Metabolic Regulation of Trisporic Acid on Blakeslea trispora Revealed by a GC-MS-Based Metabolomic Approach

The zygomycete Blakeslea trispora is used commercially as natural source of â-carotene. Trisporic acid (TA) is secreted from the mycelium of B. trispora during mating between heterothallic strains and is considered as a mediator of the regulation of mating processes and an enhancer of carotene biosynthesis. Gas chromatography-mass spectrometry and multivariate analysis were employed to investigate TA-associated intracellular biochemical changes in B. trispora. By principal component analysis, the differential metabolites discriminating the control groups from the TA-treated groups were found, which were also confirmed by the subsequent hierarchical cluster analysis. The results indicate that TA is a global regulator and its main effects at the metabolic level are reflected on the content changes in several fatty acids, carbohydrates, and amino acids. The carbon metabolism and fatty acids synthesis are sensitive to TA addition. Glycerol, glutamine, and ã-aminobutyrate might play important roles in the regulation of TA. Complemented by two-dimensional electrophoresis, the results indicate that the actions of TA at the metabolic level involve multiple metabolic processes, such as glycolysis and the bypass of the classical tricarboxylic acid cycle. These results reveal that the metabolomics strategy is a powerful tool to gain insight into the mechanism of a microorganism’s cellular response to signal inducers at the metabolic level.     

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.     

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.     

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.     

Trisporoids and carotenogenesis in Blakeslea trispora

The recent data on the pathways of carotenoid biosynthesis, which resulted in revision of the earlier concepts, are analyzed. Trisporoid diversity and their role in the sexual process of mucoraceous fungi, resulting in formation of sexual cells (zygospores) are discussed. Special attention is paid to the role of trisporic acids in carotenogenesis of combined culture of the (+) and (?) strains, in which zygospores are not formed.     

卡那霉素抗性基因在三孢布拉霉菌种中的表达

利用一个带有自主复制子,但缺失自身启动子的源于转座子Tn-5的卡那霉素抗性基因的质粒PVB32,在大肠杆菌中克隆丝状真菌三孢布拉霉DNA中有启动子功能的DNA片段;通过原生质体转化,获得了三孢布拉霉对卡那霉素抗性的表达,且抗性表现稳定,可通过孢子无性繁殖稳定遗传下去。     

Mutants of carotene production in Blakeslea trispora

The filamentous fungus Blakeslea trispora, an industrial carotene source, contains -carotene and precursors of its synthesis — phytoene, phytofluene, lycopene, and -carotene. Strain improvement through mutagenesis is difficult because all life stages are multinucleate. Mutants have been obtained following exposure of wild-type spores to N-methyl-N-nitro-N-nitrosoguanidine. Changes in the colour of the mycelia reflect variations in the accumulation of various precursors and the final product. Quantitative analysis of the mutants leads to the conclusion that the biosynthetic pathway is similar to that of the related fungus Phycomyces blakesleeanus, but the regulation is completely different. In particular, interruption of the pathway does not lead to overacummulation of precursors.     

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.     

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.     

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 -carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene producion results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on -carotene synthesis.     

三孢布拉霉发酵产番茄红素的研究进展

番茄红素是一种重要的类胡萝卜素,在生物体中具有抗氧化、抗衰老、提高免疫力等生理功能。虽然已经有部分企业实现了番茄红素的工业化生产,但产量仍然是制约三孢布拉霉发酵生产番茄红素的重要因素。在本实验室研究的基础上,本文结合近年来国内外学者的研究成果,从遗传育种、发酵工艺优化、化学调控等方面综述了提高三孢布拉霉中番茄红素产量的研究进展,并展望了未来的研究方向。