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.     

A review of factors affecting biosynthesis of carotenoids by the order Mucorales

This is a review of factors affecting carotenogenesis by the order Mucorales which includes Phycomyces blakesleeanus, Choanephora cucurbitarum and Blakeslea trispora. The Mucorales have opposite sex types and when mated, -carotene production is increased 15 to 20 times. Trisporic acids are the substances produced upon mating which stimulate carotenogenesis. Structural analogs have been shown to mimic the actions of the trisporic acids. The common denominator of the stimulators is the ionone ring and the hydrocarbon side chain. Secondary metabolism is discussed as well as the use of food byproducts to stimulate, specifically, the production of -carotene by B. trispora.     

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.     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.     

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.     

(−) Mating Type-Specific Mutants ofPhycomycesDefective in Sex Pheromone Biosynthesis

Sutter, R. P., Grandin, A. B., Dye, B. D., and Moore, W. R. 1996. (−) Mating type-specific mutants ofPhycomycesdefective in sex pheromone biosynthesis.Fungal Genetics and Biology20,268–279. We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants inPhycomyces blakesleeanusappear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (−) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of β-carotene. Separate studies with β-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis inPhycomycesis extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (−) pheromone—a reaction missing in the (−) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.     

Preparation of (25R)- and (25S)-26-functionalized steroids as tools for biosynthetic studies of cholic acids

A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3beta-hydroxy-Delta(5)- or a Delta(4)-3-keto-functionality in ring A is described starting from stigmasterol or (20S)-3beta-acetoxy-pregn-5-en-20-carboxylic acid. The obtained compounds are useful as standards for studies of cholic acids. Construction of the side chain was achieved by linkage of steroidal 23-iodides to sulfones prepared from (2R)- and (2S)-3-hydroxy-2-methylpropanoates. Oxidation of intermediate 26-alcohols into the corresponding carboxylic acids ensuring preservation of stereochemistry at C-25 and functional groups in the cyclic part was achieved with sodium chlorite catalyzed by TEMPO and bleach.     

The effect of trisporic acids on the composition of components of the cell wall in Blakeslea trispora Thaxter

The cell wall of Blakeslea trispora was found to contain chitin similar to crustaceaous chitin in physico-chemical properties; this was confirmed by the data of IR spectroscopy and X-ray diffraction analysis. Trisporic acids, hormonal regulators of reproduction, hardly affected the content of chitin, xylan, and other polysaccharides of the cell wall of Blakeslea trispora, but increased the content of protein.     

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.     

Methyl trisporate E. A sex pheromone in Phycomyces blakesleeanus

Combined mating type cultures of Phycomyces blakesleeanus accumulate 41 mg of trisporic acids/l of medium, of which 30% is trisporic acid E. The methyl ester of trisporic acid E exhibits the same zygophore -inducing activity in bioassays with P. blakesleeanus and Mucor mucedo as does the pheromone methyl trisporate C. The structure of methyl trisporate E is 1,5-dimethyl-2-hydroxyl-4-oxo-6-(2'-hydroxyl-6'- methylocta -5',7'-d ien-8'-yl) -5-cyclohexene-1-carboxylic acid methyl ester.     

Identification of 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi, 25 xi,26-hexol and partial characterization of the bile alcohol profile in urine

The nature of the bile alcohols present in urine of an infant with neonatal cholestasis has been investigated. Urine was extracted with Sep-Pak C18 cartridges and a glucuronide fraction was isolated by ion exchange chromatography on Lipidex-DEAP. Following enzymatic hydrolysis and purification on Lipidex-DEAP, the bile alcohols were isolated by high performance liquid chromatography. Fourteen compounds were studied by a combination of microchemical reactions and capillary column gas-liquid chromatography-mass spectrometry. Both C26 and C27 bile alcohols were present. Among the former, three additional isomers of the previously identified 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi-pentol were detected. A new C26 bile alcohol, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi,26 -hexol, was identified, and a 27-norcholestane-pentolone with hydroxyl groups at C-24 and C-25 and a keto group in the ring system was partially characterized. The C27 bile alcohols consisted of cholestanepentols, -tetrolones, and -pentolones. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol (5 beta-bufol), one of its isomers and an isomer of cholestane-3,7,12,24,26-pentol were present. Two cholestanetetrolones and two cholestanepentolones having the keto group in the ring system were partially characterized. The hydroxyl groups in the side chain of the tetrolones were at C-24,26 and C-25,26, respectively, whereas the pentolones had hydroxyl groups at C-24,25 and C-25,26, respectively. The excretion of glucuronidated bile alcohols in urine is suggested to reflect an alternative metabolism of intermediates in the normal biosynthesis of bile acids.     

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.     

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.     

Further study on synthesis and evaluation of 3,16,20-polyoxygenated steroids of marine origin and their analogs as potent cytotoxic agents

A series of new polyoxygenated steroid derivatives with various steroid skeleton moieties were synthesized. Antitumor activity of the compounds against three tumor cell lines (Breast cancer MCF7, lung cancer NCI and oral cancer KB) were evaluated. Compounds with aromatic A ring of this series exhibited the most potent cytotoxicities in all tested cells. The absence of OH at C-16 or lack of cholesterol like side chain at C-20 in the steroid skeleton apparently result in decreased cytotoxicity. The compound became inactive when the side chain contains double bond at C-24-C-25. When hydroxyl group at C-3 was protected no cytotoxicities against MCF7 and NCI and considerable low cytotoxicity against KB cell lines were observed.     

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.     

Physicochemical and biological properties of natural and synthetic C-22 and C-23 hydroxylated bile acids

In order to define the effect of a side chain hydroxy group on bile acid (BA) physicochemical and biological properties, 23-hydroxylated bile acids were synthesized following a new efficient route involving the alpha-oxygenation of silylalkenes. 22-Hydroxylated bile acids were also studied. The synthesized bile acids included R and S epimers of 3 alpha,7 alpha,23-trihydroxy-5 beta-cholan-24-oic acid (23R epimer: phocaecholic acid), 3 alpha,12 alpha,23-trihydroxy-5 beta-cholan-24-oic (23R epimer: bitocholic acid), and 3 alpha,7 beta,23-trihydroxy-5 beta-cholan-24-oic acid. A 3 alpha,7 alpha,22-trihydroxy-5 beta-cholan-24-oic acid (haemulcholic acid) was also studied. The presence of a hydroxy group on the side chain slightly modified the physicochemical behavior in aqueous solution with respect to common BA: the critical micellar concentration (CMC) and the hydrophilicity were similar to naturally occurring trihydroxy BA such as cholic acid. The pKa value was lowered by 1.5 units with respect to common BA, being 3.8 for all the C-23 hydroxy BA. C-22 had a higher pKa (4.2) as a result of the increased distance of the hydroxy group from the carboxy group. When the C-23 hydroxylated BA were intravenously administered to bile fistula rats, they were efficiently recovered in bile (more than 80% unmodified) while the corresponding analogs, lacking the 23- hydroxy group, were almost completely glycine- or taurine-conjugated. On the other hand, the C-22 hydroxylated BA were extensively conjugated with taurine and less than 40% of the administered dose was secreted without being conjugated. In the presence of intestinal bacteria, they were mostly metabolized to the corresponding 7-dehydroxylated compound similar to common BA with the exception of bitocholic acid which was relatively stable. The presence of a hydroxy group at the C-23 position increased the acidity of the BA and this accounted for poor absorption within the biliary tree and efficient biliary secretion without the need for conjugation. 3 alpha,7 beta-23 R/S trihydroxy-5 beta-cholan-24-oic acids could improve the efficiency of ursodeoxycholic acid (UDCA) for gallstone dissolution or cholestatic syndrome therapy, as it is relatively hydrophilic and efficiently secreted into bile without altering the glycine and taurine hepatic pool.     

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.     

Substrate specificity for the hydroxylation of polyoxygenated 4(20),11-taxadienes by Ginkgo cell suspension cultures

Three C-14 oxygenated taxanes isolated from callus cultures of Taxus spp., 2alpha,5alpha,10beta,14beta-tetra-acetoxy-4(20),11-taxadiene 3, 2alpha,5alpha,10beta-triacetoxy-14beta-propionyloxy-4(20),11-taxadiene 4, 2alpha,5alpha,10beta-triacetoxy-14beta-(2-methylbutyryl)-oxy-4(20),11-taxadiene 5, and three deacetylated derivatives of 3, 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene 6, 14beta-hydroxy-2alpha,5alpha,10beta-triacetoxy-4(20),11-taxadiene 7, 10beta,14beta-dihydroxy-2alpha,5alpha-diacetoxy-4(20),11-taxadiene 8, could all be regio- and stereo-selectively hydroxylated at the 9alpha-position by Ginkgo cell suspension cultures to yield a series of new 9alpha,14beta-dihydroxylated taxoids. The effects of functional groups, especially at C-14 of the substrates, on the biotransformation were also investigated. The results revealed that substrates with an acetoxyl group at C-14 could be more efficiently 9alpha-hydroxylated than those with a longer ester chain or a hydroxyl group at C-14. An acetoxyl or hydroxyl group at C-10 had no effect on the conversion rates of the substrates, but substrates with the hydroxyl group (compared with the acetoxyl analogues) could be converted into 9alpha-hydroxylated products more easily.