Carotene-superproducing strains of Phycomyces

Vol. 36, No. 5 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, p. 639-642 0099-2240/78/0036-0639$02.00/0 Copyright © 1978 American Society for Microbiology Carotene-Superproducing Strains of Phycomyces F. J. MURILLO, I. L. CALDERON, I. LOPEZ-DIAZ, AND E. CERDA-OLMEDO Departamento de Genetica, Facultad de Ciencias, Universidad de Sevilla, Seville, Spain Received for publication 20 August 1978 Production of fl-carotene by wild-type Phycomyces blakesleeanus can be stimulated by light, chemicals, regulatory mutations, and sexual interaction between mycelia of opposite sex. Through genetic manipulations, we have isolated strains which have simultaneously and constitutively incorporated several of these stimulatory effects. In the dark and in a simple medium, some of the strains produce up to 25 mg of f-carotene per g (dry weight), or about 500 times the wild-type production under the same conditions. High lycopene-producing strains have also been isolated by using carR mutants, which are blocked in the conversion of lycopene to fl-carotene. These strains should be useful in both industrial production of these pigments and basic research related to carotenogenesis. The production of fl-carotene by Phycomyces blakesleeanus depends on media and culture conditions but is generally low. In the dark, this fungus produces on the order of 50 Ag of ,Bcarotene per g (dry weight), making it inappropriate for industrial use (16). ,f-Carotene production can be stimulated in several ways. Photoinduction results in an accumulation of about 500 ,ig/g under blue-light intensities of 2 W/m2 (3), but light stimulation presents considerable practical difficulties in large-scale application. Several chemicals stimulate carotenogenesis when added to the medium (22); up to 2,000 /Lg/g is accumulated in the presence of vitamin A (14), but the required concentrations of the vitamin are prohibitively large; up to 4,000 ,lg/g has been observed under the best conditions in the presence of f8-ionone (16). Mutations in the gene carS result in fl-carotene contents of up to 4,000 lig/g in the dark. The carS mutants are still sensitive to vitamin A, but a double mutant, strain S106, has been obtained which reaches 6,000 ,ug/g. The new mutation, car-102, makes S106 insensitive to vitamin A. The stimulatory channel activated by vitamin A has thus become constitutive in this strain (18). In the Mucorales, the interaction between mycelia of opposite sex leads to increased carotenogenesis through formation of trisporic acids (2, 6; A. Prieto, C. Spalla, M. Bianchi, and G. Briffi, Commun. Int. Ferment. Symp. London, p. 38, 1964). Mixed cultures of Blakeslea trispora strains of opposite sex in the presence of fl-ionone have been considered promising for ficarotene production. However, it is difficult to maintain appropriate sex ratios in large cultures (10). In Phycomyces, sexual stimulation occurs in single mycelia, called intersexual heterokaryons, which contain a mixture of nuclei of opposite sex (5). Such heterokaryons produce more than 400 ,ug of fl-carotene per g (18), have a peculiar morphology, with formation of small aerial hyphae or pseudophores, and are unstable; they tend to segregate the components in homokaryotic form. The genetics of carotene biosynthesis in Phycomyces has been reviewed (9; E. Cerdfi-Olmedo and S. Torres-Martinez, Pure Appl. Chem., in press). Mutants in gene carB form phytoene, and mutants in gene carR produce lycopene (17, 21). All the intermediates from phytoene to f8carotene may be obtained in Phycomyces, either through the use of certain genetic combinations (1, 12) or through the addition of inhibitors (13, 19). The objective of this work was to obtain Phycomyces strains that would yield high contents of f8-carotene or other carotenes when grown on simple media in the dark. MATERIALS AND METHODS The strains of P. blakesleeanus used in this work are listed in Table 1. References to details about isolation, genotypes, and carotene production are given in the same table. Heterokaryons were produced by using a previously described method (20). In the text, the two heterokaryon components are separated by an asterisk. Cultures of homokaryons and heterokaryons were initiated as reported earlier (18). For all quantitative studies, cultures were grown on solid minimal medium 639 Printed in U.S.A. TABLE 1. Strains of P. blakesleeanus used in this work Strain Genotype Main carotenoid produced Reference (Lug/g, dry wt) NRRL1554 Wild type (+) fl-Carotene (56) 18 Ml carS43 (+) fl-Carotene (4,160) 18 S106 carS42 car-102 mad-103 (-) fl-Carotene (5,595) 18 H7 carR51 (+) Lycopene (1,200) This work S136 carR127 (+) Lycopene (650) This work C9 carR21 (-) Lycopene (2,470) 21 and this work (15), with glucose as carbon source, at 22°C for 4 days. In a few cases, cultures were grown on potato-dextrose agar (7), since its low cost would be an advantage in large-scale application. To observe distinct colonies, the minimal medium was supplemented with 1 mg of yeast extract per ml and acidified to pH 3.3. The extraction of carotenes and their chromatographic separation and identification have been described previously (11, 12, 21). Mutants were isolated after treatment with 100 ug of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) per ml in pH 7.0 citrate-phosphate buffer, as previously described (8). RESULTS AND DISCUSSION Intersexual heterokaryons with carS mutations. Sexual interaction and regulatory mutations multiply each other's stimulatory effect on carotenogenesis when acting simultaneously in the intersexual heterokaryon Ml * S106. Some of the heterokaryotic mycelia contain 500 times more f8-carotene than does the original wild type (Table 2). These heterokaryotic mycelia are highly variable in f-carotene content, presumably reflecting variations in nuclear ratios. They are much less stable than the heterokaryons described by Heisenberg and Cerda-Olmedo (15), since they tend to separate into mycelial patches of different colors and produce unexpectedly high proportions of homokaryotic spores. Sexual type is only one of the many differences in genetic background among the constituent nuclei, which are descended from very different wild types. The reasons for the instability are, thus, unclear. In any case, instability is a major drawback for the practical application of a strain. Intersexual heterokaryons with balanced lethal mutations. The disadvantages of the intersexual heterokaryon Ml * S106 would presumably disappear in the diploid M1/S106. We have made considerable, but fruitless, efforts to isolate diploids from different Phycomyces heterokaryons. An alternative would be to introduce recessive lethal mutations in both components of the heterokaryon. To this effect, spores from Ml * S106 mycelia were treated with NTG to a survival level of about 0.1%. Most nuclei are thus inactiTABLE 2. Production of f-carotene in intersexual heterokaryons MI * S106 and their derivatives Strain 8l-carotene (,ug/g, dry wt) a Ml * S106, mycelium A 14,340 Ml * S106, mycelium B 18,450 Ml * S106, mycelium C 20,325 Ml * S106, mycelium D 20,500 Ml * S106, mycelium E 25,342 S218 * S219 16,000 (12,620) S242 * S243 15,600 (9,000) S244 * S245 19,120 (14,120) S246 * S247 13,470 (25,230) a Numbers in parentheses correspond to potatodextrose agar cultures. The others correspond to the usual minimal medium cultures. vated, and most survivors are homokaryotic. Among the heterokaryotic survivors, there are many that cannot segregate either or both components in homokaryotic form, owing to the introduction of recessive lethal mutations (8). A total of 117 survivors of the treatment, taken from among the most brightly colored, were tested for segregation by streaking their spores on acid medium. The heterokaryons S218 * S219 and S242 * S243 were obtained in this way. They produced considerable quantities of f8-carotene (Table 2), and no homokaryons were found among their progeny, although they still showed awide range of nuclear ratios, leading to variations in fl-carotene content. Variation in nuclear ratios could be limited if the lethal mutations were not totally recessive. If both heterokaryon components had mutations or sets of mutations that made mycelia with more than 70% of the corresponding nuclei inviable, nuclear proportions would be limited to the 30 to 70% range. Heterokaryotic S218 * S219 spores were treated with NTG, and a search for stable strains led to the isolation of S244 * S245 and S246 * S247. These heterokaryons are apparently very uniform and have high fl-carotene contents (Table 2). 640 MURILLO ET AL. APPL. ENVIRON. MICROBIOL. CAROTENE-SUPERPRODUCING PHYCOMYCES STRAINS High lycopene production. The lycopene content of strains C9 and H7 (Table 3) largely exceeds the /B-carotene produced by the wild type. There is an apparent feedback regulation (18), so that the lack of the end product, /8carotene, stimulates the pathway. The heterokaryon H7 * C9 produces no pseudophores and does not surpass the lycopene content of strain C9 alone (Table 3). This total lack of sexual stimulation supports the concepts that both pseudophore formation and carotenogenesis are activated by trisporic acids and that these acids derive from /3-carotene (24). To exhibit sexual stimulation, a heterokaryon must thus be able to synthesize at least some ,8-carotene. Strain S136 was isolated after treatment of spores of strain NRRL1554 with NTG. This strain contains lycopene (Table 3) as the main carotene, but also y-carotene (110,ug/g), ,8-carotene (45 ,ug/g), and smaller amounts of phytofluene, c-carotene, and neurosporene. It presumably carries a mutation in gene carR, resulting in decreased cyclase activity, responsible for the production of the cyclated carotenes y-carotene and /3-carotene (12). The heterokaryon S136 * C9 produces pseudophores, exhibits very intense red colors, and has high lycopene content. Spores of S136 *C9 were treated with NTG, and a search for stable heterokaryons resulted in the isolation of S183 * S184, S185 * S186, and S187 * S188 (Table 3). Although stability was achieved, optimum production was not. Further mutagenesis should bring this about. Final comments. The strains obtained in this work reach very high carotene levels when grown on simple media in the absence of light or exogenous chemical stimulation. They should be particularly useful in the development of industrial processes for carotene production. There is room for further improvements and extensions. New stimulatory mutations, similar to mutation car-102 in strain S106, could be introduced in carS (+), carR (+), and carR (-) genetic backgrounds and serve as the basis for a repetition of the process described in this paper. The intersexual heterokaryons could also carry mutant and wild-type alleles of genes carB and carR so that all the intermediates from phytoene to ycarotene could be produced (1, 12). Media and culture conditions optimal for carotenogenesis in other organisms (10) should be tried out, since we have made no effort in this direction. The strains obtained in this work should be particularly useful in the development of in vitro systems for carotene biosynthesis and for the isolation of mRNA's and proteins involved in the process (4, 23). TABLE 3. Formation of lycopene in homokaryons and intersexual heterokaryons containing carR nuclei Strain * Lycopene (Ag/g, dry wt) C9 2,470 H7 1,200 S136 650 H7 * C9 mycelium A 1,950 H7 * C9 mycelium B 2,575 H7 * C9 mycelium C 2,675 S136 * C9 mycelium A 9,780 S136 * C9 mycelium B 10,550 S136 * C9 mycelium C 12,540 S136 * C9 mycelium D 12,570 S136 * C9 mycelium E 14,600 S183 * S184 6,946 S185 * S186 5,830 S187 * S188 7,101 The successive increases in carotene content described in this paper support the hypothesis of the independence of the stimulatory effects of carS mutations, vitamin A, and sexual interaction (18). ACKNOWLEDGMENTS We thank M. I. Carretero for typing the manuscript and A. Fernandez-Estefane for technical assistance. Financial support for this study came from the Fundaci6n Juan March. LITERATURE CITED 1. Arag6n, C. M. G., F. J. Murillo, M. D. De la Guardia, and E. Cerda-Olmedo. 1976. An enzyme complex for the dehydrogenation of phytoene in Phycomyces. Eur. J. 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