Yanna André , Emeline Richard , Mélanie Leroux , Isabelle Jeacomine , Eric Bayma , Sylvie Armand , Bernard Priem
{"title":"在重组大肠杆菌中生产未硫酸化的软骨素和相关软骨寡糖","authors":"Yanna André , Emeline Richard , Mélanie Leroux , Isabelle Jeacomine , Eric Bayma , Sylvie Armand , Bernard Priem","doi":"10.1016/j.carres.2024.109243","DOIUrl":null,"url":null,"abstract":"<div><p>We designed metabolically engineered non-pathogenic strains of <em>Escherichia coli</em> to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from <em>Pedobacter heparinus</em>. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-<em>threo</em>-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major “even-numbered” series was characterized by a terminal reducing <em>N</em>-acetylgalactosaminyl residue. The minor “odd-numbered” series oligosaccharides carried a terminal reducing glucuronic acid residue instead.</p><p>Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L<sup>−1</sup> chondroitin or chondroitin oligosaccharides.</p><p>All products were purified and fully characterized using NMR and mass spectrometry analyses.</p><p>This is the first report of the microbial production of large chondro-oligosaccharides.</p></div>","PeriodicalId":9415,"journal":{"name":"Carbohydrate Research","volume":"544 ","pages":"Article 109243"},"PeriodicalIF":2.4000,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0008621524002222/pdfft?md5=72d24042146c3bbaed29a40488635760&pid=1-s2.0-S0008621524002222-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Production of unsulfated chondroitin and associated chondro-oligosaccharides in recombinant Escherichia coli\",\"authors\":\"Yanna André , Emeline Richard , Mélanie Leroux , Isabelle Jeacomine , Eric Bayma , Sylvie Armand , Bernard Priem\",\"doi\":\"10.1016/j.carres.2024.109243\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>We designed metabolically engineered non-pathogenic strains of <em>Escherichia coli</em> to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from <em>Pedobacter heparinus</em>. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-<em>threo</em>-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major “even-numbered” series was characterized by a terminal reducing <em>N</em>-acetylgalactosaminyl residue. The minor “odd-numbered” series oligosaccharides carried a terminal reducing glucuronic acid residue instead.</p><p>Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L<sup>−1</sup> chondroitin or chondroitin oligosaccharides.</p><p>All products were purified and fully characterized using NMR and mass spectrometry analyses.</p><p>This is the first report of the microbial production of large chondro-oligosaccharides.</p></div>\",\"PeriodicalId\":9415,\"journal\":{\"name\":\"Carbohydrate Research\",\"volume\":\"544 \",\"pages\":\"Article 109243\"},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-08-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0008621524002222/pdfft?md5=72d24042146c3bbaed29a40488635760&pid=1-s2.0-S0008621524002222-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Carbohydrate Research\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0008621524002222\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Carbohydrate Research","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0008621524002222","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Production of unsulfated chondroitin and associated chondro-oligosaccharides in recombinant Escherichia coli
We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major “even-numbered” series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor “odd-numbered” series oligosaccharides carried a terminal reducing glucuronic acid residue instead.
Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L−1 chondroitin or chondroitin oligosaccharides.
All products were purified and fully characterized using NMR and mass spectrometry analyses.
This is the first report of the microbial production of large chondro-oligosaccharides.
期刊介绍：
Carbohydrate Research publishes reports of original research in the following areas of carbohydrate science: action of enzymes, analytical chemistry, biochemistry (biosynthesis, degradation, structural and functional biochemistry, conformation, molecular recognition, enzyme mechanisms, carbohydrate-processing enzymes, including glycosidases and glycosyltransferases), chemical synthesis, isolation of natural products, physicochemical studies, reactions and their mechanisms, the study of structures and stereochemistry, and technological aspects.
Papers on polysaccharides should have a "molecular" component; that is a paper on new or modified polysaccharides should include structural information and characterization in addition to the usual studies of rheological properties and the like. A paper on a new, naturally occurring polysaccharide should include structural information, defining monosaccharide components and linkage sequence.
Papers devoted wholly or partly to X-ray crystallographic studies, or to computational aspects (molecular mechanics or molecular orbital calculations, simulations via molecular dynamics), will be considered if they meet certain criteria. For computational papers the requirements are that the methods used be specified in sufficient detail to permit replication of the results, and that the conclusions be shown to have relevance to experimental observations - the authors'' own data or data from the literature. Specific directions for the presentation of X-ray data are given below under Results and "discussion".
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
