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: | 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. |
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| Authors: | Spencer, Garrick W. K.1,2 (AUTHOR), Li, Xu1 (AUTHOR), Lam, Kenny W. L.1 (AUTHOR), Mutch, George2 (AUTHOR), Fry, Fiona H.2 (AUTHOR), Gras, Sally L.1 (AUTHOR) sgras@unimelb.edu.au |
| Source: | Journal of Biological Engineering. 1/19/2025, Vol. 19 Issue 1, p1-12. 12p. |
| Subject Terms: | *ENZYME stability, *C-terminal residues, *GIBBS' free energy, *OPIUM poppy, *BIOCONVERSION |
| 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. [ABSTRACT FROM AUTHOR] |
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| Items | – Name: Title Label: Title Group: Ti Data: 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. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Spencer%2C+Garrick+W%2E+K%2E%22">Spencer, Garrick W. K.</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Li%2C+Xu%22">Li, Xu</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lam%2C+Kenny+W%2E+L%2E%22">Lam, Kenny W. L.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Mutch%2C+George%22">Mutch, George</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Fry%2C+Fiona+H%2E%22">Fry, Fiona H.</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Gras%2C+Sally+L%2E%22">Gras, Sally L.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> sgras@unimelb.edu.au</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+Biological+Engineering%22">Journal of Biological Engineering</searchLink>. 1/19/2025, Vol. 19 Issue 1, p1-12. 12p. – Name: Subject Label: Subject Terms Group: Su Data: *<searchLink fieldCode="DE" term="%22ENZYME+stability%22">ENZYME stability</searchLink><br />*<searchLink fieldCode="DE" term="%22C-terminal+residues%22">C-terminal residues</searchLink><br />*<searchLink fieldCode="DE" term="%22GIBBS'+free+energy%22">GIBBS' free energy</searchLink><br />*<searchLink fieldCode="DE" term="%22OPIUM+poppy%22">OPIUM poppy</searchLink><br />*<searchLink fieldCode="DE" term="%22BIOCONVERSION%22">BIOCONVERSION</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: 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. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of Biological Engineering is the property of BioMed Central and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1186/s13036-025-00477-0 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 12 StartPage: 1 Subjects: – SubjectFull: ENZYME stability Type: general – SubjectFull: C-terminal residues Type: general – SubjectFull: GIBBS' free energy Type: general – SubjectFull: OPIUM poppy Type: general – SubjectFull: BIOCONVERSION Type: general Titles: – TitleFull: 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. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Spencer, Garrick W. K. – PersonEntity: Name: NameFull: Li, Xu – PersonEntity: Name: NameFull: Lam, Kenny W. L. – PersonEntity: Name: NameFull: Mutch, George – PersonEntity: Name: NameFull: Fry, Fiona H. – PersonEntity: Name: NameFull: Gras, Sally L. IsPartOfRelationships: – BibEntity: Dates: – D: 19 M: 01 Text: 1/19/2025 Type: published Y: 2025 Identifiers: – Type: issn-print Value: 17541611 Numbering: – Type: volume Value: 19 – Type: issue Value: 1 Titles: – TitleFull: Journal of Biological Engineering Type: main |
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