Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.
We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.
Yamada
Osamu
O
National Research Institute of Brewing, Kagamiyama, Higashi-Hirshima, Japan. yamada@nrib.go.jp
Sakamoto
Kazutoshi
K
Tominaga
Mihoko
M
Nakayama
Tasuku
T
Koseki
Takuya
T
Fujita
Akiko
A
Akita
Osamu
O
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D010455:Peptides; D012233:Rhizopus / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D000900:Anti-Bacterial Agents; D004274:DNA, Recombinant; D010455:Peptides