Banana aroma is a result of acetohydroxyacid synthase and isopropylmalate synthase alternative isoforms that bypass feedback inhibition

The distinctive aroma of banana fruit (<i>Musa</i> spp.) results from the upregulation of a pathway that is, paradoxically, understood to be feedback limited. The primary character impact compounds in banana are branched-chain esters with 3-methylbutyl moieties. Recent work has established that these esters, as well as prominent "fruity" 2-methylpropyl and butyl esters, are derived from the ripening-dependent de novo synthesis of the branched-chain amino acids valine and leucine, and their respective α-ketoacid precursors. The biosynthetic pathway possesses two sequential, rate-limiting, and feedback-inhibited enzymes: acetohydroxyacid synthase and isopropylmalate synthase. We found these enzymes to be alternatively spliced in ripening banana fruit pulp. Unripe fruit and nonfruit tissues had only trace levels of alternative splicing. Revealingly, the domains corresponding to the allosteric inhibitory binding of valine and/or leucine were truncated in the altered isoforms. During ripening, the expression and frequency of the shorter splice forms increased concomitantly with production of branched-chain esters and accumulation of their α-ketoacid precursors. Purified proteins of the modified isoforms were active but relieved of feedback inhibition. Transient expression of the shortened isoforms in <i>Nicotiana benthamiana</i> led to a greater accumulation of <i>iso</i>-branched-chain metabolites than the full-length isoforms. Results indicate that a developmentally dependent and simultaneous dysregulation in two sequential steps of the branched-chain amino acid synthetic pathway enables the biosynthesis of banana fruit's unique character-impact esters.