Discovery And Biosynthesis Of Natural Products With Promising Activities From Eurotium Cristatum CB10006 And Streptomyces Sp.CB09030

Infectious diseases and inflammation are serious threats to human health.Microbial natural products are important sources of antibiotics and anti-inflammatory drugs.Eurotium cristatum is the dominant fungi species in Fuzhuan brick tea（FBT）,one of the major dark tea brands in China.However,to our knowledge,there are only limited studies on the natural products produced by E.cirstatum.Therefore,the natural product research of E.cirstatum from FBT would not only reveal its biosynthesis potential,but also shed light on the various health benefits of FBT consumption,including the antibacterial and anti-inflammatory effects.Streptomyces species produce more than 70%clinically-used antibiotics,but the traditional activity screening methods are time-consuming and relatively inefficient.Transwell-based Streptomyces solid fermentation could be more conveniently used for activity screening,leading to the discovery of natural products with interesting biological activities.Here,this thesis reported the discovery,biological activity and biosynthesis of natural products produced by E.cristatum CB10006 from FBT and S.sp.CB09030 obtained by transwell-based screening technology,which reveals the promising biosynthetic potential of those strains.The main contents are as follows:（1）Isolation,structural elucidation,and biological evaluation of natural products from E.cristatum CB10006.E.cristatum CB10001?CB10008 from different FBTs were identified by 18S r RNA sequencing.E.cristatum CB10006 was selected as the prototype for the scaled-up fermentation,resulting in the isolation of eleven natural products.They are six benzaldehydes,four anthraquinones,and one indoledione piperazine alkaloid.The spore contents of eight E.cristatum strains were also analyzed,showing the presence of abundant of benzaldehydes.Some benzaldehydes could inhibit the expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins in lipopolysaccharide-induced RAW264.7 cells,indicating that they have certain anti-inflammatory activity.Further mechanistic studies revealed that compound A6 exerted the anti-inflammatory effect by inducing the activation of adenosine monophosphate-activated protein kinase.Finally,the whole genome sequencing of CB10006 and bioinformatics analysis revealed the presence of multiple biosynthesis gene clusters.（2）Yield improvement of benzaldehydes and anthraquinones in E.cristatum CB10006.Since these benzaldehyde and anthraquinone derivatives have anti-inflammatory or antibacterial activities,the yield of catenarin（A8）was increased by 2-fold through the addition of 10 m M Na NO₃in medium.Eleven mutant strains were isolated by ribosomal engineering of CB10006 wild-type strain,and the yield of anthraquinone and benzaldehyde derivatives was increased by 1.7?2.8-fold and 1.5?2.5-fold in the mutant R2,respectively.Next,Agrobacterium tumefaciens-mediated transformation was successfully established in E.cristatum CB10006.The polyketide synthase（PKS）gene fog A responsible for benzaldehyde biosynthesis and the PKS gene erg H responsible for anthraquinone biosynthesis were knocked out through the insertion of a hygromycin resistance gene,respectively.TheΔfog A mutant no longer produced benzaldehydes,while the yield of anthraquinone A8 reached to 2.7±0.3 mg/g,which was about 2.1-fold improvement in wild-type strain.The resultingΔerg H mutant did not produce anthraquinones,while benzaldehydes were significantly increased by 8?58-fold.The yield of DAG（A1）reached to 47.8±16.8 mg/L,which was about 16.6-fold higher than the original titer of 2.9±1.3 mg/L in wild-type strain.Finally,we were able to observe the bright green fluorescence of E.cristatum mycelium by heterologous expression of the green fluorescent protein in CB10006 under a fluorescence microscope,suggesting the overexpression of the green fluorescent protein in CB10006.（3）Isolation and structural elucidation of lobophorins based on transwell activity-guided screening.Based on transwell-based strategy,we discovered that the crude extract of S.sp.CB09030 inhibited the growth of Mycobacterium smegmatis MC²155 from 16 actinomycete strains.Three lobophorin（LOB）derivatives LOBs A,B and H8（B1?B3）were obtained by the scaled-up fermentation of CB09030,followed by multiple chromatographic separation and structural analysis.The aglycone LOB H8was isolated from wild-type strain for the first time.We further obtained compound B4（O-β-d-kijanosyl-（1→17）-kijanolide）as a hydrolyzed product of LOB B,catalyzed by diluted HCl.The antibacterial activity of LOB B showed significant inhibition against M.smegmatis MC²155 and Bacillus subtilis with minimum inhibitory concentrations（MICs）4 and 1μg/m L,respectively.The MIC of LOB B against M.smegmatis MC²155was equivalent to rifamycin,a first-line clinical drug against tuberculosis.The structure-activity relationship of B1?B4 showed that the A/B/C oligosaccharide moiety is essential for the strong anti-B.subtilis activity,while the aglycon is more important in the observed anti-mycobacterial activity.In our study,11 natural products were isolated from E.cristatum CB10006 originated from FBT,and some of them possessed antibacterial and anti-inflammatory activities.The whole genome sequencing of CB10006,biosynthesis study of anthraquinone and benzaldehyde derivatives,and heterologous expression of green fluorescent protein in CB10006 all revealed that E.cristatum CB10006 has great biosynthetic potential.The isolation,structural elucidation,and anti-Mycobacterium activity evaluation of four lobophorins revealed the importance of their oligosaccharides and aglycones against different bacteria.The above study not only results in natural products with significant antibacterial or anti-inflammatory activities,but suggests that E.cristatum from FBT is worth to be developed to a powerful filamentous fungal cell factory.