The role of thioredoxin system in solvent production process of Clostridium acetobutylicum

Abstract:

[Objective] This study aimed at investigating the roles of thioredoxin dependent reduction pathway in growth and metabolism of Clostridium acetobutylicum.[Methods] We used ClosTron technology to inactivate the thioredoxin reductase genes (trxB) of Trx-dependent reduction system in C. acetobutylicum to obtain the mutant, which was further verified by Southern blotting to test the copy number of the intron. Batch fermentation was implemented in minimum medium to analyze the growth and metabolic products of the wild type and mutant. The wild type and mutant were maintained at acidogenesis and solventogenesis phase to investigate the growth and production in the phosphorus-limited continuous fermentation. Antioxidant capacity was also studied by adding different concentrations of hydrogen peroxide.[Results] The results of resistance screening and gene sequencing showed that a thioredoxin reductase inactivated mutant named C. acetobutylicum trxB::int(29) was successfully constructed. In batch fermentation, the maximum optical density (OD₆₀₀) value of mutant and wild type was similar, reaching 6.5; but the maximum OD₆₀₀ of mutant was obtained at 36 h, 12 h later than that of wild type. Nevertheless, in the acidogenic phase of continuous fermentation, the growth of wild type and mutant had little change. OD₆₀₀ was stable at 4.6 and 4.4, respectively; glucose consumption and acid production were also similar. At the solventogenenic phase, OD₆₀₀ of the mutant was 3.5 and the butanol titer decreased to 36.1 mmol/L, lower than those of the wild type (4.0 and 48 mmol/L); but the acetone titer increased to 25.5 mmol/L compared to the wild type of 18 mmol/L. Furthermore, low concentration of hydrogen peroxide had little effect on the mutant, while high concentration caused more damage to the mutant than the wild type.[Conclusion] The inactivation of thioredoxin reductase gene was not a lethal mutation. The growth and acid production ability of the mutant were similar to those of the wild type, indicating that the inactivation of thioredoxin reductase gene did not affect the metabolic process of acetogenesis. The inactivation of trxB gene mainly affected the redox balance during solventogenic phase, resulting in the increase of acetone/butanol ratio.