{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/132768"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/132768","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Microbial glucosinolate metabolism: Characterization of Enterococcus faecalis XL1 and functional analysis of a GH4 enzyme","abstract":"Glucosinolates (GSLs), sulfur-containing plant metabolites abundant in cruciferous vegetables, are hydrolyzed to isothiocyanates (ITCs) – bioactive compounds with antimicrobial, anticancer, and anti-inflammatory properties. When plant myrosinases are inactivated by cooking, gut microbes become the primary contributors to ITC formation. However, the microbial enzymes and pathways responsible for GSL metabolism remain poorly understood. This dissertation investigates the bacterial mechanisms underlying GSL hydrolysis, focusing primarily on Enterococcus faecalis XL1, a gut isolate obtained from broccoli-fed rats. Whole-genome sequencing and gene expression analyses identified multiple β-glucoside phosphotransferase system (PTS) transporters and glycoside hydrolases (GHs) responsive to sinigrin, with a GH4 enzyme Ef5510, showing the strongest induction. Functional studies revealed that Ef5510 is an NAD+/Mn2+-dependent hydrolase, and the PTS-mediated uptake step represents the rate-limiting stage of the pathway. Heterologous expression and kinetic characterization using a synthesized substrate, p-nitrophenyl-β-D-glucopyranoside-6-phosphate, confirmed Ef5510’s redox-assisted catalytic activity, suggesting a phosphorylation-coupled, GH4-mediated route distinct from classic GH1 systems. Complementary isolation studies from broccoli-fed mice revealed that GSL metabolism is not limited to ITC-forming routes but may also involve alternative pathways, including sulfatase-dependent detoxification. Together, these findings expand the understanding of microbial myrosinase-like activity, highlighting E. faecalis XL1 as a novel model for studying redox-driven GSL metabolism and providing new insights into how gut bacteria contribute to the bioactivation of dietary GSLs.","abstract_html":"Glucosinolates (GSLs), sulfur-containing plant metabolites abundant in cruciferous vegetables, are hydrolyzed to isothiocyanates (ITCs) – bioactive compounds with antimicrobial, anticancer, and anti-inflammatory properties. When plant myrosinases are inactivated by cooking, gut microbes become the primary contributors to ITC formation. However, the microbial enzymes and pathways responsible for GSL metabolism remain poorly understood. This dissertation investigates the bacterial mechanisms underlying GSL hydrolysis, focusing primarily on Enterococcus faecalis XL1, a gut isolate obtained from broccoli-fed rats. Whole-genome sequencing and gene expression analyses identified multiple β-glucoside phosphotransferase system (PTS) transporters and glycoside hydrolases (GHs) responsive to sinigrin, with a GH4 enzyme Ef5510, showing the strongest induction. Functional studies revealed that Ef5510 is an NAD+/Mn2+-dependent hydrolase, and the PTS-mediated uptake step represents the rate-limiting stage of the pathway. Heterologous expression and kinetic characterization using a synthesized substrate, p-nitrophenyl-β-D-glucopyranoside-6-phosphate, confirmed Ef5510’s redox-assisted catalytic activity, suggesting a phosphorylation-coupled, GH4-mediated route distinct from classic GH1 systems. Complementary isolation studies from broccoli-fed mice revealed that GSL metabolism is not limited to ITC-forming routes but may also involve alternative pathways, including sulfatase-dependent detoxification. Together, these findings expand the understanding of microbial myrosinase-like activity, highlighting E. faecalis XL1 as a novel model for studying redox-driven GSL metabolism and providing new insights into how gut bacteria contribute to the bioactivation of dietary GSLs.","abstract_has_math":false,"creators":["Li, Jiaxuan"],"institution":"University of Illinois Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Food Science & Human Nutrition","degree_department":null,"school":null,"contributors":["Miller, Michael J.","Banerjee, Pratik","Jeffery, Elizabeth H.","Sirk, Shannon"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-12","date_published":"2025-12","updated_at":"2026-07-22T22:25:07Z","subjects":["Glucosinolate metabolism","Isothiocyanates","Enterococcus faecalis","Glycoside hydrolase GH4","Phosphotransferase system (PTS)","Gut microbial biotransformation"],"languages":["en"],"rights":["Copyright 2025 Jiaxuan Li"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/132768","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Miller, Michael J.","Banerjee, Pratik","Jeffery, Elizabeth H.","Sirk, Shannon"]},{"key":"dc:creator","label":"Author","values":["Li, Jiaxuan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2025-12","2025-11-24"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Food Science & Human Nutrition"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Glucosinolate metabolism","Isothiocyanates","Enterococcus faecalis","Glycoside hydrolase GH4","Phosphotransferase system (PTS)","Gut microbial biotransformation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2025 Jiaxuan Li"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/132768"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Glucosinolates (GSLs), sulfur-containing plant metabolites abundant in cruciferous vegetables, are hydrolyzed to isothiocyanates (ITCs) – bioactive compounds with antimicrobial, anticancer, and anti-inflammatory properties. When plant myrosinases are inactivated by cooking, gut microbes become the primary contributors to ITC formation. However, the microbial enzymes and pathways responsible for GSL metabolism remain poorly understood. This dissertation investigates the bacterial mechanisms underlying GSL hydrolysis, focusing primarily on Enterococcus faecalis XL1, a gut isolate obtained from broccoli-fed rats. Whole-genome sequencing and gene expression analyses identified multiple β-glucoside phosphotransferase system (PTS) transporters and glycoside hydrolases (GHs) responsive to sinigrin, with a GH4 enzyme Ef5510, showing the strongest induction. Functional studies revealed that Ef5510 is an NAD+/Mn2+-dependent hydrolase, and the PTS-mediated uptake step represents the rate-limiting stage of the pathway. Heterologous expression and kinetic characterization using a synthesized substrate, p-nitrophenyl-β-D-glucopyranoside-6-phosphate, confirmed Ef5510’s redox-assisted catalytic activity, suggesting a phosphorylation-coupled, GH4-mediated route distinct from classic GH1 systems. Complementary isolation studies from broccoli-fed mice revealed that GSL metabolism is not limited to ITC-forming routes but may also involve alternative pathways, including sulfatase-dependent detoxification. Together, these findings expand the understanding of microbial myrosinase-like activity, highlighting E. faecalis XL1 as a novel model for studying redox-driven GSL metabolism and providing new insights into how gut bacteria contribute to the bioactivation of dietary GSLs.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-12-01","The student, Jiaxuan Li, accepted the attached license on 2025-11-20 at 11:30.","The student, Jiaxuan Li, submitted this Dissertation for approval on 2025-11-20 at 11:56.","This Dissertation was approved for publication on 2025-11-24 at 14:27.","DSpace SAF Submission Ingestion Package generated from Vireo submission #22911 on 2026-02-19 at 20:08:54"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Microbial glucosinolate metabolism: Characterization of Enterococcus faecalis XL1 and functional analysis of a GH4 enzyme"]}]}],"canonical_facts":{"dc:contributor":["Miller, Michael J.","Banerjee, Pratik","Jeffery, Elizabeth H.","Sirk, Shannon"],"dc:creator":["Li, Jiaxuan"],"dc:date":["2025-12","2025-11-24"],"dc:description":["Glucosinolates (GSLs), sulfur-containing plant metabolites abundant in cruciferous vegetables, are hydrolyzed to isothiocyanates (ITCs) – bioactive compounds with antimicrobial, anticancer, and anti-inflammatory properties. When plant myrosinases are inactivated by cooking, gut microbes become the primary contributors to ITC formation. However, the microbial enzymes and pathways responsible for GSL metabolism remain poorly understood. This dissertation investigates the bacterial mechanisms underlying GSL hydrolysis, focusing primarily on Enterococcus faecalis XL1, a gut isolate obtained from broccoli-fed rats. Whole-genome sequencing and gene expression analyses identified multiple β-glucoside phosphotransferase system (PTS) transporters and glycoside hydrolases (GHs) responsive to sinigrin, with a GH4 enzyme Ef5510, showing the strongest induction. Functional studies revealed that Ef5510 is an NAD+/Mn2+-dependent hydrolase, and the PTS-mediated uptake step represents the rate-limiting stage of the pathway. Heterologous expression and kinetic characterization using a synthesized substrate, p-nitrophenyl-β-D-glucopyranoside-6-phosphate, confirmed Ef5510’s redox-assisted catalytic activity, suggesting a phosphorylation-coupled, GH4-mediated route distinct from classic GH1 systems. Complementary isolation studies from broccoli-fed mice revealed that GSL metabolism is not limited to ITC-forming routes but may also involve alternative pathways, including sulfatase-dependent detoxification. Together, these findings expand the understanding of microbial myrosinase-like activity, highlighting E. faecalis XL1 as a novel model for studying redox-driven GSL metabolism and providing new insights into how gut bacteria contribute to the bioactivation of dietary GSLs.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-12-01","The student, Jiaxuan Li, accepted the attached license on 2025-11-20 at 11:30.","The student, Jiaxuan Li, submitted this Dissertation for approval on 2025-11-20 at 11:56.","This Dissertation was approved for publication on 2025-11-24 at 14:27.","DSpace SAF Submission Ingestion Package generated from Vireo submission #22911 on 2026-02-19 at 20:08:54"],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/132768"],"dc:language":["en"],"dc:rights":["Copyright 2025 Jiaxuan Li"],"dc:subject":["Glucosinolate metabolism","Isothiocyanates","Enterococcus faecalis","Glycoside hydrolase GH4","Phosphotransferase system (PTS)","Gut microbial biotransformation"],"dc:title":["Microbial glucosinolate metabolism: Characterization of Enterococcus faecalis XL1 and functional analysis of a GH4 enzyme"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Food Science & Human Nutrition"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:07Z"}