Ontology highlight
ABSTRACT:
PROVIDER: PRJNA759940 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15709767_1.fastq.gz | Fastqsanger.gz | |||
| SRR15709767_2.fastq.gz | Fastqsanger.gz | |||
| SRR15709768_1.fastq.gz | Fastqsanger.gz | |||
| SRR15709768_2.fastq.gz | Fastqsanger.gz | |||
| SRR15709769_1.fastq.gz | Fastqsanger.gz |
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Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.
2023-06-13 | PXD027027 | Pride
Project description:Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. Its relatively low catalytic constant as well as its tendency to also accept O2 as a substrate have made it a common target of enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library, without prior experimental knowledge, by coupling the phylogenetically-guided model EVmutation with “in silico evolution”. Starting with this targeted mutagenesis library, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. We identified an amino acid exchange which increased the thermostability of Gallionella Rubisco and conveyed resilience to detrimental exchanges. The establishment of this platform is a first step towards high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts. DIA proteomics was used to compare relative quantities of the native and heterologous RuBisCO, as well as the proteome at large at different gas conditions and with or without the heterologous enzyme.
2025-08-04 | PXD059631 | Pride
Project description:Background: MicroRNAs (miRNAs) play roles in various biological processes including growth, development, and defense in plants. Recent studies revealed that some plant miRNAs could produce secondary small interfering RNAs (siRNAs) such as phased, secondary siRNAs (phasiRNAs) and they regulate cascade of gene expression. Results: We performed genome-wide comparative analysis of miRNAs and their targets in Solanaceae plants in an evolutionary perspective. miRNAs were mapped onto 12 chromosomes and microsynteny analysis, based on miRNAs and their flanking genes, revealed about 86% of conserved miRNAs in pepper maintained synteny with those of tomato or potato. Degradome analysis revealed that many of genes related to transcription or defense response are regulated by miRNAs in Solanaceae plants. We found several miRNAs in pepper targeting a number of genes encoding nucleotide-binding and leucine rich repeat (NLR) or receptor-like protein (RLP), known as major players in defense responses. In addition, resistance-related miRNAs trigger phasiRNA production indicating amplification of regulation of the disease-resistant gene families. Among them, specifically evolved miRNAs in pepper, can-miR-n033a and can-miR-n026, targets many NLRs and RLPs in an expanded subgroup in pepper, respectively. Conclusions: Taken together, miRNAs might be generated and evolved to regulate diverse genes involved in plant immunity in Solanaceae. This study provides an insight into possible co-evolution between resistance-related miRNAs and defense genes in pepper.
2018-07-18 | GSE102781 | GEO
Project description:Six different Solanaceae species, Potato (Solanum tubersosum), Tomato (Lycopersicum esculentum), Pepper (Capsicum annuum), Tobacco (Nicotiana tabaccum), Petunia and Nicotiana benthiamana were grown at 25C, 16h light and 8h darkness. Mature leaves were harvested after 4-6 weeks. RNA was isolated using Qiagen RNeasy. Tomato, pepper, petunia, tobacco and N. benthamiana samples were hybridized against potato samples. Keywords: Solanaceae comparative gene expression profiling
2005-10-03 | GSE3380 | GEO
Project description:Tradeoffs Involved in Alkaloid Evolution in the Solanaceae. (Raw sequence reads)
| PRJNA1070281 | ENA
Project description:Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol-production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h-1), an engineered PRK-RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK-RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate yields by 67% and 29%, respectively. Additional C-terminal fusion of a 19 amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15x cbbm strain. These modifications did not affect glycerol production at 0.05 h-1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h-1) anaerobic batch cultures than observed for the 15x cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2x cbbm strain. At 0.05 h-1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15x cbbm strain, without affecting glycerol yield. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol yield was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK-RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth-rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.
2023-05-17 | GSE221392 | GEO
Project description:Heterologous expression of hornwort Rubisco from Anthoceros agrestis in Escherichia coliwith expression of Rubisco large and small subunits, chaperonins 60alpha/beta and chaperonin 20 as well as a suite of assembly factors, Raf1/Raf2/RbcX2/BSD2/RbcX1. Rubisco kinetics show differences in Rubisco assembled with and without RbcX1/2.
2024-11-04 | PXD052489 | Pride