Initial Study On Metabolites Regulation And Biosynthesis Of Stereum Hirsutum FP-91666

Microbial resources are one of the three biological resources tied with animal resources and plant resources.In the increasingly scarce resources today,microbes can still be said to be a treasure trove of resources with inexhaustible development potential.In this paper,Stereum hirsutum FP-91666 was used as the experimental material to study the separation of secondary metabolites.At the same time,it was induced by methylation inhibition Decitabine(DAC),according to the theory of genetics and the method of one strain many compounds(OSMAC),and the samples were detected by UPLC-MS.The results showed that there were new compounds in this strain,which laid a foundation for the development and utilization of the secondary metabolites.Vibralactone,which is the active product from S.hirsutum,is a hotspot of experts and scholars at home and abroad because of its unique ?-lactone bicyclic structure and high pancreatic lipase inhibitory activity.Studying in the biosynthetic pathway of the vibralactone,we have comfirmed that the compound did not belong to polyketones or sesquiterpenes at the source.The core carbon skeleton of vibralactone was derived from the shikimic acid pathway,and the side chain isopentenyl was derived from the MVA pathway,which it belonged to meroterpenoids in biological source.And the activity of isopentenyl transferase has also been demonstrated.The CYP450 oxidoreductase studied in this paper was the key enzyme in the step of oxidative expansion of the biotin biosynthetic pathway,and the enzyme activity was studied preliminarily.The main results of this paper were showed as follows:1.In the study of the chemical composition of S.hirsutum,utilizing the separation materials like silica gel and gel,semi-preparative HPLC,NMR(including 1D-NMR,2D-NMR),high resolution mass spectrometry(HR-ESI-MS)and ultraviolet spectroscopy,4 compounds were isolated and identified from YMG liquid fermentation products.And among them were vibralactone derivatives,which the compound 1 was a new compound and was named as Vibralactone R.2.In the YMG liquid medium,the methylation inhibitor Decitabine was added,the same amount of sterile water was used as a control to cultivate S.hirsutum strain.After 21 days of fermentation,3 mL of fermentation broth was extracted twice with equal volumes of ethyl acetate to obtain a sample.The samples were detected by UPLC-MS,and the new compounds were found in it.3.The sequence of amino acid and nucleic acid of cytochrome P450 were obtained by gene sequence analysis and NCBI comparison.Total RNA was extracted from S.hirsutum and successfully reverse transcribed into 1st cDNA by RT-PCR.And the specific PCR was performed using cDNA as template.The P450 gene was amplified and verification.The correct gene was cloned into expression vector pET28b(+)and pET32b(+),and the the recombinant plasmids were transfected into BL21(DE3)and Rosetta-gamiTMB(DE3).There were 4 expression recombinant strains.Heterologous expression was performed,and the soluble protein was obtained.4.The optimal culture temperature of pET28b(+)was 27 ?,the time was 6 h;the optimal culture temperature of pET32b(+)was 37 ?,the time was 2 h;The optimal IPTG concentration was 0.2 mM;the optimal ultrasonic response buffer was HEPES;the optimum reaction pH was 8.0.5.Using 4-(hydroxymethyl)-2-(3-methylbut-2-en-1-yl)phenol as the substrate,the target protein was subjected to catalytic reaction adopting three catalysis methods including whole cell catalysis,cell resting catalysis,in vitro crude enzyme catalysis.The products of enzyme were identified by UPLC-MS.However,the target protein did not show a clear activity.