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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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％。     

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.     

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.     

The correlation between the synthesis of beta-carotene and zygote formation by Blakeslea trispora heterothallic strains

It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.     

Trisporoids and carotenoids in <Emphasis Type="Italic">Blakeslea trispora</Emphasis> strains differing in capacity for zygote formation

The study of sexual interaction in seven (+) and four (−) Blakeslea trispora strains from the All-Russian Collection of Microorganisms showed that all the strains were capable of forming zygotes, albeit to a varying degree. Thus, pairs of strains with active zygote formation and not forming zygospores were identified. It was established that, irrespective of their capacity for zygote formation, all pairs of the (+) and (−) strains synthesized trisporoids in submerged culture. However, zygote-forming pairs accumulated significantly more trisporoids and carotenoids than the strain pairs incapable of forming zygospores. As a result, positive correlation between the capacity for zygote formation in surface culture and trisporoid and carotenoid synthesis in submerged culture was revealed in wild strains.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

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.     

Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii

To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments.