A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5' end of the RNA transcript (SMART) technique. A total of 106 clones showed halos in tricalcium phosphate (TCP) medium, and clone I-40 showed clear halos. The full-length cDNA of clone I-40 was 1355 bp with a complete open reading frame (ORF) of 1032 bp, encoding a protein of 343 amino acids. Multiple alignment analysis revealed a high degree of homology between the ORF of clone I-40 and delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) of other fungi. The ORF expression vector was constructed and transformed into Escherichia coli DH5α. The transformant (ORF-1) with the P5CDH gene secreted organic acid in medium with TCP as the sole source of phosphate. Acetic acid and α-ketoglutarate were secreted in 4 and 24 h, respectively. ORF-1 decreased the pH of the medium from 6.62 to 3.45 and released soluble phosphate at 0.172 mg·mL(-1) in 28 h. Expression of the P. oxalicum I1 p5cdh gene in E. coli could enhance organic acid secretion and phosphate-solubilizing ability.
A primary cDNA library of Aspergillus niger H1 was constructed using SMART (switching mechanism at the 5′ end of RNA transcript) technique. A total of 169 clones had halos on the insoluble phosphate medium, and clone H-47 had clear halos. The full-length cDNA of clone H-47 was 625 bp, with a complete open reading frame (ORF) of 390 bp, encoding a protein of 129 amino acids. Multiple alignment revealed a high degree of homology between the ORF of the clone and other fungi cellulosome-integrating protein (CipC-like). The expression vector of ORF was constructed and transformed into Escherichia coli DH-5α. The transformant (ORF-1) with the CipC-like gene secreted more organic acid when grown in tricalcium phosphate (TCP) medium, with TCP as the sole source of phosphate. E. coli DH5α containing the cipc-like gene secreted methanoic acid, acetic acid, malic acid, and citric acid reached 81.2, 93.3, 50.6, and 147.7 μg mL -1 , respectively, within 28 h. These results showed that the expression of the A. niger H1 CipC-like gene in E. coli could enhance organic acid secretion and improve phosphate solubilizing ability.
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