Abstract
Organic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD660, whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.
Highlights
Whole-cell catalyzed reactions offer many advantages over those catalyzed by isolated enzymes
In an octanol/water two-phase reaction system equipped with hollow-fiber membrane, 3methylcatechol is produced from toluene by an organic-solventtolerant (OST) Pseudomonas putida [3], and bioconversion of glucose to phenol is achieved in a biphasic system catalyzed by P. putida cells [4]
Adaptation of P. putida in Cyclohexane An OST strain P. putida JUCT1 was obtained through gradient adaptation in medium containing cyclohexane over 12 serial transfers
Summary
Whole-cell catalyzed reactions offer many advantages over those catalyzed by isolated enzymes. In an octanol/water two-phase reaction system equipped with hollow-fiber membrane, 3methylcatechol is produced from toluene by an organic-solventtolerant (OST) Pseudomonas putida [3], and bioconversion of glucose to phenol is achieved in a biphasic system catalyzed by P. putida cells [4]. Most organic solvents used in reaction system are toxic to microbial cells and could compromise their viability. The accumulation of various solvents in cell membrane is a major cause for the toxicity of organic solvents, which are often structurally unrelated [6,7]. Some microorganisms can assimilate toxic organic solvents when the solvent concentrations are low [8,9]. Toluene for example, concentration as low as 0.1% (v/v) was toxic enough to microbial cells. It is important to understand the mechanisms of OST in microorganisms for the development of solvent-tolerant strains of industrial interest
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