PT - JOURNAL ARTICLE AU - Veronica C. Perez AU - Ru Dai AU - Breanna Tomiczek AU - Jorrel Mendoza AU - Emily S.A. Wolf AU - Alexander Grenning AU - Wilfred Vermerris AU - Anna K. Block AU - Jeongim Kim TI - SbCYP79A61 Produces Phenylacetaldoxime, a Precursor of Benzyl Cyanide and Phenylacetic Acid in <em>Sorghum bicolor</em> AID - 10.1101/2022.05.11.491506 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.05.11.491506 4099 - http://biorxiv.org/content/early/2022/05/11/2022.05.11.491506.short 4100 - http://biorxiv.org/content/early/2022/05/11/2022.05.11.491506.full AB - Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Aldoxime biosynthesis in the cereal sorghum (Sorghum bicolor) is not well characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine, but unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses.Highlight Identification of SbCYP79A61, an enzyme catalyzing the production of phenylacetaldoxime (PAOx) in sorghum, and identification of benzyl cyanide as an intermediate of PAOx-derived phenylacetic acid (PAA) biosynthesis in monocots.Competing Interest StatementThe authors have declared no competing interest.PAAphenylacetic acidPAOxphenylacetaldoxime