Establishment And Application Of The CRISPR/Cas9-Based Targeted Cloning Technology For Natural Product Biosynthetic Gene Cluster

With rapid development in bioinformatics and sequencing technology,a wealth of microbial genomic information has been revealed.However,bioinformatic analysis indicates that currently known natural products(NPs)are only a tip of the iceberg in terms of microbial biosynthetic potential,and a variety of NP biosynthetic gene clusters(BGCs)that kept silence remain to be explored.In the genomic era,direct cloning of NP pathways for efficient refactoring and heterologous expression has become an efficient strategy for microbial NPs discovery and research,especially for those microorganisms that are genetically intractable or currently uncultivable.Accordingly,the development of convenient and efficient cloning approaches is becoming increasingly necessary.This study aims to establish a CRISPR/Cas9-based targeted cloning(CBTC)approach by combining the CRISPR/Cas9 gene editing system with in vitro packaging system,which could be applied to explore novel NPs in microbial genome and provide competent technical support for NPs research.The CBTC approach was developed by applying CRISPR/Cas9 system to release the biosynthetic pathway,which is subsequently ligated with a linearized universal vector and in vitro packaged into phage particles to infect the Escherichia coli.As proofs of concept,BGCs of Tu? 3010 biosynthetic gene cluster(stu,27.4 kb)from Streptomyces thiolactonus NRRL 15439 and sisomicin biosynthetic gene cluster(sis,40.7 kb)from Micromonospora inyoensis DSM 46123 were selected,since their genomic size approaches the lower and upper limit of this cloning approach.With high efficiency and convenience,the stu and sis have been successfully cloned with cloning efficiency of 18% and 54% respectively in 48 randomly selected colonies.It has been revealed in this research that CBTC approach facilitates cloning of NP pathways sized in a range of 30-45 kb.Moreover,above practical cases for stu and sis pathways have clearly addressed the efficiency,celerity,accuracy and convenience of the newly established approach,which is of great significance for targeted cloning of mid-sized microbial NP pathways.With coupling use of CBTC approach and in vitro CRISPR/Cas9 editing(ICE)system,we edited the stu pathway with in-frame deletion and promoter reconstruction of a putative regulator stu R respectively,and reconstructed the promoter of an un characterized thp pathway.Heterologous expression results showed stu R positively regulates the production of Tu? 3010,however,no significant effect was observed when reconstructed stu R with a constitutively expressed promoter.Moreover,promoter reconstruction of thp pathway failed to activate the production of related NP,while other strategy could give a shot to activate the expression of thp pathway.Combination of CBTC approach with transformation associated recombination(TAR)cloning has also been applied,to clone a pathway(tln,64.3 kb)from M.inyoensis DSM 46123.This part of the work is in proceeding now.