Abstract:[Objective] Most microorganisms can not grow under organic solvents of high concentration due to their toxicity. To elucidate the mechanisms of organic-solvent-tolerant (OST) of microorganism, an OST mutant P. putida JUCT1 capable of growing in the presence of 60% (V/V) cyclohexane was obtained by gradient adaptation in cyclohexane. [Methods] 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. [Results] 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. [Conclusion] In this work, proteomics analysis was proven to be an effective strategy for exploring OST mechanism of microbial cells. Importantly, this work provides molecular basis for constructing OST whole-cell catalyst for industrial applications.
