Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories
Abstract Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriou...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | Microbial Cell Factories |
| Online Access: | https://doi.org/10.1186/s12934-023-02219-7 |
| _version_ | 1797555552304234496 |
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| author | Michal Poborsky Christoph Crocoll Mohammed Saddik Motawie Barbara Ann Halkier |
| author_facet | Michal Poborsky Christoph Crocoll Mohammed Saddik Motawie Barbara Ann Halkier |
| author_sort | Michal Poborsky |
| collection | DOAJ |
| description | Abstract Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories. |
| first_indexed | 2024-03-10T16:49:09Z |
| format | Article |
| id | doaj.art-d3b50bc648df40068d2c0029f6f325a4 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-10T16:49:09Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-d3b50bc648df40068d2c0029f6f325a42023-11-20T11:22:37ZengBMCMicrobial Cell Factories1475-28592023-10-0122111010.1186/s12934-023-02219-7Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factoriesMichal Poborsky0Christoph Crocoll1Mohammed Saddik Motawie2Barbara Ann Halkier3Department of Plant and Environmental Sciences, DynaMo Center of Excellence, University of CopenhagenDepartment of Plant and Environmental Sciences, DynaMo Center of Excellence, University of CopenhagenDepartment of Plant and Environmental Sciences, Section for Plant Biochemistry, University of CopenhagenDepartment of Plant and Environmental Sciences, DynaMo Center of Excellence, University of CopenhagenAbstract Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories.https://doi.org/10.1186/s12934-023-02219-7 |
| spellingShingle | Michal Poborsky Christoph Crocoll Mohammed Saddik Motawie Barbara Ann Halkier Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories Microbial Cell Factories |
| title | Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories |
| title_full | Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories |
| title_fullStr | Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories |
| title_full_unstemmed | Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories |
| title_short | Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories |
| title_sort | systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome p450 enzymes in escherichia coli cell factories |
| url | https://doi.org/10.1186/s12934-023-02219-7 |
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