Molecular Mechanism Of Phenazine-1-carboxylic Acid Degradation By Sphingomonas Wittichii DP58 And Phenazine Degradation By Sphingbium Yanoikuyae B1

Phenazine-1-carboxylic acid?PCA?is the key component of Sheqinmycin,an antifungal pesticide widely used for the prevention of cucumber,watermelon and melon wilt,pepper root rot,rice sheath blight due to its high fungicidal efficiency,low toxicity and environmental compatibility.It is reported that PCA could be degraded in3.5 days in soil and bacterias from soil play key role in its degradation.Until now,few studies about the PCA degradation pathway and the effect of its metabolites on the environment were reported.The absence of study on PCA metabolism would hinder the application of Shenqinmycin.This study focused on the initial degradation step in PCA degradation.The result of this study would help us to explore new ways to delay the degradation of PCA in soil.Previously,strain S.wittichii DP58 which can use phenazine-1-carboxylic acid as its sole carbon and nitrogen source was isolated from pimiento rhizosoil and the genome was sequenced.Two intermediates of phenazine-1-carboxylic acid degradation,4-hydroxy-1-?2-carboxyphenyl?azacyclobut-2-ene-2-carbonitrile and4-hydroxy-1-?2-carboxyphenyl?-2-azetidinecarbonitrile,were identified.However,the genes for PCA initial degradation were still unknown.In order to investigate PCA degradation pathway,we analyzed the whole genome sequences of 26 strains in the genera Sphingomonas and Sphingobium to gain insight into dissemination of bioremediation capabilities,biodegradation potential,central pathways and genome plasticity.Transposase and IS coding genes were found in the bph and xyl gene clusters of 6 PAH-degrading,suggesting the mobility of PAH-degradation ability.?-Ketoadipate pathway with catechol as its key intermediate was the main central pathways in Sphingomonas and Sphingobium strains in aromatic compounds degradation.Moreover,a large number horizontal gene transfer related genes and prophages were predicted in the 26 strains,suggesting the ongoing evolution and dispersion of the biodegradation ability.We found that PCA degradation in strain DP58 was induced by PCA.Therefore,RNA-seq was used to find the PCA degradation related genes.The results showed that genes involved in the degradation of xenobiotics,including toluene pathway,caprolactam pathway,nitrotoluene pathway,PAHs and benzoate degradation pathway,were upregulated.The three dioxygenase encoding genes with the highest upregulation were located in the same gene cluster.The gene cluster was comprised of ten genes including two pairs of dioxygenase?pcaA1A2,pcaB1B2?,electric transfer chain?ETC,pcaA3A4?,ring-breaking dioxygenase?pcaC?,monooxynase?pcaD?,oxidordeuctase?pcaE?and regulator?pcaR?encoding genes.By partial and entire gene cluster clone and whole microbial cell transformation,it was determined that the gene cluster involved in PCA degradation.Moreover,pcaA1A2A3A4 were predicted as the genes encoding the initial dioxygenase in PCA degradation.Phylogenetic analysis was performed using the alignment of PcaA1,pcaB1 and PcaC with the?subunits of 72 characterized ring hydroxylating oxygenases.In the phylogenetic tree,PcaA1 clustered with benzoates dioxygenase and shared highest similarity with salicylate hydrolase,indicating PcaA1A2 might be involved in decarboxylation.PcaB1 clustered with dioxygenases for fluorenone,dibenzofuran,carbazole and dibenzo-dioxin degradation,implying that it might be responsible for N-heterocycle breaking.The dioxygenase encoding genes pcaA1A2 and pcaB1B2were cloned into pMD18-T and expressed in E.coli DH5?.After induced,none PCA conversion was observed.We cloned pcaA1A2 and pcaB1B2 accompanying with pcaA3A4 into pMD18-T,respectively.The biotransformation results showed that the strain only contained pcaA1A2A3A4 converted PCA into 1,2-dihydroxy phenazine.Morover,¹³C labeled CO₂ was detected in the overhead air of reaction solution when using ¹³C labeled PCA as substrate,suggesting carboxyl was converted to CO₂ instead of utilized as carbon source.The results indicate that pcaA1A2A3A4 encode the initial dioxygenase for PCA degradation?named as PCA 1,2-dioxygenase?,which convert PCA to 1,2-dihydroxy phenazine through decarboxylation and hydroxylation.To further reveal the characterization of PCA 1,2-dioxygenase,pcaA1A2 was cloned into pBBR2S and pcaA3 and pcaA4 was cloned into pET28a?+?.The recombined vectors were expressed in P.putida KT2440 and E.coli BL21?DE3?,respectively.Three components of the PCA 1,2-dioxygenase was purified using nickel column.His₆-PcaA1A2 was a polymer including two subunits which were 47and 17 kDa in size;His₆-PcaA3 and His₆-PcaA4 were approximately 49 and 15 kDa in size,respectively.All the three components were found to be essential for PCA conversion through in vitro assay,suggesting it's a type IV dioxygenase.The function of PcaA3 can be replaced by RedA2 in the in vitro analysis,indicating the low specificity of the ETC.This conclusion was also proved by the PCA conversion capability of P.putida KT2440 harboring pcaA1A2.Both NADH and NADPH can performed as electron donor for PCA 1,2-dioxygenase,while NADH supported the PCA 1,2-dioxygenase activity better than NADPH?20 folds?.A molecular mass of190 kDa was obtained for the native PcaA1A2 through FPLC analysis,indicating a?₃?₃ subunit conformation.We also found that PCA 1,2-dioxygenase converted salicylate to catechol,while it did not consume benzoate and 2-OH-PCA.Phenazine is the precursor of many phenazine dyes,pharmaceuticals and chromogens that widely used in introdustrial production.It shows toxicity to bladder and liver cell.It was found that S.yanoikuyae B1 converted PHZ to 1,2-dihy 1,2-dihydroxy phenazine.However,the genes involved in phenazine degradation were remains unknown.The genome of strain B1 was sequenced via the high throughput sequencing technology.The genome of strain B1 is 5.7 M in length.A phylogenetic analysis was performed using the amino acid sequence of ten non-horizontal house-keeping genes.The results showed that strain B1 shared the closest affinity with carbazole degradation strain XLDN2-5.It is known that,the biodegradation of PAHs in strain B1 was activated by biphenyl 2,3-dioxygenase.Therefore,we cloned bphA1fA2fA3A4 into pBbB5a-RFP and expressed in E.coli DH5?to investigate whether it is also involved in phenazine degradation.After induced,strain DH5?harboring pBbB5abphA converted phenazine to 1,2-dihyro 1,2-dihydroxy phenazine through hydrogenation and hydroxylation.1,2-dihyro 1,2-dihydroxy phenazine was unstable and could transform to 2-hydroxy-phenazine through dehydration spontaneously.Moreover,bphB and bphC were proved not to be involved in phenazine degradation by gene clone and whole microbial cell transformation.bphA1fA2f was identified to be the only two genes encoding the initial dioxygenase that responsible for PHZ degradation in strain B1 through gene inactivation and complementation.In conclusion,we identified the initial dioxygenase for PCA and phenazine degradation in S.wittichii DP58 and S.yanoikuyae B1,respectively.We also explored the enzymatic characterization of PCA 1,2-dioxygenase.Moreover,we predicted and analyzed the gene cluster for PCA degradation.The results in this thesis can provide basic knowledge for PCA and phenazine degradation.