Identification And Property Analysis Of A Metabolite Produced By A Mutant ISM Of Monascus Ruber M-7

Red fermented rice （RFR）, also known as red yeast rice or red rice, was formed by fermenting steamed rice of Monascus spp.. RFR has been applied in China for thousands of years, which contains many biologically active secondary metabolites, such as Monascus pigment （MPs）, monacolin K and y-amino butyric acid. MPs are a class of polyketides produced during metabolism of M. spp. There has been no report about the enzymes, intermediates and gene of MPs synthesis pathway up to now.Our laboratory has obtained a mutant （Intermediate secreting mutant, ISM）, when knocked-out the a subunit gene （fasa） and βsubunit gene （fasβ） of fatty acid synthase which exisit in the MPs gene cluster of M. ruber M-7. The mutant which missed the fasa and fasβby PCR and Southern hybridization, can accumulate a metabolic product （tentatively named compound X）, which may be an intermediate of MPs synthetic pathway of M. ruber M-7, because it can help ΔpksPT（a mutant which knocked-out the MPs polyketide synthase gene） generate MPs again.In current study, ISM was used as the research strain, and the structure of compound X after separated and purified, have been identified by LC-MS and NMR, the properties have also been investigated. The main results are as follows.₁ Purification and characterization of compound XThe crude extract of compound X from ISM RFR strain was prepared as followed:1g RFR powder （40 meshes） was mixed with 10 mL acetonitrile, then treated for 30min by ultrasonic after homogenized for 5 min, after that, centrifugated at 5000 r/min for 5 min, the supernatant was considered as the crude extract of compound X.The crude extract of compound X was purified with semi-preparative HPLC after concentrated by rotary evaporator, and the HPLC conditions were as below:Shimadzu Inertsil ODS-3 column （4.6 mm×250 mm, 10μm）; column temperature,30℃; mobile phase acidic （double distilled water containing 5%o H3PO4）/acetonitrile/water= 5:50:45 （/v）;flow rate;3.0 mL/min; PDA detector; detection wavelength:200 nm-500 nm. The purified compound X was analyzed by HPLC, the results showed that its purity reached chromatographic level.Physical properties of compound X were further analyzed, the results revealed that the maximum UV absorption wavelengths were 215 nm and 290nm in acetonitrile, while no fluorescence absorption characteristics was detected. The crytal of compound X was white floccule, its micro-morphologies were clustered, and single crystal was like flat, and the melting point of the compound X was 176℃.2. Structure analysis of compound XThe structure of compound X was investigated by elemental analysis, infrared spectroscopy, liquid-mass spectroscopy, MRI and so on. The results showed that the molecular mass of compound X was 274, including three elements, carbon, hydrogen, and oxygen, and their contents were 60.35%,6.14%,31.31%, respectively, which indicated that the formula of compound X was C14O5H26.IR analysis proved that compound X contained some chemical groups like-CH3,-C=O,-OH and so on.3. Biological activity analysis of compound XThe inhibitory effects of compound X on Escherichia coil, Staphylococcus aureus, Salmonella spp., Bacillus subtilis and Listeria monocytogene were researched in this study by inhibition ratioes and inhibition zones. The results showed that the inhibition ratios to Escherichia coil, Staphylococcus aureus, Salmonella spp. and Bacillus subtilis were lower than 10% in PDB at 30.3 mg/mL compound X, while that to L. monocytogene was 27.76%. In the antibacterial experiments of compound X at the same concentration on PDA, only significant inhibition zone was found for L. monocytogene. These results suggested that antimicrobial effects of compound X to the above five tested strains were poor.The inhibitory effects of compound X on the mouse myeloma cells SP2/0, mouse renalcellcarcinoma cells BHK and human renal epithelial cells 293 transfected with adenovirus El A gene was investigated. The results revealed that, the inhibition ratioes of compound X on these tumor cells were 13.32%,26.76%,23.47%, respectively, when the concentration of compound X was 100 mg/mL4. The impact of compound X on pigment production capacity of △PigE which was the deletion mutant of the gene of aryl alcohol reductase in the MPs gene cluster of M. ruber M7HPLC analysis showed that APigE which was a deletion mutant of the gene of aryl alcohol reductase in theMPs gene cluster of M7 could only produce four kinds of yellow pigments when fermented 8 days in PDB, and their retention times were approximately 4 min,11 min,15 min,20 min, respectively. When 100μL compound X at 100 mg/mL was added to ApigE’s PDB every 2 days after 6 days incubation, the types and contents of yellow pigment of ApigE in PDB were all changed after added compound X. The component at 4 min retention time was not almost affected by compound X, and the contents of the components at 11 min and 20min retention times, respectively, were increased more than twice, while the content of the component at retention time 15minwas improved more than 30%.