(408h) Proteomic Analysis of Pseudomonas Putida Reveals An Organic Solvent Tolerance-Related Gene Mmsb

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, 5 high-abundance protein spots were identified by MALDI-TOF/TOF spectra. The corresponding genes of these five proteins, arcA, PSEEN1080, mmsB, tsf, and PSEEN0851, were separately expressed in E. coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli transformants harboring mmsB (3-hydroxyisobutyrate dehydrogenase, 3-HIBDH) could grow to 1.70 OD₆₆₀, whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Other transformants also showed an increased resistance to cyclohexane as well as other organic solvents. Of these 5 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.

The OST-related function of mms and its homologous gene in E. coli, zwf (glucose-6-phosphate dehydrogenase, G6PDH), was also investigated. It was noted that the OST of E. coli host was severely impaired by zwf knockout, and its complementation resulted in significantly enhanced OST in 4% (v/v) cyclohexane than parent E. coli. Furthermore, recombinant E. coli strains carrying mmsB showed better OST capacity than that harboring zwf. To achieve a stable gene expression, mmsB was integrated into the genome of E. coli JM109(DE3) by red homologous recombination. The result strain E. coli JM109(DE3) (ΔendA::mmsB) was successfully applied as an OST host of whole-cell biocatalyst in an aqueous/butyl acetate biphasic system with 82.8% higher product yield.

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.