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This study engineered an HMB biosynthetic pathway in the probiotic Escherichia coli Nissle 1917 (EcN). The initial engineered strain exhibited low HMB titers, as carbon flux was primarily diverted toward acetate formation. Deleting the pyruvate oxidase gene (poxB) reduced acetate but caused significant pyruvate accumulation, revealing a severe kinetic bottleneck in endogenous pyruvate dehydrogenase (PDH) activity. Supplementing pantothenic acid (Vitamin B5) profoundly expanded the intracellular coenzyme A (CoA) pool, providing the essential backbone for pathway intermediates. This strategy enhanced PDH flux and improved HMB production by 5.5-fold. Through fed-batch fermentation, the engineered strain achieved an HMB titer of 8.6 g\/L with a yield of 0.14 g\/g glucose. The results of this study provided a probiotic strain that can synthesize HMB de novo and demonstrated the importance of pantothenic acid availability for EcN metabolic engineering.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c08735","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T14:02:09Z","timestamp":1788876129000},"page":"29437-29446","source":"Crossref","is-referenced-by-count":0,"title":["Biosynthesis of\n3-Hydroxy-3-methylbutyrate\nby Probiotic\n                    <i>Escherichia coli<\/i>\n                    Nissle\n1917"],"prefix":"10.1021","volume":"74","author":[{"given":"Jenny Ji-Chan","family":"Hung","sequence":"first","affiliation":[{"name":"National Yang Ming Chiao Tung University , , , ,","place":["Hsinchu City, Taiwan, 300"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tina Wen-Ting","family":"Shih","sequence":"additional","affiliation":[{"name":"National Yang Ming Chiao Tung University , , , ,","place":["Hsinchu City, Taiwan, 300"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alice Pin-Yi","family":"Du","sequence":"additional","affiliation":[{"name":"National Yang Ming Chiao Tung University , , , ,","place":["Hsinchu City, Taiwan, 300"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6658-3451","authenticated-orcid":true,"given":"Ethan I.","family":"Lan","sequence":"additional","affiliation":[{"name":"National Yang Ming Chiao Tung University , , , ,","place":["Hsinchu City, Taiwan, 300"]},{"name":"National Yang Ming Chiao Tung University , , , ,","place":["Hsinchu City, Taiwan, 300"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"2026092311093398500_cit1","volume-title":"World social report 2023: Leaving\nno one behind in an ageing world","author":"Wilmoth","year":"2023"},{"issue":"4","key":"2026092311093398500_cit2","doi-asserted-by":"publisher","first-page":"437","DOI":"10.1016\/j.cell.2005.01.027","article-title":"Understanding\nthe Odd Science of Aging","volume":"120","author":"Kirkwood","year":"2005","journal-title":"Cell"},{"key":"2026092311093398500_cit3","doi-asserted-by":"publisher","DOI":"10.1016\/j.metabol.2023.155533","article-title":"Epidemiology\nof sarcopenia: Prevalence, risk factors,\nand consequences","volume":"144","author":"Yuan","year":"2023","journal-title":"Metabolism"},{"issue":"4","key":"2026092311093398500_cit4","doi-asserted-by":"publisher","first-page":"412","DOI":"10.1093\/ageing\/afq034","article-title":"Sarcopenia: European\nconsensus on definition and diagnosis:\nReport of the European Working Group on Sarcopenia in Older People","volume":"39","author":"Cruz-Jentoft","year":"2010","journal-title":"Age Ageing"},{"key":"2026092311093398500_cit5","doi-asserted-by":"publisher","first-page":"93","DOI":"10.14283\/jfa.2019.10","article-title":"Economic\nimpact of hospitalizations in US adults with sarcopenia","volume":"8","author":"Goates","year":"2019","journal-title":"J. Frailty Aging"},{"issue":"18","key":"2026092311093398500_cit6","doi-asserted-by":"publisher","DOI":"10.3346\/jkms.2022.37.e146","article-title":"A Review of Sarcopenia Pathophysiology, Diagnosis,\nTreatment and Future Direction","volume":"37","author":"Cho","year":"2022","journal-title":"J. Korean Med.\nSci."},{"issue":"11","key":"2026092311093398500_cit7","doi-asserted-by":"publisher","first-page":"833","DOI":"10.1007\/s40266-017-0496-0","article-title":"The potential\nof \u03b2-hydroxy-\u03b2-methylbutyrate as a new strategy for the\nmanagement of sarcopenia and sarcopenic obesity","volume":"34","author":"Rossi","year":"2017","journal-title":"Drugs Aging"},{"issue":"1","key":"2026092311093398500_cit8","doi-asserted-by":"publisher","first-page":"8","DOI":"10.33879\/AMH.151.2023.11118","article-title":"Advancing sarcopenia diagnosis and treatment: Recommendations\nfrom the Taiwan Advisory Panel for Sarcopenia","volume":"15","author":"Peng","year":"2024","journal-title":"Aging Med. 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Metab."},{"issue":"2","key":"2026092311093398500_cit13","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1007\/s12603-018-1153-y","article-title":"The effect of \u03b2-hydroxy-\u03b2-methylbutyrate\n(HMB) on sarcopenia and functional frailty in older persons: a systematic\nreview","volume":"23","author":"Oktaviana","year":"2019","journal-title":"J. Nutr., Health Aging"},{"key":"2026092311093398500_cit14","unstructured":"Fact.MR, P.\n          \n          Hydroxymethylybutyrate\n(HMB) Market to Surpass US$ 90 Mn through 2030. 1997https:\/\/www.factmr.com\/media-release\/1526\/global-hydroxymethylbutyrate-hmb-market."},{"key":"2026092311093398500_cit15","unstructured":"Abbott, N.\n          \n          Beta-hydroxy beta-methylbutyrate\n(HMB). 2024."},{"issue":"11","key":"2026092311093398500_cit16","doi-asserted-by":"publisher","first-page":"2882","DOI":"10.1021\/ja01544a072","article-title":"Syntheses by Free-radical Reactions.\nV. 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Microbiol."},{"key":"2026092311093398500_cit19","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1007\/s002030050569","article-title":"Enzyme analyses\ndemonstrate that \u03b2-methylbutyric\nacid is converted to \u03b2-hydroxy-\u03b2-methylbutyric acid via\nthe leucine catabolic pathway by Galactomyces reessii","volume":"169","author":"Lee","year":"1998","journal-title":"Arch. Microbiol."},{"issue":"12","key":"2026092311093398500_cit20","doi-asserted-by":"publisher","first-page":"3712","DOI":"10.1021\/acs.jafc.1c00494","article-title":"Biosynthesis of 3-hydroxy-3-methylbutyrate from l-leucine by whole-cell\ncatalysis","volume":"69","author":"Gao","year":"2021","journal-title":"J. Agric. 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Bioeng."},{"issue":"1","key":"2026092311093398500_cit46","doi-asserted-by":"publisher","DOI":"10.1002\/biot.202300327","article-title":"Expanding the toolbox\nof probiotic Escherichia coli Nissle\n1917 for synthetic biology","volume":"19","author":"Ba","year":"2024","journal-title":"Biotechnol.\nJ."},{"issue":"13","key":"2026092311093398500_cit47","doi-asserted-by":"publisher","first-page":"7318","DOI":"10.1021\/acs.jafc.4c01075","article-title":"Development of the\nlow-temperature inducible system for recombinant protein production\nin Escherichia coli nissle 1917","volume":"72","author":"Lin","year":"2024","journal-title":"J. Agric. Food Chem."},{"issue":"15","key":"2026092311093398500_cit48","doi-asserted-by":"publisher","first-page":"8693","DOI":"10.1021\/acs.jafc.4c00827","article-title":"Constructing an Antibiotic-Free\nprotein expression system for ovalbumin\nbiosynthesis in probiotic Escherichia coli Nissle 1917","volume":"72","author":"Liu","year":"2024","journal-title":"J. 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