Initial Stages of Trisporic Acid Synthesis in Blakeslea trispora

Biotransformation of -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. Based on its spectral, chromatographic, and chemical properties, the -carotene derivative was identified as 4-hydroxy--carotene (isocryptoxanthine). One of the products of oxidative degradation of -carotene, -apo-13-carotenone, was modified in the presence of enzyme preparations from Blakeslea trispora to form trisporic acid precursors. -Apo-13-carotenone transformation proceeded more rapidly than -carotene oxidation at the carbon atom at position 4. The data suggest that, under oxidative stress, oxidative degradation of -carotene into -apo-13-carotenone leads to the formation of considerable amounts of trisporic acids.     

Characterization of beta-apo-13-carotenone and beta-apo-14'-carotenal as enzymatic products of the excentric cleavage of beta-carotene

Two new products from the incubation of beta-carotene with intestinal mucosa homogenates of human, monkey, ferret, and rat were isolated using high-performance liquid chromatography (HPLC). Identification by comparing retention times in HPLC, by monitoring ultraviolet/visible spectra, by reduction to corresponding alcohol, by oxime formation, and by mass spectrometry demonstrated that they are beta-apo-13-carotenone and beta-apo-14'-carotenal. These compounds were not found in incubations done without intestinal homogenates or with disulfiram as an inhibitor. Under standard incubation conditions, these products increased linearly for 60 min and up to a protein concentration of 1.5 mg/mL and increased along with increasing concentrations of beta-carotene. Therefore, they are enzymatic cleavage products from beta-carotene. The formation of the beta-apo-13-carotenone and beta-apo-14'-carotenal provides direct evidence for an enzymatic excentric cleavage mechanism.     

Characterization of products formed during the autoxidation of beta-carotene

The anticarcinogenic action of carotenoids such as beta-carotene has been frequently ascribed to their antioxidant properties. However, very little is actually known about the nature of the antioxidant reaction or the products that are formed. beta-Carotene was exposed to either spontaneous autoxidation conditions or to radical-initiated autoxidation conditions. The products were separated by reverse-phase HPLC, and individual peaks were characterized with an on-line diode array detector. Carbonyl products were isolated and characterized by several procedures, including borohydride reduction to the corresponding alcohols, derivatization with O-ethyl-hydroxylamine to the corresponding O-ethyl-oximes of the carbonyls, and analysis by GC-MS. Under the conditions of the experiments, the formation of a homologous series of carbonyl products was demonstrated, including beta-apo-13-carotenone, retinal, beta-apo-14'-carotenal, beta-apo-12'-carotenal, and beta-apo-10'-carotenal. Several very hydrophobic compounds were formed, which have not been previously identified. In addition, the products of NaOCl-treatment of beta-carotene were analyzed, and shown to be significantly different from the autoxidation products. This type of product analysis should be useful in determining the nature of the oxidants reacting with beta-carotene in vivo.     

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.     

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％。     

Media optimization for the production of beta-carotene by Blakeslea trispora: a statistical approach

Blakeslea trispora (+) MTCC, Blakeslea trispora NRRL 2895 (+), Blakeslea trispora NRRL 2896 (-) as well as intraspecific mating of both the strain types have been studied for optimum production of beta-carotene. Intraspecific mating of both the strain types increased the yield of beta-carotene to a considerable level (98+/-2mg/l) as compared to wild strains. Effect of different media components such as carbon, nitrogen, and sulphates, and that of process variables such as pH and inoculum size on beta-carotene production by Blakeslea trispora in shake flask culture was investigated. One factor at-a-time method was employed for the optimization of media components. Response surface methodology (RSM) was further used to determine the optimum values of process variables for maximum beta-carotene production. The fit of the quadratic model was found to be significant. A significant increase in beta-carotene production (139+/-1mg/l) was achieved using RSM.     

Superoxide dismutase and catalase activities in carotenoid-synthesizing fungi Blakeslea trispora and Neurospora crassa under the oxidative stress

The addition of menadione into the medium during cultivation of Neurospora crassa in the dark activated its constitutive superoxide dismutase. Exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. Superoxide dismutase activity in the mixed (+/-) mycelium of Blakeslea trispora synthesizing beta-carotene in the dark was much lower than that in Neurospora crassa. The superoxide dismutase activity further decreased in oxidative stress. The catalase activity decreased with an increase in the content of beta-carotene. Our results indicate that neurosporaxanthin possesses photoprotective properties in Neurospora crassa. In Blakeslea trispora (+/-) fungi, this compound acts as a major antioxidant during inactivation of enzymes that detoxify reactive oxygen species.     

Zygote formation in Blakeslea trispora: morphological peculiarities and relationship with carotenoid synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking beta-carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene production results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on beta-carotene synthesis.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of beta-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of beta-carotene (mg beta-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

A peroxidase from Lepista irina cleaves beta,beta-carotene to flavor compounds

Extracellular liquid of the edible fungus Lepista irina was found to effectively degrade beta,beta-carotene, beta-lonone, beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed as volatile breakdown products of beta,beta-carotene with mycelium-free culture supernatants, whereas beta-apo-10'-carotenal was identified as non-volatile degradation product. The key enzyme catalyzing the oxidative cleavage of beta,beta-carotene was purified with an overall yield of 63% and a purification factor of 43. Biochemical characterization showed a molecular mass of 50.5 kDa and an isoelectric point of 3.75. Fastest beta,beta-carotene degradation occurred at 34 degrees C and pH values between 3.5 and 4. Degenerate oligonucleotides were derived from N-terminal and internal amino acid sequences. By means of PCR-based cDNA-library screening a 1284 bp cDNA was identified which showed great overall similarity to Pleurotus eryngii polyvalent peroxidases. The obtained sequence contains an open reading frame of 1083 nucleotides, encoding a polypeptide of 361 amino acids. A 30 amino acid signal peptide was identified upstream of the N-terminal sequence of the mature enzyme. The L. irina versatile peroxidase represents the first microbial enzyme capable of carotenoid degradation that has been characterized on a molecular level, proving the participation of extracellular enzymes of white rot fungi in biotic carotenoid degradation processes.     

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.     

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.     

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.     

Retinoic acid can be produced from excentric cleavage of beta-carotene in human intestinal mucosa.

The hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.     

Effect of beta-carotene accumulation on spectral parameters of microscopic fungi Blakeslea trispora biomass

Parameters of reflectance spectra in visible region and colorimetry for Blakeslea trispora biomass with different content of beta-carotene were determined. Correlation level of pigment accumulation and spectral characteristics were revealed. It is established, that objective control of color characteristics for biomass is possible using measurement of colorimetric parameters.     

New insights into mechanisms of growth and beta-carotene production in Blakeslea trispora

Blakeslea trispora (Mucorales) has economic importance because of its ability to produce large amounts of beta-carotene. To shed light on the actual point of induction and to shorten the following production process, germination and growth of its two mating types, (-) and (+), were observed separately, and the mating point was investigated in lab scale experiments. The (-) mating type showed much faster germination than the (+) type on solid medium and in Erlenmeyer flasks. However, after a first period, the (-) mating type grew clearly slower than the (+) type. In addition, the (-) type branched more vividly than the (+) type. A ratio of 30:1 of (-) and (+) type at an age of 20 h was found to achieve highest beta-carotene yields. Our results provide a comprehensive overview of Blakeslea trispora growth and its product synthesis.     

Separation of β-Factor Synthesis from Stimulated β-Carotene Synthesis in Mated Cultures of Blakeslea trispora

Mated cultures of Blakeslea trispora, grown in a potato extract-glucose-thiamine medium, produced 10 to 15 times more beta-carotene than either unmated culture. Mated, but not unmated, cultures produced a family of compounds (beta factor) which stimulated carotenogenesis in unmated cultures. In fact, carotenogenesis was stimulated sixfold more in minus cultures than in plus cultures. By altering the relative amounts of plus and minus inocula used in fermentations of mated cultures, it was possible to separate the synthesis of beta factor from the synthesis of extra beta-carotene. The plus strain appeared to produce the beta factor; the minus strain appeared to produce most of the extra beta-carotene. Kinetic studies of beta-factor formation suggested that physical contact between the two strains may be required to initiate beta-factor synthesis.