TY - JOUR

T1 - Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement

AU - Leite Dias, Sara

AU - Chuang, Ling

AU - Liu, Shenyu

AU - Seligmann, Benedikt

AU - Brendel, Fabian L.

AU - Gabriel Chavez, Benjamin

AU - Hoffie, Robert E.

AU - Hoffie, Iris

AU - Kumlehn, Jochen

AU - Bültemeier, Arne

AU - Wolf, Johanna

AU - Herde, Marco

AU - Witte, Claus Peter

AU - D’Auria, John C.

AU - Franke, Jakob

N1 - We thank D. Nelson (Department of Molecular Science, University of Tennessee, Memphis) and the P450 nomenclature committee for naming CYP76M57. The group of J.F. thanks S. Krause, G. Birkenbach, K. Körner, Y. Leye, and M. Fent for excellent technical and horticultural support and M. Niehaus and L. Fischer for helpful discussions. J.F. thanks C. Hertweck (Leibniz Institute for Natural Product Research and Infection Biology, HKI) and S. O'Connor (Max Planck Institute for Chemical Ecology) for helpful discussions. The group of J.C.D. thanks E. Brueckner and M. Gerres as integral members of the technical staff of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) as well as the IPK gardener team. J.K. and R.E.H. thank S. Sommerfeld and A. Knospe for excellent technical assistance with barley transformation. The EasyClone-MarkerFree Vector Set was a gift from I. Borodina (Addgene kit no. 1000000098). Parts of figs. S7 and S9 were created with BioRender.com. Funding: J.F. acknowledges financial support from the SMART BIOTECS alliance between the Technische Universität Braunschweig and the Leibniz Universität Hannover, which is supported by the Ministry of Science and Culture (MWK) of Lower Saxony. Moreover, we thank the International Max Planck Research School for supporting S.L.D. and the Leibniz Research Alliance "Bioactive Compounds and Biotechnology" for the "GraB-ME" seed-money grant to J.C.D. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) INST 187/741-1 FUGG to C.-P.W. Author contributions: S.L.D., L.C., J.C.D., and J.F. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. S.L.D. generated the vectors for A. thaliana transformation, carried out the transformation, optimized the RP-UPLC-FLD method for metabolite measurement, and performed the chemical analysis of the Arabidopsis, Golden promise, and Tafeno plants. L.C. and J.W. performed transient expression in N. benthamiana. L.C. and B.S. designed and conducted enzyme assays and performed yeast microsome purifications. S.L. designed isotope-labeling experiments, synthesized and purified compounds, optimized analytical conditions, and performed NMR analysis. B.S. designed and performed the yeast metabolic engineering experiments. F.L.B. analyzed genome data. F.L.B., B.G.C., R.E.H., and I.H. generated vectors for gramine overexpression and knockout in barley. R.E.H. carried out barley transformations. J.K. designed and supervised barley transformation experiments. A.B., M.H., and C.-P.W. designed biochemical experiments. A.B. carried out biochemical experiments and heme quantification. M.H. and C.-P.W. assisted with MS/MS and high-resolution MS measurements. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. The coding sequence of AMIS has been deposited in GenBank under accession number OR461264.

PY - 2024/3/29

Y1 - 2024/3/29

N2 - The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

AB - The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

UR - http://www.scopus.com/inward/record.url?scp=85189272879&partnerID=8YFLogxK

U2 - 10.1126/science.adk6112

DO - 10.1126/science.adk6112

M3 - Article

C2 - 38547266

AN - SCOPUS:85189272879

VL - 383

SP - 1448

EP - 1454

JO - SCIENCE

JF - SCIENCE

SN - 0036-8075

IS - 6690

ER -