| The filamentous fungus Monascus is an important microbiological resources in china, which can produce a variety of bioactive metabolites. It’s one of the most important microorganisms which can produce the edible pigment. Monascus can also produce a toxic substance-citrinin, hindering the application of red yeast rice products.In this paper, the orf3and ctnE gene disruption mutants in Monascus aurantiacus AS3.4384were constructed using gene-knockout technology, then there secondary metabolite production were analyzed. This laid the foundation to elucidate the themetabolic pathway of citrinin.The major findings were expounded as follows:1. Through the NCBI Blast analysis, deduced that orf3gene encoding the protein of DIOX-N superfamily and the ctnE gene encoding the protein of NADB-Rossmann superfamily.2. The replacement vector of pORF3-HPH or pCTNE-HPH were respectively transfered into the protoplast of M.aurantiacus AS3.4384to disrupt target genes by PEG-CaCl2mediated protoplast transformation technology. Then, methods of hygromycin resistance screening, Dot blot analysis and Southern blot analysis were performed on selected clones. Ultimately, two orf3gene and one ctnE gene disruption mutants were gained.3. The citrinin of orf3and ctnE gene disruption mutants were analysed by HPLC and the pigments were analysed by UV spectrophotometer. Eventually citrinin production of two orf3gene disruption mutants were reduced by87%and86%compared with the original strain, but the pigment production were increased by13%and15%. It indicated that the orf3gene was a citrinin synthesis-related gene, but the deletion of the gene does not affect the production of pigment; The ctnE gene disruption mutants citrinin production were reduced by96%compared with the original strain, but pigment production were increased by40%. Confirmed that ctnE was a citrinin synthesis-reletived gene and the gene was conducived to the synthesis of pigment. |
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