Abstract
Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied “self-sufficient” CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters.
Key points
• Identified CYP505Es share > 70% amino acid identity.
• CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position.
• CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter.
Graphical Abstract
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Data availability
The data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Financial support by the South African Department of Science and Technology/National Research Foundation Centre of Excellence in Catalysis is gratefully acknowledged. We are very grateful to Prof David Nelson for naming all previously unclassified CYP505s and for supplying additional relevant sequences. We also thank Sarel Marais for technical assistance with GC analyses.
Funding
This study was funded by the National Research Foundation, South Africa (grant number 132500) and Department of Science and Technology/ National Research Foundation Centre of Excellence in Catalysis, South Africa (grant number PAR02).
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MS and DO conceived and designed the study. JvM cloned the genes and JvM and MM did the experiments. MS analyzed the data, collected the CYP505 sequences, and wrote the original draft. JA did the phylogenetic analysis. All authors read and approved the manuscript.
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Smit, M.S., Maseme, M.J., van Marwijk, J. et al. Delineation of the CYP505E subfamily of fungal self-sufficient in-chain hydroxylating cytochrome P450 monooxygenases. Appl Microbiol Biotechnol 107, 735–747 (2023). https://doi.org/10.1007/s00253-022-12329-8
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DOI: https://doi.org/10.1007/s00253-022-12329-8