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Relation between carotene overproduction and lipid accumulation in Blakeslea

A method was developed to facilitate the isolation of mutants with an increased lipid content, expected to result in a reduced cell density. The method is based in the centrifugation of spores from colonies surviving to a mutagenesis treatment in a ficoll solution of appropriate density. Simulation experiments with carotenoid overproducing mutants, previously shown to contain a higher lipid content, suggested the centrifugation at 4000 rpm in a 45% ficoll solution as the one suitable to separate wild type spores from low density spores. A search under these selecting conditions allowed the isolation of individual strains producing floating spores in further centrifugation tests, while the wild type spores in parallel experiments accumulated as a pellet in the bottom of the tubes. Some of the mutant strains exhibited morphological or pigmentation alterations. Carotenoid and lipid analyses were carried out with 19 candidate mutants. Most of them contained slight increases in carotenoid content, except one, that exhibited an strong increase (four-fold). Most of the mutants did not show major differences in lipid content, indicating that there are other cellular processes affecting cell density. New experiments were done under more stringent selection conditions (40% ficoll), requiring a lower density of the spores to float on the top of the solution. Two out of 13 floating strains isolated contained about 40% more lipids than the wild type. As in the previous set of experiments, most of the strains contained more carotenoids than the wild type.

- Strains with improved carotene production have been obtained by mutation and heterokaryosis from two selected wild strains of opposite sex, F921 and F986. Following exposure of their spores to a chemical mutagen, high-carotene mutants were identified after a screening of about 200,000 colonies. The best mutant accumulated five times the amount produced by the wild type. Further increases in carotene content were obtained after a new round of mutagenesis in the best mutant. Highest production was achieved in intersexual heterokaryons with mutant nuclei of opposite sex. These contained up to 39 mg of beta-carotene or up to 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of beta-carotene per g dry mass. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains.

- Chemical analyses of a correlation between lipid and carotenoid by HPLC-PDA and GC were performed on submerged cultures of the Blakeslea strains F986 and F921 and their crosses at 3 and 7 days post inoculation. The mated cultures had elevated levels of lipids and carotenoids, which appeared earlier during development than the parental strains. The more than five-fold increase in the beta-carotene content of the mated strains in submerged culture did not correspond with an equally large increase in fatty acid content. Beta-carotene content was observed to increase during development whereas the fatty acid content did not. The mutants SB52 and SB38 accumulated high levels of carotenoids in surface cultures but did not have corresponding increases in fatty acids, as observed by TLC. In submerged culture, these strains did not pigment nor grow as well as the parental strains and were not used in subsequent analyses. Analyses were also performed on the low-density mutants M24 and M26 and compared to their wild type background F921. These mutants accumulated less carotenoids and fatty acids than the parental strain, indicating that fatty acid content was not responsible for the buoyant spore phenotype. No relationship between the biosynthesis and accumulation of carotenoids and lipids could be found in any analysis of Blakeslea. Investigations on inhibitors of carotenoid and fatty acid biosynthesis were therefore not performed. PUFA accumulation in the mycelia was analysed, and fatty acids identified by co-migration with known standards. Lipid globules were isolated and purified as part of sub cellular fractionation process. Carotenoids were found to accumulate within these organelles and internal membrane systems were observed during the early stages of development. Carotenoids from industrial Blakeslea biomass were visualised as crystalline deposits within the globules. Globule size appeared to correspond with increased carotenoid production in the mycelia though globule number did not seem to alter greatly. TLC analysis of lipids in the globules showed the presence of beta-carotene, fatty acids, diglycerides, monoglycerides and phospholipids. A cell-free system was established in Blakeslea strains F986, F921, SB52 and SB38. The incorporation of radiolabelled isotopes from MVA was observed in crude extracts but not in isolated lipid globules of 7-day post inoculation cultures.

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Universidad de Sevilla
Departamento de Genetica. Apartado 1095.
41080 Sevilla
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