Study On Screening Of High-yielding Monacolin K Monascus And Construction Of Knockout Plasmid About PKS Gene

Monascus spp.are filamentous fungi famous for their fermented products.Especially red mold rice,was a traditional fermented food in China or even in East Asian areas with a very long edible history.Monascus species are known as producers of various secondary metabolites with polyketide structures,and the well-known ones are GABA(y-Aminobutyric Acid),monascus pigments,monascus polysaccharide and Monacolin K.But citrinin,a nephrotoxic metabolite,cannot be ignored.With the development of living standards,the fact that Monacolin K can suppress the synthesis of chelesterol has been widely noted in pharmacological circles.Therefore,through fermentation improvement and molecular modification,monascus with the nature of higher-yielding Monacolin K as well as lower-yielding citrinin have a great significance.This manuscript firstly screened Monascus purpureus M-24 with the property of high-yielding Monacolin K as well as low-yielding citrinin.Secondly,the optimum fermentation conditions were determined by six single-factor experiments and these optimal factors were used in fermenter.Thirdly,through the isolation and purification of broth after fermentation in fermenter,relatively high-purity Monacolin K was gained finally.Fourthly,knockout plasmid was constructed to konck PKS gene out.So the research was finished by a four-step procedure.?.278 strains of Monascus spp.were obtained from Monscus-fermented products,of which 10 strains were chosen as experimental strains by HPLC(High Performance Liquid Chromatography).Combinaed with TLC(Thin-Layer Chromatography)and data from full wave scanning of citrinin,Ultimately,numbered-24 was confirmed as our objective strain.Then,morphology observation,microscopical character and ITS sequence were conducted to identify numbered-24 as Monascus purpureus M-24.?.An optimum medium was gained through single-factor tests.It exhibited the optimum medium compositions were:glycerol,peptone,Carbon/Nitrogen(5:1),Culture temperture(26?),pH(5.0)and revolving speed(160 r/min).Then the consistent culture medium and fermentation conditions were appied to amplify fermentation in the 5 L fermenter.After these experiments,growth curve,curve of Monacolin K production,citrinin curve and curve of pigment about M-24 strain were draw.?.Isolaton and purification of broth was conducted.Methods of isolation were referred to Endo.Purification was performed to prepare by semi-HPLC,which was based on double-stage.?.After general information about Monacolin K production of M-24 strain,it was necessary to control or even eliminate citrinin.In this research,the knock-out plasmid of PKS gene was conducted as the procedure of Double-joint PCR.