Isolation and characterization of mutants corresponding to the <scp>MENA</scp>,<scp> MENB</scp>,<scp> MENC</scp> and <scp>MENE</scp> enzymatic steps of 5′‐monohydroxyphylloquinone biosynthesis in <i>Chlamydomonas reinhardtii</i>

Emonds-Alt, Barbara; Coosemans, Nadine; Gerards, Thomas; Remacle, Claire; Cardol, Pierre

doi:10.1111/tpj.13352

Cited by 13 publications

(9 citation statements)

References 63 publications

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“…In plants and green algae most steps of phylloquinone synthesis occur in the plastid, except three reactions from o-succinylbenzoate to dihydroxynaphthoate, which are catalyzed by peroxisomal enzymes(Emonds-Alt et al 2017;Cenci et al 2018). E. gracilis encodes a homolog of the multifunctional protein PHYLLO catalyzing all four steps of the first part of the pathway (seqid 121), but the protein is absent from the plastid proteome and lacks a targeting presequence, consistent with earlier biochemical evidence placing synthesis of o-succinylbenzoate in the cytosol(Seeger and Bentley 1991).…”

supporting

confidence: 78%

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Amg

Zoltner

Kelly

et al. 2019

Preprint

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Correspondence to mfield@mac.com (MCF, proteomics) and vlada@natur.cuni.cz (VH, plastid evolution) MCF, JL, and VH conceived the original research plans; JL and MCF supervised the experiments; ELD performed the cell fractionation; MZ and SK performed the mass spectrometry analysis; AMGNV and TGE performed protein annotation and sorting, AMGNV, KZ, and ZF interpreted the annotation results, AMGNV performed the signal domain analysis, PS performed the phylogenetic analysis, AMGNV conceived the project and wrote the article with contributions of all the authors; VH, ME, MCF, and JL supervised and complemented the writing. VH agrees to serve as the author responsible for contact and ensures communication. AbstractEuglena gracilis is a well-studied biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids. Here we describe its plastid proteome obtained by high-resolution proteomics. We identified 1,345 candidate plastid proteins and assigned functional annotations to 774 of them. More than 120 proteins are affiliated neither to the host lineage nor the plastid ancestor and may represent horizontal acquisitions from various algal and prokaryotic groups. Reconstruction of plastid metabolism confirms both the presence of previously studied/predicted enzymes/pathways and also provides direct evidence for unusual features of its metabolism including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism and the presence of two sets of the SUF pathway for FeS cluster assembly. Most significantly, one of these was acquired by lateral gene transfer (LGT) from the chlamydiae. Plastidial paralogs of membrane traffickingassociated proteins likely mediating a poorly understood fusion of transport vesicles with the outermost plastid membrane were identified, as well as derlin-related proteins that potentially act as protein translocases of the middle membrane, supporting an extremely simplified TIC complex. The proposed innovations may be also 2 linked to specific features of the transit peptide-like regions described here. Hence the Euglena plastid is demonstrated to be a product of several genomes and to combine novel and conserved metabolism and transport processes.

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confidence: 78%

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Amg

Zoltner

Kelly

et al. 2019

Preprint

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“…In this research, we designed a sgRNA targeting both GhCLA1 homeoalleles (D10G1640 and A10G2292) with a single-base difference in the target site and produced mutations in both alleles. In addition to single mutants, functional genomics research requires the production of double and multiple mutants in many occasions ( Thung et al, 2012 ; Shi et al, 2016 ; Emonds-Alt et al, 2017 ; Li P. et al, 2017 ). Simultaneous editing of multiple genes using CRISPR/Cas9 has been reported in some plant species ( Nekrasov et al, 2013 ; Xu et al, 2014 ; Fan et al, 2015 ; Sun et al, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

Genome Editing in Cotton with the CRISPR/Cas9 System

et al. 2017

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Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton (Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1, and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.

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“…Phylloquinone is synthesized by ten enzymes of which several have been characterized in Synechocystis [171,172]. The majority have been identified based on homology with proteins synthesizing menaquinone (Vitamin K 2 ) and characterized in other bacteria [173]. The second last enzyme in the pathway, MenA, utilizes phytyl diphosphate, while the last enzyme requires that dimethylphylloquinone be reduced via NAD(P)H dehydrogenase NdbB to dimethylphylloquinol, prior to synthesis of phylloquinone by MenG [174].…”

Section: Phylloquinone and Plastoquinone Biosynthesismentioning

confidence: 99%

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

2020

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Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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Isolation and characterization of mutants corresponding to the MENA, MENB, MENC and MENE enzymatic steps of 5′‐monohydroxyphylloquinone biosynthesis in Chlamydomonas reinhardtii

Cited by 13 publications

References 63 publications

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Genome Editing in Cotton with the CRISPR/Cas9 System

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

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