Cyclo(l-Leucyl-l-Prolyl) Produced byAchromobacter xylosoxidansInhibits Aflatoxin Production byAspergillus parasiticus

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Abstract
Aflatoxins are potent carcinogenic and toxic substances that are produced primarily byAspergillus flavusandAspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, byA. parasiticus. This bacterium was identified asAchromobacter xylosoxidansbased on its 16S ribosomal DNA sequence and was designatedA. xylosoxidansNFRI-A1.A. xylosoxidansstrains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-leucyl-l-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production byA. parasiticusSYS-4 (= NRRL2999) was 0.20 mg ml−1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml−1) of cyclo(l-leucyl-l-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(d-leucyl-d-prolyl) and cyclo(l-valyl-l-prolyl), whereas cyclo(d-prolyl-l-leucyl) and cyclo(l-prolyl-d-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-leucyl-l-prolyl) repressed transcription of the aflatoxin-related genesaflR,hexB,pksL1, anddmtA. This is the first report of a cyclodipeptide that affects aflatoxin production.