Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.
The industrial production of -carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of -ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N-nitro-Nnitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having -carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of -carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of -carotene per g (dry mass). -Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.
The isolation, characterization, and regulation by light and nitrogen of the lycopene b-cyclase gene from Chlorella zofingiensis Dönz (CzlcyB), involved in the biosynthesis of astaxanthin and lutein, have been performed in this work. These carotenoids are of high commercial value as dyes in food and as nutraceuticals. The open reading frame (ORF) of CzlcyB encoded a polypeptide of 546 amino acids. A single copy of CzlcyB has been found in C. zofingiensis. The chararacteristic Rossmann or dinucleotide binding fold, present in most lycopene cyclases, has been also identified in the LCYb of C. zofingiensis (CzLCYb). Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to cycle both lycopene and d-carotene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the lycopene b-cyclases (LCYb) of the chlorophycean microalgae studied, being very closely related to LCYb of plants. Transcript levels of CzlcyB were increased under nitrogen deprivation, but no increase was observed under high-light conditions. However, high irradiance triggered astaxanthin synthesis, while nitrogen deprivation by itself could not induce it. The combination of high irradiance and nitrogen deprivation led to a significant enhancement of the astaxathin accumulation.
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