Genetic Regulation Of Penicillium Sclerotiorum SD-36 And Analysis Of Its Secondary Metabolites

Penicillium sclerotiorum SD-36 can produce a variety of secondary metabolites.These secondary metabolites have special functions and diverse structures,which play important roles in antibacterial,anti-oxidant,and anti-tumor aspects.In this thesis,the third-generation sequencing technology was used to sequence the whole genome of P.sclerotiorum SD-36,and 53 gene clusters in the fungus were predicted by bioinformatics methods.Through epigenetic means,the silent gene clusters were activated by knocking out HDAC gene,and thus two indole compounds were produced.At the same time,the overexpression of a transcription regulator of a specific double PKS gene cluster up-regulated the expression of core genes on the cluster.The research results of this paper mainly include the following aspects:1.Genome sequencing and bioinformation miningIn this experiment,the whole genome of P.sclerotiorum SD-36 was sequenced through the third-generation sequencing technology.The total size of the genome was calculated to be 37242374 bp,and the average G+C content was 46.74%.53 gene clusters of secondary metabolites were annotated in the genome of P.sclerotiorum SD-36 using the anti SMASH program.These gene clusters include PKS,NRPS,TCs,indole,PKS-NRPS,TC-PKS,TC-NRPS and other types of gene clusters.2.Generation and growth phenotype of gene knockout mutantsIn this experiment,three gene knockout mutants(P.sclerotiorum SD-36 ?1628-5,-6,-7)were generated using the hygromycin B selection marker system,In the same culture time,the colonies of wild-type strain and mutant strain P.sclerotiorum SD-36?1628-5,-6,-7 on PDA medium are very different in color and spore morphology.The wild-type strain is green,while the mutant strain is greenish.At the same time,the back of the PDA plate shows that the wild-type strain is yellow,which is a little more obvious than the yellow of the mutant strain.3.Generation and structure identification of new compounds.Fermentation experiments and HPLC analysis were performed on extracts of wild-type strains and P.sclerotiorum SD-36 ?1628-5,-6,-7 mutant strains.Compared with the wild-type strain,the secondary metabolism profiles of the three mutant strains have undergone significant changes.Several new peaks(containing peak 1 and peak 2)were observed from the HPLC spectrum of the mutant extract.We finally separated two of the compounds and identified them as meleagrin and roquefortine C,respectively.4.Production and growth phenotype of overexpression mutant strainsIn this experiment,the hygromycin B selection marker system was used to generate three transcriptional regulator overexpression mutant strains.In the same culture time,The wild-type strain and the mutant strain P.sclerotiorum SD-36OE::PsAza1-1,-2,-3,4,5 have very different colonies on the PDA medium in morphology and color.The wild-type strain is green,while the mutant is orange-red.5.Increased production of compounds and structural identificationFermentation experiments and HPLC analysis were performed on extracts of wild-type strains and P.sclerotiorum SD-36 OE::PsAza1-1,-2,-3 mutant strains.Compared with the wild-type strain,the secondary metabolism profiles of the three mutant strains have undergone significant changes.From the HPLC spectrum of the mutant extract,an increase in yield was observed compared to the liquid phase spectrum of the wild strain.We finally separated two of the compounds and identified them as isochromophilone ? and sclerotiorin C.6.Increased expression of core genes in dual PKS gene clustersq RT-PCR analyzed the expression levels of three core genes(HR-PKS,NR-PKS,Psaza1)in the WT strain and the OE::PsAza1 mutant strain.Compared with the wild strain,the expression of HR-PKS,NR-PKS and transcription factor Psaza1 was up-regulated by 60-80 times.The innovations of this experiment are:1.Three g1628 deletion mutants were produced through gene knockout,and new peaks of wild strains appeared through HPLC analysis of the fermentation broth of the mutant strains,and the secondary metabolites were separated and purified and finally identified as two types.Indole alkaloids: meleagrin and roquefortine C.As far as we know,this is the first example of obtaining meleagrin and roquefortine C regulating HDAC protein in Penicillium sclerotinum.Our experimental results show that epigenetic modification has great potential in guiding the discovery of biologically active natural products in fungi.2.We conducted gene mining on P.sclerotiorum SD-36 to find a special double PKS gene cluster,and successfully obtained five overexpression mutant strains by overexpression of transcription regulators PsAza1,which were prepared by HPLC separation of the crude substance of the mutant strain's fermentation broth.Two azaphilones compounds with increased yields compared to wild strains.Azaphilones compounds have a wide range of biological activities and interesting structural features.At the same time,they have good anti-cytotoxicity,anti-viral,anti-microbial,and anti-microbial properties.The anti-inflammatory and anti-cancer activities of these compounds have attracted more and more attention from researchers.