Study On The Molecular Mechanism Of Decolorization Of Azo Dyes By Shewanella Putrefaciens CN32

Azo dyes are currently the most commonly used type of commercial synthetic dyes.They are widely used in printing and dyeing,textile,leather manufacturing and other industries due to their bright color,wide variety,and firm color fixation.However,a large amount of untreated azo dye wastewater is discharged every year,which not only causes serious environmental pollution,but also poses a threat to the health of animals,plants and even human beings.Among the current methods for treating azo wastewater,compared with physical methods and chemical methods,biological methods have many advantages such as low cost,simple and efficient,and environmentally friendly,so they have good application prospects.Biological methods usually use the metabolic process of microorganisms to degrade azo dyes.A variety of bacteria have been proved to be able to degrade azo dyes.The complete degradation of azo dyes by bacteria is generally divided into two steps.First,the azo bonds in the dye molecule are destroyed to decolorize the dye and convert it into colorless amines.Then these intermediate products are completely degraded and mineralized under aerobic conditions.Decolorization is the previous step for the complete degradation of azo dyes.Shewanella shows a good application potential in the decolorization and detoxification of azo dye wastewater.However,the molecular mechanism of decolorization is still incomplete.In this study,it was found that Shewanella putrefaciencs CN32 had good decolorization ability for a variety of azo dyes,and by constructing a transposon insert library,the global regulatory factors Crp(c AMP receptor protein)and the electron transfer protein Cym A are necessary for S.putrefaciencs CN32 to decolorize the azo dye Acid Yellow 36(AY).Then,we further explored the molecular mechanism of Crp regulating AY decolorization.The results of Quantitative Real-time PCR and Electrophoretic Mobility Shift Assay show that Crp can directly bind to the promoter region of the cym A gene and promote its expression.Subsequently,the specific location of Crp binding in the promoter region of cym A gene was determined by DNase I footprinting as the-375 to-338 nt region upstream of the initiation codon of cym A gene.In addition,riboflavin as an electron shuttle can accelerate the decolorization efficiency of S.putrefaciencs CN32 wild-type strain,but it has no effect on the decolorization efficiency of the Δcrp mutant and the Δcym A mutant,which further confirms that Crp promotes the decolorization by regulating the electron transport chain.Compared with the wild-type strain,overexpression of the cym A gene can slightly improve the AY decolorization ability of the strain and the genetically engineered strain with more decolorization potential was obtained.At the same time,this study also found that Mtr A,Mtr B and Mtr C contribute to partial electron transfer from Cym A to dye molecules,but Mtr pathway does not act as the main pathway.Other main electron transfer pathways need to be further determined in future experiments.This study reveals the molecular mechanism of the global regulatory factor Crp regulating the decolorization process of azo dyes,enriches the theoretical basis of Shewanella in decolorization of azo dyes,and will help to understand the relationship between the decolorization process and other metabolic processes in S.putrefaciencs CN32.