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Cited by 1Enzyme Catalysis and ImmobilizationPlant biochemistry and biosynthesisMicrobial Metabolic Engineering and Bioproduction

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Directed evolution of a cytochrome P450 monooxygenase for improved perillyl alcohol biosynthesis via a tailored genetically encoded biosensor

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Odhiambo, Catherine A., Malico, Alexandra A., Williams, Gavin J.·2026·RSC Advances
DOI 10.1039/d6ra03129cPMC PMC13329868

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Abstract

A tailored CymR-S77L biosensor enabled high-throughput evolution of a P450 for selective perillyl alcohol production from limonene, establishing a platform for terpene hydroxylation engineering.

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Cites

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Figures

Fig. 1 P450-mediated biosynthesis of 2. (A) P450-mediated oxidation biosynthetic route to 2via GPP and the terpene synthase, LimS. (B) Reporting the P450-mediated conversion of 1 to 2via a transcription-factor-based biosensor. (C) A 2-selective transcription factor biosensor enables directed evolution of CYP153A6.
Fig. 1 P450-mediated biosynthesis of 2. (A) P450-mediated oxidation biosynthetic route to 2via GPP and the terpene synthase, LimS. (B) Reporting the P450-mediated conversion of 1 to 2via a transcription-factor-based biosensor. (C) A 2-selective transcription factor biosensor enables directed evolution of CYP153A6.CC BY · PubMed Central
Fig. 2 Performance of wild-type and CymR-S77L with 1 and 2. (A) Scheme illustrating the CymR synthetic genetic circuit. (B) Normalized GFP fluorescence of the WT and S77L biosensor strains with 2.5 mM of 1 and 2 and fold activation. (C) Dose–response curves of WT and S77L with varying concentrations of 1 and 2. Error bars (where visible) represent the standard error of the mean (n = 3). (D) Wild-type CymR homology model with 2 (magenta sticks) docked. Residues within a 5 Å radius of the ligand are shown as green sticks, with those targeted for saturation mutagenesis shown in yellow (Tyr70, Ser77, Phe102, Ser110, and Asn134). (E) Model of CymR S77L with 2 docked. Residues within a 5 Å radius of the ligand are shown as blue sticks, with those targeted for saturation mutagenesis shown in yellow (Tyr70, Phe102, Ser110, and Asn134), and S77L is shown in magenta. Residues within a 5 Å radius of the ligand in the wild-type model are shown as green sticks.
Fig. 2 Performance of wild-type and CymR-S77L with 1 and 2. (A) Scheme illustrating the CymR synthetic genetic circuit. (B) Normalized GFP fluorescence of the WT and S77L biosensor strains with 2.5 mM of 1 and 2 and fold activation. (C) Dose–response curves of WT and S77L with varying concentrations of 1 and 2. Error bars (where visible) represent the standard error of the mean (n = 3). (D) Wild-type CymR homology model with 2 (magenta sticks) docked. Residues within a 5 Å radius of the ligand are shown as green sticks, with those targeted for saturation mutagenesis shown in yellow (Tyr70, Ser77, Phe102, Ser110, and Asn134). (E) Model of CymR S77L with 2 docked. Residues within a 5 Å radius of the ligand are shown as blue sticks, with those targeted for saturation mutagenesis shown in yellow (Tyr70, Phe102, Ser110, and Asn134), and S77L is shown in magenta. Residues within a 5 Å radius of the ligand in the wild-type model are shown as green sticks.CC BY · PubMed Central
Fig. 3 Effector profile of the wild-type and S77L CymR biosensor strains. (A) Possible products from P450-based oxidation of 1. Structures labeled with an asterisk were not available for testing. (B) Normalized GFP fluorescence (corrected by subtracting fluorescence with no effector) of wild-type and S77L with available monoterpenes at 2.5 mM. Error bars represent the standard error of the mean (n = 3).
Fig. 3 Effector profile of the wild-type and S77L CymR biosensor strains. (A) Possible products from P450-based oxidation of 1. Structures labeled with an asterisk were not available for testing. (B) Normalized GFP fluorescence (corrected by subtracting fluorescence with no effector) of wild-type and S77L with available monoterpenes at 2.5 mM. Error bars represent the standard error of the mean (n = 3).CC BY · PubMed Central
Fig. 4 Biosynthesis and detection of 2 in an engineered E. coli strain. (A) Scheme showing the four-enzyme pACYC vector consisting of: CYP153A6 (ahpG), FdR (ahpH), and Fd (ahpI) for the hydroxylation of 1. Production of 2 is monitored by the engineered CymR-S77L biosensor (bottom plasmid). (B) Biosensor fluorescence responses of a representative portion of the CYP153A6 error-prone PCR library. Each point represents an individual clone ranked by normalized fluorescence signal. Wild-type CYP153A6 is shown in black. Variants exceeding the statistical cutoff were selected for secondary screening and GC-MS validation. (C) Extracted ion count (EIC) chromatograms of 2 (m/z = 152) determined by GC-MS analysis of the extracted culture media. Error bars represent the standard error of the mean (n = 3).
Fig. 4 Biosynthesis and detection of 2 in an engineered E. coli strain. (A) Scheme showing the four-enzyme pACYC vector consisting of: CYP153A6 (ahpG), FdR (ahpH), and Fd (ahpI) for the hydroxylation of 1. Production of 2 is monitored by the engineered CymR-S77L biosensor (bottom plasmid). (B) Biosensor fluorescence responses of a representative portion of the CYP153A6 error-prone PCR library. Each point represents an individual clone ranked by normalized fluorescence signal. Wild-type CYP153A6 is shown in black. Variants exceeding the statistical cutoff were selected for secondary screening and GC-MS validation. (C) Extracted ion count (EIC) chromatograms of 2 (m/z = 152) determined by GC-MS analysis of the extracted culture media. Error bars represent the standard error of the mean (n = 3).CC BY · PubMed Central
Fig. 5 Sequence analysis and structural mapping of CYP153A6 variants identified via biosensor-guided screening. (A) Heatmap sequence alignment of selected hit variants, with CYP153A6 wild-type residues shown in gray and substitutions shown in blue. Relative fold improvements in product formation, determined by GC-MS analysis, are summarized to the right. (B) Model of CYP153A6 highlighting sites of confirmed functional mutations in the top confirmed hits (shown as spheres). 1 is shown as magenta sticks. Heme is shown as green sticks. Cys367 and Thr260 are shown as green sticks below and above the heme, respectively. The BC and FG loops are labeled. (C) Model of CYP153A6 with predicted substrate channels shown in shades of red. (D) Predicted CYP163A6:FdR:Fdx complex structure. FdR is shown in yellow. Fdx is shown in red. Selected residues are highlighted as spheres.
Fig. 5 Sequence analysis and structural mapping of CYP153A6 variants identified via biosensor-guided screening. (A) Heatmap sequence alignment of selected hit variants, with CYP153A6 wild-type residues shown in gray and substitutions shown in blue. Relative fold improvements in product formation, determined by GC-MS analysis, are summarized to the right. (B) Model of CYP153A6 highlighting sites of confirmed functional mutations in the top confirmed hits (shown as spheres). 1 is shown as magenta sticks. Heme is shown as green sticks. Cys367 and Thr260 are shown as green sticks below and above the heme, respectively. The BC and FG loops are labeled. (C) Model of CYP153A6 with predicted substrate channels shown in shades of red. (D) Predicted CYP163A6:FdR:Fdx complex structure. FdR is shown in yellow. Fdx is shown in red. Selected residues are highlighted as spheres.CC BY · PubMed Central

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Citations

Cites

Show all 64 references

Research works printed but not linked: 5

  1. Zhang, 2017, Nat. Prod. Rep., 1
  2. Chen, 2015, Am. J. Cancer Res., 5, 1580
  3. Kasey, 2018, R. Soc. Chem., 203
  4. Juárez, 2018, Nat. Commun., 9, 1
  5. Evans, 2022, bioRxiv, 2021, 463034

Metadata

Authors
Odhiambo, C. A., Malico, A. A., & Williams, G. J.
Affiliations
North Carolina State University (US)
Corresponding author
Williams, Gavin J.
ORCID
Venue
RSC Advances
Publisher
Royal Society of Chemistry (RSC)
Published
2026
Volume
16
Issue
35
Pages
35590-35600
ISSN
2046-2069 (ISSN-L)
DOI
10.1039/d6ra03129c
PMC
PMC13329868
OpenAlex
W7167252865
Cited by
1
References
64
Citations by year
2026: 1
FWCI
2.04
Type
journal article
Language
en
Open access
Licence
Locations
  • RSC Advances (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
National Institutes of Health (GM143450, T32GM133366, T32GM141887)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
directed evolution, cytochrome P450 monooxygenase, genetically encoded biosensors, regioselectivity, chemoselectivity, fluorescence-based screening

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Odhiambo, C. A., Malico, A. A., & Williams, G. J. (2026). Directed evolution of a cytochrome P450 monooxygenase for improved perillyl alcohol biosynthesis via a tailored genetically encoded biosensor. RSC Advances. https://doi.org/10.1039/d6ra03129c

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