Screening, Isolation And Stucture Elucidation Of Microbial Metabolitee Extracts For Regulating NF-κB Pathway

Objective: Nuclear factor κB (NF-κB) is a ubiquitous transcription factor inthe cytoplasm. many diseases are related to the activation or inhibition of NF-κBsignaling pathway.We established a set of methods for fermentation,extraction,and screening microbial metabolites, the active strains were fermented and thebroth was isolated, main compounds were purified and identified.Methods: Microorganisms were incubated at30℃and180rpm for3days,and then lysed with a final concentration of70%ethanol overnight. The lysateswere centrifuged, and the supernatant was concentrated by rotary evaporationand freeze-dried to obtain metabolite extracts. Luciferase activity of525metabolite extract samples was assayed using the Dual-luciferase reporter assaysystem, The HEK293cells were transfected with pNF-κB-luc and pRL-TKplasmid. After6h transfection, cells were treated with metabolite extracts orTNF-αfor24h, and Luciferase activity was assayed using the Dual-luciferasereporter assay system with the pRL-TK vector as the internal reporter control. a large-scale fermentation of3092#Strains was developed, the culturemedium contained soluble starch1%,glucose2%, beef extract0.1%,yeastextract1%, NaCl0.2%,K2HPO40.025%,CaCO30.2%,PH=7.0. Thefermentation was developed in shake flask and tranfered to tank for maintain of50L. After the extraction of the fermentation broth，the crude extract waspurifiad by Silica gel column chromatography, Sephadex LH-20,TLC and HPLC.The structure of compounds was identified by HR-MS,13C-NMR,1H-NMRetc.Biological activity of extracts and compounds were tested byDual-luciferase reporter assay system.Results: In the first round of screening, we found30kinds of metabolitesthat significantly activated NF-κB pathway and28kinds of extracts inhibitedNF-κB pathway. There were13kinds of metabolites that significantly activatedNF-κB pathway and no metabolites inhibited NF-κB pathway. And finally,6kinds of metabolite extracts of activating the NF-κB pathway were screened inthe third round of screening and validation.A series of lipopeptide Lichenysin(L31-L37) were isolated from crudeextract of the3092#fermatation broth and were identified to beL32：cyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (isoC13)å'Œcyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (anteisoC13)L34：cyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (isoC14)。L36：cyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (isoC15)å'Œcyclo-[L-Gln1â†'L-Leu2â†'D-Leu3 â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (anteisoC15).L33：cyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (isoC13或anteisoC13)å'Œcyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Val7]-β-OH fatty acid(isoC14或anteisoC14)L35：cyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Ile7]-β-OH fatty acid (nC14)å'Œcyclo-[L-Gln1â†'L-Leu2â†'D-Leu3â†'L-Val4â†'L-Asp5â†'D-Leu6â†'L-Val7]-β-OH fatty acid (nC15)Other five compounds were isolated, three of them were identified to beLeucine, n-hexadecanoic acid,3-hydroxy decanoic acid.