Quantitative Proteomics Analysis Of Rhodococcus Ruber And Fusion Expression And Catalytic Properties Of Diguanylate Cyclase

Rhodococcus ruber with organic tolerance has potential applications in biotransformation and bioremediation.Excavation of the possible response related proteins and functional of R.ruber SD3 to organic solvent are helpful to understand the organic tolerance mechanism,and also promote the application in the field of organic phase catalytic reaction.The main contents and results are as follows:1)The response of R.ruber SD3 to toluene and phenol was investigated using a quantitative proteomics approach with isobaric tag for relative and absolute quantification?iTRAQ?and liquid chromatography-tandem mass spectrometry.A total of 362 and 488 differentially expressed proteins were identified in the toluene treatment group and the phenol treatment group as compared to the control group,respectively.Stress proteins,membrane proteins,regulatory systems,and metabolic pathways associated with the organic solvent tolerance of the R.ruber SD3 strain were also elucidated.The quantitative real-time polymerase chain reaction?qPCR?experiment indicated the mRNA levels of CspA-family stress proteins,4-nitrophenol2-monooxygenase,diguanylate cyclase?DGC?in the toluene or phenol treatment group were higher than the counterpart in the control.2)Bioinformatics methods were used to analyze the physicochemical properties and structural characteristics of DGC protein.The preliminarily predicted results indicate that DGC protein is a hydrophobic protein with 6-segment transmembrane region,with more than 50%stable?-helical structure,possessing a typical GGDEF catalytically active domain.Phylogenetic tree analysis showed that DGC protein of R.ruber SD3 and R.aetherivorans were closely related,which means that the DGC protein also belongs to the Class III nucleotidyl cyclases family with similar molecular functions.The tertiary structure of DGC protein was obtained by I-TASSER modeling tool,and applied to the molecular docking.It was simulated the interaction between substrate GTP and DGC protein through hydrophobic interaction and hydrogen bonding to form a stable binding conformation.3)The function and activity of synthetic c-di-GMP of diguanylate cyclase in R.ruber SD3 response to organic solvent were further studied in this paper.The dgc gene from R.ruber SD3 was applied for codon optimization.The pPGH-Dgcs recombinant plasmicd was constructed,which was transformed into E.coli BL21?DE3?for heterologous expression.The GST tag was fused to help correct folding in the heterologous expression of DGC membrane protein under the conditions of 16°C and 0.1 mM IPTG induction by optimizing expression conditions.Here,a full-length DGC from R.ruber SD3 fused with glutathione-S-transferase?GST?was purified by glutathione agarose resin and identified as the target protein by liquid chromatography coupled with tandem mass spectrometry?LC-MS/MS?.The apparent molecular mass of one subunit of the purified diguanylate cyclase with GST tag?GST-DGC?was estimated to be 71.9 kDa by SDS-PAGE,which was approximately in accordance with the theoretical value of 73.0 kDa.The blue native PAGE indicated that GST-DGC formed octamer.The optimum pH and temperature for GST-DGC activity were 8.0 and 47°C,respectively.The fusion protein exhibited high thermostability,and 94%of activity was retained when the protein was incubated at 87°C for 1 h.Moreover,the fusion protein showed pH stability.The Km,Vmax and Kcat values for GST-DGC enzyme were 9.8?M,0.7?M/min and 1.3 S^-1.Some metal ions such as Zn²⁺,Mn²⁺,Fe²⁺,Ni²⁺and Co²⁺had inhibition effects on the protein,while other metal ions such as Mg²⁺,K⁺and Na⁺and chelating agent EDTA activated the protein.The fusion protein also showed rather high stability in the presence of toluene,cyclohexane and n-hexane.And the possible metal ions and organic solvents activation or inhibition sites of GST-DGC protein were predicted by molecular docking.It was provided targets for site-directed engineering of enzyme molecules to increase c-di-GMP production.Generally speaking,the relationship between diguanylate cyclase and microbial organic tolerance was demonstrated by proteomics and qPCR.In this study,the complete diguanylate cyclase containing 6-segment transmembrane region in Rhodococcus was first heterologously expressed and the efficiency of GST-DGC fusion protein synthesis c-di-GMP was improved by 30 times compared with the reported Kcat value of 2.6 min^-1 of thermophilic diguanylate cyclase from Thermotoga maritime.The study provided possibility to catalyze the synthesis of c-di-GMP in a large-scale,and laid a foundation for revealing the relation mechanism of diguanylate cyclase and microbial organic tolerance.