Xyloglucan Oligosaccharides Hydrolysis by Exo-Acting Glycoside Hydrolases from Hyperthermophilic Microorganism Saccharolobus solfataricus

Curci, Nicola; Strazzulli, Andrea; Iacono, Roberta; Lise, Federica De; Maurelli, Luisa; Fenza, Mauro Di; Cobucci‐Ponzano, Beatrice; Moracci, Marco

doi:10.3390/ijms22073325

Cited by 12 publications

(5 citation statements)

References 49 publications

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“…In addition, we have previously reported that in S. solfataricus the α-xylosidase XylS and the β-glycosidase Ssβ-gly, hydrolyzed tamarind seed xyloglucan oligosaccharides in vitro [48]. More recently, we observed that the α-L-fucosidase is able to remove the fucose residues from fucosylated xyloglucan oligosaccharides [59], suggesting that the three enzymes cooperate for the hydrolysis of xyloglucan oligosaccharides [17] like it has been suggested in T. maritima [49].…”

Section: Discussionsupporting

confidence: 58%

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo

et al. 2021

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Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

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Section: Discussionsupporting

confidence: 58%

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo

et al. 2021

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“…Jonuscheit et al, 2003; Wagner et al, 2012), including promoter investigations (Berkner et al, 2007; 2010; Peng et al, 2012; van der Kolk et al, 2020) or analyses of oligosaccharide hydrolysis (e.g. Curci et al, 2021). In contrast to esterase Saci_1116, LacS exhibits substantial expression levels even without 5’-UTR.…”

Section: Resultsmentioning

confidence: 99%

5’-untranslated region sequences enhance plasmid-based protein production inSulfolobus acidocaldarius

Kuschmierz,

Wagner,

Busche

et al. 2024

Preprint

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Sulfolobus acidocaldarius, a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of thesaci_2122promoter (Para), providing low basal activity and high pentose-dependent induction. However, available expression vectors lack a 5′-terminal untranslated region (5′-UTR), which is a typical element in bacterial expression vectors, usually significantly enhancing protein production in bacteria. To establishS. acidocaldariusas a production strain in biotechnology in the long-term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration ofS. acidocaldarius5′-UTRs into Paraexpression plasmids. Using the esterase Saci_1116 as a reporter protein, we observed a fourfold increase in soluble and active protein yield upon insertion of thesaci_1322(alba) 5′-UTR. Screening of four additional 5′-UTRs from other highly abundant proteins (thα, slaA, slaB, saci_0330) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within thealba5′-UTR revealed its significance for protein synthesis. Ultimately, thealba5′-UTR optimized expression vector demonstrated successful applicability in expressing various proteins, exemplified by its utilization for archaeal glycosyltransferases. Our results demonstrate that the integration of SD-motif containing 5′-UTRs significantly boosted plasmid-based protein production inS. acidocaldarius. This advancement in recombinant expression not only broadens the utility ofS. acidocaldariusas an archaeal expression platform but also marks a significant step toward potential biotechnological applications.

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“…Consistent with this, fucosidases belonging to clusters 2, 3, and 4 have been reported to have promiscuous activities for α1,2/3/4/6 fucosyl linkages (Table S1 ). Fucosidases in clusters 2 and 3 have been reported to release Fuc from xyloglucans 8 , 14 , 34 , 35 . Cluster 2 also contained the newly found exo-α- l -galactosidase BpGH29 from Bacteroides plebeius DSM 17135 36 .…”

Section: Resultsmentioning

confidence: 99%

Exploring the sequence-function space of microbial fucosidases

Martínez Gascueña,

Wu,

Wang

et al. 2024

Commun Chem

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Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages and can perform transglycosylation reactions. Based on sequence identity, α-l-fucosidases are classified in glycoside hydrolases (GHs) families of the carbohydrate-active enzyme database. Here we explored the sequence-function space of GH29 fucosidases. Based on sequence similarity network (SSN) analyses, 15 GH29 α-l-fucosidases were selected for functional characterisation. HPAEC-PAD and LC-FD-MS/MS analyses revealed substrate and linkage specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated oligosaccharides and glycoconjugates, consistent with their SSN clustering. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1,6 linkages and FA2G2 N-glycan was determined by X-ray crystallography and STD NMR. The capacity of GH29 fucosidases to carry out transfucosylation reactions with GlcNAc and 3FN as acceptors was evaluated by TLC combined with ESI–MS and NMR. These experimental data supported the use of SSN to further explore the GH29 sequence-function space through machine-learning models. Our lightweight protein language models could accurately allocate test sequences in their respective SSN clusters and assign 34,258 non-redundant GH29 sequences into SSN clusters. It is expected that the combination of these computational approaches will be used in the future for the identification of novel GHs with desired specificities.

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Xyloglucan Oligosaccharides Hydrolysis by Exo-Acting Glycoside Hydrolases from Hyperthermophilic Microorganism Saccharolobus solfataricus

Cited by 12 publications

References 49 publications

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo

5’-untranslated region sequences enhance plasmid-based protein production inSulfolobus acidocaldarius

Exploring the sequence-function space of microbial fucosidases

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