Biochemical mechanisms of temperature adaptation in the (+) and (-) strains of Blakeslea trispora

Evidence obtained with industrial beta-carotene-superproducing (+)T and (-)T strains, which fail to form zygotes, suggests that the lipids in the mycelium of the (-) strain of Blakeslea trispora lack linolenic acid. This circumstance apparently accounts for the fact that the (+) and (-) strains of B. trispora use different adaptive mechanisms to cope with an increase or decrease in cultivation temperature. In the (+) strain, temperature adaptation is based on changes in the ratio between linoleic and linolenic acyls and, also, involves shortening of acyl chains. In addition, the (+) strain contains a larger amount of protective carbohydrates, such as arabitol and trehalose. This strain is characterized by the presence of glycerol, a cryothermoprotector that protects fungal cells at low temperatures. The (-) strain lacks these biochemical mechanisms, but its neutral lipids contain a comparatively high amount of sterols and their esters. These facts enable us to interpret the enhanced thermotolerance of the (-) strain and its capacity to grow at high temperatures in terms of biochemical adaptation. In the light of the data obtained with wild-type and industrial strains, it is suggested that the lack of linolenic acid in the lipids should be considered an essential sex-specific property of the heterothallic strains of Blakeslea trispora.     

Lipid Composition of Cells of Heterothallic Strains in the Developmental Cycle of <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Lipid compositions in mycelium and spores of Blakeslea trispora heterothallic strains were studied. Distinctions between the strains in the ability to synthesize linolenic acid and in optimal growth temperature were demonstrated. The (−) strain grew at a higher temperature and was unable to synthesize linolenic acid, whereas the (+) strain accumulated this acid up to 20% of total fatty acids. The distinctions between the strains remained at different developmental stages (mycelium and spores). A higher thermotolerance of the (−) strain correlated with a high sterol content, which is typical of thermophilic fungi. The lipid compositions of heterothallic strains studied differed in lipid content, their fractional composition, the degree of unsaturation, and carotenoid composition.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 449–453.Original Russian Text Copyright © 2005 by Tereshina, Memorskaya, Feofilova.     

Volatile constituents of Plus and minus strains of Blakeslea trispora

Identification of the predominant constituents produced by the plus and the minus strains of Blakeslea trispora is described. The occurrence of xylenes in the volatiles produced by the plus strain is reported. Additionally, production of 3-hydroxy-3-methylbutanal by the plus strain and dimethyl allyl alcohol by the minus strains were confirmed. Isoamyl alcohol, 1-octen-3-ol, 3-octanol and β-phenethyl alcohol were identified in volatiles from both strains.     

Stimulation of carotenogenesis by penicillin in Blakeslea trispora

Penicilin(1 mg/ml) specifically caused an increase in the synthesis of lipids, (carotenes and sterols) in Blakeslea trispora. The increase in carte     

The correlation between the synthesis of β-carotene and zygote formation by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> 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.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

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.     

Lipid composition of cells of heterothallic strains in the developmental cycle of Blakeslea trispora

Lipid compositions in mycelium and spores of Blakeslea trispora heterothallic strains were studied. Distinctions between the strains in the ability to synthesize linolenic acid and in optimal growth temperature were demonstrated. The (-) strain grew at a higher temperature and was unable to synthesize linolenic acid, whereas the (+) strain accumulated this acid up to 20% of total fatty acids. The distinctions between the strains remained at different developmental stages (mycelium and spores). A higher thermophilicity of the (-) strain correlated with a high sterol content, which is typical of thermophilic fungi. The lipid compositions of heterothallic strains studied differed in lipid content, their fractional composition, the degree of unsaturation, and carotenoid composition.     

Specificity and transformations of the trisporic acid series of fungal sex hormones

Chromatographic and spectroscopic characterizations and comparative bioassay data are given for trisporic acid A, the separate 9-cis- and 9-trans-isomers of trisporic acids B and C, and trisporol C, all obtained from 'mated' (plus and minus) cultures of Blakeslea trispora. All five acids show comparable levels of hormone activity on both the mating types of Mucor mucedo, whereas natural trisporol C more specifically affects a plus strain and the laboratory-derived methyl esters are minus-specific. Similarly plus and minus strains of B. trispora convert trisporol C and the esters into trisporic acids at different rates, and they effect different transformations of administered methyl ¹⁴C-trisporate C.     

Stimulation of carotenogenesis in Blakeslea trispora by cupric ions

Growth of mated Blakeslea trispora in the presence of trace amounts of cupric ions resulted in increases in (a) the amount and rate of initiation of carotenogenesis, (b) the utilization of glucose and Pi and (c) growth. It also caused an increase in trisporic acid synthesis and a two-fold increase in mevalonate kinase activity.     

Sexuality in mucorales

Using varied strains of Blakeslea trispora it is shown that, contrary to earlier reports, a limiting role in hormone synthesis in mated cultures cannot be distinctively assigned to either the plus or the minus partner.     

Biotechnological lycopene production by mated fermentation of <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

A semi-industrial process (800-l fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+) and minus (–) strains has been developed. The culture medium was designed at the flask scale, using a program based on a genetic algorithm; and a fermentation process by means of this medium was developed. Fermentation involves separate vegetative phases for (+) and (–) strains and inoculation of the production medium with a mix of both together. Feeding with imidazole or pyridine, molecules known to inhibit lycopene cyclase enzymatic activity, enhanced lycopene accumulation. Different raw materials and physical parameters, including dissolved oxygen, stirring speed, air flow rate, temperature, and pH, were checked in the fermentor to get maximum lycopene production. Typical data for the fermentation process are presented and discussed. This technology can be easily scaled-up to an industrial application for the production of this carotenoid nowadays widely in demand.     

Protease and carotenogenesis in Blakeslea trispora

Carotene production by single and mated Blakeslea trispora has been studied. On mating and on the addition of trisporic acid to minus cultures there was an increase in the membrane bound neutral protease (MW 126 000) activity. The protease probably acts by inactivating the inhibitory protein of carotene biosynthesis resulting in increased carotenogenesis.     

Trisporoids under the stimulation of carotenogenesis in Blakeslea trispora

The patterns of trisporoid synthesis in joint cultivation of Blakeslea trispora mates have been studied. The pair of the not-zygospore-forming carotenoid overproducer strains T(+) and T(?) was found to synthesize a large amount of trisporoids, which did not differ in biological activity from those in the wild type strains. While the ??-carotene synthesis stimulator ??-ionone increased the amount of trisporoids, the share of trisporic acids in their composition decreased considerably. The lycopene synthesis stimulator 2-amino-6-methylpyridine caused a decrease in the content of trisporoid which had no trisporic acids in their composition. Emergence of a new substance with the maximum absorption at 250 nm, which accounted for up to 45% of the sum of trisporoids, was a general regularity in the action of both stimulators. The combined action of these two effectors resulted in additional stimulation of lycopene synthesis and was accompanied by the disappearance of trisporic acids. The aggregate findings indicate that both carotenogenesis stimulators inhibit the synthesis of trisporic acids, i.e., their action is not mediated by stimulation of trisporoid synthesis.     

Effect of trehalose on the viability of sporangiospores of the mucorous fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Sporangiospores of Blakeslea trispora are in a state of exogenous dormancy, and water is the key factor controlling their germination. A wide range of carbohydrates, ammonium salts, and yeast extract had a weak stimulating effect (less than 50%) on spore germination, whereas amino acids could significantly inhibit this process. Cultivation of B. trispora on sporogenous sucrose- and trehalose-containing media (S and T spores, respectively) resulted in significant changes in spore formation, as well as in the chemical composition of spores and their viability. In the presence of trehalose, the amount of spores increased twofold; spore viability during storage increased as well. All changes in the carbohydrate composition of the cytosol and in the composition of the spore membrane lipids indicated that the dormancy of T spores was deeper than that of S spores, which has a favorable effect on their viability.     

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.     

Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

Carotenoid synthesis and phytoene synthase activity during mating of Blakeslea trispora

Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (−) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional phytoene synthase and lycopene cyclase.