Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield
Abstract The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morph...
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2025-01-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-025-00477-0 |
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| author | Garrick W. K. Spencer Xu Li Kenny W. L. Lam George Mutch Fiona H. Fry Sally L. Gras |
| author_facet | Garrick W. K. Spencer Xu Li Kenny W. L. Lam George Mutch Fiona H. Fry Sally L. Gras |
| author_sort | Garrick W. K. Spencer |
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| description | Abstract The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morphinan alkaloids by O-demethylation and has been applied in single step biotransformation reactions or as part of larger biosynthetic cascade, however, the productivity for these reactions remains low and suboptimal enzyme properties could be improved. This mutagenesis study targeted non-conserved N-and C-terminal residues, which were replaced with the equivalent residues from enzyme thebaine 6-O-demethylase. Whole cell biotransformation performance was significantly improved in Escherichia coli expressing codeine 3-O-demethylase mutants, with a ~ 2.8-fold increase in the production of oripavine from thebaine and ~ 1.3-fold increase in the production of morphine from codeine. Statistical analysis of biotransformation yield, enzyme expression and stability, predicted using changes in Gibbs free energy (ΔΔG) with deep-learning-based model DDmut, suggested that altered enzyme stability and/or expression of soluble protein may contribute to the observed improvements in biotransformation. This approach could be beneficial for screening future codeine 3-O-demethylase mutations and for other enzymes. |
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| institution | Kabale University |
| issn | 1754-1611 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
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| spelling | doaj-art-dda0f1fe517144278c035edff703c09e2025-01-26T12:37:28ZengBMCJournal of Biological Engineering1754-16112025-01-0119111210.1186/s13036-025-00477-0Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yieldGarrick W. K. Spencer0Xu Li1Kenny W. L. Lam2George Mutch3Fiona H. Fry4Sally L. Gras5The Department of Chemical Engineering and the Bio21 Molecular Science and Biotechnology Institute, The University of MelbourneThe Department of Chemical Engineering and the Bio21 Molecular Science and Biotechnology Institute, The University of MelbourneThe Department of Chemical Engineering and the Bio21 Molecular Science and Biotechnology Institute, The University of MelbourneSun Pharmaceutical Industries Australia Pty LtdSun Pharmaceutical Industries Australia Pty LtdThe Department of Chemical Engineering and the Bio21 Molecular Science and Biotechnology Institute, The University of MelbourneAbstract The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morphinan alkaloids by O-demethylation and has been applied in single step biotransformation reactions or as part of larger biosynthetic cascade, however, the productivity for these reactions remains low and suboptimal enzyme properties could be improved. This mutagenesis study targeted non-conserved N-and C-terminal residues, which were replaced with the equivalent residues from enzyme thebaine 6-O-demethylase. Whole cell biotransformation performance was significantly improved in Escherichia coli expressing codeine 3-O-demethylase mutants, with a ~ 2.8-fold increase in the production of oripavine from thebaine and ~ 1.3-fold increase in the production of morphine from codeine. Statistical analysis of biotransformation yield, enzyme expression and stability, predicted using changes in Gibbs free energy (ΔΔG) with deep-learning-based model DDmut, suggested that altered enzyme stability and/or expression of soluble protein may contribute to the observed improvements in biotransformation. This approach could be beneficial for screening future codeine 3-O-demethylase mutations and for other enzymes.https://doi.org/10.1186/s13036-025-00477-0PoppyE.coliBioconversionOripavineMorphineDDmut. |
| spellingShingle | Garrick W. K. Spencer Xu Li Kenny W. L. Lam George Mutch Fiona H. Fry Sally L. Gras Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield Journal of Biological Engineering Poppy E.coli Bioconversion Oripavine Morphine DDmut. |
| title | Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield |
| title_full | Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield |
| title_fullStr | Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield |
| title_full_unstemmed | Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield |
| title_short | Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield |
| title_sort | codeine 3 o demethylase catalyzed biotransformation of morphinan alkaloids in escherichia coli site directed mutagenesis of terminal residues improves enzyme expression stability and biotransformation yield |
| topic | Poppy E.coli Bioconversion Oripavine Morphine DDmut. |
| url | https://doi.org/10.1186/s13036-025-00477-0 |
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