The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope-incorporation experiments using 13 C-labelled glucose or 2 H 2 O. 13 C enrichments, 2 H enrichments and 13 C 13 C coupling patterns identify isovaleryl-CoA, malonyl-CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.

Fermentation experiments with Streptomyces toxytricini were performed using (5Z,8Z)-[10,11,12,12-(2)H]tetradeca-5,8-dienoic acid or a mixture of [2,2-(2)H(2)]- and [8,8,8-(2)H(3)]octanoic acid as supplements. (2)H NMR and mass spectroscopy confirmed the incorporation of (5Z,8Z)-[10,11,12,12-(2)H]tetradeca-5,8-dienoic acid into the C(13) side chain as well as into the C(6) side chain of lipstatin. Moreover, deuterium was incorporated into the C(6) side chain of lipstatin from the 8-position but not from the 2-position of octanoate. The data establish that the beta-lactone moiety of lipstatin is formed by condensation of a C(8) and a C(14) fatty acid with a concomitant exchange of the H-2 atoms of the C(8) fatty acid.

The lipase inhibitor lipstatin is biosynthesized in Streptomyces toxytricini via condensation of a C 14 precursor and a C 8 precursor, which are both obtained from fatty acid catabolism. To study the mechanism of this reaction in more detail, S. toxytricini was grown in medium containing a mixture of U-

Three putative intermediates in the biosynthesis of the lipase inhibitor lipstatin were synthesized in stable isotope-labeled form and were added to fermentation cultures of Streptomyces toxytricini. Biosynthetic lipstatin was isolated and analyzed by NMR spectroscopy. [3,10,11,12-(2)H]-(3S,5Z,8Z)-3-hydroxytetradeca-5,8-dienoic acid (9) was shown to serve as a direct biosynthetic precursor of lipstatin. [7,8-(2)H(2)]Hexylmalonate (11) was also incorporated into lipstatin, albeit at a relatively low rate. The leucine moiety of [(13)C-formyl,(15)N]-N-formylleucine (10) was diverted to lipstatin under loss of the (13)C-labeled formyl residue.