The novel potent antifungal metabolite UK-2A, isolated from Streptomyces sp. 517-02, contains a nine-membered dilactone skeleton composed of N-(3-hydroxy-4-methoxypicolinyl)serine and 2-benzyl-3-isobutyryloxy-4-hydroxypentanoic acid. Mild alkaline hydrolysis of UK-2A afforded 2-benzyl-3-isobutyryloxy-4-methyl-4-butanolide 1 and N-(3-hydroxy-4-methoxypicolinyl)serine 2. The absolute configuration of UK-2A was determined by elucidating the configurations of 1 and 2 via asymmetric synthesis. Using methyl (S)-(+)- and (R)-(-)-lactate as starting materials, diastereoisomers of butanolide 1 were prepared. The major diastereoisomer was converted to 2-benzyl-3-hydroxy-4-methyl-4-butanolide 3a, whose optical rotation ([α]26D-88.42°) and NMR spectra matched those of 3a derived from UK-2A, establishing the configuration of 1 as (2R,3R,4S). For the serine moiety, methyl N-(3-hydroxy-4-methoxypicolinyl)serine 4 synthesized from (S)-(+)-serine showed consistent optical rotation ([α]28D+13.86°) and spectral data with the degradation product of UK-2A, confirming the C-7 configuration as S. Thus, the absolute configuration of UK-2A was elucidated as (+)-(2R,3R,4S,7S). Additionally, NMR studies revealed temperature-dependent conformation of the nine-membered dilactone skeleton: broad signals at ambient temperature, 40℃, and 50℃, while sharp spectra were observed at 0, -20, and -40℃ in CDCl3. MM2 calculations provided a sole stable conformation, and the calculated dihedral angles supported the coupling constants observed in 1H NMR at -20℃.