Cloning and Molecular Analysis of Lovastatin Biosynthesis Related Genes from Monascus Purpureus
|School||Kunming University of Science and Technology|
|Keywords||lovastatin gene clone bioinformatics overexpression|
Monascus is considered to be a filamentous ascomycetes, which is a important microbial resources of our country. It has been already used for food industry and brewing industry for thousands of years. Now, Monascus-fermented rice has commonly been used as food additives and health-care food., which is to be sold not only in tradition market, such as China, Korea,Japan, Southeast Asia and so on, but also in European and American market increasingly. Monascus-fermented product has notable medical value and edible security. The discovery of many active compounds, especially lovastatin in Monascus, make many Domestic and foreign scholars keep high enthusiasm in the study of Monascus, whose research fields has been gradually into the pharmacological mechanism of active metabolite, the related molecular mechanism of biosynthetic pathway and the metabolic regulation. Along with the deepening of the study on the structure and function of lovastatin biosynthetic genes and enzymes, using genetic engineering means to improve lovastatin yield will be a important research direction. At the same time, a further study about the structure, function and the mutual action of related enzymes, by the means of gene expression, protein purification, in vitro model test and so on, will be favorable for better understanding the biosynthesis process of lovastatin and guiding the molecular operation of related genes, in order to improve the lovastatin synthetic ability of Monascus strains fundamentally.Now, M. purpureus is commonly used for the production of red kojic rice. In this study, the full nucleotide sequence of MokH, MokF and MplaeA and the partial nucleotide sequence of hsp70, about 1kb, were amplified from M. purpureus genome by homologous method. The four genes may have an influence on the Lovastatin synthetic ability of Monascus strains in different level. The partial nucleotide sequence of M. purpureus hsp70 show the best homology to the sequence of Aspergillus niger hsp70 and the highest identity is 90%. The highest identity of MokH, MokF and MplaeA between M. purpureus and M. pilosus is 100%,100% and 99%. It indicate that the three genes is extremely conservative between Monascus strains.The four amino acid sequences encoded by the nucleotide sequences were analysed by a series of bioinformatics methods. The results of Sub-cellular localization analysis showed that MokF and MplaeA were localized in the cytoplasm, and MokH was localized in the nucleus; Hydrophobicity analysis predicted that MokH、MokF and MplaeA are all belong to hydrophilic protein, and MplaeA shows higher hydrophilic property than MokH and MokF. The results of domain function analysis indicat that MokH is a member of the Zn2Cys6 binuclear cluster motif superfamily, MokF is a member of theβ-Lactamase family and MplaeA is one of Arginine methyltransferases. We further investigated the homologous sequences of the four genes from 18 different fungi species by means of multiple alignment analysis. The phylogenetic trees were constructed based on the sequence of hsp70, MplaeA and MokF. The phylogenetic tree clearly showed that Monascus was closed to Aspergillus and Penicillium and is alienated to Ajellomyces. The dimensional structure prediction of MokF and MplaeA as well as their homologous protein showed that the more close strains are, the more similar 3D structure are.Then the constitutive fungus overexpresssion vector pCAMBIA1300M-MokH was constructed by DNA recombination technique, and transferred into Agrobacterium tumefaciens (LBA4404 and EHA105) by freeze-thawed method. Preliminary study on a transformation system of M.purpureus mediated by Agrobacterium tumefaciens showed the proper concentration of hygromycin B and cephalothin for screening of transformant is respectively 40μg/mL and 300μg/mL or 400μg/mL. In order to chose a suitable medium for transformation, we use rice flour medium, PDA medium and Czapex-Dox agar medium to culture M.purpureus.