Reduction of acetoin to 2,3-butanediol in Klebsiella pneumoniae: a new model

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724, formerly known as Aerobacter aerogenes) carried out in our laboratory yields 2,3-butanediol as the major product. Experimental data obtained in this work cannot be explained by the model presently in the literature for the formation of 2,3-butanediol isomers from acetoin isomers. A new model is proposed with the existence of two acetoin reductases and an acetoin racemase. The two reductases were separated and their stereospecificity determined. Extension of the model of other microorganisms is discussed.     

Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.     

Optimized production of 2,3-butanediol by a lactate dehydrogenase-deficient mutant of Klebsiella pneumoniae

To obtain high-yield production of 2,3-butanediol (2,3-BD) from glucose, we optimized the culture conditions for a lactate dehydrogenase-deficient mutant (ΔldhA) of Klebsiella pneumoniae using response surface methodology. 2,3-BD production was successfully improved by optimizing pH (5.6), aeration (3.50 vvm) and concentration of corn steep liquor (45.0 mL/L) as a nitrogen source, resulting in a maximum level of 2,3-BD production of 148.8 g/L and productivity of 2.48 g/L/h. 2,3-BD was also obtained with high concentration (76.24 g/L) and productivity (2.31 g/L/h) from the K. pneumoniae mutant strain using sugarcane molasses as a carbon source.     

Production of 2,3-butanediol by Klebsiella pneumoniae grown on acid hydrolyzed wood hemicellulose

Summary Previously steam explosion had been used to enhance the enzymatic hydrolysis of lignocellulosic substrates to glucose. The conditions for pretreating aspen wood chips were optimized so that highest amounts of undegraded hemicellulose could be obtained after washing the steam exploded chips. The hemicellulose rich water soluble fractions showing highest pentosan yields were then acid hydrolysed to their composite sugars. Approximately 65–75% of the total reducing sugars detected in the wood hydrolysates were in the form of monosaccharides with D-xylose being the major component. Klebsiella pneumoniae was grown in media containing these wood hydrolysates as the substrate and 2,3-butanediol yields of 0.4–0.5 g per g of monosaccharide utilised were obtained.     

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.     

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.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Deletion of the budBAC operon in Klebsiella pneumoniae to understand the physiological role of 2,3-butanediol biosynthesis

Klebsiella pneumoniae is known to produce 2,3-butanediol (2,3-BDO), a valuable chemical. In K. pneumoniae, the 2,3-BDO operon (budBAC) is involved in the production of 2,3-BDO. To observe the physiological role of the 2,3-BDO operon in a mixed acid fermentation, we constructed a budBAC-deleted strain (SGSB109). The production of extracellular metabolites, CO₂ emission, carbon distribution, and NADH/NAD⁺ balance of SGSB109 were compared with the parent strain (SGSB100). When comparing the carbon distribution at 15 hr, four significant differences were observed: in 2,3-BDO biosynthesis, lactate and acetate production (lactate and acetate production increased 2.3-fold and 4.1-fold in SGSB109 compared to SGSB100), CO₂ emission (higher in SGSB100), and carbon substrate uptake (higher in SGSB100). Previous studies on the inactivation of the 2,3-BDO operon were focused on the increase of 1,3-propanediol production. Few studies have been done observing the role of 2,3-BDO biosynthesis. This study provides a prime insight into the role of 2,3-BDO biosynthesis of K. pneumoniae.     

Analysis of 2,3-butanediol production with Klebsiella pneumoniae using sugar mixture under different oxygen supply conditions

以筛选的肺炎克雷伯氏菌(Klebsiella pneumoniae UV-86)为对象,考察供氧条件分别对菌体生长、葡萄糖和木糖双底物利用和产物合成的影响.研究发现生物量随氧供应量增加而增加.不同供氧条件对菌体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强.微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1.5 vvm下达到最高为30.1 g/L,是好氧时的2.5倍,最大体积产率为0.485 g/(L·h).不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强.因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations.

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

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.     

Production of 2,3-butanediol from wood hydrolysate byKlebsiella pneumoniae

2,3-Butanediol was produced at 12g/l (27% w/w yield) from the reducing sugars in wood hydrolysate byKlebsiella pneumoniae NRRL B-199. Ethanol was present at 4 g/l and acetoin at 0.4 g/l. Optimum conditions for a 48 h fermentation were pH 6.0, 30°C and 50 g reducing sugars/l. Adding 1% (w/v) malt extract to the medium-enhanced butanedlol production to 13.3 g/l (yield 29%) without changing ethanol production.

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Résumé On a produit 12 g/l de 2,3-butanediol (27% de rendement en poids) à partir des sucres réducteurs dans un hydrolysat de bois au moyen deKlebsiella pneumoniae NRRL B-199. L'éthanol était présent à raison de 4 g/l et l'acétoïne de 0.4 g/l. Les conditions optimum pour une fermentation de 48 h étainent les suivantes: pH 6.0, 30°C, et 50 g de sucres réducteurs par litre. L'ajout de 1% (p/v) d'extrait de malt au millieu accroît la production de butanediol jusqu'à 13.3 g/l (29% de rendement) sans changer la production d'éthanol.

     

Study of 2,3-butanediol formation by serratiamarcescens

Summary Serratia marcescens can be used for the fermentation of sugar for the formation of 2,3-butanediol. Presence of 1% calcium carbonate increases the formation of the diol and 0.292% of phosphate gives the maximum percentage yield of the diol. The optimumph for the formation of the diol has been found to be 7.     

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.     

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.     

Identification and characterization of a short-chain acyl dehydrogenase from Klebsiella pneumoniae and its application for high-level production of l-2,3-butanediol

Klebsiella pneumoniae synthesize large amounts of l-2,3-butanediol (l-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an l-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward l-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of l-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.     

Optimization of 2,3-butanediol production by Klebsiella oxytoca through oxygen transfer rate control

Production of 2,3-butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy-coupled growth, during which cell growth and oxygen supply are coupled; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3-butanediol productivity occurs during the energy-coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy-coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasing the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3-butanediol productivity of 18%. (c) 1993 John Wiley & Sons, Inc.     

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.     

Complete genome sequence of Klebsiella oxytoca KCTC 1686, used in production of 2,3-butanediol

Here we report the full genome sequence of Klebsiella oxytoca KCTC 1686, which is used in production of 2,3-butanediol. The KCTC 1686 strain contains 5,974,109 bp with G+C content of 56.05 mol% and contains 5,488 protein-coding genes and 110 structural RNAs.