Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories
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Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories. / Poborsky, Michal; Crocoll, Christoph; Motawie, Mohammed Saddik; Halkier, Barbara Ann.
In: Microbial Cell Factories, Vol. 22, 219, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories
AU - Poborsky, Michal
AU - Crocoll, Christoph
AU - Motawie, Mohammed Saddik
AU - Halkier, Barbara Ann
N1 - Publisher Copyright: © 2023, BioMed Central Ltd., part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1186/s12934-023-02219-7
DO - 10.1186/s12934-023-02219-7
M3 - Journal article
C2 - 37880718
AN - SCOPUS:85174965717
VL - 22
JO - Microbial Cell
JF - Microbial Cell
SN - 1475-2859
M1 - 219
ER -
ID: 382557197