Production and derivate composition of trisporoids in extended fermentation of Blakeslea trispora.

Trisporic acid, its precursors and derivatives are used within zygomycete fungi as communication signals and sexual regulators, and also influence the production rate of the parent compound, beta-carotene. Cultivation parameters during growth and the trisporoid production phase of Blakeslea trispora were studied in two-step shake flask cultures and up-scaled fermentations. Comparison of various fermentation protocols allowed the definition of parameters governing trisporoid production. Highest yields were obtained when the initial growth phase allowed for both rapid growth and fast exhaustion of nitrogen and phosporous sources. Onset of trisporoid production is accompanied by a pH drop in the medium and triggered by nutrient limitation, nitrogen depletion being the most important factor. Supplementation of cultures with carbon at low concentration after onset of trisporoid production led to prolonged growth and higher final product accumulation. B. trispora produces trisporoids in two major series, B and C. During a first peak in trisporic acid accumulation, production of trisporic acid B exceeds that of trisporic acid C, which later accumulates at the expense of the trisporic acid B, indicating a variable regulation of the ratio between these metabolites. These data are valuable for tailoring production systems for enrichment of specific intermediates of this complex signal family.

Carotene derivatives in sexual communication of zygomycete fungi.

Recognition between mating partners, early sexual morphogenesis and development are regulated by a family of beta-carotene derived signal compounds, the trisporoids, in zygomycete fungi. Mating type-specific precursors are released from the hyphae and exert their physiological effects upon compatible mating partners. In a cooperative synthesis pathway, later intermediates and finally trisporic acid are formed. All trisporoids occur in a number of derivatives. Trisporic acid and some precursors directly influence the transcription of genes involved in sexual development. This has been demonstrated for TSP3, encoding the carotene oxygenase involved in sexually induced cleavage of beta-carotene. Species specificity of mating despite a common and commonly recognized signaling system is maintained by several factors. Specific distribution and recognition patterns of the trisporoid derivatives and the proposed divergence in trisporoid synthesis pathways in diverse species play a role. The derivatives elicit vastly differing, partially mating type-specific responses during early sexual development. Another specificity factor is the realization of different regulation levels for the trisporoid synthesis enzymes in different species. Enzymes in the trisporoid synthesis pathway show remarkable variations in mating type-specific activity and the exact activation time during sexual development. This allows for the observed complex network of possible interactions, but at the same time forbids successful mating between dissimilar partners because the necessary transcripts or gene products are not available at the appropriate developmental stage.

4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

Cooperative biosynthesis of Trisporoids by the (+) and (-) mating types of the zygomycete Blakeslea trispora.

The fungal phylum zygomycota uses trisporic acids (TSAs), a family of apocarotenoids, during sexual reproduction to synchronize and control activity between the mycelial hyphae of opposite mating types. Separate as well as mixed cultures of Blakeslea trispora were systematically supplemented with putative, deuterium-labeled precursors downstream of beta-carotene en route to the bioactive TSAs. Analysis of the isolated metabolites allowed the reconstruction of the complete biosynthetic sequence between the first apocarotenoid, D'orenone (1), and the different series of TSAs B (8) and C (13). Both mating types produced a similar spectrum of early metabolites upstream of trisporols B (7) and C (12), while only the (+) type was able to further oxidize trisporols B (7) and C (12) to the corresponding methyltrisporoid B (5) and C (11), respectively. A novel 4-dihydrotrisporic acid B (14) that was not formed from the labeled precursors was isolated from mated strains; this compound might be derived from oxygenated beta-carotene by a parallel pathway. The ester accumulated in the culture broth of the (+) strain and was only hydrolyzed by mycelia of the (-) strain; this corresponds to a synchronization of the biosynthetic activities of both mating types.

Cleavage of beta-carotene as the first step in sexual hormone synthesis in zygomycetes is mediated by a trisporic acid regulated beta-carotene oxygenase.

Carotene cleavage is the necessary initial step in the biosynthesis of trisporic acid, the sexual signal in zygomycete fungi. Two genes encoding putative carotene oxygenases, designated tsp3 and tsp4, were identified in the genome of the zygomycete Rhizopus oryzae. Using heterologous primers, tsp3 was cloned and sequenced also from Blakeslea trispora. tsp3 transcription correlates with sexual development in both species. Northern hybridization of B. trispora mRNA revealed strong induction of tsp3 transcription in mated cultures. A very strong and direct transient induction of transcription by trisporic acid was proven by quantitative real-time PCR analysis. In R. oryzae, transcriptional induction is also inducible by stimulation with trisporoids and depends on the developmental stage of the mycelium. The functionality of the tsp3 gene product as carotene cleavage enzyme was shown as loss of carotene in an Escherichia coli strain transformed to carotene production and tsp3 expression.

4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types.

4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.

Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-).

In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of structurally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete structural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor mucedo. The activity screening contributed to establish general structure-function relationships for trisporoid action. Most crucial for activity were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The overall biological activity apparently results from the combination of the various structural elements.

Sexuality and parasitism share common regulatory pathways in the fungus Parasitella parasitica.

Parasitella parasitica, a facultative mycoparasite of zygomycetous fungi, forms cytoplasmic fusions with its hosts during infection. Thus, the organism is an efficient donor of genetic material in parasexual host-parasite interactions. Recognition between parasite and host is mediated by trisporoids, which are also responsible for sexual communication. The TDH gene for one of the key enzymes of trisporic acid biosynthesis, 4-dihydromethyl-trisporate dehydrogenase, was cloned and its transcription analysed. TDH was cloned on a 6175-bp insert and was found to map in a complex cluster of genes that suggest post-transcriptional antisense regulation. Histochemical TDH analysis in developing parasitic or sexual structures shows high enzymatic activity in Parasitella. TDH is linked to a gene for a putative acyl-CoA thioesterase (ACT). Two ORFs were identified in the 5'-region of the TDH gene, a third one, coding for 176 amino acids overlaps the ACT gene in antisense direction completely. Expression levels of ACT and ORF1 depend on parasitic and sexual interactions.

Sexual reactions in Mortierellales are mediated by the trisporic acid system.

Several species of Mortierella (Mortierellales, Zygomycota) were examined for substances regulating their sexual reactions. Compounds isolated from both mated and single growing Mortierella strains were purified by thin layer chromatography. Some of these compounds showed UV absorbance-characteristics similar to those of trisporoids, a group of compounds involved in sexual regulation in Mucorales. A compound with a 4-dihydromethyltrisporate-like absorbance spectrum was detected. To test for the interspecific sexual responses typically induced by trisporoids, the compounds extracted from Mortierella spp. were tested against the Mucorales Mucor mucedo and Phycomyces blakesleeanus and were found to induce sexual reactions in both tester strains. A gene encoding 4-dihydromethyltrisporate dehydrogenase was identified in several Mortierella species and the activity of the gene product was shown using a histochemical assay. We suggest that the regulation of sexual processes by trisporoids is common to both Mucorales and Mortierellales and may be more widespread within the Zygomycota.