Study On Five Non-ribosomal Peptide Synthase Genes And Thermolides Biosynthetic Gene Cluster In Thermomyces Dupontii NRRL 2155

Thermomyces dupontii produces macrolide Thermolides with strong nematicidal activity.Gene knockout confirmed that PKS12 is a key gene for the synthesis of Thermolides,but it is not clear whether the six biosynthetic genes in this cluster are involved in the synthesis of Thermolides.In order to explore the six biosynthesis genes(Glycosyltransferase(GT),Glycosylhydrolase(GH),Acetyltransferase(AT),Nonribosomal peptide synthetase(NRPS4),Short chain dehrdrogenase(SDR),Monooxygenase(MO))and 6 other nonribosomal peptide syntheses(NRPS1,NRPS2,NRPS3,NRPS5,NRPS6,NRPS7)gene biological function in T.dupontii,based on the CRISPR/Cas9 gene editing technology developed by our laboratory in T.dupontii in the previous stage,10 mutant strains(?GT,?GT,?GH,?AT,?NRPS4,?SDR,?MO,?NRPS3,?NRPS5,?NRPS6,?NRPS7)and 2 mutant strains with AT gene overexpression(OE-AT1 and OE-AT3),due to inappropriate selection of the Protospacer site of NRPS1 and NRPS2 A mutant strain with loss of function.The biological functions and significance of these biosynthetic genes were preliminarily explored by analyzing the differences in metabolism,phenotype,stress resistance and transcription levels between mutant strains and wild-type strains.The main results and innovations are as follows:1.Metabolite difference analysis: Compared with the metabolites of wild-type strains(WT),the ?GT mutant strain can detect Thermolides,indicating that GT does not participate in the synthesis of the compound;the Thermolides compound in the?GH mutant strain is delayed in synthesis,and the content of this compound was 12 times higher than that of WT in 5-6 days;Thermolides A-C with acyl group is not detected in the ?AT mutant strain,and the content of Thermolides D-E without acyl group is increased by 7 and 18 times in YGP fermentation broth,confirming that AT gene is responsible for the acetylation modification of Thermolides,In order to increase the content of acetyl Thermolides A-B with higher nematicidal activity,the AT gene was overexpressed.The detection of Thermolides A-B in the over-expressing OE-AT strain was 70 times higher than that of WT,and the total content of Thermolides A-E was increased by more than 556 times;Thermolides were not detected in the ?NRPS4 mutant strain,indicating that NRPS4 is involved in the synthesis of Thermolides;Thermolides were not detected in the ?SDR mutant strain,and the possible intermediate C-6 dehydrogenated Thermolides,C-6,8dehydrogenation Thermolides,C-6,8,the side chain of the hydroxydehydrogenated Thermolides and the ring-opening derivatives of Thermolides,indicating that the SDR gene in the original host is not only responsible for the reduction of the C6 ketone group in the Thermolides to a hydroxyl group,but also participates in the Thermolides backbone.It forms and participates in at least three hydroxyl reduction and even macrocyclic skeleton cyclization processes;metabolic analysis of ?NRPS3 mutant strains found that a characteristic peak is missing,and NRPS3 encodes three amino acids Trp,Met,and Cys through primary and secondary mass spectrometry and absorbance.Compared with WT,the metabolites of ?NRPS5,?NRPS6 and ?NRPS7mutant strains only increased in individual peak areas.It may be that the expression of these three genes is lower in T dupontii.2.Phenotypic difference analysis: Compared with the phenotype of WT,GH and GT are involved in the cell wall synthesis,anti-osmosis ability,sporulation and germination of T dupontii;AT is involved in regulating mycelial growth and is related to temperature,and also participates in antioxidant capacity,Sporulation and germination,and excessively high or low acetylation levels are not conducive to sporulation and spore germination;SDR participates in fungus' s anti-osmosis ability,cell wall synthesis ability,mycelial growth,sporulation and germination;NRPS3participates in sporulation and germination And the regulation of mycelium growth at45?;NRPS4 participates in the antioxidant capacity;NRPS5 and NRPS7 affect the formation of brick red pigments,participate in the antioxidant capacity,sporulation and germination of T dupontii,and the sporulation and germination are related to temperature;NRPS6 participates in the resistance Oxidation ability,spore number and germination;the colony growth of mutant strains on Thermolides biosynthesis gene cluster and Tlala gene cluster at 37?,40?,45?,50?,55? and 60?,indicating that these genes are involved in the regulation of mycelial growth and are affected by temperature,but the specific regulation mechanism remains to be further studied.3.Transcription level difference analysis: Compared with wild-type strains,the five NRPS mutant strains have very few up-regulated and down-regulated genes.NRPS3 positively regulates a zinc finger CCCH transcription factor;NRPS4 knockout did not significantly up-regulate and down-regulated differential genes;NRPS5,NRPS6,and NRPS7 knocked down 7,13 and 7 methyltransferase in the top 20,respectively Enzyme genes are all down-regulated,indicating that their three functions are the same,and NRPS6 gene expression is up-regulated after NRPS5 and NRPS7 gene knockout,suggesting that NRPS6 may be their remedy pathway.KEGG enrichment analysis found that the expression differential genes of these five NRPS are all enriched in the metabolic pathway.The most significant degree of GO enrichment is the acetic acid metabolism process,and the most enriched number is the carbohydrate metabolism process,carboxylic acid and ketone.The metabolic process of acid and organic acid.The knockout of these five NRPS genes will affect the processes involved in metabolism,oxidation,and transport,but the specific regulatory mechanism,the biological and metabolic processes involved are still unclear,and the more in-depth biological functions of these genes need to be further explored and studied.