Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938

Abstract The gene ( aba 1) encoding the NRPS complex responsible for the synthesis of the cyclic peptide antibiotic Aureobasidin A (AbA) in Aureobasidium pullulans BP-1938, was cloned using a combination of PCR and library screening approaches. The aba 1 gene was found to consist of a single, intronless open reading frame (ORF) of 34,980 bp, encoding an 11,659 amino acid protein with a calculated molecular mass of 1,286,254 Da. Putative promoter and translation start elements were identified upstream from the putative ATG in the aba 1 gene, and a consensus poly(A) addition signal (AATAAA) was identified 191 bp downstream of the translation termination codon (TGA). As predicted by the structure AbA, the aba 1 gene encodes an enzyme composed of nine biosynthetic modules, eight of which contain adenylation domains with recognizable amino acid specificity-conferring code elements, and four of which contain embedded methylation domains. The biosynthetic module located at position one in the aba 1 gene lacks recognizable specificity-conferring code elements, consistent with it being responsible for incorporation of the 2-hydroxy-3-methylpentanoic acid residue at that position in AbA. An unusual feature of the aba 1 gene sequence is a very high degree of shared identity among eight of the biosynthetic modules, at both the nucleotide and amino acid level. The majority of the modules share better than 70% nucleotide identity with another module in the complex, and modules with the same amino acid adenylation specificity share up to 95% identity. Insertion of a hygromycin B phosphotransferase ( HPT ) gene cassette in place of the module 4 sequence in aba 1 resulted in a cessation of AbA production, thus validating that the isolated gene encodes the AbA biosynthesis complex.