| Summary: | <p>Abstract</p> <p>Background</p> <p>Acetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria.</p> <p>Results</p> <p>The present study reports introducing an exogenous NAD<sup>+</sup> regeneration sysytem into a 2,3-butanediol producing strain <it>Klebsiella pneumoniae</it> to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in <it>K. pneumoniae</it> resulted in large decreases in the intracellular NADH concentration (1.4 fold) and NADH/NAD<sup>+</sup> ratio (2.0 fold). Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L) by <it>Klebsiella</it> species was obtained.</p> <p>Conclusions</p> <p>The present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD<sup>+</sup> ratio in <it>K. pneumoniae</it>. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD<sup>+</sup>-dependent microbial metabolite.</p>
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