An Effective and Simplified Fed-Batch Strategy for Improved 2,3-Butanediol Production by Klebsiella oxytoca

Nie, Zhi-Kui; Ji, Xiao‐Jun; Huang, He; Du, Jun; Li, Zhiyong; Liang, Qi; Zhang, Qi; Ouyang, Pingkai

doi:10.1007/s12010-010-9098-6

Cited by 26 publications

(13 citation statements)

References 21 publications

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“…The model predicted that the wild-type strain of K. oxytoca produces 2,3-BD along with large amounts of byproducts. This prediction was well supported by the previous studies that the wild-type strains yielded not only 2,3-BD but also lactic acid, formic acid, and ethanol as fermentation end-products (Figure 4C) [9,10,19]. Thus, genetic manipulations like gene knockout have been attempted to prevent the formation of byproducts in order to enhance 2,3-BD production [9,13].…”

Section: Resultssupporting

confidence: 76%

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

et al. 2013

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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.

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Section: Resultssupporting

confidence: 76%

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

et al. 2013

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“…The seeds were cultivated at 37°C and 200 rpm on a rotary shaker for 24 h [26]. Seed culture (5%, v/v) was then inoculated into the fermentation medium and batch and fed-batch fermentation were carried out in a 3-L stirred fermenter (BioFlo 100; New Brunswick Scientific Co., NJ, USA) with a an initial broth volume of 2 L. The fermentation medium for K. pneumoniae was composed of (g/L): glucose, 100; K 2 HPO 4 , 13.7; KH 2 PO 4 , 2.0; (NH 4 ) 2 HPO 4 , 3.3; (NH 4 ) 2 SO 4 , 6.6; MgSO 4 ·7H 2 O, 0.25; FeSO 4 ·7H 2 O, 0.05; ZnSO 4 ·7H 2 O, 0.001; MnSO 4 ·H 2 O, 0.001; CaCl 2 ·2H 2 O, 0.01; EDTA, 0.05 [8].…”

Section: Methodsmentioning

confidence: 99%

Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

Xia

et al. 2013

Biotechnol Biofuels

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BackgroundAcetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria.ResultsThe present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold) and NADH/NAD+ 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 Klebsiella species was obtained.ConclusionsThe present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. 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+-dependent microbial metabolite.

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“…Efficient fed-batch strategies could enhance the concentrations of the aim products52526. To obtain a higher concentration of (2 S ,3 S )-2,3-BD, fed-batch bioconversion was carried out using E. coli BL21 (DE3) (pETDuet- bdhfdh ), with an initial DA concentration of 20.0 g/L and adding 5.0 g/L of DA at 2, 4, and 6 h. Formate was used as the driving force for NADH regeneration and added at 0, 4 and 6 h. As shown in Figure 4a, after 14 h of reaction, 31.7 g/L of (2 S ,3 S )-2,3-BD (purity > 99.0%) was produced from 35.3 g/L of DA with a productivity of 2.3 g/(L · h).…”

Section: Resultsmentioning

confidence: 99%

Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl

Wang

et al. 2013

Sci Rep

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(2S,3S)-2,3-Butanediol ((2S,3S)-2,3-BD) is a potentially valuable liquid fuel and an excellent building block in asymmetric synthesis. In this study, cofactor engineering was applied to improve the efficiency of (2S,3S)-2,3-BD production and simplify the product purification. Two NADH regeneration enzymes, glucose dehydrogenase and formate dehydrogenase (FDH), were introduced into Escherichia coli with 2,3-BD dehydrogenase, respectively. Introduction of FDH resulted in higher (2S,3S)-2,3-BD concentration, productivity and yield from diacetyl, and large increase in the intracellular NADH concentration. In fed-batch bioconversion, the final titer, productivity and yield of (2S,3S)-2,3-BD on diacetyl reached 31.7 g/L, 2.3 g/(L·h) and 89.8%, the highest level of (2S,3S)-2,3-BD production thus far. Moreover, cosubstrate formate was almost totally converted to carbon dioxide and no organic acids were produced. The biocatalytic process presented should be a promising route for biotechnological production of NADH-dependent microbial metabolites.

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An Effective and Simplified Fed-Batch Strategy for Improved 2,3-Butanediol Production by Klebsiella oxytoca

Cited by 26 publications

References 21 publications

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

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

Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl

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