Discovery, Herbicidal Activity and Biosynthesis of a Novel Natural Tetramic Acid from <i>Alternaria Species</i>

The tetramic acid moiety is a pivotal structural unit in numerous natural products. As an analogue of the simplest tetramic acid compound tenuazonic acid (TeA), sec-pentyl-TeA (S-TeA) exhibits double herbicidal activity of TeA. Here, this work identifies S-TeA as a novel natural product synthesized by Alternaria alternata and three other filamentous fungi. Chiral analysis confirm the absolute configuration of natural S-TeA as (5S, 6S). Configuration-bioactivity studies reveal that natural (5S, 6S)-S-TeA is the eutomer and possesses the highest herbicidal activity among all tested diastereomers. Biosynthetic analyses demonstrate that threonine is the precursor to S-TeA, beginning with the production of 2-amino-3-methylhexanoic acid (AMHA) via eight enzymes from the branched-chain amino acid (BCAA) biosynthetic pathway, including threonine deaminase, 2-isopropylmalate synthase (IPMS), 3-isopropylmalate dehydratase (IPMDH), isopropylmalate dehydrogenase (ISMD), acetolactate synthase, ketol-acid reductoisomerase, dihydroxy acid dehydratase, and BCAA aminotransferase. Subsequently, AMHA undergoes acetylation and cyclization by non-ribosomal peptide synthetases to form S-TeA. Distinct differences in the biosynthetic pathways of S-TeA and TeA are identified. In vitro studies confirm the critical roles of three unique enzymes IPMS, IPMDH, and ISMD in S-TeA biosynthesis, which are absent in TeA biosynthesis. These findings provide a solid basis for developing S-TeA as a natural product herbicide.