Secondary Metabolites Gene Cluster Analysis Of Penicillium Citrinum And Exploration Of It's Product Of The Non-ribosomal Polypeptide (ATRR)

Bioactive secondary metabolites of fungi have always been an important source of new drug development,many of the current compounds and their derivatives are already sold on the market as medicine.Penicillium citrinum is a kind of fungus that can produce the antihypertensive drug—mevastatin,it has attracted the attention of researchers.Currently,there are about 200 secondary metabolites isolated from Penicillium citrinum,including furanone,quinoline,pyrrolidine alkaloids,etc.This suggests that Penicillium citrinum has a strong ability to produce secondary metabolites.There are significant bioactive compounds in the secondary metabolites of Penicillium citrinum,but only the biosynthesis gene clusters of mevastatin and citricin have been analyzed in detail.The whole genome sequencing of Penicillium citrinum and the analysis of the gene cluster of the secondary metabolites of Penicillium citrinum were important for the targeted exploration of the active secondary metabolites of Penicillium citrinum.The natural products of fungi have a class of compounds called non-ribosomal peptide compounds.Non-ribosomal polypeptides are a series of complex and diverse compounds synthesized under the action of Non-ribosomal peptide synthase.Non-ribosomal peptide synthase is a kind of multifunctional protein complex,which can recognize and activate the amino acid substrate to synthesize Non-ribosomal peptide in a specific order.According to the structure of Non-ribosomal peptide synthase,it can be divided into single-module Non-ribosomal peptide synthase and multi-module Non-ribosomal peptide synthase.The single-module synthase has a much simpler structure,often missing the condensation domain,and it's very conservative in the fungus.At present,there are two types of fungal single-module non-ribosomal peptide synthases?NRPS:A-T-TE and NRPS:A-T-R?,and the related secondary metabolites excavated have certain biological activities.This study explored the existence of single-module non-ribosomal peptide synthase NRPS:A-T-R-R through genomic analysis of Penicillium citrinum ATCC38065,and explored its metabolites by molecular biological means Objective:Sequencing of the whole genome sequence and obtain the related information of Penicillium citrinum ATCC38065,and the secondary metabolite gene clusters will be analyzed in detail.Establish the genetic transformation system of Penicillium citrinum ATCC38065.Used the molecular biological methods to expore the gene products of single-module non-ribosomal peptides?ATRR?.Methods:1.The genome of Penicillium citrinum ATCC38065 will be extracted.After sequencing,the sequence will be assembled and predicted by software,and all genes will be classified.The whole genome sequence will be uploaded to fungalSMASH to predict its secondary metabolites gene cluster,and obtain related protein sequences,and analyse about the function of secondary metabolites gene clusters of Penicillium citrinum ATCC38065 by blastp.2.Excavate special NRPS gene cluster,compare the amino acid sequence of NRPS:A-T-R-R with other strains,determine the scope of adenylation domain and reductase domain,then do the evolutionary tree analysis.3.Exploration of the three important experimental conditions:the spore germination time,the formulation of lysozyme,enzymolysis time,to introduce antiplasmid to protoplast,antagonistic screening and genomic validation of transposons,to establish the genetic transformation system of Penicillium citrinum ATCC38065.4.Investigate the expression of the regulation gene tf and core gene atrr in Penicillium citrinum ATCC38065 by RT-PCR;construct the over expression vector for Penicillium citrinum ATCC38065's genetic transformation,to obtain the transformats;detect the metabolite of transformats by HPLC,identify the structure of the compound by mass spectrometry and nuclear magnetic identification;investigate the expression quantity of the regulation gene tf and core gene atrr in transformats by RT-qPCR.5.To construct the heterologous expression vector for Penicillium citrinum ATCC38065's genetic transformation,to obtain the transformats of Aspergillus nidulans;detect the metabolite of transformats by HPLC.Results:1.The total genome sequence of the Penicillium citrinum ATCC38065 with31.34Mb was obtained,and the GC content was 48.26%,including 17,473 genes.The gene cluster prediction of the secondary metabolites of Penicillium citrinum ATCC38065 was determined that there were 119 secondary metabolites gene clusters in Penicillium citrinum ATCC38065.We found 10 non-ribosomal peptide gene clusters,15polyketide gene clusters,6 heterozygous gene clusters of polyketide and non-ribosomal peptide,4 terpene gene clusters and 84 other gene clusters.And the function of the secondary metabolism-related genes in each gene cluster was analyzed.2.We found the gene sequence of the special single-module NRPS:A-T-R-R in Penicillium citrinum ATCC38065 and other penicilliums and aspergillus.According to the amino acid sequence comparison and evolutionary tree analysis,the NRPS:A-T-R-R are quite conservative in those penicilliums and aspergillus preserved in our laboratory.The adenylation domain of NRPS:A-T-R-R significantly different from that of other single-module non-ribosomal peptide synthase NRPS:A-T-TE and NRPS:A-T-R.The first reductase domains are similar with the reductase domain in PKS and PKS-NRPS,the second reductase domain are different with others.3.T the genetic transformation system was successfully constructed.The optimal germination time of Penicillium citrinum ATCC38065 spore was 16.5h,and the conversion effect was the best when the enzyme solution was found in Lysing enzyme3mg/mL and Yatalase 2mg/mL.4.The RT-PCR results showed that tf and atrr were expressed in the P.citrinum.Obtained the transformats P.citrinum OE::tf and P.citrinum OE::atrr.The metabolite of P.citrinum OE::tf is the same with their wild type.There is a compound product peak increases in P.citrinum OE::atrr,we named this compound as compound A,the structure of compound A is?4E?-4-[?4-hydroxy-3-methyl-2-butenyl?oxy]benzoic acid,its yield as 11.7 times as the wild type.The result of RT-qPCR verified that the expression of tf and atrr in P.citrinum OE::tf are 4.0 and 7.2 times of wild type respectively,the expression of atrr in P.citrinum OE::atrr is 8.8 times of wild type.5.Obtained the transformats A.nidulans::P.citrinum atrr,the metabolites are different with the wild type,obtain the target product peak.Conclusions:In Penicillium citrinum ATCC38065,there are abundant gene clusters of secondary metabolites,which are higher than the average level of penicillium.Through the analysis of the Penicillium citrinum ATCC38065 gene cluster,we found that only pks10produced the known compound mevastatin,the compounds produced by other clusters are unknown.The acquisition of a large number of gene clusters provides an important basis for digging the biosynthesis products of Penicillium citrinum ATCC38065.Penicillium citrinum ATCC38065 is the ideal strain of our genome mining.In this study,the protoplast conversion meditated by CaCl₂-PEG of Penicillium citrinum ATCC38065was established,which made the gene manipulation of Penicillium citrinum ATCC38065 become more convenient and beneficial to the further exploration of the Penicillium citrinum ATCC38065 genome.In this study,NRPS:A-T-R-R is successful heterologous expressed in the Aspergillus nidulans,and it is expected to obtain the synthesis product of the gene cluster,and rich the types of fungi secondary metabolites.