Gene Cluster Mining Of Saccharosporone From Umezawaea Sp.NO4W-1645

Saccharosporone is a new class of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase inhibitors,which can inhibit the activities of IDO and TDO at the molecular and cellular levels,and play an important role in the treatment of tumors,mental diseases,heart and blood vessels.Although some studies have indicated that some actinomycetes can accumulate saccharosporone,the related genes,gene clusters and metabolic pathways of saccharosporone have not been described.In this study,we sequenced the genomes of Umezawaea sp.NO4W-1645 and predicted the biosynthetic gene cluster of saccharosporone,in the meantime,we constructed Umezawaea sp.NO4W-1645 fosmid library and the fosmid clones containing saccharosporone biosynthetic gene clusters were screened,then the inserted fragments from the positive clones were recombined into the plasmid p SET152.These results can provide a basis for further study of saccharosporone biosynthetic gene cluster.The main research contents and results are as follows:(1)The complete genome of Umezawaea sp.NO4W-1645 was sequenced,and the genome size of strain was about 7 Mb.Gene annotation and alignment of the genome with biosynthetic gene cluster analysis of all secondary metabolites deposited at the anti SMASH,the results showed that the genome contained two type II polyketide secondary metabolite pathways and included the same genes with the three known type II polyketide biosynthetic gene clusters of actinorhodin,sch-47554 and sch-47555.Based on the analysis of saccharosporone structure analogues,we predicted that the 33 kb gene sequence from 5274853 bp to 5308044 bp in the genome of Umezawaea sp.NO4W-1645 is saccharosporone biosynthetic gene cluster;(2)The genome of Umezawaea sp.NO4W-1645 was interrupted and the 30 kb?40 kb gene fragment was recovered.,then the gene fragment was ligated into p CC2 FOS plasmid and transformation into EPI300 cells.The fosmid library of NO4W-1645 was constructed,and all fosmid clones were deposited into 96 well plates with approximately 3500 clones,covering a3.8 fold genome of Umezawaea sp.NO4W-1645.These results can provide a basis for screening secondary metabolite biosynthetic gene clusters;(3)Based on the gene sequence of Umezawaea sp.NO4W-1645 which contains the complete biosynthetic gene cluster of saccharosporone,primers for NO4W-6444,NO4W-6465 and NO4W-6492 were designed,then fosmid clones of Umezawaea sp.NO4W-1645 were screened by PCR for two positive clones containing the sequence of the complete biosynthetic gene cluster of saccharosporone,Fos-8H3 and Fos-21D3.We performed high-throughput sequencing of Fos-21D3 plasmids,it's insert fragment was approximately 42 kb in size between genes 5269167 to 5311055 in the genome of Umezawaea sp.NO4W-1645 and containing the entire saccharosporone biosynthetic gene cluster;(4)Plasmids p KD46,Fos-21d3,and Fos-8h3 were transformed into BW25113,and the linearized plasmid p SET152 digested with Eco RV was transformed into E.coli BW25113/p KD46/Fos-8H3 and BW25113/p KD46/Fos-21D3,the vector p SET152 was recombined with the fosmid plasmid under the influence of the p KD46 plasmid,so that the insert fragment containing saccharosporone biosynthetic gene cluster was successfully transformed to the p SET152 vector.Recombinant plasmids p SET152::8H3 and p SET152::21D3 were transformed into E.coli ET12567/p UZ8002 respectively,provides a basis for further expression of saccharosporone biosynthetic gene cluster in actinomycetes.