There have been several reports on the enhancement of volumetric production (mg/l) as well as cellular accumulation (mg/ g) of microbial carotenoid upon supplementation of metal ions (copper, zinc, ferrous, calcium, cobalt, alluminium) in yeasts and molds (Bhosale, 2004; Buzzini et al., 2005). Trace elements have been shown to exert a selective influence on the carotenoid profile in red yeasts. It may be explained by hypothesizing a possible activation or inhibition mechanism by selected metal ions on specific carotenogenic enzymes, in particular, on specific desaturases involved in carotenoid biosynthesis (Buzzini et al., 2005). The other explanation is based on observations that presence of heavy metals results in formation of various active oxygen radicals what, in a turn, induces generation of protective carotenoid metabolites that reduce negative behaviour of free radicals. Such strategy has been applied in several pigment-forming microorganisms to increase the yield of microbial pigments (Breierova et al., 2008; Rapta et al., 2005).

In order to achieve rapid carotenoid overproduction, various stimulants can be added to the culture broth. One group of such enhancers is based on intermediates of the tricarboxylic acid cycle which play an important role in metabolic reactions under aerobic conditions, forming a carbon skeleton for carotenoid and lipid biosynthesis in microbes. Because pigment increase is paralleled by decreased protein synthesis, restriction of protein synthesis is an important way how to shift carbon flow to carotenoid synthesis (Flores — Cotera & Sanchez, 2001). It was also proposed that high respiratory and tricarboxylic acid cycle activity is associated with production of large quantities of reactive species and these are known to enhance carotenoid production (An, 2001). It should be emphasized that the degree of stimulation was dependent on the time of addition of the citric acid cycle intermediate to the culture medium. Some fungi showed that addition of organic acids to media elevated P-carotene content and concomitantly decrease y-carotene level with complete disappearance of lycopene (Bhosale, 2004).

Chemical substances capable of inhibiting biosynthetic pathways have been applied to characterize metabolic pathways and elucidate reaction mechanisms. In general, compounds that inhibit biosynthesis can act through various mechanisms, such as inhibiting the active site directly by an allosteric effect (reversible or otherwise), altering the regulation of gene expression and blocking essential biochemical pathways or the availability of cofactors, among other possibilities. From this view, number of chemical compounds including terpenes, ionones, amines, alkaloids, antibiotics, pyridine, imidazole and methylheptenone have been studied for their effect on carotene synthesis (Bhosale, 2004). In order to obtain commercially interesting carotenoid profiles, the effect of supplementation with diphenylamine (DPA) and nicotine in the culture media of Rhodotorula rubra and Rhodotorula glutinis was investigated. DPA blocks the sequence of desaturation reactions by inhibiting phytoene synthase, leading to an accumulation of phytoene together with other saturated carotenoids and nicotine inhibits lycopene cyclase, and consequently the cyclization reactions (Squina & Mercadante, 2005). Cultivation of Xanthophyllomyces dendrorhous in the presence of diphenylamine and nicotine at 4°C was reported to trigger interconversion of P — carotene to astaxanthin (Ducrey Sanpietro & Kula, 1998).

The addition of solvents such as ethanol, methanol, isopropanol, and ethylene glycol to the culture medium also stimulate microbial carotenogenesis. It should be noted that while ethanol supplementation (2%, v/v) stimulated P-carotene and torulene formation in Rhodotorula glutinis, torularhodin formation was suppressed (Bhosale, 2004). It was proposed that ethanol-mediated inhibition of torulene oxidation must be accompanied by an increase in P-carotene content suggesting a shift in the metabolic pathway to favor ring closure. Detailed studies revealed that ethanol activates oxidative metabolism with induction of HMG-CoA reductase, which in turn enhances carotenoid production. However, stimulation of carotenoid accumulation by ethanol or H2O2 was more effective if stress factors were employed to the medium in exponential growth phase than from the beginning of cultivation (Marova et al, 2004).

Mutagenesis

Mutagenesis is an alternative to classical strain improvement in the optimization of carotenoid production. Mutagenic treatment with N-methyl-N-nitro-N-nitrosoguanidine (NTG), UV light, antimycin, ethyl-methane sulfonate, y-irradiation, high hydrostatic pressure have been used successfully to isolate various strains with enhanced carotenoid- producing activity. UV mutant R. gracilis has shown 1.8 times higher carotenoid synthesizing activity than that of the parent strain and the relative share of P-carotene in the total carotenoids was 60%. The yellow colored mutant 32 was also obtained by UV mutagenesis of the pink yeast R. glutinis and produced a large quantity of total carotenoids (2.9 mg/ g dry cells), which was 24-fold higher accumulation of total carotenoids compared with the wild-type. Mutant 32 produced 120-fold more beta-carotene (2.05 mg/ g dry cells) than the parent culture in a much shorter time (36 h), which was 82% (w/w) of the total carotenoid content. Later, after the treatments of five repeated cycles by high hydrostatic pressure of 300 MPa, the mutant R. glutinis RG6p was obtained, beta-carotene production of which reached 10.01 mg/l, increased by 57.89% compared with 6.34 mg/l from parent strain (Frengova & Beshkova, 2009).

A fivefold increase in beta-carotene accumulation was reported for yellow mutant P. rhodozyma 2-171-1 which was obtained after ethyl-methane sulfonate mutagenesis of dark red strain P. rhodozyma. This mutant is likely to be blocked in the oxidase step and therefore unable to perform the conversion of beta-carotene to echinenone and latter to astaxanthin. The UV-mutant P. rhodozyma PG 104 produced 46-fold more P-carotene (92% of total carotenoids) than the parent culture (2% of total carotenoids) and maximum beta-carotene yields were 1.08 mg/ g dry cells and 9.95 mg/l. Using NTG mutagenesis two different strains of carotenoid accumulating X. dendrourhous mutants JH1 and JH2 were also isolated. Astaxanthin-overproducing mutant JH1 produced 4.03 mg astaxanthin/ g dry cells, and this value was about 15-fold higher than that of wild-type. Mutant JH2 produced 0.27 mg beta — carotene/ g dry cells, and this was fourfolds increase from that of wild-type and the mutant

X. dendrourhous JH1 produced maximum astaxanthin concentration of 36.06 mg/l and 5.7 mg/g dry cells under optimized cultivation conditions (Kim et al., 2005).

To isolate a carotenoid-hyperproducing yeast, P. rhodozyma 2A2 N was treated by low-dose gamma irradiation below 10 kGy and mutant 3A4-8 was obtained. It produced 3.3 mg carotenoids/ g dry cells, 50% higher carotenoid content than that of the unirradiated strain (antimycin NTG-induced mutant 2A2 N). Gamma irradiation produces oxygen radicals generated by radiolysis of water and could induce mutation of P. rhodozyma through a chromosomal rearrangement. A primary function of carotenoids in P. rhodozyma is to protect cells against singlet oxygen and these compounds have been demonstrated to quench singlet oxygen. Oxygen radicals have been known to cause changes in the molecular properties of proteins as well as enzyme activities. Thus, oxygen radicals generated by gamma irradiation might modify the pathway in astaxanthin biosynthesis of P. rhodozyma and cause an increase in carotenoid production of the mutant 3A4-8 isolated by gamma irradiation (Frengova & Beshkova, 2009).