Cloning Of Hydroxylase Genes Involved In 3-Bromo-4-Hydroxybenzoate Degradation And Its Genetic Redundancy In Pigmentiphaga Sp.Strain H8

Meta-halogenated-4-hydroxybenzoates are usually used in chemical industry as precursor for chemical synthesis,among which the 3-bromo-4-hydroxybenzoate(BHB)and 3-chloro-4-hydroxybenzoate(CHB)are widely distributed in environment,the high concentration chemical residue in the environment is causing pollution as well as serious threat to human health.Only two aerobic biodegradation pathways of BHB and CHB are reported in microbes,which are hydrolysis dehalogenation and reduction dehalogenation.It is urgent and of great significance to elucidate new degradation pathways and study the molecular mechanisms.In a large number of reports of microbial degradation of contaminants,it can be found that there are often multiple degradation genes in the host function.The genetic redundancy of this degradation gene is very common,but because of the limitations and excavation of traditional experimental methods for the study of redundant genes and the difficulty of selective advantage of redundant genes,the research on the genetic redundancy of degradation genes is relatively lagging,which limits people's understanding of microbial degradation.Therefore,studying the genetic redundancy of pollutant degradation genes is of great significance in terms of microbial degradation and selective advantages of redundant genes.According to the previous study of our lab,the strain Pigmentiphaga sp.H8,which is isolated from soil sample,can dissimilate BHB with high efficiency.BHB metabolic pathway in H8 is induced by BHB,so we predicted two p-hydroxybenzoate hydroxylase genes phbh1 and phbh2 are involved into the initial step of BHB degradation pathway based on the transcriptome and proteome analysis,as well as bioinformatic analysis.There is one more phydroxybenzoate hydroxylase phbh3 located on the genome,but it is only responsible for the 4-hydroxybenzoate(4-HB)metabolism based on the bioinformatic analysis.The results of gene deletion/complement experiments indicate that phbhl and phbh2 is responsible for the initial step of BHB degradation in H8.Both PHBHl and PHBH2 can convert BHB with a high speed when phbhl and phbh2 are heterogeneous expressed in E.coli by vector pBBR1MCS-2,the product is Br-PCA according to LC-MS and MRI analysis,whereas PHBH3 can only transfer 4-HB to protocatechuate.By same means,we confirmed that Br-PCA and protocatechuate can be ring-lysed by protocatechuate 3,4-dioxygenases PcaAB and PcaA2B2.Further study of both gene clusters phbhlpcaAB and phbh2pcaA2B2 are required.In order to study the genetic redundancy of phbh1?phbh2 and phbh3,we studied the characteristics of PHBH1,PHBH2 and PHBH3.We get the proteins by heterogeneous expression via plasmid pET29a(+)in E.coli BL21(DE3),and then purify the three enzymes utilizing Co affinity chromatography column,but the purify result of PHBH1 is not so good.PHBH1 and PHBH3 shows the highest activity in 40? but PHBH2 is 35?,the best pH of PHBH1 and PHBH2 is 8.0 and for PHBH3 is 7.5.The best co-factor of the three proteins is FAD but not FMN,the best electric donor is NADPH.Km(PHBH1 cell extracts)of 4-HB and BHB are 34.8 ± 2.4 ?M and 22.1± 1.8 ?M respectively,and the specific enzyme activity of which are 6.6 ± 1.4 ? 25.9 ± 6.1 nmol min-1 mg-1 respectively,means the best substrate of PHBH1 is BHB.For PHBH2,Km and Kcat/Km of 4-HB are 186.4 ± 18.6 ?M and(11.2 ± 0.7)×10-3 ?M-1 min-1 respectively,Km and Kcat/Km of BHB are 43.1±3.4 ?M and(3.5±0.1)×10-1 ?M-1 min-1 respectively,indicates PHBH2 has higher activity to BHB but very less to 4HB:for PHBH3.Km and Kcat/Km of 4-HB are 19.6 ± 3.9 ?M and Kcat/Km=15.0 ± 1.9 ?M-1 min-1,Km and Kcav/Km of BHB are 24.7 ± 0.9 ?M and(19.2± 0.7)×10-3?M-1 min-1,the result of which is opposite to PHBH2.So PHBH1 and PHBH2 mainly converts BHB,while PHBH3 is mainly responsible for the metabolism of 4-HB.BHB and 4-HB are analogue,and the in vitro experiments show that the catalytic activity for 4-HB of PHBH3 is inhibited by PHBH1 and PHBH2.So we measured the metabolic activity for 4-HB of H8wild type,mutant H8(?phbh1).mutant H8(?phbh2)and mutant H8(?phbh1.?phbh2),the result shows that the presence of phbh1 and phbh2 can make poster metabolic 4-HB normally.So we infer that in halogenated hydroxybenzoate contaminated soil.the prototypes of phbhl and phhh2 acquired the BHB degrade ability and the ability of preventing BHB from inhibiting 4-HB metabolism mediated by PHBH3 under selective pressure,so that the hoster can grow on both BHB and HB as carbon sources.This study found a new hydroxylation metabolic pathway of BHB from strain H8,and clarified from the molecular level,which enriched the diversity of microbial degradation of halogenated hydroxybenzoic acid.The in-depth study of the differences in the enzymatic properties and the diversity of metabolic functions of the enzymes encoded by the three redundant 4-hydroxybenzoate hydroxylase genes confirmed the selective advantages of phbhl and phbh2 to the host-ensuring the host's natural carbon The metabolic safety of the source 4-hydroxybenzoate has deepened people's understanding of the adaptive evolution of microorganisms in complex environments,and provided new evidence for the theory of"genetic redundancy gives the host selective advantage.