Genome-scale reconstruction and in silico analysis of Klebsiella oxytoca for 2,3-butanediol production

Abstract

BackgroundKlebsiella oxytoca, a Gram-negative, rod-shaped, and facultative anaerobic bacterium, is one of the most promising 2,3-butanediol (2,3-BD) producers. In order to improve the metabolic performance of K. oxytoca as an efficient biofactory, it is necessary to assess its metabolic characteristics with a system-wide scope, and to optimize the metabolic pathways at a systems level. Provision of the complete genome sequence of K. oxytoca enabled the construction of genome-scale metabolic model of K. oxytoca and its in silico analyses.ResultsThe genome-scale metabolic model of K. oxytoca was constructed using the annotated genome with biochemical and physiological information. The stoichiometric model, KoxGSC1457, is composed of 1,457 reactions and 1,099 metabolites. The model was further refined by applying biomass composition equations and comparing in silico results with experimental data based on constraints-based flux analyses. Then, the model was applied to in silico analyses to understand the properties of K. oxytoca and also to improve its capabilities for 2,3-BD production according to genetic and environmental perturbations. Firstly, in silico analysis, which tested the effect of augmenting the metabolic flux pool of 2,3-BD precursors, elucidated that increasing the pyruvate pool is primarily important for 2,3-BD synthesis. Secondly, we performed in silico single gene knockout simulation for 2,3-BD overproduction, and investigated the changes of the in silico flux solution space of a ldhA gene knockout mutant in comparison with that of the wild-type strain. Finally, the KoxGSC1457 model was used to optimize the oxygen levels during fermentation for 2,3-BD production.ConclusionsThe genome-scale metabolic model, KoxGSC1457, constructed in this study successfully investigated metabolic characteristics of K. oxytoca at systems level. The KoxGSC1457 model could be employed as an useful tool to analyze its metabolic capabilities, to predict its physiological responses according to environmental and genetic perturbations, and to design metabolic engineering strategies to improve its metabolic performance.

Effect of fluid dynamic conditions on 2,3‐butanediol production by Raoultella terrigena in SBTR: oxygen transfer and uptake rates

“…These observations suggest four culture behaviors with respect to the fluid dynamic conditions: an initial fermentation zone (100–400 rpm), where an increase in OTR favors BD and acetoin production; an intermediate region, whose border is highly sensitive to OTR (since at 550 rpm the metabolic change is evident in terms of mixed acid route products distribution); a third zone (700–1300 rpm), where the culture maximizes microbial growth, and carbon dioxide is the main by‐product; and a last agitation range (1600–2000 rpm), where the increase in stirring speed continues a decrease in microbial growth rate, in OTR ma x and in CO 2 production. According to these observations it can be suggested that R. terrigena is really sensitive to changes in dissolved oxygen availability, as occurs with other bacterial strains able to produce 2,3‐BD …”

“…For example, Park et al observed that if the stirrer speed was higher than 350 rpm, the production of 2,3‐BD decreased dramatically while the biomass growth increased in cultures with Klebsiella oxytoca using glucose as sole carbon source. In this work, the biomass profiles overlap from 550 to 750 rpm. Ji et al have proposed a two‐stage agitation speed strategy for enhancing 2,3‐BD final concentration with engineered strains of K. oxytoca , because the production declined from 300 rpm, in favor of biomass growth …”

Microbial contamination in methanol biofilters inoculated with a pure strain of Pichia pastoris: A potential limitation for waste revalorization

“…Studies have found that this microorganism is involved in the treatment of isoprene, trimethylamine, and nitrogen oxides . Moreover, metabolic reconstruction from genomic analysis has indicated that K. oxytoca can also degrade and use formaldehyde as carbon source or even use methanol if H 2 O 2 is present . Other found microorganisms were Stenotrophomonas sp., Achromobacter sp., Actinomyces sp., Alcaligenes faecalis , Burkholderia cepacia , Burkholderia sp., Enterobacter sp., Lactococcus lactis , Mesorhizobium sp., Methylopila henanense , Pseudomonas sp., Serratia sp., and Stenotrophomonas sp., which might be involved in the consumption of formaldehyde and formate (Table ) .…”

“…Moreover, metabolic reconstruction from genomic analysis has indicated that K. oxytoca can also degrade and use formaldehyde as carbon source or even use methanol if H 2 O 2 is present . Other found microorganisms were Stenotrophomonas sp., Achromobacter sp., Actinomyces sp., Alcaligenes faecalis , Burkholderia cepacia , Burkholderia sp., Enterobacter sp., Lactococcus lactis , Mesorhizobium sp., Methylopila henanense , Pseudomonas sp., Serratia sp., and Stenotrophomonas sp., which might be involved in the consumption of formaldehyde and formate (Table ) . Altogether, the cloning and sequencing results clearly suggested that most of the bacteria, most of the microorganisms, that colonized the biofilters were potentially associated to the consumption of formaldehyde.…”

Enhanced production of (R,R)-2,3-butanediol by metabolically engineered Klebsiella oxytoca

“…In order to enhance the production of 2,3-BD but reduce the formation of byproducts in K. oxytoca, many studies seeking strain improvement and fermentation optimization have been executed [10,11,13,16,23]. Recently, we developed a metabolically engineered K. oxytoca strain, in which ldhA and pflB genes were deleted (K. oxytoca ΔldhA ΔpflB), based on its in silico simulation using a genome-scale metabolic model of K. oxytoca, KoxGSC1457 [22,23]. In the K. oxytoca ΔldhA ΔpflB strain, the overall 2,3-BD yield on glucose increased remarkably (0.45 g/g, 90 % of theoretical maximum yield).…”