Molecular Response To Phenol Stress Of Burkholderia Cepacia BNS And Heterologous Expression Of Key Enzymes

Phenolic pollutants is one of the common pollutants in the production of modern industrial society.As an environmentally friendly treatment method,microbial degradation of phenols has attracted more and more attention from researchers in recent years.Thus,the response mechanism of microorganisms to phenolic pollutants stress is the focus of attention.Studying on different tolerance and biodegradation mechanism of different microbial strain to phenolic compounds is positive to artificial modification to the tolerance and degradation strategy of stains,and is of great significance to find new and effective technologies for remediation of phenolic pollutants.In this study,a strain of Burkholderia cepacian BNS was selected as experiment material,the growth and phenol degradation performance were studied.Then the phenol stress system of Burkholderia cepacia BNS was constructed and some bacteria samples of stress for 0 h,2 h,12 h and 24 h under phenol were collected for transcriptome and proteome sequencing.The response mechanism to stress under phenol of Burkholderia cepacia BNS was revealed by GO and Pathway enrichment analysis of different genes and proteins at molecular level.Finally,the two kinds of related key enzymes was cloned and expressed.The main results are as follows.1.Burkholderia cepacia BNS was strong in resistance to environmental stress.It will grow well in pH 4¹0,and still can survive at 80?for 30 min.The strain can decolorize the synthetic dye by extracellular lignin peroxidase and intracellular laccase.Meanwhile,phenol,catechol,resorcinol and?-naphthol could be degraded by strain BNS and the maximum tolerance concentration to phenol is 1.5 g/L.At low concentration,phenol may be degraded by hydroxylation,and phenol may be metabolized by both hydroxylation and carboxylation at high concentration.2.Some mRNA samples under 1.5 g/L phenol stress for 0 h,2 h,12 h and 24 h were sequenced by mRNA-Seq.A total of 7614 expression genes and 5691 significant different expression genes were obtained.Through the analysis of GO and Pathway enrichment of different expression genes,it was found that in the early stage of stress,the strain enhanced its tolerance to phenol by decreasing their metabolic activity and differently expressing of some function and stress genes related to EnvZ/OmpR,efflux pump,ABC transporter,DNA repair protein,superoxide dismutase?SOD?,cold stress protein,glutathione?GSH?,Laccase and so on.In the middle stage of stress,the expression of carbon source starvation factor CST and phenol metabolism pathway-related gene were significantly up-regulated,while efflux pump and ABC transporter continued to maintain at a high expression level and stress related genes were down-regulated.In the late stage of stress,the strain growth and phenol metabolism were much actively,the expression of phenol degradation-related continued to maintain a high level.Some genes related to oxidative phosphorylation,molecular chaperone,outer membrane protein,degradation protein,and so on were up-regulated.The strain responded to the stress and toxicity of phenol by spatiotemporally regulating expression of pathway genes at transcription level,and dominated the change from negative tolerance to positive degradation.The?-ketoadipate pathway was the main pathway of degradation.3.The different expression of the transcription factor and small RNA?sRNA?of the strain under the stress of phenol was analyzed.The information of 461 transcription factor proteins were obtained.These proteins were belong to ten protein families,and play regulation role at transcription level.The LysR family transcription factors are mainly involved in the DNA damage repair,the phenol tolerance and degradation;The AraC and TetR family transcription factors mainly regulate the expression of the efflux pump,osmotic pressure,and so on;Sigma factor regulated the tolerance and degradation of phenol by the basic transcriptional regulatory factors of?70 family and the regulation of carbon and nitrogen metabolism by?54 family and the action of anti-sigma factor.The GntR family mainly regulates the growth and metabolism of the cells and expression of the membrane-related proteins;The MarR family is related to intracellular oxidative stress;The IclR family plays an important role in the degradation of aromatic phenols such as protocatechol;The LuxR family mainly regulates the expression of the colony induction,the biological membrane and the virulence factors of the strain;The FnR family and the XylR family protein regulate the oxygen sensing pressure of the strain and the metabolism of organics.In addition,484 sRNA genes were obtained,and 1501 mRNA were regulated at the post-transcription level by sRNA which formed 16760 targeted regulation relationship.The sRNA regulated some biological processes by enrichment analysis,which were involved in acetylization of tRNA,the metabolism of the glycerophospholipid,the metabolism and activation of the protein,the metabolism of the phosphatidylinositol,and so on.4.Non-labeled quantitative proteomics technique?Label-free?was used to identify and analyze the differential expression of bacterial proteins under phenol stress.3194 protein were identified,of which 1104 proteins were significantly different,of which 684 proteins were different expression,420 proteins were new or disappear proteins.The GO functional analysis revealed that the different expression proteins were mainly involved in catalytic activity,binding,transporter activity,structural molecule activity and transcription regulator activity,which were mainly involved in the important biological processes,such as metabolic process,cellular process,localization,benzoate degradation,biological regulation process.The strain improved tolerance to phenol by regulating the different expression of the multifunctional membrane protein,the transcription regulatory protein and the stress response protein.And cell reduced the toxicity of phenol by regulating the ortho pathway and protocatechol pathway of phenol degradation.5.A laccase-like protein from Burkholderia cepacia BNS was expressed in Escherichia coli using pET32a vector and a target protein with molecular weight of about30 kDa was successfully obtained.After purification,the protein was active in the form of dimer and the relative activity was 7.81 U/mg,Km and kcat were 156.4?M and 619.44 s^-1,and the catalytic efficiency was 3.96?M^-1 s^-1 with ABTS as the substrate and the relative activity was 12.3 U/mg,Km and kcat were 100.2?M and 1162.22 s^-1,and the catalytic efficiency was 11.6?M^-1 s^-1 with 2,6-DMP as the substrate.The recombinant protein can decolorize a variety of synthetic dyes.In addition,Ppic9K vector and Pichia pastoris GS115 secretory expression system were used to express catechol 1,2-dioxygenase?cat12?from Burkholderia cepacian BNS,and a extracellular protein with molecular weight of 35kDa was successfully obtained.Activity of the protein was 22.55 U/mg with catechol as the substrate.