Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938.
The gene (aba1) 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 aba1 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 aba1 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 aba1 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 aba1 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 aba1 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 aba1 resulted in a cessation of AbA production, thus validating that the isolated gene encodes the AbA biosynthesis complex.
Slightom
Jerry L
JL
AureoGen Biosciences Inc., 6475 Technology Avenue, Suite C, Kalamazoo, MI 49009, USA.
Metzger
Brian P
BP
Luu
Hang T
HT
Elhammer
Ake P
AP
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D047630:Depsipeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D055786:Gene Knockout Techniques; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D020161:Physical Chromosome Mapping; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012995:Solubility
D017931:DNA Primers; D004271:DNA, Fungal; D047630:Depsipeptides; D005656:Fungal Proteins; C071398:aureobasidin A