Burkholderia jirisanensis sp. nov., isolated from forest soil.

A Gram-negative, catalase-positive, mesophilic, obligately aerobic bacterium designated JRM2-1T was isolated from forest soil of Jirisan Mountain, Republic of Korea, and its taxonomic position was investigated based on a polyphasic taxonomic approach. Cells of strain JRM2-1T grew optimally at pH 5.0-7.0 and at 25 °C. Strain JRM2-1T was susceptible to chloramphenicol, gentamicin, kanamycin, nalidixic acid, rifampicin, streptomycin and tetracycline. On the basis of 16S rRNA gene sequence similarity, the closest neighbour of strain JRM2-1T was Burkholderia rhizosphaerae WR43T (98.1 %). On the basis of our phylogenetic analysis, strain JRM2-1T is clearly distinguished from related species of the genus Burkholderia and is clustered with plant-associated members of the genus. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The polar lipid profile of strain JRM2-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, several unidentified aminolipids and an unidentified aminophospholipid. The isoprenoid quinone of strain JRM2-1T was Q-8 and the DNA G+C content was 63.7 mol%. On the basis of our polyphasic taxonomic investigation, strain JRM2-1T is considered to represent a novel species in the genus Burkholderia, for which the name Burkholderia jirisanensis sp. nov. is proposed. The type strain is JRM2-1T ( = AIM 0373T = KCTC 42072T = JCM 19985T).

Butyric acid production from red algae by a newly isolated Clostridium sp. S1.

OBJECTIVE: To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required. RESULTS: A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5-2 g levulinic acid/l and recovered from HMF inhibition at 0.6-2.5 g/l, resulting in 85-92% butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l. CONCLUSION: Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

Biosynthesis of pinene from glucose using metabolically-engineered Corynebacterium glutamicum.

Pinene is a monoterpenes (C10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. Herein, we have metabolically-engineered Corynebacterium glutamicum, to produce pinene and studied its toxicity in C. glutamicum. Geranyl diphosphate synthases (GPPS) and pinene synthases (PS), obtained from Pinus taeda and Abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and isopentenyl diphosphate isomerase (Idi) from C. glutamicum using CoryneBrick vector. Most strains expressing PS-GPPSs produced detectable amounts of pinene, but co-expression of DXS and IDI with PS (P. taeda) and GPPS (A. grandis) resulted in 27 µg ± 7 α-pinene g(-1) cell dry weight, which is the first report in C. glutamicum. Further engineering of PS and GPPS in the C. glutamicum strain may increase pinene production.

Conversion of levulinic acid to 2-butanone by acetoacetate decarboxylase from Clostridium acetobutylicum.

In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4-90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a ß-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation.

Association between precocious puberty and some endocrine disruptors in human plasma.

Endocrine disruptors that mimic natural hormones and inhibit the action of hormones have recently attracted attention as one of the main cause of precocious puberty. In this study, the levels of 7 EDCs and 3 isoflavones that exhibit estrogen-like actions were measured in the plasma of precocious puberty patients and compared to control subjects to determine if there is an association between the onset of precocious puberty and the levels of EDCs in the plasma. EDCs examined in this study were bisphenol-A (BPA), di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), mono(2-ethylhexyl) phthalate (MEHP), monobutyl phthalate (MBP), n-nonyl phenol (n-NP), and t-octylphenol (t-OP), and whereas the isoflavones were equol, genistein, and diadzein. The level of MBP in the plasma of patients was 1.3 times higher than that of the controls. The levels of t-OP and n-NP in the plasma of patients were respectively 1.15 and 1.2 times higher than those of the control group. Finally, the levels of the diadzein, equol and genistein were 1.37, 1.3 and 2.67 times higher than those of the control group, and genistein showed a statistically meaningful result (P = 0.0008). The results suggest that these six substances (MBP, t-OP, n-NP, daidzein, equol, and genistein) have an effect on precocious puberty.

The association between some endocrine disruptors and hypospadias in biological samples.

Hypospadias is a birth defect found in boys in which the urinary tract opening is not at the tip of the penis. The etiology of hypospadias is still unidentified, but endocrine disruptors are considered as one possible cause of hypospadias. In this study, target endocrine disruptor compounds were established for an assay. The target compounds included 5 phthalates (di-(2-ethylhexyl)phthalate (DEHP), di-n-butyl-phthalate (DBP), mono-(2- ethylhexyl) phthalate (MEHP), mono-n-butyl-phthalate (MBP) and phthalic acid (PA)), 2 alkylphenols (n-nonylphenol (n-NP) and t-octylphenol (t-OP)) and bisphenol A. The association between these 8 endocrine disruptors and hypospadias was studied. The levels of endocrine disruptors in the urine and plasma of a control group were compared with those of a patient group. DEHP (P = 0.006) and n-NP (P = 7.26e-6) in the urine samples and PA (P = 0.009) and BPA (P = 7.22e-10) in the plasma samples showed a significant association with hypospadias. The levels of endocrine disruptors in the urine and plasma of the mothers were also compared to those of the patients to investigate the metastasis of the endocrine disruptors from the mother. These levels did not, however, show a relationship with hypospadias (R(2) = 0.001-0.563).

Effect of manganese peroxidase on the decomposition of cellulosic components: Direct cellulolytic activity and synergistic effect with cellulase.

Herein, it was unearthed that manganese peroxidase (MnP) from Phanerochaete chrysosporium, a lignin-degrading enzyme, is capable of not only directly decomposing cellulosic components but also boosting cellulase activity. MnP decomposes various cellulosic substrates (carboxymethyl cellulose, cellobiose [CMC], and Avicel®) and produces reducing sugars rather than oxidized sugars such as lactone and ketoaldolase. MnP with MnII in acetate buffer evolves the MnIII-acetate complex functioning as a strong oxidant, and the non-specificity of MnIII-acetate enables cellulose-decomposition. The catalytic mechanism was proposed by analyzing catalytic products derived from MnP-treated cellopentaose. Notably, MnP also boosts cellulase activity on CMC and Avicel®, even considering the cellulolytic activity of MnP itself. To the best of the authors' knowledge, this is the first report demonstrating a previously unknown fungal MnP activity in cellulose-decomposition in addition to a known delignification activity. Consequently, the results provide a promising insight for further investigation of the versatility of lignin-degrading biocatalysts.

High serum isoflavone concentrations are associated with the risk of precocious puberty in Korean girls.

OBJECTIVE: The gradual decrease in the age of onset of puberty raises concerns about the contribution of phytoestrogen intake on sexual maturation. However, no data are available on the association between serum isoflavone (genistein, daidzein) concentration and central precocious puberty (CPP). The aim of the study was to test the association between serum isoflavone concentrations and the risk of CPP in Korean girls. STUDY DESIGN: A case-control study was conducted at Inje University Hospital in Korea. One-hundred and eight girls with CPP (aged 8·6 ± 0·8 year) and 91 age-matched controls (aged 8·5 ± 0·8 year) were examined. RESULTS: Serum concentrations of daidzein (P = 0·0202), genistein (P = 0·0021) and total isoflavones (P = 0·0009) were higher in children with CPP than in normal children. When the children were categorized into three groups according to total serum isoflavone as follows: <30, 30-70 and ≥70 nmol/l, serum concentrations of daidzein, genistein and total isoflavones significantly increased across three categories of serum isoflavone (P < 0·0001). The prevalence of CPP was significantly higher in children with serum isoflavone level of ≥30 nmol/l than those with serum level of <30 nmol/l (P = 0·0008). The adjusted OR for precocious puberty increased significantly across a range of total serum isoflavone (OR = 4·39; 95% CI: 1·83-10·51 vs OR = 5·22; 95% CI: 2·07-13·20; P = 0·001). CONCLUSION: These results suggest that elevated serum isoflavones may be associated with the risk of CPP in Korean girls.

High-temperature hydrodechlorination of ozone-depleting chlorodifluoromethane (HCFC-22) on supported Pd and Ni catalysts.

The hydrodechlorination of chlorodifluoromethane (HCFC-22) was performed by a catalytic reaction and noncatalytic thermal decomposition at high temperatures of 400-800 °C. After 47 h of time-on-stream on a supported palladium (Pd) catalyst, the gas phase composition of difluoromethane (HFC-32) is 41.0%, with 4.9% of the HCFC-22 remaining, indicating the conversion of up to 95.1% of HCFC-22. The supported nickel catalyst's deactivation is significant as it exhibits the low conversion of HCFC-22 under the same reaction conditions. The deactivation of the catalyst is caused by the polymerization of adsorbed methyl radicals, which competes with the formation of HFC-32. With concentrated reactants at high reaction temperatures, there was an increase in the catalytic activity; however, unwanted tar, methane, and trifluoromethane (HFC-23) by-products are also produced. The use of catalyst suppresses the formation of these by-products. Considering the compositions of the products of the catalytic and noncatalytic reactions, we demonstrate that the use of the supported-metal catalysts and hydrogen flow suppresses tar formation and lowers the required reaction temperature.

Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria.

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to fatty acid-derived chemicals that are widely used in the food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered for the first time to produce fatty acid ethyl esters (FAEEs) from CO2. Due to the lack of an endogenous ethanol production pathway and wax ester synthase (AftA) activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC 7942 by expressing heterologous AftA and introducing the ethanol pathway, resulting in detectable peaks of FAEEs. To enhance FAEE production, a heterologous phosphoketolase pathway was introduced in the FAEE-producing strain to supply acetyl-CoA. Subsequent optimization of the cyanobacterial culture with a hexadecane overlay resulted in engineered S. elongatus PCC 7942 that produced photosynthetic FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. This paper is the first report of photosynthetic production of FAEEs from CO2 in cyanobacteria.

Retinol, α-tocopherol, and selected minerals in breast milk of lactating women with full-term infants in South Korea.

BACKGROUND/OBJECTIVES: This study was performed to measure fat-soluble vitamins and minerals in breast milk of Korean lactating mothers who exclusively breastfed their babies. SUBJECTS/METHODS: Breast milk samples were collected from 334 mothers. Concentrations of retinol and α-tocopherol were analyzed by high performance liquid chromatography ultraviolet spectrometry while concentrations of minerals were measured by inductively coupled plasma optical emission spectrometry. RESULTS: Retinol and α-tocopherol contents of breast milk were 39.58 ± 19.64 µg/dL and 0.23 ± 0.13 mg/dL, respectively. Average sodium, potassium, calcium, phosphorus, and magnesium levels in breast milk were 11.11 ± 5.16, 38.56 ± 9.01, 27.87 ± 6.10, 13.56 ± 3.30, and 3.05 ± 0.65 mg/dL, respectively. Contents of trace elements such as iron, zinc, copper, and manganese were 40.26 ± 46.21, 98.40 ± 62.47, 24.09 ± 9.03, and 0.90 ± 1.63 µg/dL, respectively. Fat-soluble vitamin concentration was positively correlated with total fat in milk samples, but no significant differences were observed in levels of retinol, α-tocopherol, or minerals based on whether or not lactating women were taking dietary supplements. CONCLUSIONS: Micronutrient contents of breast milk samples from Korean lactating women were comparable to those of other nations. Retinol and α-tocopherol levels were correlated and also with total fat in breast milk.

Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor.

The push-and-pull strategy for metabolic engineering was successfully demonstrated in Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate synthase) of the methylerythritol phosphate pathway or the ß-subunit of phycocyanin (CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold compared to that of the control strain expressing SQS alone. To increase squalene production further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization, which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell weight). Further optimization for photobioprocessing and strain development will promote the construction of a solar-to-chemical platform.

Perspectives for biocatalytic lignin utilization: cleaving 4-O-5 and Cα-Cß bonds in dimeric lignin model compounds catalyzed by a promiscuous activity of tyrosinase.

BACKGROUND: In the biorefinery utilizing lignocellulosic biomasses, lignin decomposition to value-added phenolic derivatives is a key issue, and recently biocatalytic delignification is emerging owing to its superior selectivity, low energy consumption, and unparalleled sustainability. However, besides heme-containing peroxidases and laccases, information about lignolytic biocatalysts is still limited till date. RESULTS: Herein, we report a promiscuous activity of tyrosinase which is closely associated with delignification requiring high redox potentials (>1.4 V vs. normal hydrogen electrode [NHE]). The promiscuous activity of tyrosinase not only oxidizes veratryl alcohol, a commonly used nonphenolic substrate for assaying ligninolytic activity, to veratraldehyde but also cleaves the 4-O-5 and Cα-Cß bonds in 4-phenoxyphenol and guaiacyl glycerol-ß-guaiacyl ether (GGE) that are dimeric lignin model compounds. Cyclic voltammograms additionally verified that the promiscuous activity oxidizes lignin-related high redox potential substrates. CONCLUSION: These results might be applicable for extending the versatility of tyrosinase toward biocatalytic delignification as well as suggesting a new perspective for sustainable lignin utilization. Furthermore, the results provide insight for exploring the previously unknown promiscuous activities of biocatalysts much more diverse than ever thought before, thereby innovatively expanding the applicable area of biocatalysis.

Direct Conversion of CO2 to α-Farnesene Using Metabolically Engineered Synechococcus elongatus PCC 7942.

Direct conversion of carbon dioxide (CO2) to value-added chemicals by engineering of cyanobacteria has received attention as a sustainable strategy in food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered to produce α-farnesene from CO2. As a result of the lack of farnesene synthase (FS) activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC 7942 to express heterologous FS from either Norway spruce or apple fruit, resulting in detectable peaks of α-farnesene. To enhance α-farnesene production, an optimized methylerythritol phosphate (MEP) pathway was introduced in the farnesene-producing strain to supply farnesyl diphosphate. Subsequent cyanobacterial culture with a dodecane overlay resulted in photosynthetic production of α-farnesene (4.6 ± 0.4 mg/L in 7 days) from CO2. To the best of our knowledge, this is the first report of the photosynthetic production of α-farnesene from CO2 in the unicellular cyanobacterium S. elongatus PCC 7942.

Determination of the sequence of intersecting lines using Focused Ion Beam/Scanning Electron Microscope.

The aim of this study was to verify that the combination of focused ion beam (FIB) and scanning electron microscope/energy-dispersive X-ray (SEM/EDX) could be applied to determine the sequence of line crossings. The samples were transferred into FIB/SEM for FIB milling and an imaging operation. EDX was able to explore the chemical components and the corresponding elemental distribution in the intersection. The technique was successful in determining the sequence of heterogeneous line intersections produced using gel pens and red sealing ink with highest success rate (100% correctness). These observations show that the FIB/SEM was the appropriate instrument for an overall examination of document.

Feasibility of a facile butanol bioproduction using planetary mill pretreatment.

A facile butanol bioproduction process was developed using planetary milling, and Pinus rigida wood waste as a model substrate for fermentable sugars. The use of planetary milling as the pretreatment eliminates the need for washing and transfer of the biomass prior to enzymatic hydrolysis. Moreover, using this pretreatment process resulted in the production of only 0.072 ± 0.003 g/L soluble phenolic compounds, a concentration that was not inhibitory towards Clostridium beijerinckii NCIMB 8052. As the milling was performed in a compatible buffer (50mM acetate, pH 4.8), the enzymatic hydrolysis step was initiated by simply adding the cellulase cocktail powder directly to pretreated biomass without washing the biomass or exchanging the buffer, resulting in a glucose yield of 31 g/L (84.02%). Fermentation of the hydrolysate samples by C. beijerinckii NCIMB 8052 gave slightly better butanol yields than cultures grown in a typical lab media (P2), with final concentrations of 6.91 and 6.66 g/L, respectively.

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria.

BACKGROUND: Metabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2. RESULTS: Here, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33). CONCLUSIONS: This is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.

A dye-decolorizing peroxidase from Bacillus subtilis exhibiting substrate-dependent optimum temperature for dyes and ß-ether lignin dimer.

In the biorefinery using lignocellulosic biomass as feedstock, pretreatment to breakdown or loosen lignin is important step and various approaches have been conducted. For biological pretreatment, we screened Bacillus subtilis KCTC2023 as a potential lignin-degrading bacterium based on veratryl alcohol (VA) oxidation test and the putative heme-containing dye-decolorizing peroxidase was found in the genome of B. subtilis KCTC2023. The peroxidase from B. subtilis KCTC2023 (BsDyP) was capable of oxidizing various substrates and atypically exhibits substrate-dependent optimum temperature: 30°C for dyes (Reactive Blue19 and Reactive Black5) and 50°C for high redox potential substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid [ABTS], VA, and veratryl glycerol-ß-guaiacyl ether [VGE]) over +1.0 V vs. normal hydrogen electrode. At 50°C, optimum temperature for high redox potential substrates, BsDyP not only showed the highest VA oxidation activity (0.13 Umg(-1)) among the previously reported bacterial peroxidases but also successfully achieved VGE decomposition by cleaving Cα-Cß bond in the absence of any oxidative mediator with a specific activity of 0.086 Umg(-1) and a conversion rate of 53.5%. Based on our results, BsDyP was identified as the first bacterial peroxidase capable of oxidizing high redox potential lignin-related model compounds, especially VGE, revealing a previously unknown versatility of lignin degrading biocatalyst in nature.

Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase.

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.

High production of 2,3-butanediol from biodiesel-derived crude glycerol by metabolically engineered Klebsiella oxytoca M1.

BACKGROUND: 2,3-Butanediol (2,3-BDO) is a promising bio-based chemical because of its wide industrial applications. Previous studies on microbial production of 2,3-BDO has focused on sugar fermentation. Alternatively, biodiesel-derived crude glycerol can be used as a cheap resource for 2,3-BDO production; however, a considerable formation of 1,3-propanediol (1,3-PDO) and low concentration, productivity, and yield of 2,3-BDO from glycerol fermentation are limitations. RESULTS: Here, we report a high production of 2,3-BDO from crude glycerol using the engineered Klebsiella oxytoca M3 in which pduC (encoding glycerol dehydratase large subunit) and ldhA (encoding lactate dehydrogenase) were deleted to reduce the formation of 1,3-PDO and lactic acid. In fed-batch fermentation with the parent strain K. oxytoca M1, crude glycerol was more effective than pure glycerol as a carbon source in 2,3-BDO production (59.4 vs. 73.8 g/L) and by-product reduction (1,3-PDO, 8.9 vs. 3.7 g/L; lactic acid, 18.6 vs. 9.8 g/L). When the double mutant was used in fed-batch fermentation with pure glycerol, cell growth and glycerol consumption were significantly enhanced and 2,3-BDO production was 1.9-fold higher than that of the parent strain (59.4 vs. 115.0 g/L) with 6.9 g/L of 1,3-PDO and a small amount of lactic acid (0.7 g/L). Notably, when crude glycerol was supplied, the double mutant showed 1,3-PDO-free 2,3-BDO production with high concentration (131.5 g/L), productivity (0.84 g/L/h), and yield (0.44 g/g crude glycerol). This result is the highest 2,3-BDO production from glycerol fermentation to date. CONCLUSIONS: 2,3-BDO production from glycerol was dramatically enhanced by disruption of the pduC and ldhA genes in K. oxytoca M1 and 1,3-PDO-free 2,3-BDO production was achieved by using the double mutant and crude glycerol. 2,3-BDO production obtained in this study is comparable to 2,3-BDO production from sugar fermentation, demonstrating the feasibility of economic industrial 2,3-BDO production using crude glycerol.