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Its microbial de novo biosynthesis is severely limited by the low catalytic efficiency of O-methyltransferase. Here, we constructed a V314I mutant of caffeate O-methyltransferase via structure-guided protein engineering, which showed a 95% increase in catalytic efficiency toward 3,4-dihydroxybenzyl alcohol in Escherichia coli. Molecular simulations revealed that the mutation narrows the active-site cavity, constraining the smaller substrate into a catalytically competent orientation. To alleviate growth inhibition caused by 3,4-dihydroxybenzaldehyde accumulation, alcohol dehydrogenase gene ADH6 was introduced into the host. Moreover, S-Adenosylmethionine supply was enhanced by genomic integration of luxS-mtn and methionine supplementation. Finally, a VNA titer of 6.21 g\/L was achieved in a 3 L fermenter from glycerol. This work establishes a VNA biomanufacturing platform and demonstrates an integrative strategy for the microbial production of methylated aromatic natural products, combining enzyme active-site spatial engineering and pathway engineering.<\/jats:p>","DOI":"10.1021\/acssynbio.6c00309","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:02:27Z","timestamp":1790179347000},"page":"4112-4123","source":"Crossref","is-referenced-by-count":0,"title":["Efficient Production of Vanillyl Alcohol in\n                    <i>Escherichia coli<\/i>\n                    via Protein and Pathway Engineering"],"prefix":"10.1021","volume":"15","author":[{"given":"Jinyi","family":"Li","sequence":"first","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Du","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7218-2224","authenticated-orcid":true,"given":"Jia","family":"Wang","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6969-2952","authenticated-orcid":true,"given":"Xiaolin","family":"Shen","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4799-882X","authenticated-orcid":true,"given":"Hao","family":"Liang","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5590-6473","authenticated-orcid":true,"given":"Xinxiao","family":"Sun","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qipeng","family":"Yuan","sequence":"additional","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]},{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, China, 100029"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"issue":"5\u22126","key":"2026101610063865200_cit1","doi-asserted-by":"publisher","first-page":"317","DOI":"10.1007\/bf02491665","article-title":"Determination of gastrodin, p-hydroxybenzyl alcohol, vanillyl alcohol, p-hydroxylbenzaldehyde and vanillin in all Gastrodia tuber by high-performance liquid chromatography","volume":"55","author":"Liu","year":"2002","journal-title":"Chromatographia"},{"issue":"13","key":"2026101610063865200_cit2","doi-asserted-by":"publisher","first-page":"2130","DOI":"10.1002\/jssc.200700002","article-title":"Determination of gastrodin and vanillyl alcohol in Gastrodia elata Blume by pressurized liquid extraction at room temperature","volume":"30","author":"Ong","year":"2007","journal-title":"J. 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