Biochemical and molecular characterization of a novel glycerol dehydratase from Klebsiella pneumoniae 2e with high tolerance against crude glycerol impurities.

BACKGROUND: The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains. RESULTS: In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with Km and Vm values of 3.42 mM and 58.15 nkat mg-1, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility. CONCLUSIONS: This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.

The Method of Screening and Identification of Transcription Factor in Klebsiella.

This protocol describes a method for verifying the specific transcription factor regulating glycerol dehydratase (GDH) expression in Klebsiella. DNA pull-down accompanied with mass spectrometry is used to screen and identify the transcription factor interacting with the promoter region of the key gene in Klebsiella. EMSA method is used to validate the specific binding of the transcription factor to the promoter region in vitro. In addition, the target DNA fragments are constructed by fusion PCR to prepare competent cells from Klebsiella for electrical transformation and further transformed to obtain key gene deletion strains to verify the transcription factor responsible for the target gene expression in Klebsiella.

Elucidation of ligninolysis mechanism of a newly isolated white-rot basidiomycete Trametes hirsuta X-13.

BACKGROUND: Lignin is a complex aromatic heteropolymer comprising 15-30% dry weight of the lignocellulose. The complex structural characteristic of lignin renders it difficult for value-added utilization. Exploring efficient lignin-degrading microorganisms and investigating their lignin-degradation mechanisms would be beneficial for promoting lignin valorization. In this study, a newly isolated white-rot basidiomycete, Trametes hirsuta X-13, with capacity to utilize alkaline lignin as the sole substrate was investigated. RESULTS: The analysis of the fermentation properties of T. hirsuta X-13 using alkaline lignin as the sole substrate, including the mycelial growth, activities of ligninolytic enzymes and the rates of lignin degradation and decolorization confirmed its great ligninolysis capacity. The maximum lignin degradation rate reached 39.8% after 11 days of T. hirsuta X-13 treatment, which was higher than that of reported fungi under the same condition. Fourier transform infrared spectrometry (FTIR), gas chromatography-mass spectrometry (GC-MS) scanning electron micrographs (SEM), two-dimensional heteronuclear single quantum coherence NMR analysis (2D-HSQC NMR) collaborated with pyrolysis gas chromatography-mass spectrometry (py-GC/MS) analyses proved that lignin structure was severely deconstructed along with amounts of monomer aromatics generated. Furthermore, according to those chemical analysis, in addition to canonical Cα-Cß breakage, the cleavage of lignin interunit linkages of ß-ß might also occur by T. hirsuta X-13. CONCLUSIONS: This study characterized a newly isolated white-rot basidiomycete T. hirsuta X-13 with impressive alkaline lignin degradation ability and provided mechanistic insight into its ligninolysis mechanism, which will be valuable for the development of lignin valorization strategies.

Selective delignification of poplar wood with a newly isolated white-rot basidiomycete Peniophora incarnata T-7 by submerged fermentation to enhance saccharification.

BACKGROUND: Pretreatment is a critical step required for efficient conversion of woody biomass into biofuels and platform chemicals. Fungal pretreatment is regarded as one of the most promising technology for woody biomass conversion but remains challenging for industrial application. The exploration of potential fungus strain with high efficient delignification and less processing time for woody biomass pretreatment will be valuable for development of biorefinery industry. Here, a newly isolated white-rot basidiomycete Peniophora incarnate T-7 was employed for poplar wood pretreatment. RESULTS: The chemical component analysis showed that cellulose, hemicellulose and lignin from poplar wood declined by 16%, 48% and 70%, respectively, after 7 days submerged fermentation by P. incarnate T-7. Enzymatic saccharification analysis revealed that the maximum yields of glucose and xylose from 7 days of P. incarnate T-7 treated poplar wood reached 33.4% and 27.6%, respectively, both of which were enhanced by sevenfold relative to the untreated group. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), X-ray diffraction (XRD) and pyrolysis gas chromatography-mass spectrometry (Py-GC/MS) characterization confirmed that lignocellulosic structure of poplar wood was largely broken by P. incarnate T-7, including delignification and de-crystalline of cellulose. Meanwhile, lignin component of poplar wood was selectively degraded by P. incarnate T-7, and G-type unit of lignin was preferentially attacked by the strain. Furthermore, quantitative proteomic analysis revealed that a considerable amount of lignocellulolytic enzymes were detected in the secretory proteins of P. incarnate T-7, especially with high abundance of lignin-degrading enzymes and hemicellulases. Combination of quantitative proteomic with transcriptomic analysis results showed that most of those lignocellulolytic enzymes were highly upregulated on poplar wood substrate compared to glucose substrate. CONCLUSIONS: This study showed that P. incarnate T-7 could selectively delignify poplar wood by submerged fermentation with short time of 7 days, which greatly improved its enzymatic saccharification efficiency. Our results suggested that P. incarnate T-7 might be a promising candidate for industrial woody biomass pretreatment.

Identification and characterization of a novel bacterial carbohydrate esterase from the bacterium Pantoea ananatis Sd-1 with potential for degradation of lignocellulose and pesticides.

OBJECTIVE: Identification and characterization of a novel bacterial carbohydrate esterase (PaCes7) with application potential for lignocellulose and pesticide degradation. RESULTS: PaCes7 was identified from the lignocellulolytic bacterium, Pantoea ananatis Sd-1 as a new carbohydrate esterase. Recombinant PaCes7 heterologously expressed in Escherichia coli showed a clear preference for esters with short-chain fatty acids and exhibited maximum activity towards α-naphthol acetate at 37 °C and pH 7.5. Purified PaCes7 exhibited its catalytic activity under mesophilic conditions and retained more than 40% activity below 30 °C. It displayed a relatively wide pH stability from pH 6-11. Furthermore, the enzyme was strongly resistant to Mg2+, Pb2+, and Co2+ and activated by K+ and Ca2+. Both P. ananatis Sd-1 and PaCes7 could degrade the pesticide carbaryl. Additionally, PaCes7 was shown to work in combination with cellulase and/or xylanase in rice straw degradation. CONCLUSIONS: The data suggest that PaCes7 possesses promising biotechnological potential.

Adaptability of Klebsiella pneumoniae 2e, a Newly Isolated 1,3-Propanediol-Producing Strain, to Crude Glycerol as Revealed by Genomic Profiling.

Crude glycerol is largely generated as the main by-product of the biodiesel industry and is unprofitable for industrial application without costly purification. The direct bioconversion of crude glycerol into 1,3-propanediol (1,3-PDO) by microorganisms is a promising alternative for effective and economic utilization. In this study, Klebsiella pneumoniae 2e was newly isolated for the conversion of crude glycerol into 1,3-PDO. Batch fermentation analysis confirmed that crude glycerol and its main impurities had slight impacts on the growth, key enzyme activity, and 1,3-PDO production of K. pneumoniae 2e. The 1,3-PDO yield from crude glycerol by K. pneumoniae 2e reached 0.64 mol 1,3-PDO/mol glycerol, which was higher than that by most reported 1,3-PDO-producing Klebsiella strains. Genomic profiling revealed that K. pneumoniae 2e possesses 30 genes involved in glycerol anaerobic metabolism and 1,3-PDO biosynthesis. Quantitative real-time PCR analysis of these genes showed that the majority of the genes encoding the key enzymes for glycerol metabolism and 1,3-PDO biosynthesis were significantly upregulated during culture in crude glycerol relative to that in pure glycerol. Further comparative genomic analysis revealed a novel glycerol uptake facilitator protein in K. pneumoniae 2e and a higher number of stress response proteins than in other Klebsiella strains. This work confirms the adaptability of a newly isolated 1,3-PDO-producing strain, K. pneumoniae 2e, to crude glycerol and provides insights into the molecular mechanisms involved in its crude glycerol tolerance, which is valuable for industrial 1,3-PDO production from crude glycerol.IMPORTANCE The rapid development of the biodiesel industry has led to tremendous crude glycerol generation. Due to the presence of complex impurities, crude glycerol has low value for industry without costly purification. Obtaining novel microorganisms capable of direct and efficient bioconversion of crude glycerol to value-added products has great economic potential for industrial application. In this work, we characterized a newly isolated strain, Klebsiella pneumoniae 2e, with the capacity to efficiently produce 1,3-propanediol (1,3-PDO) from crude glycerol and demonstrated its adaptation to crude glycerol. Our work provides insights into the molecular mechanisms of K. pneumoniae 2e adaptation to crude glycerol and the expression patterns of its genes involved in 1,3-PDO biosynthesis, which will contribute to the development of industrial 1,3-PDO production from crude glycerol.

Identification and characterization of a novel bacterial pyranose 2-oxidase from the lignocellulolytic bacterium Pantoea ananatis Sd-1.

OBJECTIVE: To identify and characterize a novel bacterial pyranose 2-oxidase (P2Ox) and investigate its potential use in lignin degradation applications. RESULTS: A new bacterial P2Ox (PaP2Ox) enzyme was identified in the lignocellulolytic bacterium Pantoea ananatis Sd-1. The PaP2Ox open reading frame was cloned, and the encoded protein was heterologously expressed in an Escherichia coli expression system. Unlike another reported bacterial P2Ox enzyme, the purified PaP2Ox exhibits a homotetrameric spatial conformation that is similar to fungal P2Oxs, with each subunit having a molecular mass of 65 kDa. The recombinant PaP2Ox exhibits maximum activity at 50 °C and pH 6.5 with D-glucose as its preferred substrate. In addition, this enzyme was shown to work in combination with bacterial laccase in lignin degradation. CONCLUSIONS: The bacterial enzyme PaP2Ox has potential use in ligninolytic systems and shows promising value in industrial biotechnological applications.

Involvement of Fenton chemistry in rice straw degradation by the lignocellulolytic bacterium Pantoea ananatis Sd-1.

BACKGROUND: Lignocellulolytic bacteria have revealed to be a promising source for biofuel production, yet the underlying mechanisms are still worth exploring. Our previous study inferred that the highly efficient lignocellulose degradation by bacterium Pantoea ananatis Sd-1 might involve Fenton chemistry (Fe2+ + H2O2 + H+ â†’ Fe3+ + OH· + H2O), similar to that of white-rot and brown-rot fungi. The aim of this work is to investigate the existence of this Fenton-based oxidation mechanism in the rice straw degradation process of P. ananatis Sd-1. RESULTS: After 3 days incubation of unpretreated rice straw with P. ananatis Sd-1, the percentage in weight reduction of rice straw as well as its cellulose, hemicellulose, and lignin components reached 46.7, 43.1, 42.9, and 37.9 %, respectively. The addition of different hydroxyl radical scavengers resulted in a significant decline (P < 0.001) in rice straw degradation. Pyrolysis gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy analysis revealed the consistency of chemical changes of rice straw components that exists between P. ananatis Sd-1 and Fenton reagent treatment. In addition to the increased total iron ion concentration throughout the rice straw decomposition process, the Fe3+-reducing capacity of P. ananatis Sd-1 was induced by rice straw and predominantly contributed by aromatic compounds metabolites. The transcript levels of the glucose-methanol-choline oxidoreductase gene related to hydrogen peroxide production were significantly up-regulated (at least P < 0.01) in rice straw cultures. Higher activities of GMC oxidoreductase and less hydrogen peroxide concentration in rice straw cultures relative to glucose cultures may be responsible for increasing rice straw degradation, which includes Fenton-like reactions. CONCLUSIONS: Our results confirmed the Fenton chemistry-assisted degradation model in P. ananatis Sd-1. We are among the first to show that a Fenton-based oxidation mechanism exists in a bacteria degradation system, which provides a new perspective for how natural plant biomass is decomposed by bacteria. This degradative system may offer an alternative approach to the fungi system for lignocellulosic biofuels production.

Genomic and secretomic insight into lignocellulolytic system of an endophytic bacterium Pantoea ananatis Sd-1.

BACKGROUND: Exploring microorganisms especially bacteria associated with the degradation of lignocellulosic biomass shows great potentials in biofuels production. The rice endophytic bacterium Pantoea ananatis Sd-1 with strong lignocellulose degradation capacity has been reported in our previous study. However, a comprehensive analysis of its corresponding degradative system has not yet been conducted. The aim of this work is to identify and characterize the lignocellulolytic enzymes of the bacterium to understand its mechanism of lignocellulose degradation and facilitate its application in sustainable energy production. RESULTS: The genomic analysis revealed that there are 154 genes encoding putative carbohydrate-active enzymes (CAZy) in P. ananatis Sd-1. This number is higher than that of compared cellulolytic and ligninolytic bacteria as well as other eight P. ananatis strains. The CAZy in P. ananatis Sd-1 contains a complete repertoire of enzymes required for cellulose and hemicellulose degradation. In addition, P. ananatis Sd-1 also possesses plenty of genes encoding potential ligninolytic relevant enzymes, such as multicopper oxidase, catalase/hydroperoxidase, glutathione S-transferase, and quinone oxidoreductase. Quantitative real-time PCR analysis of parts of genes encoding lignocellulolytic enzymes revealed that they were significantly up-regulated (at least P < 0.05) in presence of rice straw. Further identification of secretome of P. ananatis Sd-1 by nano liquid chromatography-tandem mass spectrometry confirmed that considerable amounts of proteins involved in lignocellulose degradation were only detected in rice straw cultures. Rice straw saccharification levels by the secretome of P. ananatis Sd-1 reached 129.11 ± 2.7 mg/gds. Correspondingly, the assay of several lignocellulolytic enzymes including endoglucanase, exoglucanase, ß-glucosidase, xylanase-like, lignin peroxidase-like, and laccase-like activities showed that these enzymes were more active in rice straw relative to glucose substrates. The high enzymes activities were not attributed to bacterial cell densities but to the difference of secreted protein contents. CONCLUSION: Our results indicate that P. ananatis Sd-1 can produce considerable lignocellulolytic enzymes including cellulases, hemicellulases, and ligninolytic relevant enzymes. The high activities of those enzymes could be efficiently induced by lignocellulosic biomass. This identified degradative system is valuable for the lignocellulosic bioenergy industry.

An acid-stable bacterial laccase identified from the endophyte Pantoea ananatis Sd-1 genome exhibiting lignin degradation and dye decolorization abilities.

OBJECTIVES: Isolation and identification of a novel laccase (namely Lac4) with various industrial applications potentials from an endophytical bacterium. RESULTS: Endophyte Sd-1 cultured in rice straw showed intra- and extra-cellular laccase activities. Genomic analysis of Sd-1 identified four putative laccases, Lac1 to Lac4. However, only Lac4 contains the complete signature sequence of laccase and shares at most 64 % sequence identity with other characterized bacterial multi-copper oxidases. Recombinant Lac4 can oxidize non-phenolic and phenolic compounds under acidic conditions and at 30-50 °C; Km values of Lac4 for ABTS at pH 2.5 and for guaiacol at pH 4.5 were 1 ± 0.15 and 6.1 ± 1.7 mM, respectively. The activity of Lac4 was stimulated by 0.8 mM Cu(2+) and 5 mM Fe(2+). In addition, Lac4 could decolorize various synthetic dyes and exhibit the degradation rate of 38 % for lignin. CONCLUSIONS: The data suggest that Lac4 possesses promising biotechnological potentials.