Mechanism Of The Responses Of Blakeslea Trispora To Trisporic Acid

Carotenoids produced by microorganism fermentation have the advantages of low cost, high purity, and no toxic or side effect. The zygomycete Blakeslea trispora is used commercially to produce p-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. However, little information is known by far about the regulation manner of TA on carotene synthesis in B. trispora, as well as the interaction between actived carotene synthesis and other metabolitic pathway.To gain insight into the the responses of B. trispora to TA, changes in the contents of β-carotene, ubiquinone, and ergosterol in the (-) strain of B. trispora after TA addition were measured. The addition of TA to the B. trispora culture increased the production of P-carotene and ubiquinone, while the ergosterol content remained unchanged during the first48h after TA addition and decreased slightly thereafter. Then 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 γ-aminobutyrate might play important roles in the regulation of TA. The results indicate that the actions of TA at the metabolic level involve multiple metabolic processes, such as glycolysis, the bypass of the classical tricarboxylic acid cycle, and fatty acids synthesis.Proteomic approch based on two-dimensional electrophoresis was carried out to analysis the responses of B. trispora to TA. Twenty-one differentially expressed proteins were identified between the control and TA-treated groups, including adenylosuccinate synthetase, glutamine synthetase, diphosphomevalonate decarboxylase, branched-chain-amino-acid transaminase, aconitase/homoaconitase, mitochondrial FAD carrier protein, ubiquitin-protein ligase, and so on. These differentially expressed proteins participate in a variety of cellular functions, including the transport and metabolism of nucleotides, amino acids, carbohydrates, lipids, and coenzymes, energy production and conversion, posttranslational modification, protein turnover, and translation. The results indicate that these physiological processes might play crucial roles in the responses of B. trispora to TA.Transcription products of eight genes encoding enzymes in terpenoid biosynthesis were also analyzed. Four genes in the P-carotene biosynthetic pathway (ipi, carG, carRA, and carB) had increased expression following TA addition. These data suggest that TA increases terpenoid production in B. trispora by stimulation of transcription, and ipi and carG play key roles in carotene biosynthesis.The cDNAs of genes ipi and carG mentioned above were cloned. Isopentenyl pyrophosphate isomerase (IPI) and geranylgeranyl pyrophosphate synthase (GGPS) encoded by ipi and carG are key enzymes in the biosynthesis of carotenoids. Genes ipi and carG we cloned were expressed in Escherichia coli in this study. The results showed that the cDNAs of ipi and carG from B. trispora were functional in E. coli. It was also found that more GGPS activity was needed in engineered E. coli when upstream IPP activity increased. The introduction of GGPS and IPI from B. trispora made β-carotene content in E. coli increase by89.2%. The present study provides new gene sources for carotene genetic engineering.The promoter region of gene ipi and carG were also cloned by Tail-PCR and analyzed. The transcriptional factors Stell, Ste12, and Mot3were found in their promoter sequences, which are inferred to be related with the TA responses.