There is growing interest in the production of 2,3-butanediol (2,3-BD) by microbial fermentation, as it can be easily converted to methyl ethyl ketone and tetramethyl ether, blending agents for gasoline, and 1,3-butadiene, an intermediate in synthetic rubber manufacture [1–4]. Klebsiella oxytoca is known as one of the most promising 2,3-BD producers [1–10], and its whole genome sequences have been reported recently. The genome of K. oxytoca KCTC1686 consists of a chromosome of 5,974,109 bp with a 56.05% GC content, including 5,488 coding genes . The genome of K. oxytoca E718 is composed of a chromosome of 6,097,032 bp and two plasmids of 324,906 bp and 110,781 bp with a 55.5% GC content, including 5,909 coding genes . More recently, we isolated K. oxytoca KCTC12133BP from a cattle farm and sequenced its whole genome, but the sequence information, which has not yet been published, consists of a chromosome of 5,903,932 bp and a plasmid of 109,773 bp with a 55.4% GC content, including 5,793 coding genes.
The most important characteristics of K. oxytoca is to produce large amounts of C3/C4 diols, 1,3-propanediol (1,3-PD) and 2,3-BD, using various carbon sources [10, 13–20]. For example, a lactate deficient mutant of K. oxytoca coproduced 83.6 and 60.1 g/L of 1,3-PD and 2,3-BD, respectively, in a fed-batch fermentation utilizing mixed substrates of glycerol and sucrose . K. oxytoca also could produce more than 95 g/L of 2,3-BD from glucose with a yield of 0.478 g/g (95.6% of the theoretical maximum yield) and a productivity of 1.71 g/L/h in a batch fermentation, in which the agitation speed was switched from 300 to 200 rpm during the fermentation . The availability of oxygen to K. oxytoca significantly affects its physiology and 2,3-BD production [21–23]. Another striking aspect of K. oxytoca is to readily metabolize glycerol, an inevitable by-product of biodiesel production, into biomass and products of value [13, 24, 25].
The aforementioned advantages make K. oxytoca an attractive host for industrial applications. Furthermore, the U.S. National Institute of Health (NIH, Guidelines for Research Involving Recombinant DNA Molecules, 2002) has reported that K. oxytoca belongs to risk group 1 (RG 1), recognizing it as a GRAS (Generally Regarded As Safe) organism. However, in order to use this organism on an industrial scale, the strain should be further developed. Systems metabolic engineering allows the rational design of metabolic networks for the overproduction of target compounds and the creation of industrially useful microorganisms [26–31]. Here, and in silico genome-scale metabolic model of K. oxytoca, KoxGSC1457, was constructed based on genome information, databases, and experimental data. The KoxGSC1457 model is composed of 1,457 reactions and 1,099 metabolites (Additional file 1 and Additional file 2). The model was carefully examined by in silico analyses for genetic and environmental perturbations. The in silico analysis using the model predicted that the pyruvate pool is mostly important for 2,3-BD synthesis, and this was verified by fermentation of the ldhA gene knockout mutant. Also, the model showed that the availability of oxygen strongly affected the production of 2,3-BD by K. oxytoca.