The Cloning And Function Analysis Of The LaeA-like Methyltransferase Gene Llm3 In Penicillim Citrinum

Background:Fungi were the treasure of the natural products. There are more than 1500 kinds of secondary metabolites were isolated from the fungi. Half of them have antibacterial and anti-tumor activity, such as penicillin, griseofulvin, cyclosporine, cephalosporin’s, simvastatin, doxorubicin, Avi neomycin and so on. Recent studies suggest that the secondary metabolic is complex and multi-layered, including the global regulatory factor LaeA, specific pathway factor aflR and epigenetic regulatory factor. They regulated the fungi secondary metabolites and development.The global regulatory factor LaeA was discovered in Aspergillus nidulans in 2004, which have the methyltransferases domain and was highly conserved in fungi. Recent research proved that Lae A could regulate the development and the secondary metabolism. LaeA could interact with VeA and VelB protein formed the heterotrimeric velvet complex in nuclear and regulated the development and secondary metabolism in A. nidulans in 2008. The LaeA-like methyltransferases Llmf were identified in Aspergillus nidulans in 2013. Llm F was found in A. nidulans to control production of ST as well as influence sexual development. Thus, Lae A and LaeA-like methyltransferases protein have become a hot and the research focused on the global network in fungi.P. citrinum is the strain which produced Mevastatin. The complete genome sequences of P. citrinum became available and this has led to the identification of a large number of secondary metabolic genes. This indicated that P. citrinum has the potential to produce many secondary metabolites. However, the regulatory factor and mechanism still unclear. Our laboratory reported that the laeA and veA overexpression strain P. citrinum could regulate the biosynthesis of mevastatin and obtained the P. citrinum genome framework map. But they were still puzzles whether there had the LaeA-like methyltransferases and if the LaeA-like methyltransferases regulated secondary metabolites in P. citrinum. Based on the genome framework map, the llm3 gene was cloned in P. citrinum. To investigate the function of llm3 in secondary metabolism regulation and development regulation, we constructed the overexpression strain and knockout strain of llm3 in P. citrinum. Objective:To understand the function of llm3 on secondary metabolism and development regulation P. citrinum, we compared the difference between WT strain, OE::llm3 strain and Δllm3 strain in mycelium, colony, conidia production and the biosynthesis of mevastatin. Methods:1. Using the Illumina Hiseq Seq sequencing technology, obtain the framework map of the genome of Penicillium citrinum.2. Using the local blast analysis the llm3 sequence, and the coding sequences of llm3 were amplified from the cDNA of Penicillium citrinum.3. Construction llm3 knockout plasmid pYEU-Δllm3 and overexpress plasmid pGiHTGi-llm3. Using PCR, enzyme digestion and sequencing methods to verify the constructed plasmid. The OE::llm3 strain was constructed by the agrobacterium mediated transformation platform and certificated by PCR and culturing on medium with hygromycin. The Δllm3 strain was constructed by protoplast transformation and certificated by PCR and culturing on medium with hygromycin.4. To explain the regulation of llm3 in development, the mycelium, colony and conidia production of WT strain, OE::llm3 strain and Δllm3 strain were detected under microscope. Meanwhile, the transcription level of abaA and brlA were analyzed by RT-qPCR.5. The WT strain, OE::llm3 strain and Δllm3 strain were cultured in Potato glycerol medium broth for 8 days. The regulation of llm3 in mevastatin biosynthesis process was explained with fermentation and HPLC. Meanwhile, the transcription level of the transcriptional activator mlcR and the mevastatin biosynthesis gene mlcB were analyzed by RT-qPCR. Results:1. The genome was assembled using 31.34 M paired-end reads, producing 716 contigs for P. citrinum.2. The full length of llm3 was obtained by RT-PCR3. The knockout plasmid pYEU-Δllm3 and overexpress plasmid pGiHTGi-llm3 were constructed. The overexpress strain(OE::llm3) was constructed by the agrobacterium mediated transformation platform and was affirmed by the PCR. The llm3 knockout strain(Δllm3) was constructed by the protoplast transformation and certificated by PCR.4. Llm3 inhibited asexual propagation in P. citrinum. The color of OE::llm3 strain colony was much shallower than WT strain colony, and the Δllm3 was much deeper than WT strain colony. The conidia production of OE::llm3 strain was less than WT strain, and the Δllm3 was much more than WT strain. These results showed that llm3 could negatively regulate the asexual reproduction in P. citrinum.5. Llm3 increased the biosynthesis of mevastatin. At the d4 of fermentation, the mevastatin production of WT strain was reach to the maximum while the mevastatin production of OE::llm3 was still increased until the eight days. The mevastatin production of the Δllm3 strain was much lower than WT strain. Conclusion:Llm3 regulated the secondary metabolic and development; llm3 inhibited the asexual reproduction and positively regulates mevastatin biosynthesis. Llm3 inhibited asexual propagation in P. citrinum though regulating the expression abaA and brlA. Llm3 activated the biosynthesis of mevastatin through activating the expression of the mlcR and mlcB. The global regulation study of llm3 in mevastatin biosynthesis, condition in P. citrinum could support a theoretical basis for the new secondary metabolite and the illumination of its mechanism.