Development And Application Of Bacterial Multiple Genome Editing

Microbial secondary metabolites are not only an important source of drugs and biological pesticides,but also play an important role in microbial ecology.Pseudomonas and burkholderia are deformation bacteria.Natural products synthesized by Pseudomonas play a vital role in disease prevention in humans,animals and plants,and Burkholderia is a new source of bioactive natural products.Their genomes contain a large number of unknown biosynthetic gene clusters(BGCs),indicating great potential for new structures in biosynthesis.Traditional genome mining strategies include single promoter replacement,transcriptional regulatory factor manipulation,exogenous functional gene addition,and non-target metabolic pathway inactivation.Due to the complexity of the structure and regulatory network in BGCs,it is often difficult to activate silenced gene clusters using a single strategy,which leads to the inefficiency of genome mining and a large number of biosynthetic gene cluster resources still need to be mined.Deep mining of gene cluster resources requires systematic "rewriting" of multiple mining strategies on microbial genomes with the help of efficient multiple genome editing technology,guiding gene cluster resources to be transformed into natural product resources.At present,the widely used bacterial multi-genome editing techniques mainly include CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated nuclease)assisted multi-genome editing method and Red/ET recombineering assisted multi-genome iterative editing method.The CRISPR/Cas system has some problems,such as the cytotoxicity caused by heterologous exonuclease mediated DNA double-strand breaks(DSBs)and the complex expression vectors of crRNAs for multi-site editing of the genome,which have limited its use in Pseudomonas and Burkholderia.Traditional Red/ET recombineering assisted multiplex genome editing generally uses single-stranded DNAs(ssDNA)as substrates,which is efficient for elucidating the genetic basis of traits and constructing optimized microbial cell factories.However,due to the low integration efficiency and laborious acquisition of kilobase-scale ssDNA,the multiple editing technology mediated by ssDNA recombineering has some problems,such as restriction of insertion fragment length,low screening efficiency of mutants,genome preediting,and narrow application scope.In this work,we established a double-stranded DNA recombineering-assisted multiplex genome engineering(dReaMGE)strategy through intracellular dNTP concentration regulation mediated by ribonucleotide reductase(RNR)overexpression and deoxynucleoside triphosphate(dNTP)addition.This technique has the advantages of non-ssDNA-dependent,flexible editing position,high sensitivity of screening method and wide application range.dReaMGE can mediate multiplex genome editing in many bacteria such as Escherichia coli,Pseudomonas and Burkholderia.dReaMGE enables rapid and flexible simultaneous replacement of multiple kilobase-scale genome sequences(0.25-95 kb)as well as deletions and insertions of such sequences avoiding in vitro preparation of ssDNA,construction of complex guide plasmids,multiplex double-strand breaks in genomes,and pre-interference of mismatch repair systems.dReaMGE can complete months-long genome engineering works that would have taken months with traditional recombineering,e.g.,multiple biosynthetic pathways inactivation,promoters or genes insertion,genome reduction and transcriptional regulators combinatorial modification within a few days,demonstrating that dReaMGE is a convenient tool for probing of genotype-phenotype relationships and systematic modification of bacterial genomes.It can promote the deep mining of microbial gene cluster resources and the construction of cell factories,which is an ideal supplement to the current genomic engineering technology.Complex genome editing projects using technologies such as dReaMGE involve a large number of knockout,replacement,and insertion of genes and metabolic pathways.Therefore,site-specific recombinase system(SSRs)are required for screening marker deletion to maintain genomic stability or prevent the generation of superbacteria.However,the existing SSRs sites are far from meeting the needs.The SSRs consists of site-specific recombinases and recombinase recognition sites.Recombinase can specifically recognize and bind the recognition sites,and then edit the target DNA between two recognition sites,such as excision,integration,inversion and displacement.Previous works had shown that mutated recombinase recognition sites can still be effectively recognized and bound by recombinase,but cannot react with wild-type recognition sites,which expanded the application of SSRs.The most commonly used SSRs are the Cre/lox system and the Flp/FRT system.Cre/lox system is most widely used due to its high efficiency,but it has strong cytotoxicity as well as high efficiency.A series of mutated lox sites have been developed and used in genome editing.Vika/vox system from Vibrio coralliilyticus is a noval SSRs discovered in recent years,which consists of site-specific recombinase Vika and recognition site vox.Compared with Cre/lox and Flp/FRT systems,this system has the advantages of low cytotoxicity,wide application range and high safety.In this study,we mutated the vox site and obtained several vox mutated sites(vox2261,vox2272,and vox226172),which were more rigorous and significantly enhanced in recombination than wild type vox site.This study provides an effective tool for multiple editing of bacterial genomes and further expands the application range of SSRs.Based on Red/ET recombineering,dReaMGE,Vika/vox system and the development of tool components,this study explored the biosynthetic potential of one Pseudomonas and two Burkholderia species by single or multiple promoter insertion,combined editing of transcriptional regulatory factors,and gene cluster inactivation.Three silenced gene clusters were activated,one active gene cluster was excavated,the yield of one target compound was increased,the anti-tumor compound epothilone was highly expressed heterologously and six new natural products were obtained.Among them,massetolides from Pseudomonas parafulva CRS01-1 showed anti-Bacillus subtilis activity.Haereomegapolitanin A from Paraburkholderia megapolitana DSM 23488 has surfactant activity.Luminmycin G obtained by multi-promoter substitution of glidobactin gene cluster from DSM 7029 showed antitumor activity against human breast cancer cell.The above results indicate that the bacterial genome multiple editing technology and tool elements developed in this paper,including dReaMGE and Vika/vox,are an expansion and enrichment of the existing bacterial genome editing toolbox,and can greatly accelerate the transformation of the development for microbial gene cluster resources.