Optimization Of ExoCET Gene Editing Method And Its Application In Cloning And Engineering Large Gene Clusters

Microbial secondary metabolites are an important source of antibacterial,antitumor,immune regulation,insecticide and other drugs.Using gene editing technology to clone and engineer secondary metabolite biosynthetic gene clusters is an important method for discovering new drugs,increasing drug production and improving drug structure.Microbial secondary metabolite biosynthetic gene clusters are generally larger than 20 kb,and generally contain a lot of repetitive sequences,which are difficult to modify.Therefore,the development of efficient gene editing technology for cloning and modifying gene clusters has always been a concern of natural drug synthesis biology.ExoCET combines in vitro exonuclease and annealing with the remarkable capacity of full-length RecET homologous recombination.It can not only grab large fragments>100 kb from the microbial genome(GC content>70%)but also efficiently assemble 13 GC-rich fragments(GC content>65%)with high efficiency in one step,which is a powerful tool for DNA cloning and assembly.Cloning and assembly biosynthetic gene clusters by using ExoCET technology can be modified in Escherichia coli through the Red/ET recombination,such as inserting transfer elements,replacing promoters,and deleting/inserting regulatory genes to express of biosynthetic gene clusters in a variety of hosts.Red/ET recombination can perform domain point mutations or module substitutions,and combinatorial biosynthesis to obtain more derivatives.So far,there is no report of using ExoCET technology to clone large fragments from genomes with AT content>63%.With the discovery of more and more microorganisms with high AT content and genome sequencing,a lot of biosynthetic gene clusters encoding natural products have been revealed,including encoding polyketides(PKS),non-ribosomal peptides(NRPS),ribosomally synthesized and posttranslationally modified peptides(RiPPs),and terpenoids.Marine cyanobacteria have been proven to be one of the important sources of natural products.Natural products isolated from marine cyanobacteria include anti-tumor compounds Apratoxins,Coibamides,Curacins,and Largazoles.However,a lot of marine cyanobacteria have a high AT content(>60%)in their genomes.Difficult genetic manipulation or low efficiency in obtaining large DNA fragments limit the further mining of their genome resources.To explore the biosynthetic resources in these high AT content genomes,more versatile and convenient cloning techniques are required.Further study also continued to explore the best conditions for the cloning and assembly of large fragments of the genome with high AT content using ExoCET technology.To explore whether ExoCET technology is suitable for direct cloning and multi-fragment assembly of large fragments of genes with high AT content.In this study,the genome of the marine cyanobacteria Prochlorococcus MIT 9301 strain with 69%AT content was used to optimize the ExoCET conditions for cloning of large AT-rich DNA fragments.The results showed that:(1)Higher cloning efficiency was obtained when the Gibson assembly method was used for the in vitro homologous recombination than the T4 polymerase method;(2)The single-copy bacterial artificial chromosome(BAC)vector should be used and the multi-copy plasmid vector cannot clone them;(3)The ExoCETBAC strategy can not only capture fragments larger than 80 kb from the Prochlorococcus genome but also assemble 11 pieces of DNA fragments with 100%accuracy;(4)ExoCET-BAC can capture 4 pieces of genomic fragments ranging 7-20 kb simultaneously in one step.Genome sequencing revealed that AT-rich organisms account for more than 30%.The ExoCET-BAC strategy established in this study provides efficient enabling technology for genome functional research of AT-rich organisms.In addition to optimizing the acquisition efficiency of ExoCET technology for cloning and assembling large fragments of high AT content DNA,this study also successfully used ExoCET technology to assemble a high GC content multioperon artificial gene cluster,achieving the heterologous expression of salinomycin.Salinomycin is a polyether antibiotic isolated from Streptomyces albus.Its selective activity against cancer stem cells has highlighted the potential of salinomycin as a chemotherapeutic compound in cancer treatment.Cancer stem cells are a distinct cancer cell subpopulation that exists in many liquid and solid tumors.They are resistant to various therapies and drive tumor initiation,progression,metastasis and recurrence.The activity of salinomycin to breast cancer stem cells is 100-fold higher than that of paclitaxel.However,the potential toxic and side effects of salinomycin on neural stem cells and hematopoietic stem cells are the main obstacles that limit its clinical application.A series of structural derivatives of salinomycin with different activities is the key to study the structure-activity relationship for the development of anticancer drugs with high therapeutic effect and low toxicity.Due to the complex structure of salinomycin,it is difficult to modify its structure by chemical synthesis.Therefore,modifying biosynthetic gene cluster is an important method to obtain more derivatives.In this study,we first transferred the salinomycin gene cluster cloned using ExoCET technology into three heterologous expression hosts S.lividans K4-114,S.albus J1074 and S.coelicolor CH999,and found that the yield of salinomycin in S.albus J1074 was 0.5 mg L-1,and almost no product was detected in S.lividans K4-114 and S.coelicolor CH999.To improve the heterologous expression yield of salinomycin,we used ExoCET assembly technology to construct an artificial salinomycin gene cluster of 106 kb with 6 operons.The artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid ?-oxidation genes into one operon:(1)salA?-? PKS genes;(2)ethylmalonyl-CoA synthase genes(salP,salQ)and thioesterase genes(salG?,salG?);(3)the salX gene coding for a possible acyl carrier protein,the epoxidase gene salC,and epoxide hydrolase/cyclase genes salB?-?;(4)the cytochrome P450 gene salD,the ferredoxin gene salF,the dehydratase gene salE,and exporter genes(salH,sall);(5)regulatory genes salJ and salO;(6)five p-oxidation genes SLNWT5323,SLNWT5008,SLNWT850,SLNWT400,and SLNWT812 from S.albus DSM41398;SA15p,SA6p,SA2p,SA31p,SA13p,and SA28p promoters were placed upstream of each group.The yield of the multi-operon artificial gene cluster achieved 14.3 mg L-1 and 10.3 mg L-1 in the heterologous expression hosts S.lividans K4-114 and S.coelicolor CH999,respectively.The yield in S.albus J1074 reached 19.3 mg L-1,which was 37.6-fold higher than that of the original gene cluster.In addition,the functions of the regulatory genes sail,salN and salO in the heterologous host S.albus J1074 were also studied.The artificial gene cluster established in this study laid an important foundation for the structural derivation of salinomycin.In summary,this study has successfully applied ExoCET technology to the cloning and assembly of large fragments of the microbial genome with high AT content,and has achieved the efficient acquisition of large fragments of the prochlorococcus genome with high AT content.This study has also successfully applied ExoCET technology to assembling the 106 kb salinomycin biosynthetic gene cluster with high GC content and significantly increased salinomycin heterologous expression yield.This study layed an important foundation for the cloning and engineering of the gene cluster to obtain more derivatives for structure-activity relationship research.Gene editing technology is an important tool for the research of microbial secondary metabolites.This study further expands the application scope of ExoCET technology and provides an important enabling technology for the development of microbial natural product drugs.