Mining And Precursor-directed Biosynthesis Of Secondary Metabolites From Sponges Associated Microorganisms

Marine microorganisms represent a promising and sustainable source of natural products,which exhibit a vast potential for new secondary metabolites.Sponges are primary contributors of marine microorganisms due to the high abundance and diversity of symbiotic microbial communities.Sponge associated microorganism plays a vital role in chemical defense for its host,thus usually producing a wide array of novel secondary metabolites with excellent bioactivities.However,the development of these secondary metabolites from marine microorganisms is usually limited by the traditional methods of cultivation and isolation due to the difficulties of production and repetitive purification,etc.Strategies that integrate production,structure prediction,and dereplication of secondary metabolites will provide resolutions of above-mentioned difficulties.Meanwhile,precursor-directed biosynthesis in microorganisms will facilitate the structural modification of secondary metabolites to obtain “unnatural” secondary metabolites with better bioactivity.In this study,we first confirmed the sponge associated microorganisms with the potential for producing functional molecules.The integrated strategies of OSMAC(One Strain Many Compounds)and dereplication of HRMS(high resolution mass spectrum)revealed that sponge associated fungus Acremonium persicinum F10 had the potential of producing new secondary metabolites when cultured in rice medium;The integrated strategies of epigenetic modifiers induction cultivation and dereplication of HRMS showed that new secondary metabolites were produced in sponge associated fungus Pestalotiopsis maculans 16F-12 by bortezomib-inducing cultivation.The potential of producing bioactive secondary metabolites by sponge associated actinomycetes Streptomyces sp.MB044 and Streptomyces rochei MB037 was disclosed by mining their genome and prediction by HRMS database.Moreover,the integrated strategies of OSMAC and dereplication of HRMS predicted that sponge associated fungus Eurotium rubrum 13J1-4 had the potential of producing bioactive secondary metabolites.Subsequently,the detailed analysis of secondary metabolites from potential strains was carried out by scale-up fermentation,chemical profiles guided-separation,and identification by spectra of HRMS,1D/2D NMR,IR,and UV,etc.Seven new cyclopeptides(compounds 1-6,and 9)and two known compounds(compounds 10 and 11)were traced from strain A.persicinum F10.Two new chelate compounds(compounds 7 and 8),with Ga3+ as the central ion,were synthesized in vitro;Six purified compounds(12-17),including four new compounds(13–16)and two known compounds(17–18),were isolated by tracing the extra peaks of secondary metabolites from strain P.maculans 16F-12;Eight known compounds(18-25)were obtained from sponge associated fungus E.rubrum 13J1-4,actinomyces Streptomyces sp.MB044 and S.rochei MB037.New compounds 1,5,7,and 8 exhibited excellent anti-fungal activities,comparable with the positive control,amphotericin B(MIC at1.0 ?M).Moreover,borrelidin(25)exhibited potent activity against tumor cells,the IC50 values of which against lung cancer cells A549,H446,and H1688 were at sub-micromolar level(0.4 ?M,0.3 ?M,and 0.3 ?M,respectively).The clinical utilization of borrelidin has been hampered by its high cytotoxicity,which results in a narrow therapeutic index,against normal cells.We developed an efficient method of biosynthesizing the low-toxic compound BC194 with antiangiogenic activity via precursor-directed biosynthesis by borrelidin-producing strain S.rochei MB037.The Radix astragali(RA)medium was found to be the optimum medium for producing borrelidin,and the precursor-directed biosynthesis of BC194 by the borrelidin-producing strain S.rochei MB037 was implemented by adding 5 m M exogenous precursor,trans-1,2-cyclobutanedicarboxylic acid(trans-1,2-CBDA).We analyzed the biosynthesis of the endogenous precursor,trans-1,2-cyclopentanedicarboxylic acid(trans-1,2-CPDA)and found that3,4-dihydroxyphenylacetic acid(DHPAO)was a key enzyme in this biosynthetic pathway.By screening inhibitors and substrate analogs of DHPAO,we observed that potassium ferricyanide was a possible inhibitor of DHPAO,which could reduce the yield of trans-1,2-CPDA.By adding 5 m M potassium ferricyanide in the precursor-directed biosynthesis of BC194,the yield of borrelidin was significantly reduced and the yield of BC194 increased by 112.5%;By increasing the trans-1,2-CBDA concentration and using the macroporous adsorbent resin DA-201,the yield of BC194 was increased by 69.7% and 36.6%,respectively,reaching 27.59 ±1.15 mg/L;We developed an effective method for separation and purification of BC194 by employing HSCCC,and the recovery reached 93% from the previous 56%.In summary,by combinations of induction,prediction,and dereplication of microbial secondary metabolites,we quickly found the potential sponge associated microorganisms which could produce functional molecules.Then,twenty-five compounds were traced and prepared from these potential sponge associated microorganisms,including thirteen new structural compounds(1-9 and 12-15)and high cytotoxic borrelidin.Subsequently,the precursor-directed biosynthesis and yield optimization of low-cytotoxic BC194 with antiangiogenic activity,from borrelidin,were carried out by sponge associated actinomyces S.rochei MB037.These studies disclose the application potential of comprehensive mining strategies in discovery and biosynthesis of secondary metabolites,and provide the reference for efficient mining and utilization of secondary metabolites from sponge associated microorganisms.