Screening And Functional Validation Of Citrinin Related Genes In Monascus Purpureus

Monascus spp. are traditional Chinese fermentation microorganism, and widely used in the food industry. Monascus pigments are important natural colorants that allowed to be added into food. And in recent years, people found that Monascus can produce a variety of secondary metabolites, such as monacolin, ergosterol, β-aminobutyric acid, glucosamine and a variety of enzymes, which expanded the applications of Monascus. However, in1995the French scientist Blanc reported that a fungal toxin was isolated from Monascus products, which was identified as Citrinin later. Citrinin is harmful to human kidneys. The existence of Citrinin becomes a bottleneck of Monascus industry, and has seriously affected the production and export of Monascus products. In order to obtain the genes involved in citrinin biosynthesis, in this study, we successfully constructed a mutant library containing927mutants using Agrobacterium-mediated transformation method. After inhibition zone test selection and PCR verification, we get11Citrinin lower yields mutant. Using Tail-PCR amplification, the T-DNA flanking sequence of a mutant was obtained. Sequence analysis indicated that it exhibited75%similarity with oryzae/allyl ester reductase gene (RIB40) of Aspergillus oryzae.In order to obtain a modified Monascus strain that is defective in citrinin production, gene knockout technique was used to silence the ORF3of citrinin gene cluster. Primers were synthesized and used to amplify the1021bp upstream region and1009bp downstream region of the target gene. After being digested with ApaI and XhoI, SmaI and XbaI, the two flanking regions were inserted the plasmid pUCH2-8, which were digested using the same enzymes. The recombinant plasmid was further confirmed using PCR and restrictive digestion. After transformation into Monascus purpureus strain and screening using selective plate and PCR, a mutant was obtained. Pigments and citrinin producing abilities of the mutant were examined. The results indicated that citrinin yield decreased to3.52%of the wild-type strain. By contrast, the yellow pigment production increased89.9%, the orange pigment improved172.1%, and the red pigment increased140.6%. These results demonstrated that, after ORF3being knocked out, the common substrate was used to produce pigments instead of citrinin in the mutant.In this study, a gene involved in citrinin production was obtained and the function of a gene located in citrinin biosynthesis gene cluster were verified using gene knock out procedure. This work is helpful to further researches on citrinin biosynthetic pathway and construction strains defected in citrinin producing.