Streptomyces rochei 7434AN4 predominantly produces lankacidin and lankamycin under normal culture conditions, thus suggesting that other biosynthetic gene clusters for secondary metabolites are silent. To identify the silent metabolites of 7434AN4, we constructed mutant KA57 with multiple disruptions of the transcriptional repressor srrB and the biosynthesis genes for both antibiotics. KA57 accumulated a compound (KA57A) with a strong UV absorption at 235 nm, not detected in the parent strain or other mutants. Various spectroscopic analyses revealed that KA57A is an azoxyalkene compound with the molecular formula C₁₀H₂₀N₂O₃ and with the R configuration at C-2. Biosynthesis of KA57A was also studied by feeding with labeled acetates, amino acids, and 1-hexylamine. The hexenyl moiety (C1′–C6′) was derived from fatty acid, whereas the 3-aminobutan-1,2-diol moiety (C1–C4) was derived from C-2 of acetate (C1) and serine (C2–C4). Incorporation of [1,1-²H₂]-1-hexylamine indicated that C1′–C2′ dehydrogenation occurs as the final step of biosynthesis.

New signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei were extracted from the culture filtrate and purified by Sephadex LH20 and silica gel chromatography with the help of bioassay. Chiral HPLC and ESI-MS analyses indicated the presence of two active components—SRB1 and SRB2—and their molecular formulas were established to be C₁₅H₂₄O₅ and C₁₆H₂₆O₅, respectively. By extensive NMR analysis, SRB1 and SRB2 were determined to be 2-(1′-hydroxy-6′-oxo-8′-methylnonyl)-3-methyl-4-hydroxybut-2-en-1,4-olide and 2-(1′-hydroxy-6′-oxo-8′-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide, respectively. These structures were finally confirmed by chemical synthesis and the absolute configuration at C-1′ was determined to be R in each case. The synthetic 1′R isomers induced production of lankacidin and lankamycin at around 40 nM concentrations. SRB1 and SRB2 are therefore distinct from the well-known 2,3-disubstituted γ-butyrolactone molecules such as A-factor, virginia butanolide, and SCB1 and and belong, like avenolide, recently isolated from Streptomyces avermitilis, to the γ-butenolide family.