Biological Control Efficacy and Action Mechanism of Klebsiella pneumoniae JCK-2201 Producing Meso-2,3-Butanediol Against Tomato Bacterial Wilt.

Bacterial wilt caused by Ralstonia solanacearum is a fatal disease that affects the production of tomatoes and many other crops worldwide. As an effective strategy to manage bacterial wilt, biological control agents using plant growth-promoting rhizobacteria (PGPR) are being developed. In this study, we screened 2,3-butanediol (BDO)-producing PGPR to control tomato bacterial wilt and investigated the action mechanism of the disease control agent. Of the 943 strains isolated from soil, Klebsiella pneumoniae strain JCK-2201 produced the highest concentration of 2,3-BDO. The culture broth of K. pneumoniae JCK-2201 did not show any direct activity on R. solanacearum in vitro, but a 100-fold dilution effectively controlled tomato bacterial wilt with a control value of 77% in vivo. Fermentation utilizing K. pneumoniae JCK-2201 was optimized to produce 48 g/L of meso-2,3-BDO, which is 50% of the sucrose conversion efficiency. In addition, the control efficacy and mechanism of meso-2,3-BDO produced by JCK-2201 in tomato bacterial wilt were determined by comparative analysis with Bacillus licheniformis DSM13 producing meso-2,3-BDO and B. licheniformis DSM13 ΔalsS that did not produce 2,3-BDO, as the step of converting pyruvate to α-acetolactate was omitted. Tomato seedlings treated with the K. pneumoniae JCK-2201 (500-fold dilution) and B. licheniformis DSM13 (100-fold dilution) culture broth produced meso-2,3-BDO that significantly reduced R. solanacearum-induced disease severity with control values of 55% and 63%, respectively. The formulated meso-2,3-BDO 9% soluble concentrate (SL; 1,000-fold dilution) showed 87% control against tomato bacterial wilt in the field condition. Klebsiella pneumoniae JCK-2201 and B. licheniformis DSM13 treatment induced the expression of plant defense marker genes, such as LePR1, LePR2, LePR5, LePR3, and PI-II, in the salicylic acid and jasmonic acid signaling pathways at 4 days after inoculation. These results show that 2,3-BDO-producing bacteria and 2,3-BDO are potential biological control agents that act through induction of resistance for controlling tomato bacterial wilt.

Enhancement of Disease Control Efficacy of Chemical Fungicides Combined with Plant Resistance Inducer 2,3-Butanediol against Turfgrass Fungal Diseases.

Turfgrass, the most widely grown ornamental crop, is severely affected by fungal pathogens including Sclerotinia homoeocarpa, Rhizoctonia solani, and Magnaporthe poae. At present, turfgrass fungal disease management predominantly relies on synthetic fungicide treatments. However, the extensive application of fungicides to the soil increases residual detection frequency, raising concerns for the environment and human health. The bacterial volatile compound, 2,3-butanediol (BDO), was found to induce plant resistance. In this study, we evaluated the disease control efficacy of a combination of stereoisomers of 2,3-BDO and commercial fungicides against turfgrass fungal diseases in both growth room and fields. In the growth room experiment, the combination of 0.9% 2R,3R-BDO (levo) soluble liquid (SL) formulation and 9% 2R,3S-BDO (meso) SL with half concentration of fungicides significantly increased the disease control efficacy against dollar spot and summer patch disease when compared to the half concentration of fungicide alone. In field experiments, the disease control efficiency of levo 0.9% and meso 9% SL, in combination with a fungicide, was confirmed against dollar spot and large patch disease. Additionally, the induction of defense-related genes involved in the salicylic acid and jasmonic acid/ethylene signaling pathways and reactive oxygen species detoxification-related genes under Clarireedia sp. infection was confirmed with levo 0.9% and meso 9% SL treatment in creeping bentgrass. Our findings suggest that 2,3-BDO isomer formulations can be combined with chemical fungicides as a new integrated tool to control Clarireedia sp. infection in turfgrass, thereby reducing the use of chemical fungicides.

Exogenous Bio-Based 2,3-Butanediols Enhanced Abiotic Stress Tolerance of Tomato and Turfgrass under Drought or Chilling Stress.

Among abiotic stresses in plants, drought and chilling stresses reduce the supply of moisture to plant tissues, inhibit photosynthesis, and severely reduce plant growth and yield. Thus, the application of water stress-tolerant agents can be a useful strategy to maintain plant growth under abiotic stresses. This study assessed the effect of exogenous bio-based 2,3-butanediol (BDO) application on drought and chilling response in tomato and turfgrass, and expression levels of several plant signaling pathway-related gene transcripts. Bio-based 2,3-BDOs were formulated to levo-2,3-BDO 0.9% soluble concentrate (levo 0.9% SL) and meso-2,3-BDO 9% SL (meso 9% SL). Under drought and chilling stress conditions, the application of levo 0.9% SL in creeping bentgrass and meso 9% SL in tomato plants significantly reduced the deleterious effects of abiotic stresses. Interestingly, pretreatment with levo-2,3-BDO in creeping bentgrass and meso-2,3-BDO in tomato plants enhanced JA and SA signaling pathway-related gene transcript expression levels in different ways. In addition, all tomato plants treated with acibenzolar-S-methyl (as a positive control) withered completely under chilling stress, whereas 2,3-BDO-treated tomato plants exhibited excellent cold tolerance. According to our findings, bio-based 2,3-BDO isomers as sustainable water stress-tolerant agents, levo- and meso-2,3-BDOs, could enhance tolerance to drought and/or chilling stresses in various plants through somewhat different molecular activities without any side effects.

CRISPR-Cas9 mediated engineering of Bacillus licheniformis for industrial production of (2R,3S)-butanediol.

Bacillus lichenformis is an industrially promising generally recognized as safe (GRAS) strain that can be used for the production of a valuable chemical, 2,3-butanediol (BDO). Conventional gene deletion vectors and/or methods are time-consuming and have poor efficiency. Therefore, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 mediated homologous recombination was used to engineer a newly isolated and UV-mutagenized B. licheniformis 4071-15 strain. With the help of a CRISPR-Cas9 system, this one-step process could be used for the deletion of ldh gene within 4 days with high-efficiency exceeding 60%. In addition, the sequential deletion of target genes for engineering studies was evaluated, and it was confirmed that a triple mutant strain (ldh, dgp, and acoR) could be obtained by repeated one-step cycles. Furthermore, a practical metabolic engineering study was carried out using a CRISPR-Cas9 system for the stereospecific production of (2R,3S)-BDO. The predicted (2R,3R)-butanediol dehydrogenase encoded by the gdh gene was selected as a target for the production of (2R,3S)-BDO, and the mutant was successfully obtained. The results show that the stereospecific production of (2R,3S)-BDO was possible with the gdh deletion mutant, while the 4071-15 host strain still generated 26% of (2R,3R)-BDO. It was also shown that the 4071-15 Δgdh mutant could produce 115 g/L of (2R,3S)-BDO in 64 hr by two-stage fed-batch fermentation. This study has shown the efficient development of a (2R,3S)-BDO producing B. licheniformis strain based on CRISPR-Cas9 and fermentation technologies.

Engineering a newly isolated Bacillus licheniformis strain for the production of (2R,3R)-butanediol.

Several microorganisms can produce 2,3-butanediol (BDO), an industrially promising chemical. In this study, a Bacillus licheniformis named as 4071, was isolated from soil sample. It is a GRAS (generally recognized as safe) strain and could over-produce 2,3-BDO. Due to its mucoid forming characteristics, UV-random mutagenesis was carried out to obtain a mucoid-free strain, 4071-15. As a result, capabilities of 4071-15 strain in terms of transformation efficiency of bacillus plasmids (pC194, pUB110, and pUCB129) and fermentation performance were highly upgraded compared to those of the parent strain. In particular, 4071-15 strain could produce 123 g/L of 2,3-BDO in a fed-batch fermentation in which the ratio of (2R,3S)- to (2R,3R)-form isomers was 1:1. To increase the selectivity of (2R,3R)-BDO, budC gene was deleted by using temperature-sensitive gene deletion process via homologous recombination. The 4071-15 △budC mutant strain dramatically increased selectivity of (2R,3R)-BDO to 91% [96.3 g/L of (2R,3R)-BDO and 9.33 g/L of (2R,3S)-BDO], which was 43% higher than that obtained by the parent strain. This study has shown the potential of an isolate for 2,3-BDO production, and that the ratio of 2,3-BDO can be controlled by genetic engineering depending on its industrial usage.

Microbial production of 2,3-butanediol for industrial applications.

2,3-Butanediol (2,3-BD) has great potential for diverse industries, including chemical, cosmetics, agriculture, and pharmaceutical areas. However, its industrial production and usage are limited by the fairly high cost of its petro-based production. Several bio-based 2,3-BD production processes have been developed and their economic advantages over petro-based production process have been reported. In particular, many 2,3-BD-producing microorganisms including bacteria and yeast have been isolated and metabolically engineered for efficient production of 2,3-BD. In addition, several fermentation processes have been tested using feedstocks such as starch, sugar, glycerol, and even lignocellulose as raw materials. Since separation and purification of 2,3-BD from fermentation broth account for the majority of its production cost, cost-effective processes have been simultaneously developed. The construction of a demonstration plant that can annually produce around 300 tons of 2,3-BD is scheduled to be mechanically completed in Korea in 2019. In this paper, core technologies for bio-based 2,3-BD production are reviewed and their potentials for use in the commercial sector are discussed.

Isolation and Evaluation of Bacillus Strains for Industrial Production of 2,3-Butanediol.

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis, which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

Metabolic engineering of Klebsiella pneumoniae based on in silico analysis and its pilot-scale application for 1,3-propanediol and 2,3-butanediol co-production.

Klebsiella pneumoniae naturally produces relatively large amounts of 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD) along with various byproducts using glycerol as a carbon source. The ldhA and mdh genes in K. pneumoniae were deleted based on its in silico gene knockout simulation with the criteria of maximizing 1,3-PD and 2,3-BD production and minimizing byproducts formation and cell growth retardation. In addition, the agitation speed, which is known to strongly affect 1,3-PD and 2,3-BD production in Klebsiella strains, was optimized. The K. pneumoniae ΔldhA Δmdh strain produced 125 g/L of diols (1,3-PD and 2,3-BD) with a productivity of 2.0 g/L/h in the lab-scale (5-L bioreactor) fed-batch fermentation using high-quality guaranteed reagent grade glycerol. To evaluate the industrial capacity of the constructed K. pneumoniae ΔldhA Δmdh strain, a pilot-scale (5000-L bioreactor) fed-batch fermentation was carried out using crude glycerol obtained from the industrial biodiesel plant. The pilot-scale fed-batch fermentation of the K. pneumoniae ΔldhA Δmdh strain produced 114 g/L of diols (70 g/L of 1,3-PD and 44 g/L of 2,3-BD), with a yield of 0.60 g diols per gram glycerol and a productivity of 2.2 g/L/h of diols, which should be suitable for the industrial co-production of 1,3-PD and 2,3-BD.

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens.

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600 of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.

Effects of mutation of 2,3-butanediol formation pathway on glycerol metabolism and 1,3-propanediol production by Klebsiella pneumoniae J2B.

The current study investigates the impact of mutation of 2,3-butanediol (BDO) formation pathway on glycerol metabolism and 1,3-propanediol (PDO) production by lactate dehydrogenase deficient mutant of Klebsiella pneumoniae J2B. To this end, BDO pathway genes, budA, budB, budC and budO (whole-bud operon), were deleted from K. pneumoniae J2B ΔldhA and the mutants were studied for glycerol metabolism and alcohols (PDO, BDO) production. ΔbudO-mutant-only could completely abolish BDO production, but with reductions in cell growth and PDO production. By modifying the culture medium, the ΔbudO mutant could recover its performance on the flask scale. However, in bioreactor experiments, the ΔbudO mutant accumulated a significant amount of pyruvate (>73mM) in the late phase and PDO production stopped concomitantly. Glycolytic intermediates of glycerol, especially glyceraldehyde-3-phosphate (G3P) was highly inhibitory to glycerol dehydratase (GDHt); its accumulation, followed by pyruvate accumulation, was assumed to be responsible for the ΔbudO mutant's low PDO production.

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.

Metabolic engineering of Klebsiella pneumoniae and in silico investigation for enhanced 2,3-butanediol production.

OBJECTIVES: To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated. RESULTS: Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation. CONCLUSIONS: K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

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

Microbial fermentation produces a racemic mixture of 2,3-butanediol ((R,R)-BD, (S,S)-BD, and meso-BD), and the compositions and physiochemical properties vary from microorganism to microorganism. Although the meso form is much more difficult to transport and store because of its higher freezing point than those of the optically active forms, most microorganisms capable of producing 2,3-BD mainly yield meso-2,3-BD. Thus, we developed a metabolically engineered (R,R)-2,3-BD overproducing strain using a Klebsiella oxytoca ΔldhA ΔpflB strain, which shows an outstanding 2,3-BD production performance with more than 90 % of the meso form. A budC gene encoding 2,3-BD dehydrogenase in the K. oxytoca ΔldhA ΔpflB strain was replaced with an exogenous gene encoding (R,R)-2,3-BD dehydrogenase from Paenibacillus polymyxa (K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH strain), and then its expression level was further amplified with using a pBBR1MCS plasmid. The fed-batch fermentation of the K. oxytoca ΔldhA ΔpflB ΔbudC::PBDH (pBBR-PBDH) strain with intermittent glucose feeding allowed the production of 106.7 g/L of (R,R)-2,3-BD [meso-2,3-BD, 9.3 g/L], with a yield of 0.40 g/g and a productivity of 3.1 g/L/h, which should be useful for the industrial application of 2,3-BD.

2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation.

This study presents a new and effective downstream process to recover 2,3-butanediol (2,3-BD) from fermentation broth which is produced by a recombinant Klebsiella pneumoniae strain. The ldhA-deficient K. pneumoniae strain yielded about 90 g/L of 2,3-BD, along with a number of by-products, such as organic acids and alcohols, in a 65 h fed-batch fermentation. The pH-adjusted cell-free fermentation broth was firstly concentrated until 2,3-BD reached around 500 g/L by vacuum evaporation at 50°C and 50 mbar vacuum pressure. The concentrated solution was further treated using light alcohols, including methanol, ethanol, and isopropanol, for the precipitation of organic acids and inorganic salts. Isopropanol showed the highest removal efficiency, in which 92.5% and 99.8% of organic acids and inorganic salts were precipitated, respectively. At a final step, a vacuum distillation process enabled the recovery of 76.2% of the treated 2,3-BD, with 96.1% purity, indicating that fermentatively produced 2,3-BD is effectively recovered by a simple alcohol precipitation and vacuum distillation.

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production.

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.

Metabolic engineering of a novel Klebsiella oxytoca strain for enhanced 2,3-butanediol production.

Fermentative 2,3-butanediol (2,3-BD) production has been receiving increasing interest for its potential as a platform chemical intended for the production of synthetic rubbers, plastics, and solvents. In this study, Klebsiella oxytoca GSC 12206, a 2,3-BD native hyper-producing and nonpathogenic bacterium, was isolated from a cattle farm. Since this isolate produced a significant amount of lactic acid along with 2,3-BD, its mutant deficient in lactic acid formation was constructed by disrupting the ldhA gene which encodes lactate dehydrogenase. The ldhA gene was deleted precisely by using the pKGS plasmid. When compared to the wild-type strain, the mutant deleted with the ldhA gene in glucose fermentation resulted in an increase of 54%, 13%, 60%, and 78% of 2,3-BD titer, productivity, yield, and selectivity, respectively. A fed-batch fermentation by this mutant with intermittent glucose feeding produced 115 g/L of 2,3-BD with an yield and productivity of 0.41 g 2,3-BD per g glucose and 2.27 g/L h, respectively, indicating the usefulness for the industrial production of 2,3-BD.

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

BACKGROUND: Klebsiella 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. RESULTS: The 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. CONCLUSIONS: The 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.

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei.

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.

Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum.

Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1(D485G) gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD(**)) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer. IMPORTANCE Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineered Clostridium acetobutylicum strain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

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.