Study On The Functions Of Secondary Metabolites And Genes Involved In Low Temperature Response Of Thermomyces Species

Fungi are one of the most diverse groups.Currently,nearly half of the secondary metabolites of biologically active microorganisms are produced by filamentous fungi.According to the source of biosynthesis,fungal secondary metabolites are mainly divided into polyketides(PK),non-ribosomal peptides(NRP),terpenoids and alkaloids.Fungi can respond to environmental stimuli using metabolic exchange which influences development and survival of the fungal community.Predominant thermophilic fungi T.lanuginosus and its sister T.dupontii have very similar biosynthetic gene clusters with similar typical characteristics.However,these two fungi displayed distinct pigment variation in response to temperature changes.T.lanuginosus has commonly been reported to be the most dominant thermophilic fungus in diverse geothermal systems while T.dupontii was not.In order to clarify that different compounds accumulated in response to temperature changes are an important reason for the different ecological status of the two thermophilic fungi,the phenotypic differences of the two thermophilic fungi at the lowest growth temperature(37℃)and the optimum growth temperature(50℃)were compared,and the differences in metabolism and transcriptional levels were detected at different temperatures.In order to further explore the molecular mechanism of low temperature response to different compounds,the differences in phenotype,sporulation,spore germination ability and stress resistance between mutant strains and wild-type strains at different temperatures were compared by knocking out the target genes,and the functions of these genes in host response to low temperature were expounded.The main findings were described as follows:1.Through LC-MS detection,it was found that Thermomyces species accumulated compounds 1-8 at low temperature,compounds 1 and 2 were PKS compounds,and compounds 3-8 belonged to another special compound TPS-NRPS hybrid compound.Compound 1 belonged to a specific compound in T.lanuginosus,and compounds 3-8 were the specific compounds in T.dupontii.Combined with transcription level analysis,it was speculated that compound 1 is DHN-melanin,which is synthesized by PKS31;compound 2 is Carviolin A,which is synthesized by PKS30;compounds 3-7 were synthesized by TPS-NRPS.2.The T.lanuginosus strain with PKS31 gene mutation was constructed by CRISPR-Cas9 system.Phenotypic analysis showed that PKS31 in T.lanuginosus could maintain the stability of spore morphology and normal germination at low temperature,and maintain the hydrophobicity of hyphae.In the environment,compound 1 can also promote the germination of host seeds and the growth of bud length;PKS31 in T.dupontii is different from that in T.lanuginosus.The main function of PKS31 in T.dupontii is mainly involved in anti-oxidation and anti-osmotic stress,which has no significant relationship with cell wall interference.3.After knock-out,the loss of TPS-NRPS had opposite effects on T.dupontii spore germination at different temperatures.ΔNRPS accumulated DOPA melanin at 37℃,and showed stronger infection ability for maize seeds at 37℃ than at 50℃.These results indicate that isopentenyl indole alkaloids play a negative role in conidiospore formation,spore germination,infection ability and interaction with host of T.dupontii.Isopentenyl indole alkaloids play a key role in regulating the size of conidia and the morphology of vacuoles in hyphae at 37℃.4.After knocking out PKS30 in T.lanuginosus,the spore germination of mutant only occured at low temperature,while the loss of PKS30 gene in T.dupontii could lead to the decrease of spore yield and germination rate of the mutant;the loss of PKS30 gene in T.lanuginosus could lead to the change of mutant sensitivity to NaCl and H2O2 stress,while the loss of PKS30 gene in T.dupontii could lead to the change of mutant sensitivity to SDS and Congo Red mediated cell wall synthesis interference;PKS30 in two thermophilic fungi showed the same function,that is,resistance to Bacillus;after knocking out PKS30,the two thermophilic fungi could not produce antibacterial effect in the presence of spores.5.By analyzing the stress resistance of the two strains with PKS25 mutation,it was found that the sensitivity and sporulation of the mutant strains to NaCl stress changed.Saccharomyces cerevisiae with heterologous PKS25 expression can produce pungent odor compounds,and the ethyl acetate phase is yellow,but these compounds are easy to degrade,so the structure cannot be analyzed.6.After knocking out Ccg-8,the mutant strain produced obvious sporulation delay at 50℃,and the hyphal hydrophobicity decreased.Therefore,it is speculated that Ccg8 can regulate the expression of PKS31 through the regulation of temperature,and then regulate the rhythm of T.lanuginosus.In addition,the expression of mutant PKS30 is up-regulated,and Ccg-8 may have opposite regulatory effects on the two pigment genes.7.After knocking out AvrBs2 gene,a large number of plasmolysis appeared in hyphae and increased sensitivity to NaCl stress.Therefore,it is speculate that the function of AvrBs2 gene participates in maintaining the balance of host osmotic pressure,and the down-regulation of AvrBs2 gene at low temperature results from the fact that T.lanuginosus adapts to the environment in order to avoid strong immune response of the host.In summary,through gene knockout,metabolism and phenotypic analysis,it is found that the accumulation of different compounds in the thermophilic genus at low temperature may cause their ecological status to be different.The product of PKS31 in T.lanuginosus is 4,8-Dihydroxy-1-tetralone(4-DHT)and most of the ascomycetes DHN-melanin intermediate product scytalone has a slight difference in the position of the hydroxyl group on the naphthalene ring structure.4-DHT can not only maintain the structure and function of T.lanuginosus spores,help T.lanuginosus to colonize corn,but also promote the germination and growth of corn seeds.The products of TPS-NRPS in T.dupontii were isopentenyl indole alkaloid talathermophilins.This type of compounds has similar functions to similar compounds in Aspergillus fumigatus.It can bind to iron and may participate in maintaining the ions steady state of T.dupontii at low temperatures,although not DHN-melanin related compounds are detected,T.dupontii can accumulate DOPA-melanin at low temperature to resist stress.It was verified that Carviolin A in T.dupontii and T.lanuginosus had the function of resisting bacillus,and successfully expressed the PKS-NRPS gene precursor compound in Saccharomyces cerevisiae.T.lanuginosus is an important model for resaerch fungal heat-resistance mechanism and xylanase.Through multi-omics analysis and gene editing,it aims at the changes of four secondary metabolites and two regulatory genes in the thermophilic genus.The differences in the environmental distribution and ecological status of thermophilic fungi are explained.The study of the distribution of thermophilic fungi helps to design biofuel production and optimization.The focus of this research is that the versatility of thermophilic fungi depends on the adaptability of secondary metabolites.From an ecological point of view,this research can provide a reference for research in the field of metabolic engineering.