Construction And Application Of The Inducible Transformation-associated Recombination In Saccharomyces Cerevisiae

The microbial genomes are a rich source of secondary metabolites which have high research and application value in the field of new drug development.However,the gene clusters are responsible for the synthesis of secondary metabolites range from tens of to hundreds of kilobases,which hinders the efficient cloning and leads to the discovery of only a scarcity of secondary metabolites.Transformation-associated recombination(TAR)cloning is a strategy that captures the large gene clusters by means of the efficient homologous recombination machinery in Saccharomyces cerevisiae.The application of TAR has been limited due to the high ration of false-positive clones,due to the vector self-ligation and low-efficiency of co-transformation with both vector and genomic fragments.To address these issues,here the inducible transformation-associated recombination(iTAR)system was constructed based on TAR cloning.The mechanism of iTAR lies in linearization of the iTAR vector by galactose inducing the expression of the endonuclease I-SceI,thus the target DNA fragment is integrated into the linearized vector by homologous recombination.Here we applied the CRISPR/Cas9 technology for precise and efficient cleavage at the boundaries of the target gene cluster in vitro,in the purpose of stringently match the homologous regions with the iTAR cloning vector,thus facilitating homologous recombination.The digested genomic fragments were then transformed into the galactose treated yeast host that harbors the specific iTAR cloning vector,and resulted in a positive cloning rate of 73.2%,which is significantly higher than the efficiency of TAR cloning(0.5-2%).Sequencing analysis revealed that 70.4%of the false positive clones were damaged at the I-SceI site,probably due to the self-ligation of the linearized vector by Non-Homologous End Joining(NHEJ).Deletion of the nejl gene in Saccharomyces cerevisiae BY4742 further improved the iTAR cloning efficiency to 83.1%,thus paving the way for the large-scale capturing of long size DNA fragments.The iTAR procedure was then applied for the capture of the polymyxin gene cluster from Paenibacillus polymyxa SQR-21S,which was then integrated into the genome of the model strain Bacillus subtilis 168 for expression.The engineered strain not only exhibited antibacterial activity,but also produced the compound with molecular weight that is consistent with polymyxin E1.Above all,we provided a novel procedure for the cloning of large fragments of DNA using iTAR cloning technology,which is simple and effective.Furthermore,we successfully transferred the polymyxin gene cluster from Paenibacillus polymyxa to Bacillus subtilis and produced the compound polymyxin El.Therefore,this research opened a new avenue for the synthetic biology and metabolic engineering regarding the microbial secondary metabolites.