The Research And Application Of Recombineering System In Burkholderiales Species

Bacteria represent an important source of medicines and pesticides.Current genome-sequencing projects have revealed a large number of biosynthetic gene clusters(BGCs),but their natural products have not yet been identified.The enzymes encoded by these unexploited biosynthetic genes may synthesize metabolites that could serve as a reservoir of novel scaffolds for drug screening.Two major genome mining strategies,featuring homologous and heterologous expression,were employed to mine cryptic BGCs.Homologous expression in the native producers ensures proper substrates,precursors,cofactors,and accessory factors for biosynthesis,which could maximize generation of cryptic BGC biosynthesis products.However,this strategy requires efficient genetic manipulation tools in the producers to meet the demand of high-throughput genome mining in the postgenomic era.Red/ET recombineering is an in vivo homologous recombination-based genetic engineering method employed primarily in Escherichia coli.The Red?? system from E.coli has been directly applied to several closely related Gram-negative bacteria for genome engineering,e.g.,Salmonella enterica,Klebsiella pneumoniae,Agrobacterium tumefaciens,Pantoea ananatis,and naturally transformable Burkholderia species.However,it is not functional in other Burkholderiales strains.In more distant species,host-specific Red?? or RecET homolog systems are mandatory.The Burkholderiales are an order of Proteobacteria,and they include a genus Burkholderia containing pathogenic organisms,such as B.mallei(glanders)B.pseudomallei(melioidosis)and B.cepacia complex,and a genus Paraburkholderia harboring environmental species,for examples,Plant Growth-Promoting Endophyte Paraburkholderia phytofirmans Strain PsJN,and P.rhizoxinica.an Endosymbiont of Rhizopus microspores.Above mentioned each Burkholderia and Paraburkholderia species contain a remarkable large number of putative natural product biosynthetic genes clusters as predicted by antiSMASH pipeline.Those gene clusters are predicted to encode diverse compounds,say,lantipeptides,nonribosomal peptides,polyketides,siderophores and terpenes.Statistically,the percentage content of the polyketide synthases(PKSs)and nonribosomal peptide synthetases(NRPSs)in Burkholderia and Paraburkholderia genomes is only next to that of actinobacteria,higher than those of bacilli,cyanobacteria,myxobacteria and fungi.Thus Burkholderiales species have emerged as a new source of various natural products.Although numerous natural products from Burkholderiales species have been discovered.a plethora of gene clusters are still cryptic or orphan.To access these biosynthetic gene cluster using homologous expression strategy,a reliable genetic tools is necessary.The E.coli derived Red?? system can easily knock-out(KO)and pull-out(PO)chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and B.pseudomallei,but it is not workable in other Burkholderia strains.Thus,a native Red?? homo logs system from Burkholderiales species need to be developed for efficient genetic manipulation.Here,we describe the discovery of a pair of novel bacteriophage recombinases and application of recombinase-assisted promoter engineering to rapidly identify and activate several cryptic biosynthetic gene clusters in two Burkholderiales strains that currently lack effective genetic tools.Construction of an efficient genome engineering platform in a natural product producer expedites mining of cryptic BGCs in their native backgrounds,and host melioration for yield or structure optimization.We activated a crptical BCG(BGC 6A)by in situ constructive promoters insertions and confirmed the products of one unexplored BCGs by the comparative analysis of BCGs activated and inactivated mutant strains mediated by the native Red?? homologs system.We also used this system for deletion of large chromosomal DNA fragments(up to 200 kb),cryptical BCGs activation and product confirming of cryptical BCGs.This system can also be extend to application in P.phytofirmans PsJN for gene replacement with high efficiency.This strategy enables potentially scalable discovery of novel metabolites with intriguing bioactivities from many other bacteriaThe construction and optimization of the genetic operation system of DSM 7029.BlastP analysis using the amino acid sequences from E.coli Red?? or RecET as queries revealed seven Red??-like and ten RecET-like recombinase pairs in?1000 Burkholderialesstrains.A Red??-like operon in DSM 7029 and A pair of RecET-like recombinases(ET-BDU)in Burkholderia sp.BDU8 were choosed as the objects of study.After the functional verification,we constructed Red?-Red??7029 based on the native phage protein of DSM 7029 for the rapid genetic manipulation of Burkholderiales.We also optimized work condition of this system and finally improved the efficiency of this genetic manipulation system.Under the optimal condition A 200 kb genome sequence(3054675-3254511)encoding two BGCs was successfully replaced using 50 bp homology arms.Red?-Red?7029 was competent at single-stranded DNA recombination,especially when using the lagging ssDNA,the efficiency is substantially improved,13 times higher than the Red?-Red??7029 mediated dsDNA recombination.Genetic Tools for seamless mutagensis in DSM 7029The previous work indicated that mutant pheS gene that could be selected out in the presence of cPhe has enormous potential to be utilized as a counter-selected marker in Burkholderia species.The similar A311G mutation of 7029 pheS gene was synthesized in DSM 7029 and named as 7029 pheS*,this cassette was preliminary utilized in the deletion of glidobactin biosynthesis gene cluster,there was no selection marker or "scar" sites left on the chromosome of DSM7029?glb.Bioprospecting of DSM 7029 by in situ activation of biosynthsis gene clustersThe successfully development and optimization of efficient recombineering in DSM 7029 allows us to scrutinize its cryptic biosynthetic gene clusters.We activated oneunexplored silent BCG 6A of DSM 7029 by introducing of heterologus constitutive promoters upstream of silent BCGs.The product of BGC 6 A,a novel class of lipopeptides-glidopeptins was identified through extensive spectroscopic characterization.Extended applications of Red??7029 recombinases to Burkholderia phytofirmans sp PsJN for genome miningRed?-Red??7029 recombineering was also effective in P.phytofirmans strain PsJN for BGC manipulation.Site-specific recombination systerms(SSRs)are gene targeting technologies which were developed from the recognition of the recombination sites by the site-specific recombinases.The general application of the SSRs includes the expression of a recombinase in a precise type of cells,catalyzing recombination at the corresponding sites,resulting in target gene extirpation or reporter gene expression.The mostly used recombinase all belong to the T-SSR protein family.Recombination sites typically range between 30 to 200 nucleotides in length and consist of two motifs with a partial inverted-repeat symmetry,to which the recombinase binds,and which flank a central crossover sequence,often referred to as the spacer sequence,at which the recombination takes place.Dre is site-specific recombinase from phage D6 which was isolated from Salmonella oranienburg.Dre recombinase was reported could mediate precise site-specific recombination between target sites termed rox which are 32 base pair long.The identification of the spacer region of the rox siteIn the previous work,the rox site was considered to be 32 base pair long and based on 14 base pair palindromes separated by 4 base pairs asymmetric sequence,in this work we identified the spacer region with 8 bp for the first time.The comparative analysis of the toxicity of different site-specific recombinases in E.coliThe toxicity of the Cre recombinase is mainly because of the existence of recombinase recognition sites in the cell chromosomes.In this work we compared the growth curves of E.coli which carring expression plasmids of different site-specific recombinase respectively.The results of the growth curves determination revealed that both Cre and Flp are in position of toxicity,Dre did not show any detectable toxicity.The construction of mutant rox sitesDre can mediate both intramolecular(excision or inversion)and intermolecular(integrative)recombination.However,the efficiency of the integration is very low because the integrated DNA which has rox sites of identical orientation at two ends that can be easily eliminated through excursive recombination with the present of the Dre recombinase.The mutant rox sites can promote site-specific integration events much more efficiently than wild-type rox because they can avoid subsequent rounds of recombination after the initial integration has accrued.In this part of work we constructed a mutant rox sites library which contains more than 90 mutant rox sites with single-,double-and five-base(s)substitutionScreenning of mutant rox sites with high specificity and efficiency.We identified several mutant rox sites that are incompatible with wild type rox site,but can efficiently recombine with the same mutant rox site in vivo.The incompatibility among each mutated rox sites allowed the deletion of multiple markers by one Dre in the genetic engineering of prokaryotic or eukaryotic genomes.