Complete genome sequence of the 2,3-butanediol-producing Klebsiella pneumoniae strain KCTC 2242

Here we report the full genome sequence of Klebsiella pneumoniae KCTC 2242,consisting of a 5.26-Mb chromosome (57.6% GC%; 5,035 genes [4,923 encoding known proteins, 112 RNA genes]) and a 202-kb plasmid (50.2% GC%; 229 genes [229 encoding known proteins]).     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724

It is known that 2,3-butanediol is a potentially valuable chemical feedstock that can be produced from the sugars present in hemicellulose and celluose hydrolysates. Klebsiella oxytoca is able to ferment most pentoses, hexoses, and disaccharides. Butanediol appears to be a primary metabolite, excreted as a product of energy methabolism. The theoretical maximum yield of butanediol from monosaccharides is 0.50 g/g. This article describes the effects of pH, xylose concentration, and the oxygen transfer rate on the bioconversion of D-xylose to 2,3-butanediol. Product inhibition by butanediol is also examined. The most important variable affecting the kinetics of this system appears to be the oxygen transfer rate. A higher oxygen supply favors the formation of cell mass at the expense of butanediol. Decreasing the oxygen supply rate increases the butanediol yield, but decreases the overall conversion rate due to a lower cell concentration.     

Production of 2,3-butanediol by Klebsiella oxytoca

Summary High glucose concentrations result in high levels of 2,3-butanediol, improved yield and productivity, and a decrease in cell growth in batch cultures of Klebsiella oxytoca. A maximum of 84.2 g butanediol/l and a yield of 0.5 was obtained with an initial glucose concentration of 262.6g/l. Adding the substrate in two steps in a modified fed-batch operation resulted in 85.5 g butanediol/l, 6.4 g acetoin/l and 3.4 g ethanol/l with a net yield of 0.5. Increasing the cell density to 60g/l resulted in productivities as high as 3.22 g/l.h.     

Effects of aeration on 2,3-butanediol production from glucose by Klebsiella oxytoca

Higher cell concentrations and greater 2,3-butanediol production were observed in aerobic cultures of Klebsiella oxytoca than with anaerobic cultures. The concentration of butanediol inhibitors such as ethanol and lactic acid are partially suppressed by adequate aeration-agitation. Excessive aeration-agitation leads to the formation of acetoin and acetic acid at the expense of butanediol. With 94.3 g/l of glucose in the media, aerobic batch cultures produced 38.1 g/l butanediol with complete substrate use and a productivity of 0.39 g/l/h.     

2,3-Butanediol production from starch by engineered Klebsiella pneumoniae G31-A

2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.     

2,3-Butanediol production from glucose by Klebsiella pneumoniae in a cell recycle system

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae from glucose was studied in a cell recycle system with total recycle of biomass. Under these conditions productivity greater than batch or continuous system were obtained. However, when the cell concentration in the bioreactor built up to 40 g l⁻¹, the production of 2,3-butanediol started decreasing. The coefficient of mass transfer for oxygen decreased significantly and the viscosity increased rapidly after this cell concentration was reached. The increase in viscosity was partially due to production of polysaccharides. This appears at high cell concentration, due to severe oxygen limitation, when the organism may switch from 2,3-butanediol to polysaccharide production.     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.     

Enhanced activity of meso-secondary alcohol dehydrogenase from Klebsiella species by codon optimization

Meso-secondary alcohol dehydrogenases (meso-SADH) from Klebsiella oxytoca KCTC1686 and Klebsiella pneumoniae KCTC2242 were codon optimized and expressed in Escherichia coli W3110. The published gene data of K. pneumoniae NTUH-K2044 (NCBI accession number AP006725), K. pneumoniae 342 (NCBI accession number CP000964), and K. pneumoniae MGH 78578 (NCBI accession number CP000647), were compared with the meso-SADH sequences of each strain, respectively. Codon-optimized meso-SADH enzymes of K. oxytoca and K. pneumoniae showed approximately twofold to fivefold increased enzyme activities for acetoin reduction over native enzymes. The highest activities for each strain were obtained at 30–37 °C and pH 6–7 (yielding 203.1 U/mg of protein and 156.5 U/mg of protein, respectively). The increased enzyme activity of the codon-optimized enzymes indicated that these modified enzymes could convert acetoin into 2,3-butanediol with a high yield.     

The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis

A variety of microorganism species are able naturally to produce 2,3-butanediol (2,3-BDO), although only a few of them are suitable for consideration as having potential for mass production purposes. Klebsiella pneumoniae (K. pneumoniae) is one such strain which has been widely studied and used industrially to produce 2,3-BDO. In the central carbon metabolism of K. pneumoniae, the 2,3-BDO synthesis pathway is dominated by three essential enzymes, namely acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase, which are encoded by the budA, budB, and budC genes, respectively. The mechanisms of the three enzymes have been characterized with regard to their function and roles in 2,3-BDO synthesis and cell growth (Blomqvist et al. in J Bacteriol 175(5):1392–1404, 1993), while a few studies have focused on the cooperative mechanisms of the three enzymes and their mutual interactions. Therefore, the K. pneumoniae KCTC2242::ΔwabG wild-type strain was utilized to reconstruct seven new mutants by single, double, and triple overexpression of the three enzymes key to this study. Subsequently, continuous cultures were performed to obtain steady-state metabolism in the organisms and experimental data were analyzed by metabolic flux analysis (MFA) to determine the regulation mechanisms. The MFA results showed that the seven overexpressed mutants all exhibited enhanced 2,3-BDO production, and the strain overexpressing the budBA gene produced the highest yield. While the enzyme encoded by the budA gene produced branched-chain amino acids which were favorable for cell growth, the budB gene enzyme rapidly enhanced the conversion of acetolactate to acetoin in an oxygen-dependent manner, and the budC gene enzyme catalyzed the reversible conversion of acetoin to 2,3-BDO and regulated the intracellular NAD⁺/NADH balance.     

Effect of succinic acid on 2,3-butanediol production by Klebsiella oxytoca

Summary The effect of succinic acid on the growth of Klebsiella oxytoca and its production of 2,3-butanediol was studied. Increasing succinic acid from 0 g/L to 30 g/L increased the final butanediol concentration. The maximum butanediol productivity occurred at an initial succinic acid concentration of approximately 10 g/L.     

Production of 2,3‐butanediol by Klebsiella oxytoca from various sugars in microalgal hydrolysate

A new fermentation process using a mixed sugar medium is proposed in this study for 2,3‐butanediol (2,3‐BDO) production. The medium contained seven different monosugars known to be present in Nannochloropsis oceanica hydrolysate. The performance of each sugar when existing alone or together with glucose was evaluated. All the sugars except fucose were successfully metabolized for 2,3‐BDO production. A 2,3‐BDO yield of 0.31g/g was achieved with the mixed sugar medium, which was very close to that with the glucose‐only medium. However, the 2,3‐BDO productivity (0.28 g L^?1 h^?1) was found to be about 30% lower than that with glucose, implying, as expected, the existence of glucose repression on the uptake of other sugars. Strain development is in need to remove such negative effect of glucose for improved process efficiency. Fucose with the lowest uptake rate and no contribution to 2,3‐BDO production can be a high value‐added byproduct, once recovered and purified. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1669–1675, 2015     

产酸克雷伯氏杆菌发酵产2，3-丁二醇的培养基优化

采用不同设计方法相结合的策略对耐高糖产酸克雷伯氏杆菌（Klebsiella oxytoca）ME—UD-3-4发酵产2,3-丁二醇的培养基进行优化。首先在单因素实验的基础上采用Plackett—Burrnan设计法对影响ME—UD-3-4发酵产2,3-丁二醇的相关因素进行研究,筛选到3种有显著效应的因素（P〈0．05）：葡萄糖、玉米浆和MgSO4·7H2O。然后利用响应曲面法（Response Surface Methodology,RSM）对这3种因素的最佳水平范围进一步探讨;对得到的回归模型进行分析,得最佳条件（g／L）：葡萄糖220、玉米浆19和MgSO4·7H2O 0．4;在最佳条件下,发酵80h,2,3-丁二醇产量从原来的57．3 g／L提高到86．1 g／L,生产强度由0．72g／（L·h）提高到1．08g/（L·h）。     

Colonization of the botanical environment by Klebsiella isolates of pathogenic origin.

Growth, survival, and pathogenicity of Klebsiella growing in and on environmental foci were examined. Total coliforms present in raw wastes from pulp mills were in excess of 10(5)/ml, and 60 to 80% were Klebsiella. Fecal coliform counts ranged from 10(1) to 10(5)/ml. Klebsiella isolates from industrial effluents and a variety of human and bovine mastitis origins multiplied in pulp waste and commonly exceeded 10(6) cells per ml. Pathogenic isolates also multiplied in dilute aqueous extracts of sawdust to comparable levels. Klebsiella strains from vegetable surfaces and human infections grew rapidly on the surfaces of potatoes and lettuce and exceeded 10(3) organisms per g of surface peel and leaf after a 24h incubation at room temperature. After 7 weeks on potatoes stored at 5 degrees C, some 10 to 30% of the day 1 Klebsiella counts were recoverable. Three Klebsiella isolates of pathogenic origin were passed 45 times through sterile pulp effluent (270 generations), and mean lethal dose levels in mice were periodically monitored. In two instances, a significant decrease in virulence was noted after 15 to 26 passes (90 to 156 generations). The third culture, of bovine mastitis origin, retained its original mean lethal dose value. Botanical milieu provided suitable habitats for the multiplication and colonization of Klebsiella isolates of disease origins in the same manner as indigenous isolates. Aquatic environments polluted with botanical material served as potential reservoirs for perpetuating the growth and spread of opportunistic Klebsiella pathogens that may ultimately colonize animals, humans, and aquatic organisms.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Neural network predictive control by MIMS monitored 2,3-butanediol fermentation by Klebsiella oxytoca

With the aid of a membrane introduction mass spectrometer (MIMS), the major product 2,3-butanediol (2,3-BDL) as well as the other metabolites from the fermentation carried by Klebsiella oxytoca can be measured on-line simultaneously. A backpropagation neural network (BPN) being recognized with superior mapping ability was applied to this control study. This neural network adaptive control differs from those conventional controls for fermentation systems in which the measurements of cell mass and glucose are not included in the network model. It is only the measured product concentrations from the MIMS that are involved. Oxygen composition was chosen to be the control variable for this fermentation system. Oxygen composition was directly correlated to the measured product concentrations in the controller model. A two-dimensional (number of input nodes by number of data sets) moving window for on-line, dynamic learning of this fermentation system was applied. The input nodes of the network were also properly selected. Number of the training data sets for obtaining better control results was also determined empirically. Two control structures for this 2,3-BDL fermentation are discussed and compared in this work. The effect from adding time delay element to the network controller was also investigated.     

Cloning, expression and characterization of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae

The budC gene encoding the meso-2,3-BDH from Klebsiella pneumoniae XJ-Li was expressed in E. coli BL21 (DE3) pLys. Hypothetical amino acid sequence alignments revealed that the enzyme belongs to the short chain dehydrogenase/reductase family. After purification and refolding, the recombinant enzyme had activities of 218 U/mg for reduction of acetoin and 66 U/mg for oxidation of meso-2,3-butanediol. Highest activities were at pH 8.0 and 9.0 respectively. These are higher than other meso-2,3-butanediol dehydrogenases from K. pneumoniae. The low K (m) value (0.65 mM) for acetoin indicated that the enzyme can easily reduce acetoin to meso-2,3-butanediol. There were no significant activities towards 2R,3R-2,3-butanediol, 1,4-butanediol and 2S,3S-2,3-butanediol, suggesting that the enzyme has a high stereospecificity for the meso-dihydric alcohol.