The Global Regulation And Mechanism Of VeA In Penicillim Citrinum

Background:At present, the industrial application of natural products is mainly derived from biological organisms (animals, plants and microorganisms) of primary metobolites and secondary metabolites. In all of these reported natural products, about 20-25% of them endow bioactive. In addition, in the 22500 bioactive compounds deriving from microorganisms, about 45% comes from actinomycetes,38% from fungi,17% from single-celled bacteria. Most of these fungi sources active compounds derive from filamentous fungi, such as beta-lactam antibiotics, griseofulvin, fusidic acid, cyclosporine, insecticide and statins, etc. Recent studies suggest that the secondary metabolic regulation of filamentous fungi is a multilevel control process that is composed of global regulatory factors and specifically regulating factors, for example, the sterigmatocystin biosynthesis is regulated by the global regulatory factor LaeA and specifically regulating factor AflR in Aspergillus nidulans.The global regulatory factor VeA was discovered as an inhibitor of conidiation in Aspergillus nidulans in 1965. Now, VeA and its homologues have been discovered in Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans and Penicillium chrysogenum, etc. Subsequent research found that the regulation of VeA was regulated by light. VeA could combine with VelB to form VeA/VelB complex and regulated the development of filamentous fungi and that VeA/VelB complex could combine with LaeA to form VelB/VeA/LaeA and regulated secondary metabiolism of filamentous fungi. According these researches, researchers discovered VosA, VelC and LlmF in vinous filamentous fungi. These studies indicated that VeA and its homologues regulate secondary metabiolism and development in filamentous fungi.The Penicillium citrinum has been used for the commercial production of mevastatin. However, the regulation study of Penicillium citrinum is rare. The regulation of mevastatin biosynthesis is limited to the analysis of mevastatin biosynthesis cluster. And no research reported about the regulation of secondary metabiolism in Penicillium citrinum. Our laboratory reported that the laeA overexpression strain Penicillium citrinum could regulated the biosynthesis of mevastatin, as well as cloned the full length of veA in Penicillium citrinum. To investigate the function of veA in secondary metabiolism regulation, development regulation and osmotic stress tolerance regulation, etc, we constructed the overexpression strain of veA in Penicillium citrinum.Obejective:To understand the function of veA on secondary metabiolism regulation, development regulation and osmotic stress tolerance regulation, etc. in Penicillium citrinum, we compared the difference between WT strain and OE::veA strain in mycelium, colony and conidia production.Methods:1. The coding sequences of veA were amplified from the cDNA of Penicillium citrinum using primers tailed with BamHI and SacI (veA-F and veA-R). This product was digested by the corresponding restriction enzymes and ligated into plasmid pGiHTGi (containing a hygromycin resistant gene and a glycerol phosphate aldehyde dehydrogenase promoter PgpdAi).The OE::veA strain was constructed by the agrobacterium mediated transformation platform and verificated by PCR cloning hygromycin gene and culturing on medium with hygromycin.2. To investigate the relationship of light and veA transcription level, the difference of veA transcription level between dark and light was analyzed by RT-qPCR.3. To explain the regulation of veA in development, the mycelium, colony and conidia production of WT strain and OE::veA strain were detected under microscope. Meanwhile, the transcription level of abaA and brlA were analyzed by RT-qPCR.4. The WT strain and OE::veA strain were cultured in Patato glycerol broth for 8 days. The regulation of veA in mevastatin biosynthesis process was explained with fermentation and HPLC. Meanwhile, the transcription level of the mevastatin biosynthesis gene mlcB and the transcriptional activator mlcR were analyzed by RT-qPCR.5. The difference of colony diameter and conidia production of WT strain and OE::veA strain wree analyzed in medium with Nacl/Kcl/Glucose/H2O2. Meanwhile, the transcription level of trxB, a gene which involved in stress response in filamentous fungi was analyzed by RT-qPCR.Results:1. The combinant plasmid pGiHTGi-veA was constructed. The veA overexpression strain (OE::veA) was constructed by the agrobacterium mediated transformation platform and was affirmed by the hygromycin gene. 2. veA inhibited asexual propagation in Penicillium citrinum. When the veA transcription level of OE::veA strain was higher than WT strain, the colour of OE::veA strain colony was much shallower than WT strain colony, the mycelium development of OE::veA strain was slower than WT strain and the conidia production of OE::veA strain was less than WT strain. These results showed that veA could negatively regulate the asexual reproduction in Penicillium citrinum.3. Light could activate veA expression and inhibit colony growth, mycelium development and asexual reproduction. Meanwhile, the the transcription level of veA is lower in dark than in light.4. veA activated the biosynthesis of mevastatin. In the first four days fermentation, the mevastatin production of OE::veA strain was 115.88mg/L while the mevastatin production of WT strain was 39.96mg/L. After four days fermentation, WT strain did not produce mevastatin while OE::veA strain began to degradate mevastatin to 68mg/L. The dry weights of strains were no significant difference between WT strain and OE::veA strain.5. veA was associated with environment strss in Penicillium citrinum. The spores reduction phenomenon in OE::veA strain could be neutralized by adding Nacl/Kcl/Glucose. veA enhanced the H2O2 tolerance of Penicillium citrinum. These phenomenons were connected with the trxB gene of HOG pathway.Conclusion:Light inhibits the asexual reproduction by activating veA expression in Penicillium citrinum. The overexpression of veA resulted veA express increase, and inhibited the mycelium development and asexual reproduction. veA positively regulates mevastatin biosynthesis and enhances the tolerance to Nacl/Kcl/Glucose/H202 in Penicillium citrinum. There exists a velvet family regulation net which was based on veA gene in Penicillium citrinum. The regulation network of velvet family combined secondary metabiolism regulation, development regulation and osmotic stress tolerance regulation.The global regulation study of veA in mevastatin biosynthesis, conidiation and stress response tolerance in Penicillium citrinum could supply a theoretical basis for the development of high yield strain and new secondary metabolite.