{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,19]],"date-time":"2026-07-19T01:32:47Z","timestamp":1784424767680,"version":"3.55.0"},"reference-count":32,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2022,2,2]],"date-time":"2022-02-02T00:00:00Z","timestamp":1643760000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["2021R1A4A1022206"],"award-info":[{"award-number":["2021R1A4A1022206"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotechnology Journal"],"published-print":{"date-parts":[[2022,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background and aim<\/jats:title><jats:p>Difucosyllactose (Di\u2010FL) has strong antimicrobial activity against various pathogens, including group B <jats:italic>Streptococcus<\/jats:italic>, identified as the leading cause of neonatal sepsis. In this study, we sought to develop <jats:italic>Escherichia coli<\/jats:italic> as a microbial cell factory for efficiently producing Di\u2010FL as well as 2\u2032\u2010fucosyllactose (2\u2032\u2010FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the <jats:italic>salvage<\/jats:italic> guanosine 5\u2032\u2010diphosphate (GDP)\u2010<jats:sc>l<\/jats:sc>\u2010fucose biosynthetic pathway.<\/jats:p><\/jats:sec><jats:sec><jats:title>Main methods and major results<\/jats:title><jats:p>The biosynthetic pathway for producing fucosylated oligosaccharides via the <jats:italic>salvage<\/jats:italic> pathway requires two enzymes, <jats:sc>l<\/jats:sc>\u2010fucokinase\/GDP\u2010<jats:sc>l<\/jats:sc>\u2010fucose phosphorylase (FKP) from <jats:italic>Bacteroides fragilis<\/jats:italic> and \u03b1\u20101,2\u2010fucosyltransferase (FucT2) from <jats:italic>Helicobacter pylori<\/jats:italic>. To decrease the intracellular accumulation of 2\u2032\u2010FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the \u0394<jats:italic>LFAR\u2010YA<\/jats:italic>\/FF+P\u2010PLA<jats:sub>2<\/jats:sub> strain expressing phospholipase A<jats:sub>2<\/jats:sub> (PLA<jats:sub>2<\/jats:sub>) from <jats:italic>Streptomyces violaceoruber<\/jats:italic>, whose native signal peptide was replaced with the PelB signal peptide (P\u2010PLA<jats:sub>2<\/jats:sub>), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di\u2010FL from lactose and fucose. As a result, a batch fermentation with the \u0394<jats:italic>LFAR\u2010YA<\/jats:italic>\/FF+P\u2010PLA<jats:sub>2<\/jats:sub> strain resulted in the production of 1.22\u00a0\u00b1 0.01\u00a0g\u00a0L<jats:sup>\u22121<\/jats:sup> Di\u2010FL and 0.47\u00a0\u00b1 0.01\u00a0g\u00a0L<jats:sup>\u22121<\/jats:sup> 2\u2032\u2010FL, whereas the control strain could only produce 0.65\u00a0\u00b1 0.01\u00a0g\u00a0L<jats:sup>\u22121<\/jats:sup> 2\u2032\u2010FL.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions and implications<\/jats:title><jats:p>This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and\/or products across the cell membrane is limited.<\/jats:p><\/jats:sec>","DOI":"10.1002\/biot.202100629","type":"journal-article","created":{"date-parts":[[2022,1,24]],"date-time":"2022-01-24T12:03:54Z","timestamp":1643025834000},"source":"Crossref","is-referenced-by-count":30,"title":["Simultaneous production of 2\u2032\u2010fucosyllactose and difucosyllactose by engineered <i>Escherichia coli<\/i> with high secretion efficiency"],"prefix":"10.1002","volume":"17","author":[{"given":"Hyun\u2010Jae","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Food Science and Biotechnology Chung\u2010Ang University, Anseong Gyeonggi 17546 Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong Joo","family":"Shin","sequence":"additional","affiliation":[{"name":"Department of Agricultural Biotechnology Seoul National University  Seoul 08826 Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kanghee","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Food Science and Biotechnology Chung\u2010Ang University, Anseong Gyeonggi 17546 Republic of 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