Characterization of the Formation of Branched Short-Chain Fatty Acid：CoAs for Bitter Acid Biosynthesis in Hop Glandular Trichomes

Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop （Humulus lupulus） glandular trichomes. Branched short-chain acyI-CoAs （e.g. isobutyryI-CoA, isovaleryl- CoA and 2-methylbutyryI-CoA）, derived from the degradation of branched-chain amino acids （BCAAs）, are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyI-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HICCL2 （carboxyl CoA ligase） protein had high specific activity for isovaleric acid as a substrate （Kcat/Km = 4100 s-~ M-l）, whereas recombinant HICCL4 specifically utilized isobutyric acid （Kcat/Km = 1800 s-1 M-1） and 2-methylbutyric acid （Kcat/ Km = 6900 s-1 M-~） as substrates. Both HICCLs, like hop valerophenone synthase （HIVPS）, were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HICCL2 and HICCL4 with HIVPS in yeast led to significant production of acylphloroglucinols （the direct precursors for bitter acid biosynthesis）, which further confirmed the biochemical function of these two HICCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid：CoA ligases in planta are also discussed.