Capsular Polysaccharide Biosynthesis from Recombinant E. coli and Chondroitin Sülfate Production

2019 ◽  
Vol 65 (6) ◽  
pp. 17
Author(s):  
Ayse Sebnem Erenler
Keyword(s):  
Chondroitin Sulfate ◽  
Capsular Polysaccharide ◽  
Polysaccharide Biosynthesis ◽  
E Coli ◽  
Sulfate Production

Cloning and Expression vgb-kfo Genes in E. coli and Microbial Chondroitin Sulfate Production

2019 ◽  
Vol 11 (12) ◽  
pp. 1745-1754
Author(s):  
Ayse Sebnem Erenler ◽  
Hikmet Geckil ◽  
Aysun Bay Karabulut ◽  
Nusret Akpolat ◽  
Resit Sevimli ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Research Literature ◽  
Cloning And Expression ◽  
Limited Area ◽  
E Coli ◽  
Sulfate Production ◽  
Hemoglobin Gene ◽  
Drug Potency ◽  
First Time ◽  
Published Research

This study focused on a new plasmid and new recombinant strain developed for the production of microbial chondroitin sulfate a new and limited area of study the strategies we applied for the production of microbial chondroitin sulfate, and the possible contributions of this study to published research literature. In this study, pETM6-PACF, which carries the genes responsible for capsular chondroitin synthesis [kfA, kfoC, kfoF] was used as the basic plasmid. The Vitreoscilla hemoglobin gene region was transformed into this basic plasmid and the common expression of both gene groups was added to research literature for the first time. This plasmid was transferred to non-pathogenic E. coli (C2987) to produce a completely new chondroitin source specific to this study. Following the transformation by chondroitin synthesis, and the subsequent microbial production of chondroitin by the application of purification protocols, microbial chondroitin sulfate was produced in sulfate form. Consequently, in comparison to published literature, a product with a low molecular weight value of 269 Daltons was developed. This product, which has significant potential drug potency, can be used in many different areas as a novel and unique biomedical product.

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

scholarly journals Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Sheila Patrick ◽  
Simon Houston ◽  
Zubin Thacker ◽  
Garry W. Blakely
Keyword(s):  
Capsular Polysaccharide ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Phase Variable ◽  
Multiple Gene ◽  
Polysaccharide Biosynthesis ◽  
Group 1

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

scholarly journals Identification and Characterization of thecps Locus of Streptococcus suis Serotype 2: the Capsule Protects against Phagocytosis and Is an Important Virulence Factor

1999 ◽  
Vol 67 (4) ◽  
pp. 1750-1756 ◽  
Author(s):  
Hilde E. Smith ◽  
Marloes Damman ◽  
Joeke van der Velde ◽  
Frans Wagenaar ◽  
Henk J. Wisselink ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Capsular Polysaccharide ◽  
Streptococcus Suis ◽  
Open Reading Frames ◽  
Transcriptional Unit ◽  
Polysaccharide Biosynthesis ◽  
Capsule Production ◽  
Important Virulence Factor ◽  
Isogenic Mutants

ABSTRACT To study the role of the capsule of Streptococcus suisserotype 2 in virulence, we generated two isogenic mutants disturbed in capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis (cps) locus of S. suis serotype 2. Based on the established sequence, 14 open reading frames (ORFs), designated Orf2Z, Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged to a single transcriptional unit. The gene products of 11 of these ORFs showed similarity to proteins involved in polysaccharide biosynthesis of other gram-positive microorganisms. Nonencapsulated isogenic mutants were generated in the cps2B and cps2EF genes by insertional mutagenesis. In contrast to the wild-type S. suis serotype 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro. More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These observations indicate that the capsule of S. suis serotype 2 plays an essential role in the pathogenesis of S. suisserotype 2 infections.

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