Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea
Abstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the mar...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2020-09-01
|
| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-020-01439-5 |
| _version_ | 1831835300236623872 |
|---|---|
| author | Junheng Liang Huimin Wang Xiaoying Bian Youming Zhang Guoping Zhao Xiaoming Ding |
| author_facet | Junheng Liang Huimin Wang Xiaoying Bian Youming Zhang Guoping Zhao Xiaoming Ding |
| author_sort | Junheng Liang |
| collection | DOAJ |
| description | Abstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives. |
| first_indexed | 2024-12-23T04:33:39Z |
| format | Article |
| id | doaj.art-3e02dcae5f6844d095aea0bc411218a6 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-23T04:33:39Z |
| publishDate | 2020-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-3e02dcae5f6844d095aea0bc411218a62022-12-21T17:59:57ZengBMCMicrobial Cell Factories1475-28592020-09-0119111310.1186/s12934-020-01439-5Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitaleaJunheng Liang0Huimin Wang1Xiaoying Bian2Youming Zhang3Guoping Zhao4Xiaoming Ding5Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan UniversityCollaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan UniversityShandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong UniversityShandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong UniversityCollaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan UniversityCollaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan UniversityAbstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.http://link.springer.com/article/10.1186/s12934-020-01439-5Epothilone BEpoK (CYP167A1)Electron transport partnerGene overexpressionWhole-cell biotransformation |
| spellingShingle | Junheng Liang Huimin Wang Xiaoying Bian Youming Zhang Guoping Zhao Xiaoming Ding Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea Microbial Cell Factories Epothilone B EpoK (CYP167A1) Electron transport partner Gene overexpression Whole-cell biotransformation |
| title | Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea |
| title_full | Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea |
| title_fullStr | Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea |
| title_full_unstemmed | Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea |
| title_short | Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea |
| title_sort | heterologous redox partners supporting the efficient catalysis of epothilone b biosynthesis by epok in schlegelella brevitalea |
| topic | Epothilone B EpoK (CYP167A1) Electron transport partner Gene overexpression Whole-cell biotransformation |
| url | http://link.springer.com/article/10.1186/s12934-020-01439-5 |
| work_keys_str_mv | AT junhengliang heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea AT huiminwang heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea AT xiaoyingbian heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea AT youmingzhang heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea AT guopingzhao heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea AT xiaomingding heterologousredoxpartnerssupportingtheefficientcatalysisofepothilonebbiosynthesisbyepokinschlegelellabrevitalea |