| Monascus pigments(MPs)as an important class of secondary metabolites from Monascus spp.are a complex mixture mainly including 3 kinds of(orange,red and yellow)MPs constituents and have been widely utilized for several centuries in China,especially in Southeast Asian countries.Up to now,even though about 90 MPs components have been identified,MPs biosynthesis pathway is still dispute.Studies have shown that,MPs biosynthetic pathway is regulated by pigment gene cluster,so analyszing the pigments from the knockout strains or heterologous expression strains is an effective method to reveal the pigment gene function and to explore biosynthesis pathway of MPs.The RFR(Red fermented rice)of Monascus ruber M-7,11 kinds of knockout strains(△pig A,△pig B,△pig C,△pig D,△pig E,△fasα,△fasβ,△pks10,△fmds,△fao,ISM);Czapek medium of 3 kinds of Aspergillus oryzae heterologous expression pigment-related genes strains(A.oryzae-A,A.oryzae-A-O/N,A.oryzae-A-O/N-D)and 2 mutant strains(A.oryzae-ACP1、A.oryzae-ACP2)are studied in this experiments.HPLC conditions are established to analyze the pigments among these strains.It is an effective method to explore the possible biosynthesis pathway of MPs by combining HPLC results,the gene prediction function and feeding experiment.The main findings are as follows:1.Establishment of HPLC for analyzing MPsIt is important for establishing a uniform HPLC detection method to analyze the various and complicated MPs in Monascus ruber M-7,gene knockout strains and heterologous expression strains.In this experiment,the strains of M-7 and △fasα were used for establishing the HPLC methods due to the more weak polar compounds in M-7 and the more strong polar compounds in △fasα.The red fermented rice(RFR)of M-7 and △fasα were extracted by methanol with solid-liquid ratio 1:5(g/m L),then ultrasonic extracted for 30 min after mixed thoroughly and at last the supernatant was taken out after 12 000 r/min centrifugation for 10 min.The extracting solution was filtrated through a 0.22 μm membrane,then 10 μL sample diluted 300 times was separated by Shimadzu Inertsil ODS-3 at 25℃,the flow rate was set as 0.8 m L/min and detected at 520 nm,470 nm,380 nm and 360 nm by PDA.The establishment of G1 and G2 can be applied to analyze the weak polar component and a polar component respectively.The results show that the main pigments in M-7,△pks10,△fmds,△fao,△pig E can be separated following a gradient elution of G1 program and the main pigments in△pig C,△pig D,△fasα,△fasβ,△pig A,△pig B,heterologous expression strains can be separated following a gradient elution of G2 program.2.The separation and identification of pigments in Monascus ruber M-7The pigments from RFR of M-7 were separated by thin layer chromatography(TLC),which was performed on silica gel G with the mobile phase of toluene: ethyl acetate: formic acid =7:3:1(V/V/V).The result shows that according to the maximum absorption wavelength(MAW)14 kinds of MPs are separated including 6 kinds of red pigments(R3-R8)whose MAW is 490 nm～530 nm,6 kinds of yellow pigments(R9-R10,Y1-Y2,BF1-BF2)whose is MAW 330 nm～450 nm,and 2 kinds of orange pigments(O1-O2)whose MAW is 460 nm～480 nm,.Their Rt under G1 analysis are(min): 9.223,13.224,7.150,7.494,8.262,12.307,6.892,10.564,16.727,22.201,14.245,19.802,17.499,23.505 respectively.After collected the 14 kinds of pigments and combined the results of MS,spectrum with references,Red Pigments A(R5),Red Pigments B(R6),Rubropuncta-mine(R7),Monascorubramine(R8),Monasfluol A(R9),Monasfluol B(R10),Monascine(Y1),Ankaflavin(Y2),Monasfluore A(BF1),Monasfluore B(BF2),Monascorubrin(O1),Rubropunctatin(O2)are identified.The structure of R3(m/z=482)and R4(m/z=510)are unidentified.3.The pigments isolation and identification in RFR of M-7 pigment-related gene knockout strains and heterologous expression strains of Czapek mediumThe analysis of TLC,HPLC(G1)and MS indicates that the major kinds of indentified pigments in RFR of △pks10,△fmds,△fao and M-7 are same;The Rt of major5 kinds of indentified yellow pigments in RFR of △pig E: FK17-P2b2(YP1,m/z=236),Monasfluore A(YP2,m/z=356),Monasflouore B(YP3,m/z=384),Monascin(YP4,m/z=358),Ankaflavin(YP5,m/z=386)are 4.553,13.779,19.459,16.318,21.893;The analysis of TLC,HPLC(G2)and MS indicates that there were no pigments in RFR of △pig A and △pig B;the RFR of △fasα and △fasβ contains 1 kinds of yellow pigment named △fasα-1(Monascusone A)whose Rt is 10.435 min.;The Rt(min)of△pig D(m/z=250)in RFR of △pig D and M7pks-1(m/z=252)the main substance in RFR of ISM are 9.060 and 17.722 respectively;the RFR of △pig C contains 3 kinds of components named △pig C-1(m/z=248),△pig C-2(m/z=232)and △pig C-3(m/z=246)whose Rt(min)are 17.975,21.386 and 24.620 respectively under the same condition.The pigments in 5 kinds of Aspergillus heterologous expression strains at Czapek medium for 5 d were choosen to analyze.The pigments were extracted by methanol with solid-liquid ratio of 1:4(g/m L),then 20 μL extraction was separated by HPLC(G2)condition.The results shows major new metabolites in A.oryzae-A,A.oryzae-A-O/N are△pig C-2 and △pig C-3 respectively by LC-MS analysis compared with the pigments in△pig C.There is no detectable pigment in A.oryzae-ACP1,A.oryzae-ACP2 and A.oryzae-A-O/N-D.4.Feeding experiment to monitor the kinds of pigments in △pig AOur previous studies have show that M7pks-1 the main subtances in RFR of ISM might be an important precursor in MPs biosynthesis pathway.The final concentration of M7pks-1 added to Czapek solid medium of △pig A cultured at 28℃ was 0.005,0.1,0.5(mg/m L),new pigments in Czapek medium(including mycelium)of △ pig A were chosen to analyze under the condition of G1.Different concentrations of M7pks-1 added to the medium cultured for 6 d at28℃.The results show that when the concentration of M7pks-1 is 0.005 mg/m L,△pig A can generate 6 kinds of yellow pigment including R9-R10,Y1-Y2,BF1-BF2;When the concentration of M7pks-1 is 0.1 mg/m L,△pig A can generate 4 kinds of yellow pigments including Y1-Y2,BF1-BF2 and 4 kinds of red pigments including R7-R8 and 2 kinds of unidentified red pigments whose Rt is the same as R9-R10;The main pigments are same between the 0.1 mg/m L concentration of M7pks-1 and 0.5 mg/m L concentration of M7pks-1.Minimum concentration of M7pks-1(0.1 mg/m L)that can prompte △ pig A to produce a stable pigments was chosen to detect MPs in △pig A cultured for different days at 28℃.The results show that when Czapek solid medium of △pig A cultured for 3 d-5 d,the main pigments are Y1,Y2,BF1 and BF2;while at the same concentration for 6 d and12 d,besides the 4 kinds of yellow pigments,there are 4 kinds of red pigments including R7-R8 and 2 kinds of red pigments whose Rt are same as R9,R10 but the structure are unidentified.There are no any orange pigments in this adding experiment.Therefore M7pks-1 is a precursor in the MPs synthesis pathway and the formation of yellow pigments is earlier than the formation of red pigments. |
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