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Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras

ABSTRACT: In this study, we identified the O-glycosylation sites in deletion constructs of recombinant Clostridium botulinum flagellin (CbFla) expressed from the E. coli strain, EV136 (K1:K12 strains engineered by Prof. Eric Vimr’s group to accumulate CMP-sialic acid in the cytosol). The recombinant flagellins were expressed with or without co-expression of motility associated factor (Maf) (flagellin nonulosonic acid glycosyltransferase) in these strains, purified and subjected to MS/MS analysis at Taplin Biological Mass Spectrometry facility. We also studied the O-glycosylation of recombinant Clostridium botulinum flagellin (CbFla) co-expressed with Geobacillus kaustophilus Maf. We also studied the O-glycosylation of flagellin chimeras made by grafting mini-flagellin sequences on to an unrelated protein with an F-type lectin domain from Streptosporangium roseum SrNaFLD.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Clostridium Botulinum Str. Langelan F

SUBMITTER: Ramya Chakravarthy

LAB HEAD: Ramya Chakravarthy

PROVIDER: PXD044191 | Pride | 2024-08-22

REPOSITORIES: Pride

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Project description:Irani2015 - Genome-scale metabolic model of P.pastoris N-glycosylation This model is described in the article: Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins. Irani ZA, Kerkhoven E, Shojaosadati SA, Nielsen J. Biotechnol. Bioeng. 2015 Oct; Abstract: Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better predictions of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. This article is protected by copyright. All rights reserved. This model is hosted on BioModels Database and identified by: MODEL1510220000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:The members of the genus Clostridium, such as the other spore-forming anaerobic bacteria, have a complex and strictly regulated life-cycle but very little is known about genetic pathways involved at different stages. Clostridium sporogenes, Gram positive bacterium usually involved in food spoilage and frequently isolated from late blowed cheese is genetically indistinguishable from proteolytic Clostridium botulinum and is the non-neurotoxigenic counterpart often used as exemplar for the toxic subtypes. In this work we have performed a microscopy study combined with a custom array-based analysis of C. sporogenes cycle, from dormant spores to early stationary phase. We identified in spores a total of 211 transcripts validating the ipothesis that mRNAs are abundant and strikingly different from those present in growing cells. The spores transcripts included genes responsible of different life-sustaining functions, suggesting the theory of transcripts entrapment or of a basic poly-functional genes activation for future steps. In addition, after three hours from the beginning of the germination process, a 20% of the total up-regulated genes were temporally expressed in germinating spores. The vegetative condition appears to be the more active in terms of gene transcription and protein synthesis and also genes for germination and sporulation factors seem to be expressed at this point. These results suggest that spores are not silent entities and a wider knowledge about genetic pathways involved in Clostridium life cycle could be of help for a better understanding also of pathogenic clostridia types. RNA was isolated from spores, germinating spores (3h), outgrowth/transitional mid-log vegetative cells and early stationary cells of Clostridium sporogenes UC9000 previously isolated from milk. There were 3 biological replicates (independent cultures) for each condition. Probes corresponding to 3400 genomic CDSs of C. botulinum A ATCC 3502 were synthesized in three replicates randomly distributed on the chip.

Project description:The two-component system CBO0366/CBO0365 was recently demonstrated to have a role in cold tolerance of Group I Clostridium botulinum ATCC 3502. The mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. A transcriptomic analysis with DNA microarrays was performed to identify the differences in global gene expression patterns of the wild-type ATCC 3502 and a derivative mutant with insertionally inactivated cbo0365 at 37 M-BM-0C and 15 M-BM-0C. Altogether 150 or 141 chromosomal CDSs were found to be differently expressed in the cbo0365 mutant at 37 M-BM-0C or 15 M-BM-0C, respectively, and thus considered to be under direct or indirect transcriptional control of the response regulator CBO0365. Of the differentially-expressed CDSs, expression of 141 CDSs was similarly affected at both temperatures investigated, suggesting that the putative CBO0365 regulon was practically not affected by temperature. The regulon involved genes related to acetone-butanol-ethanol (ABE) fermentation, motility, to arsenic resistance, and phosphate uptake and transport. Deteriorated growth at 17 M-BM-0C was observed for mutants with disrupted ABE fermentation pathway components (crt, bdh and ctfA), arsenic detoxifying machinery components (arsC and arsR), or phosphate uptake mechanism components (phoT), suggesting roles for these mechanisms in cold tolerance of Group I C. botulinum. Electrophoretic mobility shift assays showed recombinant CBO0365 to bind to the promoter regions of crt, arsR, and phoT, as well as to the promoter region of its own operon, suggesting direct DNA-binding transcriptional activation or repression as means for CBO0365 in regulating these operons. The results provide insight to the mechanisms Group I C. botulinum utilize in coping with cold. C. botulinum ATCC 3502 cbo0365 mutant vs. wild type; 3 replicates of each strain; growth at 37C in TPGY broth batch culture and subjected to cold shock to 15C; sampling at mid-log growth phase before cold shock, and 1 h after temperature downshift to 15C (= 2 time points). Dye-swapped hybridization.

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	64809_63291.mzXML	Mzxml
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Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras

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