Metabolic engineering has been widely applied in modifying metabolic pathways to improve the properties of microbial strains, including manipulation of enzyme levels through the amplification, disruption, or addition of a metabolic pathway . These approaches have been proven to be powerful in developing microbial strains for the commercial production of organic acids, amino acids, biofuels, and pharmaceuticals . Nevertheless, over-expression, deletion, or introduction of heterologous genes in target metabolic pathways do not always result in the desired phenotype . In recent years, cofactor engineering, which is considered as a new branch of metabolic engneering, has attracted increasing attention. Manipulations of the cofactor form and level have become a useful tool for metabolic engineering to redistribute/enhance carbon flux in metabolic networks . Nicotinamide adenine dinucleotides (NADH and NAD+), as one pair of key cofactors play an important role in over 300 biochemical reactions involving oxidation and reduction [2, 5]. Therefore, this cofactor pair (NADH and NAD+) has a critical effect on maintaining the intracellular redox balance, which is a basic condition for microorganism to metabolize and grow . Regulation of the NADH/NAD+ ratio can be achieved by either weakening the metabolic pathways competing for NADH or NAD+[7–9], or introducing an NADH or NAD+ regeneration system. Intracellular concentrations of NADH and NAD+ can be changed by expressing an NAD+-dependent formate dehydrogenase (EC 184.108.40.206; FDH), an NADH oxidase (EC 220.127.116.11; NOX), or a nicotinic acid phosphoribosyl transferase (EC 18.104.22.168; NAPRTase). Increase of intracellular NADH availability by overexpressing FDH in bacteria provoked a significant metabolic redistribution [10, 11]. Heterologous expression of NOX was conducted in Lactococcus latis, resulting in a low NADH/NAD+ ratio and the shift from homolactic fermentation to mixed-acid fermentation under aerobic conditions . Overexpressing the gene of pncB encoding NAPRTase in E. coli, was observed to increase the total NAD+ level and decrease the NADH/NAD+ ratio . These studies showed that the expression of the NADH or NAD+-related enzymes could lead to a dramatically altered NADH/NAD+ ratio and a significantly changed spectrum of metabolic products .
Cofactor engineering has been successfully applied for the prodcution of many bio-based chemicals, such as 1,3-propanediol [9, 11], pyruvic acid , and succinic acid . In the present work, this novel and powerful technology will be applied to the production of another important bio-based chemical, acetoin. Acetoin is defined as one of the high value-added platform compounds and selected by the U. S. Department of Energy as one of the potential top 30 chemical building blocks from sugars . A number of microorganisms are able to accumulate acetoin, including the genera Klebsiella, Paenibacillus, Bacillus, Serratia, etc. [16, 17]. However, these strains are widely known as good producers of 2,3-butanediol (2,3-BD) which is another important bio-based platform chemical . Acetoin is only generated as a minor by-product. Although some Bacillus strains have been used for acetoin production [19–21], the long fermentation period generally needed in fermentative acetoin using Bacillus strains still hinder its large-scale production.
In bacteria, acetoin and 2,3-BD are produced by the mixed acid-2,3-BD fermentation pathway. In the presence of NAD+, 2,3-BD dehydrogenase (EC 22.214.171.124; BDH), which is a NADH-dependent dehydrogenase, can catalyze 2,3-BD to acetoin  (Additional file 1: Figure S1). Due to the reversible transformation between acetoin and 2,3-BD coupled with the NADH/NAD+ conversion, the synthesis of these two products have been considered involving in regulation of the NADH/NAD+ ratio in bacteria . That is to say, these two products exist in the same branch of the metabolic pathway linked with NADH and NAD+ transformation. Considering that Klebsiella species are the most powerful 2,3-BD producers, they could be engineered to obtain high acetoin producing ability. In this work, an NAD+ regeneration system (nox-2 gene from Streptococcus pneumoniae, encoding NOX) was introduced into a 2,3-BD producing strain Klebsilla pneumoniae to manipulate the intracellular NAD+ level and NADH/NAD+ ratio for acetoin overproduction. The consequent effect on the distribution of metabolites in K. pneumoniae, especially, on the production of compounds that require NADH or NAD+ for their synthesis, including acetoin, 2,3-BD, and other by-products, such as ethanol, lactic acid, and acetic acid, was investigated.