Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil.
Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids
solubilizing phosphate.
Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can
solubilize P in tricalcium phosphate (TCP) medium. We aimed to obtain the gene expressed in Escherichia coli, which can
enhance organic acid secretion.
Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5’-end of
RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in
TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid,
fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid
secretion ability of the transformed E. coli DH5α™.
Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length
cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of
233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which
the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg.l-1 acetic acid within 48 h.
The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™
clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone
I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert.
Conclusions. We obtained a novel gene from Penicillium oxalicum I1 whose overexpression in E. coli DH5α™ increased the
secretion of acetic acid. This observation should help to understand what is the function of the gene isolated from P. oxalicum
as well as that of its homologs found in several other species of the Penicillium genus.