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Acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), which catalyzes the interconversion between acetoin and 2,3-butanediol, plays an important role in distribution of the products pools. This work characterized the  Bacillus subtilis  AR/BDH for the first time. The enzyme showed very different pH preferences of pH 6.5 for reduction and pH 8.5 for oxidation. Based on these above results, a two-stage pH control strategy was optimized for acetoin production, in which the pH was controlled at 6.5 for quickly converting glucose to acetoin and 2,3-butanediol, and then 8.0 for reversely transforming 2,3-butanediol to acetoin. By over-expression of AR/BDH in the wild-type  B. subtilis  JNA 3-10 and applying fed-batch fermentation based on the two-stage pH control strategy, acetoin yield of  B. subtilis  was improved to a new record of 73.6 g/l, with the productivity of 0.77 g/(l·h). The molar yield of acetoin was improved from 57.5% to 83.5% and the ratio of acetoin/2,3-butanediol was switched from 2.7∶1 to 18.0∶1.

Two-Stage pH Control Strategy Based on the pH Preference of Acetoin Reductase Regulates Acetoin and 2,3-Butanediol Distribution in Bacillus subtilis