Organic-solvent-tolerant Mechanisms Of Pseudomonas Putida Based On Proteomic Analysis

Organic solvent are toxic to most microorganism, but some organic-solvent-tolerant (OST) bacteria overcome the destructive effects of organic solvent through all kinds of accommodative mechanisms.In this work, Pseudomonas putida was adapted in cyclohexane, and an OST mutant P. putida JUCT1 capable of growing in the presence of 60% (V/V) cyclohexane was obtained. Two-dimensional gel electrophoresis (2-DE) was used to compare and analyze the total cellular protein of JUCT1 when growing in the presence of 60% (V/V) cyclohexane or not. From 22 proteins whose intensity values show over 50% discrepancies under different solvent conditions, 3 high abundance protein spots were identified by MALDI-TOF/TOF spectra as 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase, and their corresponding genes mmsB, tsf, and PSEEN0851 were expressed in the E. coli respectively. These the solvent tolerance of JM109 was significantly increase by these three proteins, particularly 3-hydroxyisobutyrate dehydrogenase.Amino acid sequence alignment shows that the similarity between isochorismatase superfamily hydrolase in P. putida JUCT1 and E. coli is more than 94%. It is supposed that the two enzymes may share similar activity as well as OST-related functions by unknown mechanisms. Protein chain elongation factor EF-Ts is another protein identified from 2-DE images based on its intensity discrepancy. Our result shows that the OST of E. coli was slightly enhanced by its over-expression. It suggests that the protein chain elongation factor EF-Ts might assist in the expression of certain stress-response proteins to improve the solvent tolerance.The enzymatic mechanism and evolutionary origin of 3-hydroxyisobutyrate dehydrogenase is similar to that of 6-phosphogluconate dehydrogenase, and both of them belong to the 3-hydroxyacid dehydrogenase family. The 6-phosphogluconate dehydrogenase encoding gene zwf belongs to the mar-sox regulon genes, which are important for the regulation of a number of stress response genes. It is therefore speculated that 3-hydroxyisobutyrate dehydrogenase is involved in the OST regulation in P. putida and dedicates to the enhanced solvent tolerance.In E. coli, homology search showed that two genes ycaC, tsf is similar to tsf and PSEEN0851 from P. putida JUCT1. Besides, the enzymatic mechanism and evolutionary origin of 3-hydroxyisobutyrate dehydrogenase (mmsB) is similar to that of 6-phosphogluconate dehydrogenase (zwf) from E. coli, and both of them belong to the 3-hydroxyacid dehydrogenase family. Three genes (ycaC, tsf, zwf) in E.coli JM109 were deleted respectively by Red recombination. In the presence of 4% (V/V) decalin, the solvent tolerance of recombinant strains was significantly increase by the over-expression of three genes compared with their knockout strains, particularly zwf. It suggests that these three proteins from E. coli could also assist to improve the solvent tolerance of microbial cells.In this work, proteomics analysis was proven to be an effective strategy for exploring OST mechanism of microbial cells. Simultaneously, several proteins and their coding genes were found to contribute to increase the OST of microorganism. Importantly, this work provides molecular basis for constructing OST whole-cell catalyst for industrial applications.