Combinatorial Biosynthesis Of Syrbactins And Heterologous Expression Of The Prenvlisatin Biosynhetic Gene Cluster

Microorganisms are the main source of important pharmaceutical compounds of medicinal.In order to discover more natural compounds against tumors and bacterial infections,the proteasome-inhibiting syrbactin-family compounds and prenylated indole derivatives were studied from the perspective of combinatorial biosynthesis and genome mining.1 Genome mining and combinatorial biosynthesis of syrbactinsCancer threat has been a global health issue.Proteasomes able to regulate cell cycle and cell apoptosis become essential targets for antitumor drug screening.Syrbactins isolated from microorganisms target the active site of proteasomes.Syrbactins including syringolins,glidobactins,cepafungins and luminmycins show a common mode and are considered to be leading compounds as a new generation of proteasome inhibitors.They show similarity at the level of structure and biosynthesis:their 12-membered lactam cyclic skeleton was composed of a series of substrates assembled via modular-assembly mechanism mediated by a nonribosomal peptide synthetase(NRPS)-polyketide synthase(PKS)complex,allowing to engineer their biosynthetic pathways to generate improved leading compounds utilizing synthetic biological techniques.1.1 Genome mining and heterologous expression of syrbactins gene clustersIn this study,we used the Red/ET DNA recombination engineering technology platform(direct cloning of large fragments,orderly assembly of multiple fragments,etc.)to carry out genome mining and obtained syrbactins biosynthetic gene clusters glb,syl and Pglb from different sources.But Pglb could be activated in its native host or heterologous host.These syrbactins biosynthetic gene clusters were used as materials.We tried to express the syrbactins biosynthetic gene cluster in different hosts,hoping to find a best suitable host to effectively express the"artificial"biosynthetic gene cluster.We found that Glb could not express in S.albus J1074,syl could be expressed in in both S.coelicolor M145 and S.lividans TK24.But,at present,there is no common host that can express both glb and syl gene clusters.1.2 Blocking and replenishing of glidobactin gene clusterWe knocked out glidobactins biosynthetic genes glbC and glbF on the DSM 7029 genome using the genetic operation system established by our team in Burkholderia DSM 7029,and obtained two mutants blocking glidobactins biosynthetic pathway.Then,we constructed glbC and glbF replenishment expression plasmids and electrotransformed them into the above knockout mutants to obtain replenishment strains.Gene complementation experiments showed that the intraspecific replenishment could restore the production of glidobactins,but the yield decreased to 10%of wild type DSM 7029.1.3 Replacement of heterologous structural genes in glidobactin gene clusterWe also tried to replace the structural gene.The structural genes sylD and sy1C corresponding to glbC and glbF,the structural gene Pg1bC homologous to glbC in Pglb were electrotransformed into the corresponding DSM 7029 knockout mutants by RK2-apra plasmids.The replacement experiments of knockout genes showed that the substitution of interspecific structural genes could not produce expected hybrid compounds.1.4 Module and domain swappingBased on the glidobactins biosynthetic gene cluster glb,we attempted to make modules and domains swapping.The modules and domains used to swap originated from two gene clusters,syl and Pglb.Although no expected heterozygous compounds were produced,dipeptide-like compounds were detected,indicating that module and domain substitution disrupted glidobactins biosynthetic pathway.In order to eliminate the effects of GC content and codon preference in syrbactins biosynthetic gene clusters from different sources,we optimized the codon of the module used for modules and domains swapping.However,substitution of module and domains did not result in the detection of expected hybrid compounds.2 Direct cloning and biosynthesis of prenylisatin gene clusterPrenylisatin is a bacterial-derived prenylated indole compound and has the characteristics of anti-bacterial,anti-fungal and cytotoxicity.It is formed by isoprenyl substitution of tryptophan in different positions under the catalysis of isoprenyl transferase.At present,the complete biosynthetic pathway of these compounds has not yet been elucidated.The aim of this study is to identify the prenylisatin biosynthetic gene cluster by genomic mining using Red/ET direct cloning technology,and to study its biosynthetic pathway.2.1 Direct cloning and heterogeneous expression of prenylisatin gene clusterWe used antiSMASH to predict the Streptomyces rochei D74 genome and found that the strain contained a large number of secondary metabolic gene cdusters.The silent gene cluster 2 was cloned directly and introduced into Streptomyces coelicolor A3(2).The results of HPLC-MS showed that heterologous expression successfully activated the gene cluster,and the compound(m/z 215.6[M+H]+)was consistent with the literature reports.2.2 Functional study of single gene in prenylisatin biosynthetic gene clusterSeventeen genes in the prenylisatin gene cluster were inactivated y knock out and then analyzed by LC-MS.The results showed that the three genes isa8,isa9 and sa10 were key genes in the prenylisatin biosynthesis pathway,and their deletion resulted in no accumulation of target products.Homologous analysis showed that isa8 and isa9 were tryptophan synthase arnd aromatic isoprenetransferase genes,respectively.Function of isa10 is uncertain(might encode a regulator).3 ConclusionWe tried many strategies to modify the biosynthetic pathway of syrbactins,but did not synthesize the expected heterozygous compounds.The reason may be tmat the NRPS and PKS systems themselves are relatively complex,even minor changes can have a huge impact on the whole biosynthetic pathway,syrbactins are synthesized through NRPS/PKS hybrid pathway,the protein-protein interaction is more difficult to predict than single NRPS or PKS.At present,there is no successful case of NRPS/PKS hybrid gene cluster for module swapping has been reported.We captured prenylisatin biosynthetic gene cluster directly by Red/ET direct cloning technique from Streptomyces rochei D74 genome.By knockout experiments on single gene of prenylisatin biosynthetic gene cluster,we identified three key genes(tryptophan synthase,aromatic isopentene transferase and uncharacterized protein),They will be as important gene resources for combinatorial biosynthesis of natural product.