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.
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Project description:The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 μg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.
| S-EPMC11436758 | 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:Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.
2025-04-12 | PXD062870 | Pride
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.
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