Identification And Analysis Of Polycyclic Aromatic Hydrocarbon Degrading Bacteria And Dioxygenase Mining And Application

Polycyclic aromatic hydrocarbons(PAHs)are common organic pollutants,which are carcinogenic and teratogenic.Remediation of contaminated sites has received more and more attention.In this work,the strain SJTF8 with high-efficiency degradation of PAHs was isolated and identified from environmental soil,and was identified as Sphingobium yanoikuyae with the identification of 16SrDNA and average nucleotide identity(ANI)analysis.The degradation efficiency,environmental tolerance,the whole-genome sequencing and metabolic pathway analysis,and key enzymes mining and application have been well studied.S.yanoikuyae SJTF8 could use typical PAHs(naphthalene,phenanthrene,and anthracene)and heterocyclic and halogenated aromatic compounds(dibenzothiophene and 9-bromophenanthrene)as the sole carbon source.Strain SJTF8 degraded more than 98%of 500 mg/L phenanthrene in 4 days and showed great tolerance to heavy metals and acidic conditions.Adding 0.30 mM Cu²⁺,1.15 mM Zn²⁺or 0.01 mM Cd²⁺had little effect on the cell growth and degradation efficiency of phenanthrene;250 mg/L phenanthrene can still be completely degraded within 48 hours.In addition,through whole-genome sequencing,the whole genome sequence of S.yanoikuyae SJTF8 was obtained,and three plasmids were found.Annotated genes related to stress tolerance and PAHs degradation were found that they were mainly distributed in plasmid 1 and plasmid 2,respectively.36 genes related to heavy metal resistance were found in the genome of strain SJTF8,such as the genes encoding the periplasmic heavy metal sensor enzyme and the efflux transporter,in particular,18 related genes were found in plasmid 1.Elimination of the plasmid 2 in the strain SJTF8 will result in the loss of the ability to degrade PAHs.Therefore,the genes for the degradation of aromatic compounds by the strain SJTF8 is closely related to the plasmid 2.In addition,according to the results of genome mining,the possible degradation pathway and the metabolites degraded by strain SJTF8 were predicted.The salicylic acid degradation pathway is the pathway that the strain SJTF8 tends to utilize.Using RT-qPCR to detect the transcription level of 21 RHDs genes predicted in plasmid 2 of wild-type strain SJTF8 with phenanthrene as an inducer,13genes were up-regulated 17–105 times.Furthermore,5 groups of recombinant E.coli with target ring-hydroxylating dioxygenases(RHDs)genes were constructed.By HPLC analysis,E.coli BL21(RHD-1)had the ability to catalyze the conversion of phenanthrene and other PAHs.Therefore,genes 03570 and 03075 were identified as the?and?subunits of RHD,gene 03235 encoded ferredoxin of RHD,and gene 03385 encoded ferredoxin reductase of RHD.Multiple sequence alignment analysis revealed that the dioxygenase sequence is highly conserved among the similar bacteria.With the S.yanoikuyae B1 dioxygenase?and?subunits of BphA1fA2f as the optimal template,the three-dimensional structure of the dioxygenase?and?subunits of BphA1fA2f-F8 in strain SJTF8 was simulated,and it conformed to the mushroom-like?₃?₃ hexamer complex structure.In addition,the high-value 2-hydroxydibenzofuran plays an important role in drug synthesis.Based on a group of RHD genes identified in this study,the recombinant E.coli BL21(RHD-1)could catalyze the benzofuran to generate the corresponding cis-1,2-dihydroxy-1,2-dihydrodibenzofuran.After simple acidification,the target product 2-hydroxydibenzofuran can be obtained with a total yield of 90.7%.At the same time,E.coli BL21(RHD-1)could hydroxylate a variety of aromatic compounds,and generate corresponding hydroxylated aromatic compounds as precursors for fine chemical synthesis such as drugs and chemical light emitting materials.This work comprehensively studied the characteristics of strain Sphingobium yanoikuyae SJTF8 to degrade PAHs,enriched the PAHs degrading strain library,and can be promoted for bioremediation of truly polluted environment.At the same time,the potential applications of the identified dioxygenases were explored,providing ideas for the green synthesis of related high-value hydroxylated aromatic compounds.