Use of metabolic stimulators and inhibitors for enhanced production of beta-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896

This work investigates the potential of metabolic stimulators, firstly to enhance the production of beta-carotene, and later use of inhibitors of lycopene cyclase so as to accumulate lycopene in the fermentation medium. Various non-ionic surfactants, natural oils, stimulators such as amino-acids, tricarboxylic acid cycle (TCA) intermediates, vitamin A and antibiotics were investigated for improved production of beta-carotene using the zygomycete fungus Blakeslea trispora. Span 20 at 0.2% increased the beta-carotene production from 139 mg/l to 318 mg/l. Examination of the mycelial morphology of the B. trispora with span 20 showed a shorter mycelial length, which allowed a well-dispersed growth of B. trispora. Supplementation of the medium with 1000 ppm vitamin A acetate gave highest concentration of beta-carotene (830+/-6 mg/l). Several chemical inhibitors such as imidazole, pyridine, triethylamine, piperidine, and nicotinic acid were then evaluated to block the biosynthesis at lycopene. Piperidine at 500 ppm gave a 7.76-fold improvement, and produced high titers of lycopene (775+/-5 mg/l) in a medium supplemented with vitamin A acetate.     

Hormonal Interactions in Mucor mucedo and Blakeslea trispora

Evidence is presented that progametangia in both the plus and the minus mating types of Mucor mucedo can be induced by one substance, namely (-)-trisporic acid B. A method is described for the determination of the concentration of the sex factors (trisporone, trisporic acid B, trisporic acid C) in mated cultures of Mucorales by polarography. It can be demonstrated that the amount of plus mycelium is limiting for the production of the sex factors in Blakeslea trispora. It is shown that the minus type of this organism is able to synthesize the sex factors when incubated in the filtered medium of a mated culture. Cycloheximide and 5-fluorouracil inhibit strongly the sex factor production in a mated culture of B. trispora at any time. This result suggests that sexual activity comprises the synthesis of proteins which are involved in the production of the sex factors.     

Cultures of separated mating types of Blakeslea trispora make D and E forms of trisporic acids

Trisporic acids are end products of the sex-specific pheromones in mucoraceous fungi. We have found three new trisporic acids in cultures of Blakeslea trispora in which (+) and (-) mating types were separated by a membrane with 0.45-microns pores. Two of the trisporic acids were new compounds; the structure of the third [previously described by Miller and Sutter [(1984) J. Biol. Chem. 259, 6420] as methyl trisporate-E with a hydroxyl group at C-2] was revised. Trisporic acid-E(3R), trisporic acid-E(3S), and trisporic acid-D(2S) were in a 1:1:2 ratio, accounted for 9% of the total trisporic acids, and differed by the position and configuration of a hydroxyl group on the ring at C-2 or C-3, the conformation of the ring, the extent of rotation of the side chain relative to the ring, and either a carbonyl or hydroxyl group on the side chain at C-13. These three compounds accounted for only 0.5% of the total trisporic acids in combined mating type cultures. Since the combined cultures did not metabolize trisporic acid-E(3R), its biosynthesis apparently ceases when opposing mating types contact each other physically. We speculate that B. trispora and Phycomyces blakesleeanus utilize different pheromones to regulate an early event (possibly zygotropism) in sexual development.     

Mutants and intersexual heterokaryons of Blakeslea trispora for production of beta-carotene and lycopene

The industrial production of beta-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of beta-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-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having beta-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 beta-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 beta-carotene per g (dry mass). Beta-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.     

Production and derivate composition of trisporoids in extended fermentation of Blakeslea trispora

Trisporic acid, its precursors and derivatives are used within zygomycete fungi as communication signals and sexual regulators, and also influence the production rate of the parent compound, β-carotene. Cultivation parameters during growth and the trisporoid production phase of Blakeslea trispora were studied in two-step shake flask cultures and up-scaled fermentations. Comparison of various fermentation protocols allowed the definition of parameters governing trisporoid production. Highest yields were obtained when the initial growth phase allowed for both rapid growth and fast exhaustion of nitrogen and phosporous sources. Onset of trisporoid production is accompanied by a pH drop in the medium and triggered by nutrient limitation, nitrogen depletion being the most important factor. Supplementation of cultures with carbon at low concentration after onset of trisporoid production led to prolonged growth and higher final product accumulation. B. trispora produces trisporoids in two major series, B and C. During a first peak in trisporic acid accumulation, production of trisporic acid B exceeds that of trisporic acid C, which later accumulates at the expense of the trisporic acid B, indicating a variable regulation of the ratio between these metabolites. These data are valuable for tailoring production systems for enrichment of specific intermediates of this complex signal family.     

初级代谢产物和有性生殖促进剂对番茄红素发酵的影响

考察几种初级代谢中间产物对三孢布拉霉发酵生产番茄红素的影响,以及卵磷脂对三孢布拉霉正负菌接合孢子及发酵的影响。结果表明：发酵24h分别添加2．0％的柠檬酸和2．0％的三孢酸,番茄红素的产量分别达到0．99g／L和1．26g/L,比对照分别提高39．43％和32．63％;卵磷脂的添加能促进三孢布拉霉两性接合孢子的形成,进而促进番茄红素的合成,当大豆卵磷脂的添加量为0．3％时,番茄红素的产量为1．58g/L,比对照提高56．44％。     

Initial stages of trisporic acid synthesis in Blakeslea trispora

Biotransformation of beta-carotene with enzyme preparations isolated from the mycelium of Blakeslea trispora resulted in the formation of its hydroxylated metabolite and apocarotenals, products of oxidative degradation of this compound. By spectral, chromatographic, and chemical properties, the beta-carotene derivative was identified as 4-hydroxy-beta-carotene (isocryptoxanthine). One of the products of oxidative degradation of beta-carotene, beta-apo-13-carotenone, underwent modification in the presence of enzyme preparations from Blakeslea trispora with the formation of trisporic acid precursors. It should be emphasized that beta-apo-13-carotenone transformation proceeded more rapidly than beta-carotene oxidation by carbon in the 4-position. Our findings suggest that under conditions of oxidative stress, oxidative degradation of beta-carotene into beta-apo-13-carotenone leads to the formation of considerable amounts of trisporic acids.     

Light induction of the carotenoid biosynthesis pathway in Blakeslea trispora

A gene of Blakeslea trispora has been cloned by heterologous hybridization with the Mucor circinelloides crgA gene, a repressor of light-inducible carotenogenesis. This gene is the ortholog of the M. circinelloides crgA, since it was able to restore the wild-type phenotype of a null crgA mutant of M. circinelloides. The expression of B. trispora crgA gene is light-induced and photoadapted, as occurs for M. circinelloides crgA. Light induction and photoadaptation of B. trispora crgA was also observed in M. circinelloides, which suggests that the mechanisms involved in light regulation are basically conserved between these filamentous fungi. Conservation of the regulatory pathway that controls carotene biosynthesis was supported by the light-induced and photoadapted expression of all structural carotenogenic genes of B. trispora. Consequently, the beta-carotene content of dark grown mycelia of B. trispora increased upon illumination with white light.     

Chemical regulators of carotenogenesis by Blakeslea trispora

The activation of the carotene biosynthetic pathway in Blakeslea trispora was found to occur by trisporic acid and many other compounds such as abscisic acid, β-ionone, α-ionone and vitamin A which share significant structural similarity with trisporic acid. The magnitude of stimulatory activities of these effectors was in the order trisporic acid > abscisic acid > β-ionone > α-ionone > vitamin A. Comparison of structures and stimulatory activities of all the effectors indicated that the short length of the side chain and the presence of a keto group in the ring structure of the trisporic acid molecule contributed significantly to the biological activity towards carotenogenesis.     

Trisporic Acid Biosynthesis in Separate Plus and Minus Cultures of Blakeslea trispora: Identification by Mucor Assay of Two Mating-Type-Specific Components

Separate plus and minus cultures of Blakeslea trispora synthesize small amounts of trisporic acids under specific conditions. These amounts are expressed as a percentage of the trisporic acids (50 mg/liter of medium) synthesized by mixed plus-minus cultures in 5 days. Plus cultures, without additives from minus cultures, synthesize 0.1% trisporic acids. Plus cultures synthesize 0.4% trisporic acids when stimulated by M-factor, a mating-type-specific component synthesized by minus cultures. Minus cultures, without additives from plus cultures, do not synthesize even 0.0001% trisporic acids. Minus cultures synthesize 1% trisporic acids when stimulated by P-factor, a mating-type-specific component synthesized by plus cultures. Minus cultures synthesize M-factor when stimulated by pi, a component synthesized by plus cultures. We speculate that (i) minus cultures synthesize a component, mu, which stimulates P-factor synthesis in plus cultures, and (ii) both M-factor and P-factor are precursors of trisporic acids.     

Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-)

In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of structurally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete structural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor mucedo. The activity screening contributed to establish general structure-function relationships for trisporoid action. Most crucial for activity were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The overall biological activity apparently results from the combination of the various structural elements.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

Blakeslea trispora mutants with a disrupted sexual process

Three groups of Blakeslea trispora (+) and (-) mutants were obtained and their phenotypical characteristics were studied as well as biochemical changes in the course of mating and the ability to synthesize carotenoids when the sexual process of these mutants was disordered. The first group of mutants synthesized carotenoids at the control level, the second group produced them below the control level, and the third group accumulated less than 1% of carotenoids as compared to the control. The difference in the yields of carotenoids among the three groups of mutants in the mated culture is attributed to the presence (or absence) of the ability to synthesize trisporic acids in them.     

Heterothallism of mucoraceous molds: a review of biological implications and uses in biotechnology

The phenomenon of heterothallism in filamentous fungi has been reviewed, with emphasis on the discussion of hormonal regulation of heterothallic strains of mucoraceous molds. This process is viewed from the viewpoint of current understanding that fungal cells communicate with each other using a special "language", i.e., signaling chemicals (hormones, or pheromones). Physiological and biochemical criteria of distinguishing heterosexual strains, which make it possible to draw analogies with higher eukaryotes, are set forth for the first time, based on experimental data obtained with Blakeslea trispora. The synthetic pathway to trisporic acids (a zygogenic sex hormone of Mucorales), their relation to carotenoids, and biological functions are described. The similarity (both structural and functional) between fungal, plant, and animal hormones is another topic dealt with. Current understanding of the role of terpenoids in the evolution of sexual communication and transduction is presented, with an excursion into microbial endocrinology, a novel field of research in biology. The concluding part of the review analyzes data on the biotechnological implications of the phenomenon of heterothallism. Specifically, it may be used for obtaining a series of isoprenoid compounds, such as beta-carotene and licopin (which exhibit pronounced antioxidant activity), as well as sterols and trisporic acids.     

Relationship Between Sexuality and Carotene Synthesis in Blakeslea trispora

When stimulated by equivalent amounts of progametangia-inducing hormones, cultures of the minus mating type of Blakeslea trispora produce about the same quantities of carotenoids as mated cultures of the fungus, which suggests that the stimulation of carotene synthesis during the sexual activity of mated cultures is the result of hormonal action. These hormones were isolated and purified. From spectroscopic analysis of purified samples, it appears that the hormones are identical with trisporic acids B and C. When both mating types of B. trispora were cultivated in one vessel but were kept apart by membrane filters, the formation of sex hormones was not inhibited. Physical contact between the mating types is obviously not required for the induction of sexual activity. The sex hormones also formed in combined cultures of B. trispora-plus and Zygorhynchus moelleri (a homothallic species), but not in combined cultures of B. trispora-minus and Z. moelleri. This is evidence for the hypothesis that the hormones are produced by B. trispora-plus only.