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However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate\n                    <jats:italic>N<\/jats:italic>\n                    -deacetylase\/\n                    <jats:italic>N<\/jats:italic>\n                    -sulfotransferase. Here, we introduce the monosaccharide\n                    <jats:italic>N<\/jats:italic>\n                    -trifluoroacetylglucosamine into\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type\n                    <jats:italic>N<\/jats:italic>\n                    -sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU\/mg) and anti-FIIa (48.62 IU\/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.\n                  <\/jats:p>","DOI":"10.1038\/s41467-024-48193-5","type":"journal-article","created":{"date-parts":[[2024,5,4]],"date-time":"2024-05-04T06:01:47Z","timestamp":1714802507000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies"],"prefix":"10.1038","volume":"15","author":[{"given":"Jian-Qun","family":"Deng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Jia","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ya-Lin","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si-Yu","family":"Xin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin-Yu","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui-Min","family":"Xi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng-Shan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2856-9162","authenticated-orcid":false,"given":"Ju-Zheng","family":"Sheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,5,4]]},"reference":[{"key":"48193_CR1","doi-asserted-by":"publisher","first-page":"eaan5954","DOI":"10.1126\/scitranslmed.aan5954","volume":"9","author":"Y Xu","year":"2017","unstructured":"Xu, Y. et al. 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