ARTP Mutagenesis And Genomic Study Of Griseofulvin Producing Strain D-756

Griseofulvin is an antifungal antibiotic that is clinically used to treat dermatophyte infections and can also be used to control crop fungal diseases.The research object of this study is Penicillium griseofulvum D-756,which is a high-yield mutant of griseofulvin obtained from the multi-generation compound mutagenesis of the wild strain,Penicillium griseofulvum 4541,and has strong ability to utilize starch raw materials and chlorine resistance,is an early industrial strain.In order to better understand the cell operation process and metabolic mechanism of the strain,and to provide guidance for further improvement of industrial strains,we used the mutant strain D-756 as the research strain,and carried out genome-wide sequencing to understand its griseofulvin biosynthesis gene clusters and key regulatory factors;D-756 was also subjected to plasma mutagenesis,and it is expected to further screen excellent mutants.The research contents are as follows:1.ARTP mutagenesis of Penicillium griseofulvum D-756: Mutagenesis of D-756 strain was carried out by ARTP(atmospheric and room temperature plasma)mutagenesis instrument.Rice spore method was used for primary screening the mutants and diazonium salt method was used to measure the titer of griseofulvin.The results showed that the positive mutation rate of ARTP mutagenesis was 19.5%,and the optimal mutagenesis time was 150 s.After the second screening by shake flask,there were 6mutant strains with higher titer than the starting strain.Among them,the titer of 3-41 was23% higher than that of the starting strain D-756.2.LC/MS(liquid chromatograph-mass spectrometer)analysis of griseofulvin producted by Penicillium griseofulvum D-756 and wild strains: The fermentation products of D-756 and two wild strains were analyzed by LC/MS,and the effect of chloride ions in culture medium on the synthesis of griseofulcin was investigated.The LC/MS spectra of the fermentation products of D-756 and wild type strains 4541 and40293 were compared.The results showed that the UV absorption peak area of D-756 was nearly 170 times of that of the two wild type strains.These results are in good agreement with the titer results determined by diazo salt method.The results also showedthat the ultraviolet absorption peak area of griseofulvin to dechlorinated griseofulvin in the fermentation products of chlorine-containing medium and dechlorination medium were 16:1 and 1:3,respectively,indicating that chloride ion is important for the synthesis of griseofulvin.3.ITS and 18 S rDNA sequences of mutant D-756 and wild strain 4541,40293 were cloned and sequenced.The results showed that the ITS fragments of D-756,40293 and4541 strains were 545 bp,and the sequence was identical.The 18 S rDNA fragments of the three strains were 1715 bp in length,and the sequence similarity was more than 99%.The ITS and 18 S rDNA sequences of the three strains were searched homologously in GenBank database,and the NJ phylogenetic tree was constructed.Phylogenetic analysis showed that the three strains were highly homologous and clustered with P.griseofulvum(GU566212.1,NR103892.1,AY373917.1,etc.).4.Whole genome sequencing of Penicillium griseofulvum D-756: Whole genome sequencing of D-756 was carried out using the Illumina HiSeq PE150 and PacBio SMRT sequencing platforms.The results showed that the genome of Penicillium griseofulvum D-756 consisted of 6 scaffolds with a genome size of 28.9Mb,there are 6513 genes,188 tRNAs,47 rRNAs,3 sRNAs and 44 snRNAs,8046 repeated sequences,and GC content is 47.4%.Functional analysis of various genes showed that many genes in the genome of Penicillium griseofulvum D-756 were involved in the transport and catabolic signal conduction as well as carbohydrate and amino acid metabolism,suggesting that D-756 strain has strong capacity to utilize carbon and nitrogen source and material exchange,and possessed the capability of industrial strain.CAZy functional analysis showed that more than 60% of the enzymes in the annotated carbohydrate active enzymes belonged to glycosidic hydrolases,which well explained the phenotype of the strong utilization of starch materials in D-756 fermentation production.The composition and arrangement of the griseofulvin gene cluster in P.griseofulvum D-756 were analyzed based on the published sequence of griseofulvin gene cluster.It is found that the D-756 griseofulvin gene cluster consists of 12 genes,10 of which are in one cluster and the other two in the other scaffolds.The regulatory mechanism of this gene cluster on the synthesis of griseofulvin of D-756 needs to be further studied.