Disentangling hydroxynitrile glucoside biosynthesis in a barley (Hordeum vulgare) metabolon provides access to elite malting barleys for ethyl carbamate‐free whisky production

• Published in: The Plant journal : for cell and molecular biology Vol. 119; no. 1; pp. 364 - 382
• Main Authors: Jørgensen, Morten E., Houston, Kelly, Jørgensen, Hans Jørgen L., Thomsen, Hanne C., Tekaat, Linda, Krogh, Camilla Timmermann, Mellor, Silas B., Braune, Katarzyna Birch, Damm, Mette L., Pedas, Pai Rosager, Voss, Cynthia, Rasmussen, Magnus Wohlfahrt, Nielsen, Kasper, Skadhauge, Birgitte, Motawia, Mohammed S., Møller, Birger Lindberg, Dockter, Christoph, Sørensen, Mette
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.07.2024
• Subjects: Alcoholic beverages; Barley; Biosynthesis; CYP71L1; CYP79A12; Distillation; Enzymes; Ethyl carbamate; FIND‐IT technology; Functional morphology; Genome-Wide Association Study; Glucosides; Glucosides - metabolism; HNG‐0 barley; Hordeum - genetics; Hordeum - metabolism; Hordeum - microbiology; Hordeum spontaneum; Hordeum vulgare; MATE transporter; Metabolites; Net blotch; Nitriles - metabolism; Plant breeding; Plant Proteins - genetics; Plant Proteins - metabolism; Protein biosynthesis; Quantitative Trait Loci; Urethane - metabolism; Whiskey; Whisky; CYP71L1; CYP79A12; MATE transporter; FIND‐IT technology; HNG‐0 barley
• ISSN: 0960-7412; 1365-313X; 1365-313X
• DOI: 10.1111/tpj.16768

SUMMARY Barley produces several specialized metabolites, including five α‐, β‐, and γ‐hydroxynitrile glucosides (HNGs). In malting barley, presence of the α‐HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite‐GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock‐out (KO)‐lines, isolated from the barley FIND‐IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO‐lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG‐0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production. Significance Statement Barley produces five hydroxynitrile glucosides (HNGs). Analysis of HNG levels in more than 300 barley accessions identifies three barley accessions with altered relative HNG profiles. The HNG biosynthetic pathway is organized within a metabolon and the functional role of the individual CYPs and UGTs involved ís elucidated using FIND‐IT knock‐out mutants. The CYP79A12 knock‐out mutant does not produce HNGs and provides a barley line for use in whisky production avoiding undesired formation of HNG‐derived ethyl carbamate.