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The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-β-D-glucans
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan..ORCID iD: 0000-0001-8716-8196
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan..
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.ORCID iD: 0000-0002-3369-2440
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1Article in journal (Refereed) Published
Abstract [en]

(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

Place, publisher, year, edition, pages
Springer Nature , 2023. Vol. 14, no 1
National Category
Microbiology Biochemistry and Molecular Biology Structural Biology
Identifiers
URN: urn:nbn:se:kth:diva-333209DOI: 10.1038/s41467-023-40214-zISI: 001038888100004PubMedID: 37500617Scopus ID: 2-s2.0-85165916371OAI: oai:DiVA.org:kth-333209DiVA, id: diva2:1784617
Note

QC 20230731

Available from: 2023-07-28 Created: 2023-07-28 Last updated: 2023-11-03Bibliographically approved
In thesis
1. Biosynthesis of bacterial polysaccharides by novel glycosyltransferase enzymes
Open this publication in new window or tab >>Biosynthesis of bacterial polysaccharides by novel glycosyltransferase enzymes
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Within the intricate microbiological realm, exopolysaccharides (EPS) emerge as a significant class of high molecular weight polysaccharides, serving pivotal roles in bacterial survival, virulence, communication, and defense against environmental adversities. The versatile nature of bacterial EPS extends beyond mere biological functions, reaching into medications, cosmetics, functional food, and sustainable industries. Although EPS is a vital and much-exploited class of polysaccharide, their biosynthesis remain less explored or understood. This thesis delves into the exploration of enzymes integral to EPS synthesis, focusing specifically on the characterization of putative membrane-bound enzymes from the so-called glycosyltransferase family 2 (GT2). The initial investigations of the research are centered on (1,3;1,4)-β-D-glucans, polymers with mixed linkage backbones that are widely distributed across various biological systems. Despite their prevalence, collective understanding of the biosynthesis of these polymers in the bacterial domain, particularly in gram-positive strains, remains limited. Through extensive research, distinct genes encoding (1,3;1,4)-β-D-glucan synthases were identified in two gram-positive bacteria: Romboutsia ilealis and Clostridium ventriculi. A gain-of-function approach was employed and provided conclusive evidence of the synthase activity of these identified genes. Subsequently, the thesis shifts focus to Chitinophaga pinensis, a gram-negative bacterium with roles in maintaining ecosystem balance through its proficiency in carbohydrate breakdown and recycling. The exploration led to the discovery of an uncommon GT2 β-glucan synthase with activity in curdlan synthesis. Several unusual features of the C. pinensis enzyme highlight the extensive diversity and nuances within the GT2 polysaccharide synthase family, particularly the fact that such catalysts sometimes have close connections with carbohydrate-degrading enzymes. Characterization of these putative GT2 proteins was verified by a variety of techniques, including gene expression in E. coli and yeast host systems, enzyme-coupled oligosaccharide profiling, and in vitro radiometric activity assays. To advance the investigation, bioinformatics tools such as protein alignment, phylogenetic analysis, and model structure analysis were employed.

Abstract [sv]

Inom det komplexa mikrobiologiska området framträder exopolysackarider (EPS) som en betydande klass av högmolekylära polysackarider. De spelar centrala roller inom bakteriers överlevnad, virulens, kommunikation och försvar mot miljömässiga motgångar. Den mångsidiga naturen hos bakteriell EPS sträcker sig bortom enbart biologiska funktioner och når in i läkemedel, kosmetika, funktionell mat och hållbara industrier. Även om EPS är en vital och ofta utnyttjad klass av polysackarider, är deras biosyntes mindre utforskad eller förstådd. Denna avhandling fördjupar sig i utforskningen utav enzymer som är centrala för EPS-syntes, med särskilt fokus på karaktäriseringen av tänkta membranbundna enzymer från den så kallade glykosyltransferasfamiljen 2 (GT2). De inledande undersökningarna av forskningen kretsar kring (1,3;1,4)-β-D-glukaner, polymerer med blandade länkryggar som är allmänt fördelade över olika biologiska system. Trots deras förekomst är den kollektiva förståelsen för biosyntesen av dessa polymerer inom bakteriedomänen, särskilt i gram-positiva stammar, begränsad. Genom omfattande forskning identifierades specifika gener som kodar för (1,3;1,4)-β-D-glukansyntaser i två gram-positiva bakterier: Romboutsia ilealis och Clostridium ventriculi. Ett tillvägagångssätt för att öka funktionen användes och gav definitiva bevis för syntasaktiviteten hos dessa identifierade gener. Avhandlingen skiftar därefter fokus till Chitinophaga pinensis, en gram-negativ bakterie som spelar en roll i att upprätthålla ekosystembalansen genom sin skicklighet i nedbrytning och återvinning av kolhydrater. Utforskningen ledde till upptäckten av ett ovanlig GT2 β-glukansyntas med aktivitet inom curdlan-syntes. Flera ovanliga egenskaper hos C. pinensis-enzymet framhäver den omfattande mångfalden och nyanserna inom GT2 polysackaridsyntasfamiljen, särskilt det faktum att sådana katalysatorer ibland har nära kopplingar med kolhydratnedbrytande enzymer. Karaktärisering av dessa tänkta GT2-proteiner verifierades med hjälp av en mängd olika tekniker, inklusive genuttryck i E. coli och jästvärdssystem, enzymkopplad oligosackaridprofilering och in vitro radiometriska aktivitetsanalyser. För att fördjupa undersökningen användes bioinformatikverktyg såsom proteinjustering, fylogenetisk analys och modellstrukturanalys.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2023. p. 83
Series
TRITA-CBH-FOU ; 2023:54
National Category
Biological Sciences Microbiology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-339177 (URN)978-91-8040-765-6 (ISBN)
Public defence
2023-12-15, Kollegiesalen, Brinellvägen 6, via Zoom: https://kth-se.zoom.us/j/61815154778, Stockholm, 10:00 (English)
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Note

QC 2023-11-06

Available from: 2023-11-06 Created: 2023-11-03 Last updated: 2023-11-09Bibliographically approved

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Chang, Shu-ChiehKao, Mu-RongSaldivar, Rebecka KarmakarDiaz-Moreno, Sara MFurlanetto, ValentinaYayo, JohannesDivne, ChristinaVilaplana, FranciscoHsieh, Yves S. Y.

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Chang, Shu-ChiehKao, Mu-RongSaldivar, Rebecka KarmakarDiaz-Moreno, Sara MFurlanetto, ValentinaYayo, JohannesDivne, ChristinaVilaplana, FranciscoHsieh, Yves S. Y.
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