Cloning Of The Biosynthetic Gene Cluster Of The Marine Microbial Drug Didemnin B

Natural products derived from marine microorganisms are rich in diversity of structure and activity,which have become a new hotspot of drug discovery and development.Didemnins are a kind of natural products derived from marine microorganisms,which are firstly discovered from Mediterranean sea worms(Aplidiumalbicans)and have complex cyclic peptide structure.Currently,the identified didemnins include didemnin B,dehydrodidemnin B(aplidine),nordidemnin B and didemnin X/Y.They are produced by Tistrella mobilis and Tistrella bauzanensis of ?-Proteobacteria and have anti-tumor,antiviral and immunosuppressive activities.At present,aplidine has entered the clinical phase III,mainly used for the treatment of multiple myeloma,lymphoma,etc.Didemnins are biosynthesized by a hybrid of non-ribosomal peptide synthethase(NRPS)/polyketone synthases(PKS).The biosynthetic gene cluster(BGC)of didemnins B has been cloned,with a size of about 80 kb.However,due to the difficulty in genetic manipulation of these bacteria,the production of didemnins is very low,which greatly restricts the progress of development of these drugs.The problem of the drug source of didemnins can be effectively solved by the establishment of a microbial heterologous biosynthesis system.Therefore,this study aimed to clone the didemnin B BGC from the strain of Tistrella mobilis using the RNA-mediated CRISPR/Cas9 endonuclease combined with TAR(transformation-associated recombination)technique(Cas9-TAR),which will lay the foundation for subsequent development of the didemnin B heterologous synthesis system.First of all,two Tistrella mobilis strains were fermented and the cultures were analyzed by HPLC and LC-MS.We showed that these two strains had the synthesis ability of didemnin B.Subsequently,the genome DNA of the T.mobilis strain with a relatively high titer was extracted,and we used Cas9-TAR to clone the complete didemnin B BGC in one step.We attempted several times,but failed.To address this problem,we changed our cloning strategy.The gene cluster was divided into two segments(did L and did R),which were captured separately and ligated into a complete didemnin B BGC by TAR.Using pCAP01 as the starting plasmid,the capture plasmid pCAP-did L-HR and pCAP-did R-HR with the upstream and downstream homologous regions of did L and did R(approximately 1.5 kb each)were constructed,respectively.To enable the subsequent assembly of did L and did R,an overlapping region of about 80 bp was designed between these two DNA fragments.At the same time,sg RNA that can guide Cas9 to efficiently cleave upstream and downstream regions of did L and did R on the genome was designed and screened.Then,the genome DNA of the T.mobilis strain was cleaved by sg RNA-Cas9 to release the fragment of target gene cluster.Through protoplast transformation,the constructed capture plasmid(pCAP-did L-HR or pCAP-did R-HR)and the digested genome DNA were simultaneously introduced into Saccharomyces cerevisiae,and the positive yeast clones with the cloning of did L or did R in pCAP01 were screened and verified by auxotrophic screening and colony PCR.The plasmids were extracted and transformed into E.coli.The recombinant plasmids containing did L and did R(pCAP-did L and pCAP-did R)were obtained by enzymatic digestion,PCR and sequencing.The pCAP01 plasmid can accommodate DNA fragments less than 60 kb.Therefore,in order to obtain the entire gene cluster,based on the BAC plasmid pCC1 BAC,a modified BAC plasmid named pCL01 was constructed by the introduction of the element for screening and plasmid replication in yeast(ARSH4/CEN6-TRP1)and the element(?C31 integrase/att P-acc(3)IV)used for antibiotic screening and chromosomal integration in actinomycetes.Using pCL01,a capture plasmid pCL01-HIS-HR(containing the homologous regions of did L and did R,1.5 kb each)was constructed for the assembly of did L and did R,in which His was a histidine auxotrophic screening marker.Using the His auxotrophic screening,the vector self-ligation background could be significantly reduced.In addition,in order to reduce the background caused by ineffective digestion of pCL01-HIS-HR vector,the vector was divided into 3 segments,and the corresponding linear segments were obtained by PCR.There are approximately 500 bp homologous regions in the adjacent region of each fragment and the ends of did L and did R.At the same time,pCAP-did L and pCAP-did R were treated with Nhel/Spe I to recover the DNA fragments of did L and did R.Subsequently,three overlapping linear fragments of pCL01-HIS-HR and the partial BGC segments,did L and did R were transformed into S.cerevisiae.Similarly,the yeast clone containing the BAC recombinant plasmid with a complete didemnin B BGC was successfully screened by auxotrophic screening and colony PCR.Then,the BAC recombinant plasmid was extracted and transformed into E.coli EPI300.After PCR,enzyme digestion and sequencing verification,the BAC plasmid containing the entire didemnin B BGC(pCL01-didemnin B)was obtained.