{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:24:18Z","timestamp":1785435858898,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,24]]},"group-title":"Synthetic Biology","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T00:00:00Z","timestamp":1784851200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R21EB033892"],"award-info":[{"award-number":["R21EB033892"]}],"id":[{"id":"https:\/\/ror.org\/00372qc85","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Propel a Cure Foundation"},{"name":"Mendez Family Foundation"},{"award":["1938052"],"award-info":[{"award-number":["1938052"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,24]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Hydrogen sulfide (H\n                  <jats:sub>2<\/jats:sub>\n                  S) is a microbiota-derived metabolite in the gastrointestinal tract implicated in a number of diseases. Its volatility and reactivity make experimentally controlling H\n                  <jats:sub>2<\/jats:sub>\n                  S concentration\n                  <jats:italic>in vivo<\/jats:italic>\n                  difficult, limiting our ability to interrogate its dose-dependent effects on host physiology. Engineered bacteria present a compelling solution, yet most probiotic metabolic engineering approaches have focused on\n                  <jats:italic>in vitro<\/jats:italic>\n                  optimization, failing to account for the complex intestinal environment. Here, we engineered\n                  <jats:italic>Escherichia coli<\/jats:italic>\n                  strains to produce or consume H\n                  <jats:sub>2<\/jats:sub>\n                  S in specific intestinal regions by incorporating knowledge of the local metabolic environment and resident microbial activities into the design process. Analysis of human-derived\n                  <jats:italic>ex vivo<\/jats:italic>\n                  cultures revealed that glutathione (GSH) is inefficiently converted to H\n                  <jats:sub>2<\/jats:sub>\n                  S, suggesting GSH as a relatively stable substrate for engineered sulfide production. We thus engineered a GSH-dependent H\n                  <jats:sub>2<\/jats:sub>\n                  S producer, which increased levels 21-fold\n                  <jats:italic>ex vivo<\/jats:italic>\n                  . To target the nutrient-rich, microbially sparse environment of the small intestine, we optimized a H\n                  <jats:sub>2<\/jats:sub>\n                  S producer that uses L-cysteine as a sulfur source, demonstrating a 7-fold increase in H\n                  <jats:sub>2<\/jats:sub>\n                  S levels in mice. Finally, to develop strains capable of sequestering H\n                  <jats:sub>2<\/jats:sub>\n                  S, we leveraged the availability of fumarate and nitrate as electron acceptors in the large intestine by engineering a strain expressing sulfide:quinone oxidoreductase (Sqr). This enables oxidation of H\n                  <jats:sub>2<\/jats:sub>\n                  S to intracellular polysulfides and achieves higher consumption rates than alternative sequestration strategies reliant on resource-intensive GSH production. Together, this work developed engineered microbes as precision tools to modulate H\n                  <jats:sub>2<\/jats:sub>\n                  S levels and showcases a generalizable framework for region-targeted design of engineered probiotics.\n                <\/jats:p>","DOI":"10.64898\/2026.07.23.738723","type":"posted-content","created":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T13:35:12Z","timestamp":1784900112000},"source":"Crossref","is-referenced-by-count":0,"title":["Metabolic engineering of\n                  <i>Escherichia coli<\/i>\n                  to modulate hydrogen sulfide levels in the mammalian gut"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2531-8333","authenticated-orcid":false,"given":"Justin A.","family":"Hayes","sequence":"first","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8871-1151","authenticated-orcid":false,"given":"Brent","family":"Buchinger","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna W.","family":"Lunger","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanvi","family":"Khanvilkar","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6499-4831","authenticated-orcid":false,"given":"William","family":"Gasparrini","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-0061-4158","authenticated-orcid":false,"given":"Matthew T.","family":"Fernez","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2311-0152","authenticated-orcid":false,"given":"Madeleine","family":"Morrissette","sequence":"additional","affiliation":[{"name":"Holobiome Inc., 650 Albany St., Boston, MA 02118, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6223-3713","authenticated-orcid":false,"given":"Philip","family":"Strandwitz","sequence":"additional","affiliation":[{"name":"Holobiome Inc., 650 Albany St., Boston, MA 02118, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0433-9290","authenticated-orcid":false,"given":"Abigail N.","family":"Koppes","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"},{"name":"Department of Bioengineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3376-6358","authenticated-orcid":false,"given":"Ryan","family":"Koppes","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"},{"name":"Department of Bioengineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6570-2236","authenticated-orcid":false,"given":"Benjamin M.","family":"Woolston","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"},{"name":"Department of Bioengineering, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA"},{"name":"Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026072723500658000_2026.07.23.738723v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41575-019-0258-z"},{"key":"2026072723500658000_2026.07.23.738723v1.2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.658354"},{"key":"2026072723500658000_2026.07.23.738723v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-17956-1"},{"key":"2026072723500658000_2026.07.23.738723v1.4","doi-asserted-by":"crossref","first-page":"866","DOI":"10.3390\/microorganisms3040866","article-title":"Hydrogen Sulfide in Physiology and Diseases of the Digestive Tract","volume":"3","year":"2015","journal-title":"Microorganisms"},{"key":"2026072723500658000_2026.07.23.738723v1.5","first-page":"211","article-title":"Effects of Hydrogen Sulfide on the Microbiome: From Toxicity to Therapy","volume":"36","year":"2021","journal-title":"Antioxid. 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Advances in Gas Therapeutics for Wound Healing: Mechanisms, Delivery Materials, and Prospects. Small Struct. 5, (2024).","DOI":"10.1002\/sstr.202300151"},{"key":"2026072723500658000_2026.07.23.738723v1.17","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.2010.06580.x"},{"key":"2026072723500658000_2026.07.23.738723v1.18","doi-asserted-by":"crossref","first-page":"6108","DOI":"10.1039\/C6CS00212A","article-title":"A practical guide to working with H2S at the interface of chemistry and biology","volume":"45","year":"2016","journal-title":"Chem. Soc. Rev"},{"key":"2026072723500658000_2026.07.23.738723v1.19","first-page":"1","article-title":"Hydrogels for Gasotransmitter Delivery: Nitric Oxide, Carbon Monoxide, and Hydrogen Sulfide","volume":"24","year":"2024","journal-title":"Macromol. 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