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Pachulicz, R. J., Jovcevski, B., Bulone, V. & Pukala, T. L. (2025). Acid-catalysed esterification of anthocyanin glucosyl units by organic acids: Chemical factors and structural implications. Food Chemistry, 480, Article ID 143878.
Open this publication in new window or tab >>Acid-catalysed esterification of anthocyanin glucosyl units by organic acids: Chemical factors and structural implications
2025 (English)In: Food Chemistry, ISSN 0308-8146, E-ISSN 1873-7072, Vol. 480, article id 143878Article in journal (Refereed) Published
Abstract [en]

Anthocyanins are plant-derived pigments with diverse applications due to their favourable spectral and biological properties, which are often modulated through enzymatic addition of structural modifications. However, evidence indicates that anthocyanin glycosyl groups may be modified by organic acids in solution through an acid-catalysed esterification. Here, the anthocyanin standard cyanidin-3-glucoside is shown to undergo esterification in aqueous conditions with citric acid, acetic acid, and most efficiently, formic acid. Extended to a biological sample, red cabbage anthocyanins incubated with formic acid resulted in 53 unique species from 9 original anthocyanins. From these findings a key structural determinant to predict formylation patterns is identified: the precise glucosyl unit where hydroxycinnamic acid acylation occurs, which dictates the number of free primary alcohol groups in the anthocyanin available for formylation. This study demonstrates the unexpected structural complexity these reactions introduce into samples that necessitate careful consideration in anthocyanin handling. Harnessing these reactions in future may help produce anthocyanins with controlled structures and properties for downstream applications.

Place, publisher, year, edition, pages
Elsevier BV, 2025
Keywords
Anthocyanins, Brassica oleracea, Formylation, Glucosyl modification, Mass spectrometry, Structural analysis
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-362025 (URN)10.1016/j.foodchem.2025.143878 (DOI)001452428900001 ()40112715 (PubMedID)2-s2.0-105000167262 (Scopus ID)
Note

QC 20250425

Available from: 2025-04-03 Created: 2025-04-03 Last updated: 2025-04-25Bibliographically approved
Clayton-Cuch, D., Yu, L., McDougal, D., Burbidge, C. A., Bruning, J. B., Bradley, D., . . . Bulone, V. (2024). Biochemical and in silico characterization of glycosyltransferases from red sweet cherry (Prunus avium L.) reveals their broad specificity toward phenolic substrates. Food Chemistry: Molecular Sciences, 8, Article ID 100193.
Open this publication in new window or tab >>Biochemical and in silico characterization of glycosyltransferases from red sweet cherry (Prunus avium L.) reveals their broad specificity toward phenolic substrates
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2024 (English)In: Food Chemistry: Molecular Sciences, E-ISSN 2666-5662, Vol. 8, article id 100193Article in journal (Refereed) Published
Abstract [en]

Polyphenolic compounds are a class of phytonutrients that play important roles in plants and contribute to human health when incorporated into our diet through fruit consumption. A large proportion occur as glycoconjugates but the enzymes responsible for their glycosylation are poorly characterized. Here, we report the biochemical and structural characterization of two glycosyltransferases from sweet cherry named PaUGT1 and PaUGT2. Both are promiscuous glucosyltransferases active on diverse anthocyanidins and flavonols, as well as phenolic acids in the case of PaUGT1. They also exhibit weaker galactosyltransferase activity. The expression of the gene encoding PaUGT1, the most active of the two proteins, follows anthocyanin accumulation during fruit ripening, suggesting that this enzyme is the primary glycosyltransferase involved in flavonoid glycosylation in sweet cherry. It can potentially be used to synthesize diverse glycoconjugates of flavonoids for integration into bioactive formulations, and for generating new fruit cultivars with enhanced health-promoting properties using breeding methods.

Place, publisher, year, edition, pages
Elsevier BV, 2024
Keywords
Anthocyanins, Cherry, Flavonols, Glycosyltransferase, Phenolic compounds, Protein structure modelling
National Category
Food Science
Identifiers
urn:nbn:se:kth:diva-342619 (URN)10.1016/j.fochms.2023.100193 (DOI)001164596400001 ()2-s2.0-85182519698 (Scopus ID)
Note

QC 20240301

Available from: 2024-01-25 Created: 2024-01-25 Last updated: 2024-06-19Bibliographically approved
Hrmova, M., Zimmer, J., Bulone, V. & Fincher, G. B. (2024). Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans. Scientific Journal of Silesian University of Technology. Series Transport, 194(1), 33-50
Open this publication in new window or tab >>Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans
2024 (English)In: Scientific Journal of Silesian University of Technology. Series Transport, ISSN 0209-3324, Vol. 194, no 1, p. 33-50Article in journal (Refereed) Published
Abstract [en]

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Place, publisher, year, edition, pages
Oxford University Press (OUP), 2024
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-342388 (URN)10.1093/plphys/kiad415 (DOI)001050204900001 ()37594400 (PubMedID)2-s2.0-85181588193 (Scopus ID)
Note

QC 20240122

Available from: 2024-01-17 Created: 2024-01-17 Last updated: 2025-02-20Bibliographically approved
Pinto, S. C., Leong, W. H., Tan, H., McKee, L. S., Prevost, A., Ma, C., . . . Tucker, M. R. (2024). Germline β−1,3-glucan deposits are required for female gametogenesis in Arabidopsis thaliana. Nature Communications, 15(1), Article ID 5875.
Open this publication in new window or tab >>Germline β−1,3-glucan deposits are required for female gametogenesis in Arabidopsis thaliana
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2024 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 15, no 1, article id 5875Article in journal (Refereed) Published
Abstract [en]

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in β−1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether β−1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to β−1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a β−1,3-glucanase in the female germline transiently perturbed β−1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline β−1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

Place, publisher, year, edition, pages
Springer Nature, 2024
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-350967 (URN)10.1038/s41467-024-50143-0 (DOI)001381534800018 ()38997266 (PubMedID)2-s2.0-85198383475 (Scopus ID)
Note

QC 20240725

Available from: 2024-07-24 Created: 2024-07-24 Last updated: 2025-01-20Bibliographically approved
Carreno-Quintero, N., Tohge, T., Van Acker, R., McKee, L. S., Zhou, Q., Bolze, A., . . . Fraser, P. D. (2024). Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock. Bioresources and bioprocessing, 11(1), Article ID 12.
Open this publication in new window or tab >>Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock
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2024 (English)In: Bioresources and bioprocessing, ISSN 2197-4365, Vol. 11, no 1, article id 12Article in journal (Refereed) Published
Abstract [en]

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

Place, publisher, year, edition, pages
Springer Nature, 2024
Keywords
Nicotiana glauca, Metabolite profiling, Biorefinary, Bioproducts
National Category
Chemical Engineering
Identifiers
urn:nbn:se:kth:diva-343034 (URN)10.1186/s40643-023-00726-4 (DOI)001145230900004 ()2-s2.0-85182603856 (Scopus ID)
Note

QC 20240208

Available from: 2024-02-08 Created: 2024-02-08 Last updated: 2025-02-18Bibliographically approved
Hao, M.-S., Mazurkewich, S., Li, H., Kvammen, A., Saha, S., Koskela, S., . . . McKee, L. S. (2024). Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to β-glucans. Nature Communications, 15(1), Article ID 3429.
Open this publication in new window or tab >>Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to β-glucans
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2024 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 15, no 1, article id 3429Article in journal (Refereed) Published
Abstract [en]

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β−1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Place, publisher, year, edition, pages
Springer Nature, 2024
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-345877 (URN)10.1038/s41467-024-47584-y (DOI)38653764 (PubMedID)2-s2.0-85191077746 (Scopus ID)
Funder
Swedish Research Council Formas, 2019-00389Swedish Energy Agency, 2019-006926Swedish Research Council, 2020-03618Swedish Research Council Formas, 2019-00389Swedish Energy Agency, 2019-006926Swedish Research Council, 2020-03618
Note

QC 20240429

Available from: 2024-04-24 Created: 2024-04-24 Last updated: 2025-02-20Bibliographically approved
Lu, Z., Kvammen, A., Li, H., Hao, M., Inman, A. R., Bulone, V. & McKee, L. S. (2023). A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell walls. mSphere, 8(4)
Open this publication in new window or tab >>A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell walls
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2023 (English)In: mSphere, E-ISSN 2379-5042, Vol. 8, no 4Article in journal (Refereed) Published
Abstract [en]

In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL’s outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade.

Place, publisher, year, edition, pages
American Society for Microbiology, 2023
National Category
Biochemistry Molecular Biology Microbiology
Identifiers
urn:nbn:se:kth:diva-333943 (URN)10.1128/msphere.00244-23 (DOI)001037206600001 ()37493618 (PubMedID)2-s2.0-85168802162 (Scopus ID)
Funder
Swedish Research Council, 2017-04906Swedish Energy Agency, 2019-006926
Note

QC 20230823

Available from: 2023-08-15 Created: 2023-08-15 Last updated: 2025-03-21Bibliographically approved
Nokling-Eide, K., Tan, F., Wang, S., Zhou, Q., Gravdahl, M., Langeng, A.-M., . . . Arlov, O. (2023). Acid preservation of cultivated brown algae Saccharina latissima and Alaria esculenta and characterization of extracted alginate and cellulose. Algal Research, 71, 103057, Article ID 103057.
Open this publication in new window or tab >>Acid preservation of cultivated brown algae Saccharina latissima and Alaria esculenta and characterization of extracted alginate and cellulose
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2023 (English)In: Algal Research, ISSN 2211-9264, Vol. 71, p. 103057-, article id 103057Article in journal (Refereed) Published
Abstract [en]

Cultivated brown algae represent an important potential source of carbohydrate polymers for packaging and other biobased materials. However, their exploitation is currently limited by a short harvest season and a lack of cost-effective and sustainable methods to preserve biopolymer quality. In the present study, cultivated Saccharina latissima (SL) and Alaria esculenta (AE) were preserved with formic acid at 4, 13 and 20 degrees C for up to 16 weeks prior to extraction and characterization of alginate and cellulose. The data show up to 40 % increased yield of alginate from preserved biomass compared with fresh and non-preserved biomass, primarily due to removal of minerals and other soluble compounds during the acid wash. Acid preservation and storage caused a reduction in alginate weight average molecular weight (Mw) that was mainly dependent on storage temperature and to a lesser extent on storage time; storage at 4 degrees C maintained the Mw of alginates at 350-500 kDa. Preservation had no effect on the guluronate block structure of the extracted alginates, but guluronic acid content and block length increased in the non-preserved samples, presumably due to enzymatic degradation of the alginate's M-rich re-gions. Preservation of the seaweed resulted in an increased cellulose yield compared with fresh and non -preserved biomass, again due to the biomass being reduced during acid wash. The molecular weight and crys-tallinity of cellulose were not altered by the process. Altogether our findings demonstrate that acid preservation at low temperatures can effectively stabilize seaweed biomass while preserving alginate and cellulose quality for biomaterials and other applications.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
Acid preservation, Alginate, Brown algae, Cellulose, Seaweed cultivation
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:kth:diva-326663 (URN)10.1016/j.algal.2023.103057 (DOI)000965583600001 ()2-s2.0-85151040772 (Scopus ID)
Note

QC 20230508

Available from: 2023-05-08 Created: 2023-05-08 Last updated: 2023-05-08Bibliographically approved
Rocafort, M., Srivastava, V., Bowen, J. K., Diaz-Moreno, S. M., Guo, Y., Bulone, V., . . . Mesarich, C. H. (2023). Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis. Microbiology Spectrum, 11(3)
Open this publication in new window or tab >>Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis
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2023 (English)In: Microbiology Spectrum, E-ISSN 2165-0497, Vol. 11, no 3Article in journal (Refereed) Published
Abstract [en]

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and b-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and b-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Place, publisher, year, edition, pages
American Society for Microbiology, 2023
Keywords
apple scab, cell wall, KEYWORDS Venturia inaequalis, morphological differentiation, subcuticular infection structures
National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:kth:diva-334628 (URN)10.1128/spectrum.04219-22 (DOI)000969407400001 ()37039647 (PubMedID)2-s2.0-85163913746 (Scopus ID)
Note

QC 20230823

Available from: 2023-08-23 Created: 2023-08-23 Last updated: 2023-10-06Bibliographically approved
Shao, Z., Ampomah, O., Vieira, F. R., Dorrell, R. G., Li, S., Tirichine, L., . . . Bowler, C. (2023). Characterization of a Marine Diatom Chitin Synthase Using a Combination of Meta-Omics, Genomics, and Heterologous Expression Approaches. mSystems, 8(2)
Open this publication in new window or tab >>Characterization of a Marine Diatom Chitin Synthase Using a Combination of Meta-Omics, Genomics, and Heterologous Expression Approaches
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2023 (English)In: mSystems, E-ISSN 2379-5077, Vol. 8, no 2Article in journal (Refereed) Published
Abstract [en]

b-Chitin has important ecological and physiological roles and potential for widespread applications, but the characterization of chitin-related enzymes from b-chitin producers was rarely reported. Querying against the Tara Oceans Gene Atlas, 4,939 chitin-related unique sequences from 12 Pfam accessions were found in Bacillariophyta metatranscriptomes. Putative chitin synthase (CHS) sequences are decreasingly present in Crustacea (39%), Stramenopiles (16%) and Insecta (14%) from the Marine Atlas of Tara Oceans Unigenes version 1 Metatranscriptomes (MATOUv11T) database. A CHS gene from the model diatom Thalassiosira pseudonana (Thaps3_J4413, designated TpCHS1) was identified. Homology analysis of TpCHS1 in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP), PhycoCosm, and the PLAZA diatom omics data set showed that Mediophyceae and Thalassionemales species were potential new b-chitin producers besides Thalassiosirales. TpCHS1 was overexpressed in Saccharomyces cerevisiae and Phaeodactylum tricornutum. In transgenic P. tricornutum lines, TpCHS1-eGFP localizes to the Golgi apparatus and plasma membrane and predominantly accumulates in the cleavage furrow during cell division. Enhanced TpCHS1 expression could induce abnormal cell morphology and reduce growth rates in P. tricornutum, which might be ascribed to the inhibition of the G2/M phase. S. cerevisiae was proved to be a better system for expressing large amounts of active TpCHS1, which effectively incorporates UDP-N-acetylglucosamine in radiometric in vitro assays. Our study expands the knowledge on chitin synthase taxonomic distribution in marine eukaryotic microbes, and is the first to collectively characterize an active marine diatom CHS which may play an important role during cell division.

Place, publisher, year, edition, pages
American Society for Microbiology, 2023
Keywords
chitin, chitin synthase, diatom genomes, enzymatic activity, meta-omics, subcellular localization
National Category
Biochemistry Molecular Biology Bioinformatics and Computational Biology
Identifiers
urn:nbn:se:kth:diva-338459 (URN)10.1128/msystems.01131-22 (DOI)000937373200001 ()36790195 (PubMedID)2-s2.0-85158050419 (Scopus ID)
Note

QC 20231116

Available from: 2023-11-16 Created: 2023-11-16 Last updated: 2025-02-20Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2809-4160

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