Molecular Cloning, Expreeion And Characterization Of The Histone Deacetylase Genes Pci-RpdA And Pci-HdaA In Penicillium Citrinum

Background:Secondary metabolites of filamentous fungi are important sources of new drugs, a lot of useful antibiotics, immunosuppressants, anticancer drugs are produced by fungi. One of those is Mevastatin, a polyketide compound produced by Penicillium citrinum and has been used as a substrate for microbial conversion to pravastatin. Pravastatin has been widely used as a pharmaceutical product for the therapy of hypercholesterolemia, with the extensive market and consumer demand. Since the huge impact on economic and human health the statins have, P. citrinum, as a important pharmaceutical microbial resources, its metabolites and metabolic regulation mechanism are extensively researched. Secondary metabolites of fungi are a complex multi-level control process. Biosynthesis of secondary metabolites not only regulated by way of specific transcription factor, it is also regulated by the global regulator. Research on some fungal species in recent years shows, chromosome epigenetic state is directly related to the regulation of fungal secondary metabolite gene cluster. Chromosome epigenetic state is also known as chromosome remolding, which is controlled by histone code, including histone methylation, acetylation, phosphorylation and etc. Acetylation of histones is a dynamic process that depends on the concerted action of histone acetyltransferases and histone deacetylases. In recent years, the histone deacetylase as microbial breeding target and microbial silence gene mining attracted the attention of scientists:Treatment of a variety of fungi with HDAC inhibitors or molecular operation fungi of histone deacetylase gene (such as gene knockout) increased production of numerous natural products or even obtained novel secondary metabolic products, which provide the material basement for the discovery of new pharmaceutical research value lead compounds.Aim:Clone, identify and expression the histone deacetylase genes Pci-RpdA and Pci-HdaA, in order to provide candidate genes and molecular targets for epigenetic breeding in mevastatin-producing fungus Penicillium citrinum. Detect the relative expression of Pci-RpdA and Pci-HdaA through different fermentation periods, thus revealing the transcription characteristics of the two genes, in order to lay the foundation fpr the further gene function study. Comparison of the two genes sequence differences in the wild strain ATCC38065and industrial strain IMB002, we inferred changes of enzyme protein tertiary structure caused by the sequence differences brought about the functional differences.Methods:3’-race technology and nested-PCR technology were used in order to obtained the Pci-RpdA and Pci-HdaA DNA sequence and cDNA sequence. E. coli protein expression system was used to expreeion the combination RpdA and HdaA. Corresponding structure and functions were predicted by the bioinformatic software. qRT-PCR was used to detect the expression characteristics of Pci-RpdA and Pci-HdaA through fermentation process in wild strain. DNA and cDNA of Pci-RpdA and Pci-HdaA in industrial strain IMB002were cloned, and software was used to comparison the nucleic acid and protein sequences to the wild strain.Result:The full-length RpdA sequence is2,072bp with4exons and3introns. The open reading frame is1,920bp which encode a protein of639amino acid residues. The RpdA protein presents a conserved sequences component which belongs to Arginase-HDAC superfamily. The full-length Pci-HdaA sequence is3,053bp with an ORF of2,295bp encoding764amino acids. The Pci-HdaA protein also presents a very conserved sequences which belongs to Arginase-HDAC superfamily.Two recombinant expression vectors pET-28(+)-RpdA and pET-28(+)-HdaA were constructed successfully based on pET-28(+). Preliminary study on the expreeion of recombinant RpdA and recombinant HdaA in E. coli was carried out, and the recombinant RpdA protein was purifield.qRT-PCR analysis showed that in different fermentation stages in wild strain P. citrinum, the transcription level of Pci-RpdA was significant different. The transcription level was relatively high in the early stage of fermentation, but declined in the later stage. The transcription level of Pci-HdaA was also significant different. The transcription level was relatively low in the early stage of fermentation, but relatively active in the later stage.Analysis of the two genes’sequences differences in the wild strain ATCC38065and the industrial strain IMB002displayed that as the changes of the Pci-RpdA gene sequence in the170site and1938site, made the protein Pci-RpdA sequence57arginine and596atorvastatin in wild strain ATCC38065mutate into lysine and serine in the industrial strain IMB002. The secondary structures of Pci-RpdA in the two strains showed that due to a mutation of the57amino acids,59-60amino acid residues formed curled structure in the wild strains ATCC38065changed into formed an small β-sheet structure;596amino acid mutations in industrial strain IMB002, but the mutation in596site of protein sequence did not affect the secondary structure of Pci-RpdA. As the changes of the Pci-HdaA gene sequence in the778site and1360site, made the protein Pci-HdaA sequence240valine residue and381alanine residue in wild strain ATCC38065mutate into lsoleucine and threonine in the industrial strain IMB002. The secondary structure of Pci-HdaA in the two strains showed that the mutation in240site of protein sequence did not affect the secondary structure of Pci-HdaA, but due to a mutation of the381amino acids,366-380amino acid residues formed α-helix structure in the wild strains ATCC38065shorten into366-377amino acid residues in industrial strain IMB002.Conclusion:We have cloned and analyzed histone deacetylase genes Pci-RpdA and Pci-HdaA from P. citrinum which provide a good foundation for further research on epigenetic breeding in P. citrinum. We have got the recombination RpdA protein using E. coli expression system, which provide basis for further characterization of this enzyme. We have bioinformatic analysised the feature of Pci-RpdA and Pci-HdaA and their coding products, found the important expreeion characterization of Pci-RpdA and Pci-HdaA through fermentation, which provide the theoretical basis for further gene function study. Also, we compared the sequences of Pci-RpdA and Pci-HdaA in wild strain and industrial strain respectively, which provide clues for the correlation of HDACs and high-yield metabolites mechanism, and for understanding the complicated epigenetic regulation mechanisms in eukaryotes.