Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production
The bioproduction of 1-alkenes is of significant global interest due to their potential as green commodity chemicals and next-generation ‘drop-in’ biofuels. Here, we report an engineering strategy to enhance the catalytic activity and substrate specificity of the membrane-bound metalloenzyme UndB, s...
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2025
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| _version_ | 1852015805569433600 |
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| author | Tabish Iqbal (5593025) |
| author2 | Subhashini Murugan (17407884) Jayaprakash Karupusamy (22428500) Abhishek Sirohiwal (5368541) Debasis Das (1344114) |
| author2_role | author author author author |
| author_facet | Tabish Iqbal (5593025) Subhashini Murugan (17407884) Jayaprakash Karupusamy (22428500) Abhishek Sirohiwal (5368541) Debasis Das (1344114) |
| author_role | author |
| dc.creator.none.fl_str_mv | Tabish Iqbal (5593025) Subhashini Murugan (17407884) Jayaprakash Karupusamy (22428500) Abhishek Sirohiwal (5368541) Debasis Das (1344114) |
| dc.date.none.fl_str_mv | 2025-10-14T11:48:49Z |
| dc.identifier.none.fl_str_mv | 10.1021/acscentsci.5c01099.s003 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/media/Engineering_of_Integral_Membrane_Metalloenzyme_UndB_and_Designing_of_a_Cell-Free_Biocatalytic_Platform_Enabled_Efficient_1_Alkene_Production/30354881 |
| dc.rights.none.fl_str_mv | CC BY-NC 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biophysics Biochemistry Biotechnology Immunology Inorganic Chemistry Physical Sciences not elsewhere classified green commodity chemicals generation ‘ drop enabling efficient long scale simulations unveiled free biocatalytic platform chain fatty acids bound metalloenzyme undb system achieved 98 governs substrate uptake alkene yield using highly efficient undb providing mechanistic insights promising bioproduction strategy system achieved yield medium simulations revealed offering insights highly dynamic alkene production undb ’ undb family engineered undb chain 1 biocatalytic 1 ’ biofuels undecene production total turnover substrate specificity substrate recognition structural analysis significantly improving precise modulation pair network mild conditions key determinant fold improvement evolutionary adaptability engineering strategy crucial ion catalytic activity catalyst loading 04 mol |
| dc.title.none.fl_str_mv | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| dc.type.none.fl_str_mv | Dataset Media info:eu-repo/semantics/publishedVersion dataset |
| description | The bioproduction of 1-alkenes is of significant global interest due to their potential as green commodity chemicals and next-generation ‘drop-in’ biofuels. Here, we report an engineering strategy to enhance the catalytic activity and substrate specificity of the membrane-bound metalloenzyme UndB, significantly improving its utility in biocatalytic 1-alkene production. We developed a highly efficient UndB-based cell-free biocatalytic platform for high-yield medium-chain 1-alkene production. This system achieved a 262-fold improvement in UndB activity toward 1-undecene production, with a total turnover of 3412. Through structural analysis of the UndB family of proteins, we engineered UndB by domain-swapping, enhancing its selectivity toward naturally abundant long-chain fatty acids, enabling efficient long-chain 1-alkene production. Our large-scale simulations unveiled a crucial ion-pair network that orchestrates substrate–protein interactions, providing a framework for substrate stabilization. We identified a highly dynamic and functionally pivotal Arg121 residue that governs substrate uptake and stabilization, providing mechanistic insights into UndB’s substrate recognition. Furthermore, simulations revealed that precise modulation of the substrate-binding pocket volume serves as the key determinant of substrate specificity across UndB variants, offering insights into the evolutionary adaptability of the UndB family. Our system achieved 98% 1-alkene yield using only 0.04 mol % catalyst loading under mild conditions, presenting a promising bioproduction strategy. |
| eu_rights_str_mv | openAccess |
| id | Manara_ec00aed35a550ac7ec0d58c2e19e4a53 |
| identifier_str_mv | 10.1021/acscentsci.5c01099.s003 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/30354881 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY-NC 4.0 |
| spelling | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene ProductionTabish Iqbal (5593025)Subhashini Murugan (17407884)Jayaprakash Karupusamy (22428500)Abhishek Sirohiwal (5368541)Debasis Das (1344114)BiophysicsBiochemistryBiotechnologyImmunologyInorganic ChemistryPhysical Sciences not elsewhere classifiedgreen commodity chemicalsgeneration ‘ dropenabling efficient longscale simulations unveiledfree biocatalytic platformchain fatty acidsbound metalloenzyme undbsystem achieved 98governs substrate uptakealkene yield usinghighly efficient undbproviding mechanistic insightspromising bioproduction strategysystem achievedyield mediumsimulations revealedoffering insightshighly dynamicalkene productionundb ’undb familyengineered undbchain 1biocatalytic 1’ biofuelsundecene productiontotal turnoversubstrate specificitysubstrate recognitionstructural analysissignificantly improvingprecise modulationpair networkmild conditionskey determinantfold improvementevolutionary adaptabilityengineering strategycrucial ioncatalytic activitycatalyst loading04 molThe bioproduction of 1-alkenes is of significant global interest due to their potential as green commodity chemicals and next-generation ‘drop-in’ biofuels. Here, we report an engineering strategy to enhance the catalytic activity and substrate specificity of the membrane-bound metalloenzyme UndB, significantly improving its utility in biocatalytic 1-alkene production. We developed a highly efficient UndB-based cell-free biocatalytic platform for high-yield medium-chain 1-alkene production. This system achieved a 262-fold improvement in UndB activity toward 1-undecene production, with a total turnover of 3412. Through structural analysis of the UndB family of proteins, we engineered UndB by domain-swapping, enhancing its selectivity toward naturally abundant long-chain fatty acids, enabling efficient long-chain 1-alkene production. Our large-scale simulations unveiled a crucial ion-pair network that orchestrates substrate–protein interactions, providing a framework for substrate stabilization. We identified a highly dynamic and functionally pivotal Arg121 residue that governs substrate uptake and stabilization, providing mechanistic insights into UndB’s substrate recognition. Furthermore, simulations revealed that precise modulation of the substrate-binding pocket volume serves as the key determinant of substrate specificity across UndB variants, offering insights into the evolutionary adaptability of the UndB family. Our system achieved 98% 1-alkene yield using only 0.04 mol % catalyst loading under mild conditions, presenting a promising bioproduction strategy.2025-10-14T11:48:49ZDatasetMediainfo:eu-repo/semantics/publishedVersiondataset10.1021/acscentsci.5c01099.s003https://figshare.com/articles/media/Engineering_of_Integral_Membrane_Metalloenzyme_UndB_and_Designing_of_a_Cell-Free_Biocatalytic_Platform_Enabled_Efficient_1_Alkene_Production/30354881CC BY-NC 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/303548812025-10-14T11:48:49Z |
| spellingShingle | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production Tabish Iqbal (5593025) Biophysics Biochemistry Biotechnology Immunology Inorganic Chemistry Physical Sciences not elsewhere classified green commodity chemicals generation ‘ drop enabling efficient long scale simulations unveiled free biocatalytic platform chain fatty acids bound metalloenzyme undb system achieved 98 governs substrate uptake alkene yield using highly efficient undb providing mechanistic insights promising bioproduction strategy system achieved yield medium simulations revealed offering insights highly dynamic alkene production undb ’ undb family engineered undb chain 1 biocatalytic 1 ’ biofuels undecene production total turnover substrate specificity substrate recognition structural analysis significantly improving precise modulation pair network mild conditions key determinant fold improvement evolutionary adaptability engineering strategy crucial ion catalytic activity catalyst loading 04 mol |
| status_str | publishedVersion |
| title | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| title_full | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| title_fullStr | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| title_full_unstemmed | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| title_short | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| title_sort | Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production |
| topic | Biophysics Biochemistry Biotechnology Immunology Inorganic Chemistry Physical Sciences not elsewhere classified green commodity chemicals generation ‘ drop enabling efficient long scale simulations unveiled free biocatalytic platform chain fatty acids bound metalloenzyme undb system achieved 98 governs substrate uptake alkene yield using highly efficient undb providing mechanistic insights promising bioproduction strategy system achieved yield medium simulations revealed offering insights highly dynamic alkene production undb ’ undb family engineered undb chain 1 biocatalytic 1 ’ biofuels undecene production total turnover substrate specificity substrate recognition structural analysis significantly improving precise modulation pair network mild conditions key determinant fold improvement evolutionary adaptability engineering strategy crucial ion catalytic activity catalyst loading 04 mol |