ISSN 0003-6838, Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 5, pp. 439–454. © MAIK “Nauka/Interperiodica” (Russia), 2006. Original Russian Text © E.P. Feofilova, 2006, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 5, pp. 501Ð519.

Heterothallism of Mucoraceous Fungi: A Review of Biological Implications and Uses in Biotechnology E. P. Feofilova Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, 117811 Russia e-mail: feofi[email protected] Received October 25, 2005

Abstract—The phenomenon of heterothallism in filamentous fungi is reviewed, with emphasis on the discus- sion of hormonal regulation of heterothallic strains of mucoraceous fungi. This process is viewed from the standpoint 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 between 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 communi- cation and transduction is presented with an excursion into microbial endocrinology, a novel field of research in biology. The concluding part of the review analyzes the data on biotechnological implications of the phenom- enon of heterothallism. Specifically, it may be used to obtain a series of isoprenoid compounds, such as β-car- otene and lycopene (which exhibit pronounced antioxidant activity), as well as sterols and trisporic acids. DOI: 10.1134/S0003683806050012

Microbial populations have been considered THE ESSENCE OF THE TERM recently not as separately developing cells but as a com- HETEROTHALLISM AND ITS PREVALENCE munity with a close communication between individual AMONG FUNGI cells [1, 2]. The cells communicate due to the evolu- The reproduction of the majority of fungi displays tionary emergence of the corresponding mechanisms an interesting specific feature. In 1904, Blakeslee [7] allowing signals from external space to be perceived studied the sexual reproduction of mucoraceous fungi, and transformed [3]. These signals are mainly chemical in particular, Blakeslea trispora, and found that zygotes compounds and allow the cell to adapt quickly to exter- were not formed when these fungi were grown from one spore. The main condition for sexual process is nal impacts by changing its chemical composition. In copulation of the mycelia different with respect to sex. turn, the adapted cells generate new chemical com- Blakeslee named these mycelia (+) and (Ð), and the pounds (new signals), whose synthesis is determined phenomenon itself got the name heterothallism. Some- by the stress effect. what later, heterothallism was discovered in other Muc- orales, for example, the fungi of the genus Absidia. The We still know little about the chemical “language” study demonstrated that these fungi differed in the size used by the cells to communicate. The communication and structure of their copulative organs; thus, in this of heterothallic fungal strains during sexual reproduc- case, the (+) and (Ð) designations of mycelia may be tion, when the cells interact via specific molecules replaced with female and male mycelia, and the term called hormones, is most studied in this respect [4Ð6]. heterothallism acquires the meaning of occurrence of The phenomenon of heterothallism, connected with the different sexes; correspondingly, homothallism is most important cellular process—sexual reproduction, regarded as hermaphrodism [8]. whose individual stages are controlled by special hor- Heterothallism, first discovered in Mucorales, is mones involved in the interactions between sexual part- now found in virtually all fungal groups; however, it is ners—is the best illustration of the need to study inter- prevalent and studied best for mucoraceous fungi. About 60 species of the last group are heterothallic and cellular interactions. Consideration of the heterothal- only 20 are homothallic [8]. Heterothallism is also lism in filamentous fungi from this particular widespread in Neomycota in the class Basidiomycetes. standpoint enables a deeper understanding of this Overall, 50 species of Ustilaginaceae display this phe- unique phenomenon and makes it possible to draw an nomenon; of the studied Hymenomycetes species, over analogy with multicellular eukaryotes. 50 are heterothallic.

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Heterothallism is also spread among the organisms and at particular stages of the organism’s development, that were previously ascribed to fungi, namely, hindering determination of their chemical nature. In oomycetes (now the kingdom Chromista). Of lower addition, fungal hormones can perform additional func- eukaryotes, heterothallism is met in marine alga; for tions in the cell metabolism, another factor that makes example, brown alga (genus Fucus) have motile male their identification difficult. For example, the sex hor- gametes and nonmotile eggs, while the genera Ectocar- mone of mucoraceous fungi—trisporic acids (TSA)— pus and Cutleris have motile male and female gametes was first discovered as stimulators of carotenogenesis. [9]. The sexual process of the ciliate (protozoon) Ble- Similar to other organisms, the most essential pro- pharisma intermedia involves two types of cells, type 1 cess for fungi in preserving the species is sexual repro- and type 2. duction, and nature in this case spent much trouble to The heterothallic cells differ according to the mating make this process reproducible, using for this purpose types. The fungi have bipolar and tetrapolar mating regulation with hormones. Due to the specific chemical types [11]. The former is characteristic of Zygomycetes structure, it is hormones that are able to control the but is also found in Ascomycetes and Basidiomycetes. reproduction of heterosexual organisms. It is appropri- In the last group, bipolar mating was discovered in ate to dwell here on the example of fungal hormones, Ustilago gordei. The tetrapolar mating is characteristic the majority of which are lipid-like compounds or, of Basidiomycetes, studied best for Coprinus cenereus more precisely, terpenoids [5]. These molecules are and Schizophyllum commune, and found in several now regarded as evolutionarily the most ancient com- U. maidis species. Zygomycetes have a true bipolar pounds that display “the effect of small molecules” (the mating of (+) and (Ð) mycelia, which differ sexually theory of dynamic play of small molecules in evolu- according to their ability to synthesize sex hormones tion) [14]. Presumably, it is not accidental that the hor- (see below). However, there is another way of provid- mones of fungi and pseudofungi involved in the sexual ing copulation between different species—the so-called process are terpenes or sterols. For example, the female lecherous mating. This mating type was discovered in sex hormone of the water fungus Achlia ambisexua- the parasitic fungi Parasitella, growing on Absidia, lis—antheridiol (previously called hormone A)—is a which is the host. In this case, the parasite can play the steroid C29H42O5) causing hyphal branching. The male role of the opposite-sign copulating strain, and pseu- cells in turn synthesize hormone B (oogoniol), which is dosexual structures are formed [12]. The bipolar mat- a sterol ester (C H O ), inducing the female cell to ing system of certain Ascomycetes is called ideomor- 33 54 6 develop oogonia. Hormone B is 7-keto-ë29-sterol lack- phic. This term means the sequence of the loci of cop- ing a lactone ring, characteristic of antheridiol. The ste- ulating species that do not display the homology to the rol fucosterol is the precursor of both hormones. copulating strain of the opposite sex. This system is characteristic of Neurospora crassa and several other Sterols are also involved in the reproduction of Ascomycetes [12]. pseudofungi. Pseudofungi (Phytophthora and Pythium) are incapable of synthesizing sterols and grow well in the absence of these compounds; however, they require HORMONE-LIKE COMPOUNDS ARE sterols for reproduction. For example, ergosterol stim- THE CHEMICAL LANGUAGE OF SEXUAL ulates Phymatotrichum omnivorum to develop conidia REPRODUCTION OF HETEROTHALLIC FUNGI and Phythophtora sp. to develop sporangia. Sterols are The sexual reproduction of fungi is controlled by also necessary for the sexual reproduction (oospore for- special compounds called hormones. This term was mation) of P. coctorum. ë29 The most active in these α first defined in 1935 for plants [4] and later supple- processes are ë27 sterols with an -configuration at C24 mented with reference to fungi [6]. Thus, hormones are of the side chain—sistosterols and stigmasterols. ë27 compounds whose function is the regulation of sexual sterols are less active, and ë28 sterols display an inter- process. The fungal hormones are classified according mediate activity. Cholesterol stimulates heterothallic to their specific properties into pheromones, growth strains of Phytophthora to form oospores, and this substances, morphogens, and sex factors by analogy effect is more pronounced when cholesterol is applied with sex attractants, plant auxins, and retinoic acid. to both heterothallic strains. Thus, these plant patho- There are other approaches to classifying hormones. gens (Phytophthora and Pythium) require sterols for For example, the hormones controlling the fungal sex- their sexual reproduction, as they cannot synthesize ual reproduction are called erogens; erotactins are the these compounds themselves. An analogy with the hormones that attract the motile cells (gametes) to one insect pathogen Lagenidium giganteum [15] is evident another; and erotropins determine the growth direction here. of nonmotile cells involved in copulation [6, 13]. The sexual reproduction of chytrid fungi (Allomyces Characteristic of the substances called fungal hor- species) is accompanied by development of male and mones is that they act at low concentrations, are very female gametagangia. When nutrient sources are unstable, and are easily degradable under the action of depleted, their gametogenesis results in small male the recipient cell [6]. Moreover, these substances are as gametes containing γ-carotene and colored bright a rule synthesized in very low amounts (10–8 to 10–11 M) orange and larger female gametes. Contact of these

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HETEROTHALLISM OF MUCORACEOUS FUNGI 441 cells brings about the formation of a biflagellate zygote. ogy [22, 23]. The same research direction makes it pos- The hormone of female gamete is sirenin, a sesquiter- sible to answer the question of the role of fungal hor- pene ë15H24O2 [5]; the hormone of male cells, parisin, mones in the pathogenesis of mammals, i.e., of the role is also a terpene [6]. of these signal molecules in the development of mycoses, especially, opportunistic mycoses. Study of In addition to terpenoids, other lipid-like com- the phenomenon of heterothallism in a model of lower pounds stimulate fungal reproduction. For example, eukaryotes, fungi, may form the foundation for devel- arachidonic and oleic acids stimulate zygote formation opment of the science of the physiological and bio- of Saccharomyces cerevisea in the presence of sex pher- chemical sex distinctions of higher eukaryotes and the omones [16]. Cerebrosides stimulate Schyzophyllum role of this phenomenon in stress reactions. According commune to develop fruit bodies [17]. A number of gas- to modern concepts, characteristic of stress is not only eous hormones of microorganisms acting at a concen- a biochemical diversity of reactions but also a sexual tration of –9 M are described. Among them is the 10 dimorphism, which is especially evident, for example, methyl ester of 3-oxypalmitic acid, a new autoregulator in the stress secretion of catecholamines and glucocor- controlling virulence [18]. ticosteroids in humans [24]. In addition, the phenome- As was demonstrated, the ascomycete Arthroderma non of heterothallism is widely used in biotechnology incurvatum has (+) and (Ð) factors, formed by the (+) for the manufacture of medically important iso- and (Ð) hyphae of this fungus, which induce the devel- prenoids: β-carotene, lycopene, zeaxanthin, and others, opment of protocleistothecia. These factors are small displaying valuable properties for drug design, such as readily diffusing hydrophobic molecules, possibly of a antioxidant activity [25]. lipid nature. Ascomycetes, in particular, Aspergillus nidulans, have psi-factors, which induces formation of cleistothecia by homothallic species instead of conidio- PHYSIOLOGICAL AND BIOCHEMICAL phores; in this case, a yellow pigment is secreted into DISTINCTIONS BETWEEN HETEROTHALLIC the medium. This process involves three psi-factors: the STRAINS most active psi-C-factor, 5Ð8-dioxylinolenic acid, and In 1913, Satina and Blakeslea [26] discovered the psi-A-factor (the least active component). The mutants phenomenon of heterothallism, and since that time the deficient in the ability to form sexual and vegetative biochemical distinctions between heterothallic Mucor spores synthesize these factors most actively [19]. The strains have been intensively studied. Preliminary stud- lipid extracts from hyphae of many fungi influence the ies demonstrated that one spore never developed into reproduction process. For example, the lipid extracts the mycelium capable of forming zygotes and that the (SF-factor) of cocultivated heterothallic Pyrenopeziza sexual process required the interaction of two mycelia brassicae strains inhibit the asexual spore formation of different sexes. Initially, these researchers desig- and intensify sexual reproduction. The development of nated the heterothallic strains as (+) and (Ð) and then fertile perithecia by a number of fungi (Nectria haema- demonstrated that the strains differed considerably in tococca) is also stimulated by linolenic acid, and this the biochemical activity, in particular, with respect to particular acid in the lipid extracts is presumably the the Manoilov reaction [27]. This reaction, discovered most active component [20]. Another metabolite of by E.O. Manoilov, a physician, in 1922, allowed the Fusarium and Gibberella—the mycotoxin zearale- male blood to be distinguished from female using spe- none—is a lactone of resorcylic acid, which stimulates cial reagents. It appeared later that the same reaction the development of perithecia [21]. All the compounds allowed the sex of dioecious plants to be detected and, and factors described above may be attributed to the as mentioned above, the sex of heterothallic Mucorales class of morphogenic hormones, regulating reproduc- strains. It was also found that water and alcohol extracts tion, whose chemical structure has yet to be determined from strain (+) mycelium contained larger amounts of precisely. catalase and peroxidase and more actively reduced per- The chemical structures of over 12 fungal hormones manganate and tellurium salts. As we see it, of special involved in the sexual process are already known; how- interest are the data obtained in 1927 [27] that the ever, the studies into mucoraceous fungi were the most Manoilov reaction, distinguishing between the male successful. Thus, the further review will elucidate the and female blood according to a specific staining, following issues: the physiological and biochemical allowed also for a distinct differentiation between (+) distinctions between Mucorales (+) and (Ð) strains; the and (Ð) strains. Comparing this data with the reactions biosynthesis of their zygogenic hormone—trisporic to sex in plants and humans suggested that the (+) strain acids (TSA); and the modern concept of TSA function of Mucorales corresponded to female sex and (Ð) strain from the standpoint of secondary metabolism. We will to male. Additional data indicating considerable differ- also consider the new data on the ability of fungal hor- ences between the hormonal regulation of (+) and (Ð) mones to interact with mammalian hormones. The sim- strains of Mucorales during zygote formation were ilarity between hormones of fungi and mammals and obtained in the 1970s. their interaction are so important that this has given The study of the distinctions between heterothallic birth to a new branch of biology, microbial endocrinol- fungal strains is rather difficult, because, unlike eukary-

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442 FEOFILOVA otes, the (+) and (Ð) strains of Mucorales are morpho- media tested, (+) strains form higher numbers of spores logically similar. As was mentioned above, the majority and have a shorter terminal phase (sporogenesis phase of researchers failed to find morphological differences 2); the difference in duration of this phase amounts to between the heterothallic strains of Mucorales; none- about 40 h [32]. theless, examination of mycelium [28] at the initial A stable trait that manifests itself independently of phase of intensive growth demonstrates that the myce- the medium composition is the response of heterothal- lium of (Ð) strains is morphologically more uniform lic strains to addition of sex hormone, trisporic acids, to and lacks the “ampule-like” enlargements characteris- the medium. We will detail below the synthesis of this tic of the (+) strain and cocultivated heterothallic hormone and its biological function in zygote forma- strains. In addition, the (+) strain in a liquid culture tion of heterothallic Mucorales. Here, we review the develops a hypertrophied expanded mycelium as if data on the specific aspect of TSA action, namely, their formed of connected hyphae. These specific morpho- effect on carotene synthesis, demonstrating that only logical features of B. trispora (+) strain are retained the B. trispora (Ð) strain is an active producer of this even when this strain is cocultivated with an opposite- isoprenoid. The experimental data available so far dem- sign strain of other Mucorales representatives, for onstrate that this (Ð) strain synthesizes 12- to 15-fold example, with Cunninghamella elegans. Another diffi- larger amounts of β-carotene compared with the culty is the fact that several homothallic strains display (+) strain [31Ð35]. This stable trait formed the basis for a trend for heterothallism, as well as certain heterothal- development of a specialized biotechnology for caro- lic strains, to homothallism. For example, homothallic tene production (see below). strains of Rhizopus pusillus formed mature zygotes with a heterothallic strain of this fungus [29], hindering The differences between the (+) and (Ð) strains of determination of several specific physiological and bio- B. trispora in the rates of biosynthesis of the com- chemical features. pounds that have a common precursor, acetyl-CoA, were also demonstrated for other isoprenoid com- The distinctions between heterothallic strains were pounds, in particular, sterols and ubiquinone Q9; how- discovered in their requirements for the nutrient ever, this effect appeared only with the addition of TSA. medium. The first studies were aimed at the search for For example, in the presence of TSA, the (Ð) strain pro- differences in consumption of carbon and nitrogen duced six- to sevenfold larger amounts of ubiquinone sources by the strains of different sexes. For example, Q9 compared with the (+) strain, which displayed inhib- the (+) strain of Mucor hiemalis assimilated maltose ited synthesis of this isoprenoid [36]. The difference better than its (Ð) strain, while the latter did not con- between heterothallic strains in biosynthetic rates of sume sucrose [26]. According to later data, the other lipids, namely, sterols (two- to threefold), was (−) strain of Ç. trispora is able to assimilate starch, also demonstrated. It is found that, in the biosynthetic whereas the (+) strain is not [30]; urea is more intensely sequence of carotenoid synthesis, TSA significantly consumed by (+) strains, whereas starch, peptone, lac- stimulates only phytofluin and β-carotene syntheses by tose, and trehalose are more intensely consumed by the (–) strain [37] without influencing the other colored (−) strains. Especially pronounced differences between polyenes. the consumption of carbon and nitrogen sources were detected between the commercial (+)T and (Ð)T strains, Study of the lipid compositions of mycelium and spontaneous mutants, which are β-carotene overpro- spores of the B. trispora heterothallic strains [38, 39] ducers. demonstrates that, unlike (+) strain, the phospho- and glycolipids of the (Ð) strain lack linolenic acid; more- More reliable data about the physiological and bio- over, this fatty acid in mycelium is absent even in neu- chemical distinctions between heterothallic strains tral lipids. Characteristic features of heterothallic were obtained when studying their growth intensities. It strains discovered are also the amount of synthesized was demonstrated involving a large number of (+) and lipids and the composition of phospholipids. For exam- (Ð) strains that they differed in the rate of biomass accu- ple, the spores and mycelium of the (+) strain contain mulation, and this difference depended on the compo- considerably larger amounts of lipids and form more sition of the growth medium and the ability of heteroth- phosphatidylcholine and phosphatidylethanolamine, allic strains to form zygotes [31]. For instance, whereas the neutral lipids of the mycelium of the (+) strains displayed a higher growth rate on solid nat- (−) strain contain more sterols and their esters; more- ural media. On synthetic Goodwin media in the case of over, this specific feature is more pronounced when submerged cultivation, certain (Ð) strains accumulated comparing the stylospores (STS) of heterothallic a lager amount of biomass than (+) strains. The ability strains (the difference is almost eightfold). It is also of (Ð) strains to accumulate more biomass was also shown that STS and sporangiospores (SPS) of the observed for the commercial strains, overproducers of (+) strains contain lycopene, which is undetectable in carotene, that lost their ability to form zygotes. the dormant cells of the (Ð) strain. It is demonstrated that the heterothallic strains of The composition and amount of the cytosol carbo- B. trispora also display differences in the rate of spore hydrates of these heterothallic strains are also different formation and the number of spores produced. In many [38]. For example, the mycelium of the B. trispora (+)T

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 HETEROTHALLISM OF MUCORACEOUS FUNGI 443 strain contains glycerin and larger amounts of trehalose decrease in respiration intensity, the protein content in and other protector carbohydrates compared with the mycelium is reduced and new proteins appear, which is (−) strain. STS and SPS of heterothallic strains also dif- seen as a qualitative change in the protein composition fer in the amount and composition of carbohydrates; for in electrophoretic patterns. For example, the number of example, the spores of the (+) strain contain more treh- proteins by the beginning of carotene synthesis alose than the spores of the (Ð) strain; and the trehalose decreases from 23 to 15Ð16 and reduces to 9Ð10 bands content is higher in the STS formed at a higher temper- by the beginning of TSA synthesis [28]. ature. The beginning of carotene synthesis coincides with All these distinctions in the lipid and carbohydrate the drop in the levels of SH groups, which continue compositions of heterothallic strains have an essential decreasing during TSA formation. These data sug- effect on their adaptive potential and the optimal tem- gested that the introduction of sulfhydryl groups into perature for mycelial growth. As demonstrated by the the medium for growing B. trispora had to influence the example of adaptation of the mycelium to temperature synthesis of isoprenoids. Indeed, addition of cysteine to stress [38], the ratio of acyls of linoleic and linolenic the medium for growing mycelium at the beginning of acids changes under these conditions in the (+) strain, the growth inhibition phase resulted in a decrease in and the mechanism of acyl chain shortening becomes TSA and carotene production and an intensification of involved, which assists the change in the saturation mycelial growth. In contrast, supplementing the media degree (SD) of the lipid bilayer. These biochemical with the compounds that block SH-groups intensifies mechanisms are absent in the (Ð) strain; however, carotene and TSA syntheses [40]. higher contents of sterols and their esters are detected; therefore, the main adaptation mechanism for the Later, the growth conditions were found that (−) strain due to inactive ∆12-desaturase is the change in allowed the syntheses of TSA and carotene to be sepa- lipid SD with involvement of acyls and the “sterol rated. It is demonstrated that the addition of acetic acid mechanism.” Presumably, this is the particular explana- or β-ionone to the cultivation medium and a change in tion for the fact that the (Ð) strain grows at a higher tem- the aeration degree result in a considerable decrease in perature and is incapable of adapting to lower cultiva- TSA content. For example, β-ionone increases the car- tion temperatures. These differences, in particular, the otenoid yield almost threefold with a concurrent signif- inactive ∆12-desaturase, are detected in both wild icant decrease in TSA production [41]. Note also that (−) strains and the commercial (Ð) strain, thereby indi- the inoculum amount and a certain ratio (+) to (Ð) cating that this phenomenon is of a general character B. trispora mycelia in it have different effects on bio- and may be used as a main criterion for determining the syntheses of carotene and TSA. However, the syntheses sex of heterothallic strains. of carotene and TSA are tightly connected, as is evident from the experiments on supplementing the growth medium with inhibitors of carotenogenesis—pyridine THE CONDITIONS NECESSARY derivatives and diphenylamine. These compounds pre- FOR SYNTHESIS OF ZYGOGENIC vent formation of carotenoids; in particular, 4-ami- SEX HORMONE—TRISPORIC ACIDS— nopyridine blocks carotene cyclization, while dipheny- BY HETEROTHALLIC Mucorales STRAINS lamine interferes with the dehydrogenation of colorless Trisporic acids are oxidized derivatives of β-caro- polyenes—phytoin and phytofluin. In all cases, a virtu- tene containing 18 carbon atoms. TSA are a mixture ally complete inhibition of carotene synthesis and pro- containing TSA A, B, C, E, and D. The predominant duction of TSA were observed. These data demonstrate acids are C and B, differing in that TSA C contains a that the syntheses of carotenoids and TSA occur most carbonyl group in its side chain and TSA B contains a likely at the late trophophase, when the growth inhibi- hydroxyl group. The maximal amounts of carotenoids tion commences; i.e., both processes are tightly inter- and TSA are synthesized during cocultivation of the connected [37]. B. trispora heterothallic strains in the mid- and late sta- tionary growth phase. Note that carotenoid synthesis REGULATION AND INTERCONNECTION insignificantly leaves behind the synthesis of TSA. In Β this process, the synthesis of TSA C precedes TSA B OF -CAROTENE AND TSA SYNTHESES synthesis; the later compound is detectable only in a BY HETEROTHALLIC Mucorales STRAINS 3-day-old mycelium [28]. The regulation of carotene and TSA syntheses were By the beginning of carotenoid and TSA syntheses, studied using translation and transcription inhibitors the metabolism of mycelium of cocultivated heterothal- [42]. The transcription inhibition by actinomycin D lic strains undergoes essential changes. The high respi- suppresses both TSA and carotene syntheses; note that ration level characteristic of an intensively growing the produced amount of carotene decreases 24-fold ver- mycelium drops almost twofold by the moment caro- sus a 2.5-fold decrease in the biomass. These data dem- tene is produced. This corresponds to a parallel onstrate that, under the conditions used, actinomycin D decrease in the contents of electron transfer chain com- influences the syntheses of secondary metabolites in an ponents—cytochromes a, b, and c1. Along with the addressed fashion. Analogous results were obtained

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 444 FEOFILOVA using another inhibitor, acridine orange, which blocks plates on ribosomes and the posttranslational level [28, or impairs the template in the way of RNA polymerase. 43]. The glutaramide antibiotic cycloheximide was used to block translation. Preliminary experiments with THE CONTROL OF SEXUAL DIFFERENTIATION labeled leucine demonstrated that cycloheximide at a IN Mucorales AND SYNTHESIS concentration of 50 µg/ml inhibited the protein synthe- OF SEX HORMONE (TSA) sis in B. trispora and Choanephora conjuncta more than One of the main conditions required during the sex- threefold. It was also found that cycloheximide had no ual process in Mucorales is a physical interaction nonspecific side effect, as the normal growth processes between (+) and (Ð) mycelia. The formation of of the fungal culture were restored upon removal of this zygospores is preceded by the growth of specialized inhibitor. These experiments demonstrated that even a hyphae—(+) and (–) zygophores—towards one 1-h translation inhibition by cycloheximide suppressed another, their direct contact, and the fusion of their con- the syntheses of β-carotene and TSA, also confirming tents; then the sexual spore is formed. Projections are that their syntheses are interconnected. formed on the surface of zygophores before their con- The above data suggest that the carotene-synthesiz- tact, which may be regarded as the origin of progameta- ing enzymes are inducible and, presumably, are synthe- gangia. The heterothallic mycelia interact and the zygo- sized before pigment formation under the effect of the phores grow towards one another with the help of a spe- corresponding derepressor or inducer. Indeed, compar- cial chemical language—sex hormones. This was ison of B trispora protein electrophoretic patterns and observed in experiments where heterothallic mycelia . growing on agar surface were separated with a semiper- the kinetics of carotene accumulation revealed a protein meable membrane. The fungal hyphae growing in the band (or a band containing several proteins) designated vicinity of the membrane were colored bright yellow arbitrarily as no. 7 [43]. This band is absent in 15-h-old and formed zygophores, although they did not contact mycelium and appears somewhat later, when the caro- directly. These data suggested that the (+) and tene formation in mycelium commences. With the (−) strains of heterothallic fungi synthesized certain development of B. trispora, the intensity of band no. 7 substances capable of passing through the semiperme- decreases to a complete absence in the cultures with able membrane and inducing the growth of zygophores. arrested protein synthesis. The correlation between car- otene synthesis and protein no. 7 was confirmed in the Further experiments demonstrated that volatile experiments when the growth medium was supple- compounds were involved in the sexual process of these mented with carotene inhibitors—diphenylamine and fungi in addition to the water-soluble substances able to 4-aminopyridine. These compounds inhibited carotene pass through the semipermeable membrane. They synthesis and, correspondingly, reduced protein band induced the growth of zygophores of heterothallic no. 7 in electrophoretic patterns. In contrast, addition of strains towards one another. The following experiment stimulators of carotenogenesis, for example, 5-ethyl-5- demonstrated the existence of volatile hormones. The (2-amyl)-barbiturate, which increases carotene synthe- agar blocks with (+) and (Ð) mycelia of M. mucedo sis almost twofold, and β-ionone resulted in an evident grown on the surface were placed in a closed space at a increase in band no. 7. Thus, the ability of β-ionone to distance of several millimeters from one another. These induce the synthesis of carotenogenic protein no. 7 in conditions prevented the liquid diffusion; however, the combination with the results on the effects of transla- zygophores grew towards one another [46, 47]. tion and transcription inhibitors described above sug- Thus, it was demonstrated by the 1950s that Muco- gest that β-ionone stimulates a de novo synthesis of car- rales possessed two types of sex hormones responsible otenogenic enzymes and that β-ionone acts at the level for the development of progametagangia and the vola- of template translation on ribosomes and has a stabiliz- tile compounds causing the phenomenon of zygoto- ing effect on the existing carotenogenic templates [43]. pism. The research that allowed the chemical structure of one hormone to be clarified was commenced in 1957 TSA not only increases carotene synthesis in [47]. Plempel et al. [47, 48] postulated that the sexual B. trispora, but also stimulates the syntheses of water- process of the heterothallic fungus M. mucedo com- soluble proteins and RNA and intensifies the inclusion prised three successive stages. (1) The heterothallic of labeled leucine into B. trispora proteins. Disc-PAGE mycelia grown vegetatively without the sexual partner assay demonstrated that the addition of TSA into a form specific substances—(+) and (–) progamones— growth medium increased the intensity of protein band which diffuse into the substrate. (2) The progamone no. 7 and assisted the appearance of a new protein band synthesized by the mycelium of the (Ð) strain induces designated arbitrarily as band no. 2 [44]. The experi- the mycelium of the (+) strain to produce another spe- ments with cyclohexamide and actinomycin D suggest cific compound, called a (+) gamone, and the (Ð) strain that TSA synthesis is stimulated at a posttranscriptional when meeting the (+) gamone forms a (Ð) gamone. The level. Thus, although β-ionone and TSA are stimulators (Ð) gamone induces the mycelium of the (+) strain to for β-carotene synthesis in B. trispora, their syntheses develop sexual organs, zygophores. (3) The (+) and (Ð) are regulated at different levels—translation of tem- zygophores evolve the volatile substances responsible

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 HETEROTHALLISM OF MUCORACEOUS FUNGI 445 for zygotropic reactions, which assist the growth of reach the protoplasm of the partner. In this process, the zygophores of opposite sign towards one another; the precursors and the products formed are similar for the result is copulation and zygote formation [48]. The (+) and (Ð) strains; however, the sequences of chemical attempts to separate (+) and (Ð) gamones failed, since, transformations are different, providing the possibility according to the data of Plempel, they have a similar of collaborative synthesis. structure. The molecular weight of gamones is small: The initial stages of carotene degradation are the 314 in acetone and 368 in chloroform; their total for- least studied in the sequence of biosynthetic reactions mula is ë20ç25é5. According to chemical assays, the leading to TSA production described above. The first molecule of gamones contains hydroxyl groups, an pathway, discovered in the 1970s [53, 54], implies that ester bond, and a double bond between carbon atoms. the oxidative cleavage of β-carotene at the central dou- The nature of the zygogenic sex hormone of Muco- ble bond gives retinal. According to the second path- rales was finally clarified thanks to the discovery (1964) way, proposed considerably later [57], the enzymatic of a factor that provided a multifold stimulation of hydroxylation of β-carotene at C4 followed by asym- β-carotene synthesis by B. trispora (Ð) strain [49Ð51]. metric cleavage of the polyene chain gives isocryptox- This factor, called the β-factor or the zygogenic sex anthine. This pathway is similar to the synthesis of hormone of Mucorales (ZSH), which is a mixture of abscisic acid [58] in plants and certain fungi. According trisporic acids, was presumably the analogue of Plem- to [59, 60], the particular pathway of TSA synthesis is pel’s gamones. Later, the compounds controlling the determined by the level of active oxygen species (AOS) sexual process in Mucorales were thoroughly studied in mycelium and the intensity of oxidative stress. The (as was believed at that time); this sexual process is a second pathway is predominant at a low AOS level, unique phenomenon, which has no analogues in its reg- whereas an activation of oxidative processes switches ulation complexity and involves two partners. the TSA synthesis to the first pathway to form β-apo- 13-carotenone; in this case, the level of carotene degra- The observation that the sexual process in these dation increases considerably. It is assumed that the fungi is accompanied by a considerable increase in the β first pathway, determined by a high AOS level, is pre- -carotene content in mycelium (see above) consider- dominant [60]. ably assisted the study into Mucorales ZSH. The possi- bility of TSA formation from β-carotene was confirmed The data demonstrating a similarity of fungal ZSH by the following data: (1) the absence of TSA when car- and abscisic acid in their chemical structure confirm to otene synthesis is inhibited by diphenylamine [37]; (2) a certain degree that TSA can be synthesized according the absence of TSA in the Phycomyces blakesleeanus to the second pathway. As is demonstrated, both com- mutant strains that do not synthesize β-carotene [52]; pounds stimulate the ability of several Mucorales spe- and (3) inclusion of the label from β-carotene into TSA cies to reproduce in an asexual manner; influence simi- [53]. The mechanism of TSA formation, involving the larly the growth, respiration, and seed germination of interaction of two heterothallic strains (Fig. 1), was several plant species, for example, pea cultivar Pioner; finally clarified in [53–56]. It was demonstrated that intensify the synthesis of phytoin, a biosynthetic pre- cursor of carotenoids, in carrot tissue cultures; have a TSA is a ë18-apocarotenoid formed by oxidation of the carotene molecule. The successive biosynthetic path- trend of inhibiting the development of malignant ways (Fig. 1) common for both heterothallic strains are tumors (Ehrlich ascites); and kill certain insects, for the following: (1) 14-15-oxygenase catalyzes an oxida- example, the caterpillars of the gipsy moth, without tive cleavage of the β-carotene molecule to give retinal; inhibiting other morphogenetic stages, as is typical of (2) dehydrogenase oxidizes retinal to retinoic acid; (3) other terpenoids [28]. lyase transforms retinoate to ë18-ketone via CoA deriv- The data obtained in studying other carotene-syn- ative; (4) monooxygenase oxidizes the C4 group in thesizing fungi, for example, Fusarium oxysporum cyclohexane ring to the alcohol group, and this product [61], also demonstrate that AOS influences the synthe- is reduced at C11 and C12 to form 4-dihydroxy- sis of carotenoids. According to these data, carotenoids trisporin; and (5) monooxygenases of both heterothallic of this fungus are synthesized in response to oxidative strains oxidize the methyl group at C1 of this com- stress, as is confirmed by numerous experiments where pound to alcohol to form 4-dihydroxytrisporol. The carotenogenesis was induced in the presence of the (+) strain has a specific enzyme that activates the oxida- exogenous photosensitizer methylene blue (MB) and a tion of the alcohol group at C1 by dehydrogenase to direct correlation between carotenogenesis and MS carboxyl group, forming 4-dihydrotrisporate. The concentration was demonstrated. The same dependence (−) strain also has specific enzymes, for example, was confirmed by the experiments on the effect of red NADP-dependent dehydrogenase, which oxidizes the light on the synthesis of fungal pigments and carotene C4-hydroxyl group to ketone to form trisporol. level in the F. oxysporum grown in an argon atmo- The following biological interactions of heterothal- sphere. lic cells underlie the reactions described above: the It was also shown that TSA was undetectable in the strains of opposite sexes must be in tight contact with cocultivated mucoraceous fungi lacking carotene, pre- one another and their metabolites must diffuse and sumably, because TSA was synthesized in a very small

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 446 FEOFILOVA

I 15'

15

(+) (–)

II O

(+) (–)

OH III O

(+) CH2OH (–)

OH O

IV V R R

COOH CH2OH

(+) (–) O O 4 VI 1

COOH

Fig. 1. Collaborative biosynthesis of trisporic acids by the (+) and (Ð) strains of B. trispora [111]: (I) β-carotene; (II) retinal; (III) 4 dihydrotrisporol; (IV) 4-dihydrotrisporic acid; (V) trisporol; and (VI) trisporic acid. amount compared with the carotene-synthesizing Note that the results described above are possibly fungi, for example, B. trispora. Nonetheless, the cocul- connected with the fact that only the B. trispora tivation of B. trispora (Ð) strain with the C. elegans (−) strain is able to oversynthesize carotene upon addi- strain unable to synthesize carotene resulted in produc- tion of Mucorales ZSH. Note in this connection that tion of an insignificant amount of β-carotene (41 µg per TSA is capable of displaying biological functions 1 g dry mycelium) and TSA (0.004 g/l). These data sug- depending on the changes in the chemical structure. For gest that mucoraceous fungi possess at least a part of this purpose, TSA methyl esters were produced where, the biosynthetic pathway to trisporic acids indepen- for example, the carbonyl group was reduced to dently of their ability to accumulate carotene [28]. hydroxyl, carbonyl and ester groups were transformed

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 HETEROTHALLISM OF MUCORACEOUS FUNGI 447 into hydroxyl, or carbonyl group in the cycle was posed above or by the fact that the colorless strains are replaced with hydroxyl, etc. [62]. It was shown that the able to synthesize TSA precursor despite the absence of ability of the B. trispora (Ð) strain to synthesize caro- carotene. Recalling that only (Ð) strains are capable of tene depended on the structure of TSA analogues. The intensifying the syntheses of carotene and other iso- esterification of carboxyl group in the core of the TSA prenoids under the effect of TSA, the results of the molecule decreased the intensity of the carotene-stimu- experiments in question may be regarded as a confirma- lating effect of this ester on the (Ð) strain 1.5Ð2.0-fold tion of the essential physiological and biochemical dis- compared with the effect of TSA. Reduction of the car- tinctions between heterothallic strains. In addition, this bonyl group in this TSA ester further decreased the suggests that TSA formation does not require a consid- stimulatory effect on carotene synthesis. The biological erable carotene accumulation in mycelium and that the activity of the compound with two hydroxyl groups formed carotene or a certain carotene precursor is dropped 4-fold; with three hydroxyl groups, 7- to directly used for synthesizing TSA. Appropriate here 8-fold; and with the reduced keto group in TSA, are the data that the UV-absorbing substances similar to 50-fold. Thus, the replacement of the carboxyl group in trisporoids were detected in Mortierella spp., a repre- TSA molecule with the alcohol group results in a con- sentative of the order Mucorales. These substances siderable decrease in the stimulatory effect on carotene induce zygophore formation in M. mucedo and stimu- synthesis by B. trispora culture [62]. late carotene synthesis and inhibit mycelial growth in Research into the significance of functional groups P. blakesleeanus [65]. The homology to DNA frag- in TSA molecules in connection with carotenogenesis ments of the gene encoding the key enzyme of TSA was continued in the studies of the effect of TSA natu- biosynthesis—dehydromethyltrisporate dehydroge- ral analogues on this process [63]. It was demonstrated nase [EC 1.1.1.-]—was discovered in other representa- that abscisic acid, a plant dormancy hormone, inhibited tives of the genus Mortierella; however, their TSA con- β-carotene production, whereas vitamin A, on the con- tent is so low that it does not allow a reliable conclusion trary, intensified carotene synthesis, but unlike TSA, on the presence of this substance. Nonetheless, bioas- however, only if the medium was supplemented with say demonstrated that the extracts obtained from these sunflower oil and Tween [64]. These data suggested Mortierella representatives and containing trisporoids that, in addition to TSA, the carotenogenesis in Muco- induced zygophore formation similar to the extracts rales is regulated by another isoprenoid, namely, ret- from M. mucedo and B. trispora. The above data on inol, and presumably according to a negative feedback interspecies matings of heterothallic mucoraceous pattern. fungi may be also interpreted from another standpoint. It was demonstrated [66] that the sexual process com- mences only when the growth of vegetative mycelium ARE TRISPORATES THE SIGNAL MOLECULES stops. However, strains of different sexes have different IN SEXUAL MORPHOGENESIS OF ALL growth rates, while the growing hypha is unable to pro- Mucorales FUNGI AND WHAT ARE ADDITIONAL duce pheromones (TSA precursors or trisporoids). Pre- TSA FUNCTIONS? sumably, the difference in the growth intensity prevents As was believed [55, 56], the biosynthetic pathway P. blakesleeanus and B. trispora as well as B. trispora to TSA (Fig. 1) was common for all Mucorales. How- and M. mucedo from forming zygophores during cocul- ever, a number of facts allow this statement to be sup- tivation, as a coordinated arrest of the growth is absent. plemented. First, according to the scheme in question, As was mentioned above, heterothallic strains of β-carotene is the precursor of trisporates. However, B. trispora differ in the growth rate and intensity and, several representatives of the order Mucorales, such as possibly, these distinctions are most essential, as the Cunninghamella japonica, C. echinulata, and others, do beginning of sexual reproduction is connected with the not produce carotenoids, have a colorless mycelium, synthesis of sexual precursors by the (Ð) strain, requir- but develop zygotes during cocultivation of their het- ing the arrest of mycelial growth. erothallic strains. On the other hand, the carotene-syn- The recent data described above suggest a revision thesizing Mucorales species, such as B. trispora and of the TSA role in the sexual process of Mucorales. Choanephora conjuncta, produce, respectively, 100 and First, recall that B. trispora is the only fungus display- 80 mg/l TSA and 1500 and 1000 mg/g dry mycelium ing considerable amounts of TSA. As was mentioned β-carotene on synthetic media [28]. above, a part of mucoraceous fungi does not virtually The experiments on cocultivation of heterothallic synthesize this isoprenoid; however, they form zygotes. strains belonging to different Mucorales species The study of the mutant Phycomyces strains capable revealed an interesting fact—TSA and carotene are and incapable of synthesizing carotene gives to a cer- synthesized only when the (Ð) strains of carotene-syn- tain degree the answer to this question. Three mutant thesizing fungi are cocultivated with colorless (−) strains—M3, M4, and M5—defective for synthesis (+) strains, for example, (Ð) B. trispora and (+) C. ele- of early trisporoid precursors (according to Plempel, gans. These data are explainable either by the fact that the authors call these compounds pheromones) and the (−) strain of B. trispora itself is able to synthesize incapable of synthesizing carotene were obtained [68]. TSA notwithstanding the collaborative scheme pro- It was found that mutants M3 and M4 synthesized a

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 448 FEOFILOVA previously unknown precursor of pheromones, which Although TSA synthesis and the regulation of the was then metabolized to form the already identified sexual process in Mucorales have been studied since TSA precursors. Unexpectedly, this precursor appeared the 1970s, the role of TSA is not completely clear. to be water-soluble, i.e., must have at least two polar According to one of the recent publications on trispor- functional groups containing oxygen atoms. Carotene oids of Mucorales [65], the role of TSA in regulation of derivatives with one oxygen function are known, the sexual process of these fungi was never clearly namely, retinal, retinol, and retinyl acetate, but they are determined in experiments (the extracellular function water-insoluble. Therefore, it was proposed that the and action of TSA were not studied in detail). It is new water-soluble precursor of pheromones may be assumed that the penetration of TSA through the fungal 4-dihydroxytrisporin (a trisporoid). However, attempts cell wall depends on the charge of the cell wall and that to isolate this precursor failed. Then a compound was TSA acts as a weak acid; however, the underling mech- synthesized, designated as Cpd-P regarded putatively anism is still vague [56]. The presence of precursors is as an intermediate precursor of 4-dihydroxytrisporin. sufficient for Mucorales heterothallic strains to provide The grounds for this assumption were the comparison the interaction between (+) and (Ð) partners and induce of chemical structures of three compounds: Cpd-P, sexual morphogenesis. The function ascribed to TSAÐ 4-dihydroxytrisporin, and (Ð) pheromone trisporin. All stimulation of the synthesis of sex hormone precursorsÐ these compounds differ only in the chemical groups at may be substituted with a self-induction of the hor- C4: Cpd-P has two hydrogen atoms, 4-dihydroxy- mones themselves. It is unlikely that TSA is the com- trisporin has a hydroxyl group, and trisporin has an mon sex hormone for all mucoraceous fungi, taking oxygen atom. These structural differences suggested into account the diversity of trisporoid isoforms in var- that the biosyntheses of Cpd-P and the (Ð) pheromone ious species, as well as the presence of Cpd-P in the trisporin differ in the presence of two additional enzy- colorless strains. Note also that in addition to the three matic reactions that are absent in the wild (Ð) strain of TSA types (A, B, and C), TSA D and E, synthesized by P. blakesleeanus. It appeared that the colorless mutants heterothallic strains separated by membrane, were are unable to synthesize trisporin and, presumably, pro- identified [67]. Therefore, a new approach to the role of duce the chemically synthesized compound Cpd-P, TSA from the standpoint of secondary metabolism may which is the precursor of 4-dihydroxytrisporin for col- be of certain interest. orless strains. Moreover, the strains containing caro- TSA is a secondary metabolite, i.e., the product syn- tene, in particular, (Ð) strains, do not synthesize this thesized by cells at the final stages of culture growth. compound. However, TSA precursors are formed considerably ear- Further studies confirmed that Cpd-P transforma- lier, possibly, during the early growth inhibition phase. tion into TSA occurred only in the four cocultivated (+) In addition to TSA, B. trispora culture produces and (Ð) strains incapable of synthesizing carotene. another secondary metabolite, β-carotene, a TSA pre- Moreover, the observed level of TSA production was cursor. Moreover, carotene is the starting compound for very low. This is also confirmed by the data on mutants sporopollenin [69]. Sporopollenin is a component of containing the Car R-gene, encoding a defect cyclase the cell wall of asexual and sexual cellsÐsporan- (the enzyme converting lycopene into β-carotene). The giospores, stylospores, and zygotes. These data demon- addition of Cpd-P when cultivating these strains on strate that B. trispora has a complex regulation of solid media results in the appearance of the lycopene β-carotene synthesis due to the bifunctional pathway of band. its utilization during secondary syntheses. Along with The data above suggest the following inference. The the function of the sex hormone, TSA stimulates caro- initial stages in the synthesis of TSA precursors of the tene synthesis with the (Ð) strain as the only derepressor Mucorales strains capable of synthesizing β-carotene of the process. However, a question arises here on why (SC strains) differ from those of the strains incapable of TSA is readily soluble in the hydrophilic culture liquid synthesizing this carotenoid (NSC strains). The latter of the fungi, while after extraction, these compounds strains synthesize a specific precursor, Cpd-P, formed become hydrophobic and, in this chemical modifica- from lycopene and blocking to a certain degree the syn- tion, can stimulate carotene synthesis only in the (Ð) thesis of β-carotene. Then Cpd-P is transformed into heterothallic strain. This fact so far remains unex- the precursor common for SC and NSC strains, 4-dihy- plained, and it is likely that the answer to this question droxytrisporin, and the collaborative synthesis of TSA will considerably advance the understanding of het- commences; however, TSA are synthesized in a consid- erothallism. Presumably, the hypothesis that TSA is an erably smaller amount compared with SC strains [68]. inactive product, a certain “depot” necessary for pro- Presumably, either the ratio of TSA precursors or their duction of the compounds regulating the first stages of amounts are changed in this case. For example, the sexual process [70, 71], will require an explanation at Phycomyces deficient in carotene but synthesizing the the same time. This hypothesis was proposed as early as pheromone Cpd-P form during the cocultivation of (+) in the 1970s and fit the accumulated data on the signif- and (Ð) strains 25% TSA B, 68% TSA C, and 7% TSA icance of secondary metabolites, which may function E, whereas the wild strains synthesizing carotene pro- as secondary substances utilized during germination of duce 6% TSA B, 40% TSA C, and 30% TSA E. the cells switching off from dormancy. For example, a

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 HETEROTHALLISM OF MUCORACEOUS FUNGI 449 well-known secondary metabolite, trehalose, may be a Cancer Ishemic heart depot for the substrate, such as glucose, along with the disease main protective function for membrane phospholipids; hence, this disaccharide is intensively consumed during fungal spore germination [72]. In addition to the func- Ageing Carotenoids tions of the sex hormone and carotenogenesis stimula- Photoprotection tor, TSA may presumably play the role of growth inhib- itor, similar to plant abscisin, when fungal cells influ- Immunodeficiency enced by a stress factor switch to ontogenetic anabiosis. Cataract The chemical similarity of these retinoids, abscisic acid and TSA, is a confirmation of this hypothesis. Study of Fig. 2. Use of carotenoids (β-carotene and lycopene) for the mechanism underlying the action of the compounds therapy of the diseases most widespread by the beginning of controlling the first stages of the sexual process of muc- the 21st century [76]. oraceous fungi may also contribute to our understand- ing of the role of vitamin A in higher organisms. It is known that vitamin A is not only involved in the vision ture economically sound. However, this required process but is also necessary for normal growth, devel- numerous studies that resulted in production of highly opment, and reproduction of animals. However, the active strains and allowed the corresponding cultivation precise role of this vitamin as a mediator in the last two conditions to be selected. processes is still unknown. Therefore, the discovery of The research into the cultivation conditions for this “trisporate-like” compounds, similar to analogous sub- producer which provide the maximal amount of the tar- stances of Mucorales [73, 74], in rats fed large doses of get product was of great importance for the increase in vitamin A may indicate a certain analogy in the hor- carotene yield. At the end of the 20th century, the coc- monal regulation of sexual processes of higher and ultivation of (+) and (Ð) strains of mucoraceous fungi in lower eukaryotes and confirms that Mucorales of the a medium containing soybean and corn flours, potas- overall kingdom Fungi are closest to animals [75]. sium phosphate, thiamine and less than 4% plant oil [78] gave the highest carotene yield. Numerous com- pounds, such as β-ionone, kerosene, linoleic acid, ter- USE OF HETEROTHALLIC FUNGAL STRAINS penoids, several nitrogen-containing heterocyclic com- IN BIOTECHNOLOGY FOR MANUFACTURING pounds, and antioxidants, were recommended as stim- CAROTENOIDS ulators of carotenogenesis. A number of industrial The phenomenon of heterothallism is widely used in wastes were used as stimulators, such as citrus pulp, cit- biotechnology for manufacturing a number of iso- rus molasses, and biomasses of some filamentous prenoid compounds, in particular, β-carotene and lyco- fungi, yeasts, and bacteria. The fraction of organic pene. The therapeutic effect of these carotenoids is acids in the citrus molasses is an active stimulator. The manifold, and this is the particular reason of their high biomass of filamentous fungi, yeasts, or bacteria also value for medicine. Carotene and lycopene are admin- increases the carotene yield but to a lesser degree than istered to treat the human diseases (Fig. 2) that have citrus pulp, and the stimulatory effect of the biomass become most widespread in the 21st century. For exam- stems from the action of Krebs cycle organic acids. ple, special attention is now given to carotene as an Nonionic surfactants, benzurones, hydrogen peroxide, antioxidant in treating cataract and several defects of dimethyl phthalate, veratrol, etc., were also used to the retina [76]. stimulate β-carotene synthesis by B. trispora [79Ð85]. Biotechnological method for producing b-caro- Along with chemical compounds, other factors con- tene. The first regulations for biotechnological manu- tributed to the increase in carotene yield, for example, facture of β-carotene in 20-l fermenters using exposure to light with various spectral compositions B. trispora heterothallic strains was proposed as early [86]. The white light influences the spore formation by as in 1963 [77]. However, this method failed to find any B. trispora, and this is used in commercial practice by further use, as the price of microbiological carotene illuminating the (+) and (Ð) strains for 48Ð56 h after was higher than the price of chemically synthesized keeping them in darkness for 3Ð4 days. We demon- product. However, several decades later, natural food strated [86] in the early 1990s that UV inhibited and medical preparations received a highest demand on B. trispora spore formation; blue and red light had vir- the medical market. This induced an active develop- tually no effect, and only green light stimulated the pro- ment of the microbiological technology for carotene cess. More detailed studies demonstrated that green production, and despite the fact that carotene can be light (GL) substantially influenced the spore formation manufactured from bacteria, alga, plants, and fungi, the of both (+) and (Ð) strains; however, each strain heterothallic strains of B. trispora won out. These required selection of the corresponding illumination strains are able under industrial conditions (10-m3 fer- modes, which allowed us to find that the (+) strain menters) to stably produce 3Ð4 g/l carotene; this low- formed spores more actively. An interesting fact was ered the price of this isoprenoid and made its manufac- discovered: when the spores illuminated with GL are

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 450 FEOFILOVA used as an inoculum, the carotene yield in mycelium is Producing b-carotene using only one (Ð) strain of higher compared with the mycelium grown from B. trispora. Use of only one (Ð) strain of B. trispora “dark” spores (7944.8 and 596.4 g/l, respectively, after considerably simplifies the production of β-carotene a 4-day cultivation on the medium without oil and [93]. The B. trispora (Ð) strains capable of synthesizing β-ionone) [86]. larger amounts of β-carotene were produced using N-methyl-N-nitro-N-nitrosoguanidine, ethylmethane The oxygen content in the fermenter also influences sulfonate, nitrous acid, nucleotide analogues, acridine, carotene yield. To create the necessary oxygen concen- ultraviolet, x rays, γ rays, or combinations of these fac- tration (50% higher than the saturation level), the tors. These strains are of interest for commercial pur- medium was enriched with pure oxygen and concur- poses, because the fermentations with one strain are rently supplemented with the surfactant Span 20 to pre- more stable and the price of the target product is sub- vent its foaming in fermenter. Under the control of oxy- stantially lowered [94]. gen level and foaming of the medium, a high carotene concentration—1.190 mg/l—could be obtained after a To enhance the synthesis, the growth medium was 6-day cultivation with the yield of B. trispora biomass supplemented with compounds displaying an antihy- amounting to 2.5% dry mycelium weight, i.e., approx- percholesteric activity, for example, lovastatin, imately fivefold higher compared with the conventional decreasing the level of sterols (ergosterol). It was cultivation [87, 88]. assumed that the shunt in lipid synthesis would assist an increase in β-carotene yield. Another way to increase In the 1980s, the use of spontaneous mutants the carotene production is via inhibitors of acetyl-CoA allowed the carotene yield under industrial conditions synthesis, such as propionate and butyrate analogues, to be increased by 10Ð15%. Methods utilizing chloroacetate, fluoroacetic acid, and 2-chloroaceta- mutagens were developed somewhat later. For this pur- mide. The mutant spores of B. trispora (Ð) strains were pose, for example, B. trispora spores were exposed to exposed to these compounds to test the surviving spores N-methyl-N-nitro-N-nitrosoguanidine for 30 min in as inoculum for production of β-carotene. 100 g of the mutagen (spore survival rate, about 1%) Another approach to increasing the yield of β-caro- [89, 90]. However, these mutants under industrial con- tene using only one strain is exogenous addition of ditions quickly lost their activity and were replaced by trisporic acids during fermentation. For this purpose, wild strains. Note also that the increase in β-carotene MM-1 medium, containing cottonseed oil, soybean level in B trispora by genetic methods is feasible only . extracts, sodium phosphate, and magnesium sulfate, in a more moderate range compared, for example, with was used. Upon 54 h of fermentation, TSA, inducers of P blakesleeanus and that such B. trispora strains under . β-carotene synthesis (β-ionone), and, optionally, ion- industrial conditions retain their activity for a short ized kerosene, citrus pulp, citrus oils (limonene), and period. Recently, high and stable carotene yields were α-ketoglutarate were added. The yield of carotene obtained using specially selected strains, optimal could be increased from 0.15 to 1.5 and even 3.0 g/l. media, and nonstandard carotene isolation methods, for TSA is obtained by cofermenting B. trispora (+) and example, ethanol extraction under alkaline conditions (−) strains. For this purpose, culture liquid is separated [91]. from mycelium and acidified to pH 2.0 to extract TSA It is known that the production capacity of strains with chloroform, purify additionally with sodium can be elevated by increasing the content or activity of bicarbonate solution, and extract with methylene chlo- the corresponding enzymes or changing the biosyn- ride upon acidification to pH 2.0. The E value of puri- thetic pathway utilizing genetic engineering methods to fied TSA amounts to 620. TSA is added to a 24-h-old modify the corresponding metabolic pathways by culture of the (Ð) strain. Barbiturate added to the growth recombinant DNA. The metabolic engineering media allows the TSA yield to be increased consider- approach opens the possibility to obtain carotenoids ably [95]. TSA acts on fungal phospholipids and from strains that do not synthesize these pigments [92]. increases the content of phosphatidylcholine, which is For example, strains of Candida utilis, E. coli, and accompanied by an increase in the β-carotene content. S. cerevisiae capable of synthesizing not only carotene It is demonstrated that TSA stimulates not only the pro- but also lycopene were obtained. Unfortunately, low duction of β-carotene by the B. trispora (Ð) strain but and unstable yields of the target product prevent appli- also syntheses of ubiquinone Q9 and riboflavin [96, 97]. cation of such strains for the time being. Producing lycopene by biotechnological method. Essential attention in the biotechnological manufac- The cocultivation of B. trispora heterothallic strains ture of carotene is given to the chemical characteristics provides biotechnological production of another caro- of the final product. β-Carotene must be the major com- tenoid, lycopene. As mentioned above, this carotenoid ponent in the carotenoid mixture (92Ð96%), while the is the most powerful natural antioxidant and, therefore, content of other carotenoids must amount to 8Ð4%. is of considerable interest for antitumor therapy [98]. β-Carotene is mainly found as an all-trans-isomer (90Ð Virtually all fungi synthesize lycopene; however, in 95%); the present 15cis-, 13cis-, and 9cis-isomers form a very insignificant amount. Two approaches can be together 10Ð15%. used to intensify its synthesis via inhibition of cycliza-

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 HETEROTHALLISM OF MUCORACEOUS FUNGI 451 tion: changing the cultivation conditions or acting on abilities to (1) synthesize β-carotene and (2) form cer- the enzymes cyclases. It is known that cultivation under tain TSA at a certain ratio. neutral or weakly acid conditions [99] and changes in A new precursor, Cpd-P, was discovered in the the composition of growth media [100] assist intensifi- strains of mucoraceous fungi incapable of synthesizing cation of lycopene synthesis; however, the yield of the carotene; this precursor is transformed into TSA, while target product obtained by this method is economically TSA are present in a very insignificant amount and has unsound. Lycopene cyclization and transformation into a different composition compared with the carotene- carotene can be inhibited using substituted amines and synthesizing strains [68]. The discovery of new TSA nitrogen-containing heterocycles (azines) [82]. The types, E and D [67], and the fact that TSA E is not search for stimulators of lycopene formation allowed metabolized during the sexual interaction of heterothal- the development of a technology involving azine deriv- lic strains suggested that the ratio of various TSA types atives (MAP): addition of MAP at a dose of 0.05 g/l and, especially, TSA E and D, is involved in the control increased the lycopene yield to 0.7 g/l medium. This at the initial stages of sexual morphogenesis; namely, technology met the economic requirements [101]. In they coordinate the growth of zygophores of different addition, the technology for manufacturing the prepara- sexes towards one another, i.e., the zygotropism reac- tion Mikolikopin was developed [102]. This prepara- tion. This type of sexual interaction is not new in tion contains lycopene in combination with phospho- nature; for example, it is found in the flight of butter- lipids, essential fatty acids, ubiquinone Q9, and biolog- flies of different sexes, when a distinct ratio of phero- ically active substances, valuable medicinal substances mones determines the motion of the female individual produced by B. trispora concurrently with lycopene. towards the male [107]. Genetic methods have also been recently applied to lycopene production. For example, S. cerevisiae with In addition to the discovery of species-specificity of an inserted gene from Erwinia uredovora was con- the language for communication of heterothallic structed. This strain is proposed for producing lycopene strains, new concepts of TSA functions appeared at the [103]. Yet the use of genetically constructed strains in a eve of the 21st century. It has been demonstrated that routine production of carotenoids considerably these sex hormones are the signals for arresting growth increases the price of the target product. Nonetheless, processes, representing an analogy to the plant hor- these strains may become promising for commercial mone abscisin. The growth arrest in plants caused by purposes [104Ð106] if novel fermentation methods are stress is determined particularly by accumulation of the developed instead of the previously used technologies phytohormone abscisic acid [108]. This compound is [92, 104]. very similar to TSA in its chemical structure, and in certain cases both isoprenoids can be synthesized from *** a common precursor, isocryptoxanthine [57]. Among the main problems of modern biology and, Recently, the language of heterothallic strains, rep- especially, biotechnology is the research into the “lan- resented by isoprenoid hormones, attracted the interest guage” of cell communication in the microbial popula- of researchers from two standpoints. First, it is hypoth- tion. It is doubtless that this communication is based on esized that the biosynthesis of polyisoprenoid mole- the synthesis of specific compounds involved in cell cules existed as early as 2.5 billion years ago [109]. interactions. Here the sexual reproduction is of appar- Thus, the hormonal regulation of reproduction of Muc- ent interest, as the cells communicate during this pro- orales heterothallic strains originated at early evolu- cess due to the synthesis of specific hormones. tionary stages and is a very ancient process. Moreover, the biosynthesis of isoprenoid compounds is a very The language of fungal cell communication during economical process and is widely used for communica- sexual reproduction has been studied since the begin- tion by a large number of species at various evolution- ning of the last century. The interaction between het- ary levels. If we go along, the above data suggest that erothallic cells of mucoraceous fungi is now the most mucoraceous fungi, the most ancient branch of the studied field. A scheme was proposed (Fig. 1) explain- kingdom Fungi [75], originated not 700 million years ing the interactions between the male and female ago, but considerably earlier. Second, molecules simi- strains involved in collaborative synthesis of hormones lar to mammalian hormones are discovered in both that gives the zygogenic sex hormone (trisporic acids), prokaryotes and eukaryotes. These data suggest that which are responsible for the development of sexual certain biochemical “elements” of the state-of-the-art hyphae (zygophores) in Mucorales. At the border of the endocrine system have analogues in unicellular organ- 20th and 21st centuries, this scheme was revised, and isms [111]. Moreover, recent studies [111] demonstrate novel specific features were discovered in the sexual considerable chemical and biological similarities communication of cells. Recent studies into sexual between the hormones (trisporates) of mucoraceous morphogenesis demonstrate that the language of signal fungi and plant and animal hormones (abscisin and ret- molecules is not common for representatives of the inoic acid). Therefore, it is now believed that fungal and entire order Mucorales but is species-specific. This spe- mammalian cells interact with one another “in a strik- cific feature is determined by the following factors: the ingly similar manner” [111]. The following example is

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 5 2006 452 FEOFILOVA sufficiently illustrative. The steroid 5α-andost-16-en-3- nomically sound [92]. Therefore, further studies will α-ol is a metabolite of the fungus black truffle Tubor presumably be directed to changing the fermentation melanosporum, has a musky odor, and is the main com- strategies, for example, overcoming autoselection. For ponent of the hog’s pheromone. This steroid is the rea- the time being, however, the phenomenon of heteroth- son why swine are used to search for truffles. The data allism efficiently serves biotechnology, and B. trispora indicating the similarity of the Mucorales isoprenoid heterothallic strains stably produce under industrial hormones to those of plants and animals (abscisin, a conditions 3Ð4 g/l carotene and 1.3Ð1.5 g/l lycopene dormancy hormone, and retinoic acid, a morphogenic [102, 114], which is now profitable from the economic hormone) are described above. Note also that fungi standpoint. contain a human neuromediator, serotonin, which is formed at the early trophophase. The fungi Psilocybe synthesize the serotonin derivatives psylocibin and ACKNOWLEDGMENTS psilocin during the same growth phase. The work was supported by the Russian Foundation It appeared that the ability of fungal and mammalian for Basic Research, project no. 03-04-48683 and, in cells to interact with one another via hormones is not part, project “ofi-a” no. 050408011. only of apparent theoretical interest, but also of an applied value, in particular, in medicine in the field of REFERENCES fungal infections [112]. For example, the human hor- mone progesterone inhibits the growth of a number of 1. Oleskin, A.V., Botvinko, I.V., and Tsavkelova, E.A., pathogenic dermatophytes. Estrogen inhibits the trans- Mikrobiologiya, 2000, vol. 69, no. 3, pp. 309Ð327. formation of the pathogen Paracoccidioides brasilien- 2. 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