Cloning, characterization, and computer- aided evolution of a thermostable laccase of the DUF152 family from Klebsiella michiganensis
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ABSTRACT:
PROVIDER: PRJEB80708 | ENA |
REPOSITORIES: ENA
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Project description:ra10-01_laccases; laccase mutations. We demonstrated that laccases are involved in lignin polymerisation. Mutants have already been tested on microarrays and there is few differences compared to wild-type. The laccase mutation seems surgical. We possess a new double mutant, called snips, with a semi-dwarf phenotype, and we want to determine its profile. Each mutant was compared to wild type. All plants were harvested at the same developmental stage in the same growth chamber between 10h30 and 11h. 10 dye-swaps. CATMA arrays.
2011-07-02 | E-GEOD-22907 | biostudies-arrayexpress
Project description:ra10-01_laccases; laccase mutations. We demonstrated that laccases are involved in lignin polymerisation. Mutants have already been tested on microarrays and there is few differences compared to wild-type. The laccase mutation seems surgical. We possess a new double mutant, called snips, with a semi-dwarf phenotype, and we want to determine its profile.
2011-07-02 | GSE22907 | GEO
Project description:In this project, the transcriptomic data was obtained from the 6-day and 10-day submerged cultures of Cerrena unicolor sp. 87613 under PDA media, respectively. C.unicolor is reported to be an important medicinal fungus as well as an efficient laccase producer. Interestingly, C.unicolor sp.87613 presented a highest laccase production with ~420 U/mL at fermentation day 6, while the laccase production was reduced by ~27% at fermentation day 10. Therefore, these collected data were used to unveil the potential regulatory mechanism of laccase production. Besides, these transcriptomic data also provide essential data source for a better understanding of C.unicolor in various aspects.
2023-07-10 | GSE236542 | GEO
Project description:Laccases were proposed to catalyze the oxidative polymerization of monolignols. We identified 49 laccase gene models in Populus trichocarpa, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all 9 transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an approximately 40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up Graphic Gaussian Model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content. Total twelve trees were used. Those include nine individual transgenic trees for overexpressing Ptr-miR397a, as nine biological replicates, and three wild-type trees.
2014-01-17 | E-GEOD-54153 | biostudies-arrayexpress
Project description:Filamentous fungi are promising organisms for lignin degradation and mineralization. However, novel lignin-degrading fungal species are underexplored. Here, we isolated a fungal strain of Curvularia clavata that can utilize lignosulfonate as the carbon source and exhibited a relative high laccase activity during growth on lignosulfonate. Comparative transcriptomic analysis of the WT strain grown on glucose and lignosulfonate indicates that lignosulfonate and/or its metabolites have a significant effect on the gene expression profiles of C. clavata J1. Three regulators of laccase activity were identified, including a methyltransferase CcLaeA and two transcription factors, Rpn-4 and Tah-1. When grown on lignosulfonate, the laccase activity of the CclaeA and rpn-4 disrupted mutants (ΔCclaeA and Δrpn-4) increased by 49.2% and 43.5%, respectively, compared to the wild-type (WT) strain, whereas the tah-1 disrupted mutant (Δtah-1) decreased by 59.2%.
2024-10-09 | GSE278592 | GEO
Project description:The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.
| S-EPMC5331465 | biostudies-literature
Project description:Multimaterials deposition, a distinct advantage in bioprinting, overcomes material's limitation in hydrogel-based bioprinting. Multimaterials are deposited in a build/support configuration to improve the structural integrity of three-dimensional bioprinted construct. A combination of rapid cross-linking hydrogel has been chosen for the build/support setup. The bioprinted construct was further chemically cross-linked to ensure a stable construct after print. This paper also proposes a file segmentation and preparation technique to be used in bioprinting for printing freeform structures.
| S-EPMC7294690 | biostudies-literature
Project description:Laccases were proposed to catalyze the oxidative polymerization of monolignols. We identified 49 laccase gene models in Populus trichocarpa, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all 9 transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an approximately 40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up Graphic Gaussian Model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.
2014-01-17 | GSE54153 | GEO
Project description:BACKGROUND: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. RESULTS: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg(-1) protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. CONCLUSION: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L(-1) in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.
| S-EPMC3558336 | biostudies-literature
Project description:The Ebola virus (EBOV) causes severe human infection that lacks effective treatment. A recent screen identified a series of compounds that block EBOV-like particle entry into human cells. Using data from this screen, quantitative structure-activity relationship models were built and employed for virtual screening of a ∼17 million compound library. Experimental testing of 102 hits yielded 14 compounds with IC50 values under 10 μM, including several sub-micromolar inhibitors, and more than 10-fold selectivity against host cytotoxicity. These confirmed hits include FDA-approved drugs and clinical candidates with non-antiviral indications, as well as compounds with novel scaffolds and no previously known bioactivity. Five selected hits inhibited BSL-4 live-EBOV infection in a dose-dependent manner, including vindesine (0.34 μM). Additional studies of these novel anti-EBOV compounds revealed their mechanisms of action, including the inhibition of NPC1 protein, cathepsin B/L, and lysosomal function. Compounds identified in this study are among the most potent and well-characterized anti-EBOV inhibitors reported to date.
| S-EPMC6548547 | biostudies-literature