Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli
Abstract Microbial cell factories produce valuable compounds by exploiting cytochrome P450 catalytic systems. However, the inefficient electron transfer flux (ETF) between P450 and cytochrome P450 reductase (CPR) hinders the efficient synthesis of natural products. Herein, an ETF is systematically e...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2025-08-01
|
| Series: | Advanced Science |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/advs.202414859 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849233242674167808 |
|---|---|
| author | Wenzhao Xu Pengling Wei Lirong Chen Ling Gao Xiaole Xia |
| author_facet | Wenzhao Xu Pengling Wei Lirong Chen Ling Gao Xiaole Xia |
| author_sort | Wenzhao Xu |
| collection | DOAJ |
| description | Abstract Microbial cell factories produce valuable compounds by exploiting cytochrome P450 catalytic systems. However, the inefficient electron transfer flux (ETF) between P450 and cytochrome P450 reductase (CPR) hinders the efficient synthesis of natural products. Herein, an ETF is systematically engineered by regulating the electron transfer rate, electron‐receiving rate, and electron donor NADPH availability for serotonin production. First, a putative electron transfer pathway (ETP) is identified using virtual computing and evolved based on a genetically encoded serotonin RNA biosensor. Subsequently, an intermediate site strategy is developed to shorten the electron‐hopping steps and distance in the ETP of CPR for enhancing the electron transfer rate. Next, the heme‐binding domain is engineered to reduce the distance between heme‐Fe and the substrate channel terminal in T5H for improving the electron‐receiving rate. Furthermore, the NADPH pool is enlarged to increase the electron supply for efficient catalysis of P450 systems. Finally, tryptophan‐5‐hydroxylase (T5H) activity (Kcat/KM) in the optimal mutant is 36.62‐fold than that of wild‐type. The engineered strain E. coli S11 can produce 15.42 g L−1 serotonin in a 7.5‐L bioreactor, which is 9.17‐fold of the previous reported. This strategy provides a systematic approach for regulating ETF in complex P450 catalytic systems for efficient chemical biosynthesis. |
| format | Article |
| id | doaj-art-35775f3e85554d618a36f2cc35df6a3c |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-35775f3e85554d618a36f2cc35df6a3c2025-08-20T11:56:10ZengWileyAdvanced Science2198-38442025-08-011230n/an/a10.1002/advs.202414859Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia ColiWenzhao Xu0Pengling Wei1Lirong Chen2Ling Gao3Xiaole Xia4Key Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University 1800 Lihu Road Wuxi Jiangsu 214122 ChinaKey Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University 1800 Lihu Road Wuxi Jiangsu 214122 ChinaKey Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University 1800 Lihu Road Wuxi Jiangsu 214122 ChinaKey Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University 1800 Lihu Road Wuxi Jiangsu 214122 ChinaKey Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University 1800 Lihu Road Wuxi Jiangsu 214122 ChinaAbstract Microbial cell factories produce valuable compounds by exploiting cytochrome P450 catalytic systems. However, the inefficient electron transfer flux (ETF) between P450 and cytochrome P450 reductase (CPR) hinders the efficient synthesis of natural products. Herein, an ETF is systematically engineered by regulating the electron transfer rate, electron‐receiving rate, and electron donor NADPH availability for serotonin production. First, a putative electron transfer pathway (ETP) is identified using virtual computing and evolved based on a genetically encoded serotonin RNA biosensor. Subsequently, an intermediate site strategy is developed to shorten the electron‐hopping steps and distance in the ETP of CPR for enhancing the electron transfer rate. Next, the heme‐binding domain is engineered to reduce the distance between heme‐Fe and the substrate channel terminal in T5H for improving the electron‐receiving rate. Furthermore, the NADPH pool is enlarged to increase the electron supply for efficient catalysis of P450 systems. Finally, tryptophan‐5‐hydroxylase (T5H) activity (Kcat/KM) in the optimal mutant is 36.62‐fold than that of wild‐type. The engineered strain E. coli S11 can produce 15.42 g L−1 serotonin in a 7.5‐L bioreactor, which is 9.17‐fold of the previous reported. This strategy provides a systematic approach for regulating ETF in complex P450 catalytic systems for efficient chemical biosynthesis.https://doi.org/10.1002/advs.202414859cytochrome P450 enzymeselectron transfer fluxP450 reductasesRNA‐biosensorsserotonintryptamine‐5‐hydroxylases |
| spellingShingle | Wenzhao Xu Pengling Wei Lirong Chen Ling Gao Xiaole Xia Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli Advanced Science cytochrome P450 enzymes electron transfer flux P450 reductases RNA‐biosensors serotonin tryptamine‐5‐hydroxylases |
| title | Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli |
| title_full | Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli |
| title_fullStr | Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli |
| title_full_unstemmed | Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli |
| title_short | Engineering Electron Transfer Flux between Cytochrome P450 Enzyme and P450 Reductase to Enhance Serotonin Production in Escherichia Coli |
| title_sort | engineering electron transfer flux between cytochrome p450 enzyme and p450 reductase to enhance serotonin production in escherichia coli |
| topic | cytochrome P450 enzymes electron transfer flux P450 reductases RNA‐biosensors serotonin tryptamine‐5‐hydroxylases |
| url | https://doi.org/10.1002/advs.202414859 |
| work_keys_str_mv | AT wenzhaoxu engineeringelectrontransferfluxbetweencytochromep450enzymeandp450reductasetoenhanceserotoninproductioninescherichiacoli AT penglingwei engineeringelectrontransferfluxbetweencytochromep450enzymeandp450reductasetoenhanceserotoninproductioninescherichiacoli AT lirongchen engineeringelectrontransferfluxbetweencytochromep450enzymeandp450reductasetoenhanceserotoninproductioninescherichiacoli AT linggao engineeringelectrontransferfluxbetweencytochromep450enzymeandp450reductasetoenhanceserotoninproductioninescherichiacoli AT xiaolexia engineeringelectrontransferfluxbetweencytochromep450enzymeandp450reductasetoenhanceserotoninproductioninescherichiacoli |