultivation conditions was even 5-fold higher attributed to a stronger oxygenase reaction. High oxygen up-regulated geranylgeranyl pyrophosphate synthase and phytoene desaturase messages whereas higher transcripts of phytoene synthase / lycopene cyclase and astaxanthin synthase accumulated under low oxygen. In each case, light was antagonistic.

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