Journal
CHEMISTRY & BIOLOGY
Volume 19, Issue 6, Pages 674-678Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2012.04.017
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Funding
- National Science Foundation predoctoral fellowship
- John Innes Centre
- University of East Anglia
- Biotechnology and Biological Sciences Research Council [BB/J009091/1, BBS/E/J/000CA489] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/J012947/1] Funding Source: researchfish
- BBSRC [BB/J009091/1, BBS/E/J/000CA489] Funding Source: UKRI
- EPSRC [EP/J012947/1] Funding Source: UKRI
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Opium poppy (Papaver somniferum) produces medicinally important benzylisoquinoline alkaloids, including the analgesics codeine and morphine, in the morphinan pathway. We aligned three dioxygenases that were recently discovered in P. somniferum and subsequently identified the nonconserved regions. Two of these enzymes, codeine O-demethylase (PsCODM) and thebaine O-demethylase (PsT6ODM), are known to facilitate regioselective O-demethylation in morphinan biosynthesis. We systematically swapped the residues that were nonconserved between the PsCODM and PsT6ODM sequences to generate 16 mutant PsCODM proteins that could be overexpressed in Escherichia coli. While wild-type PsCODM can demethylate both codeine and thebaine, one engineered PsCODM mutant selectively demethylates codeine. Use of this reengineered enzyme in the reconstitution of morphine biosynthesis could selectively disable a redundant pathway branch and therefore impact the yields of the downstream products codeine and morphine in subsequent metabolic engineering efforts.
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