Cloning and expression of delta-1-pyrroline-5-carboxylate dehydrogenase in Escherichia coli DH5α improves phosphate solubilization.

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.

[Construction of cDNA library of Aspergillus niger H1 and screening of phosphate-dissolving related gene].

OBJECTIVE: To obtain phosphate-dissolving genes from cDNA library of Aspergillus niger H1. METHODS: The double-stranded cDNA was synthesized using switching mechanism at 5'end of RNA transcript technique and ligated to the vector pBluescript II SK (+). We transformed recombinant plasmid into E. coli HST08, resulting in a primary cDNA library. We screened clones with phosphate-dissolving activities on the insoluble phosphate medium and blasted the sequence in National Center for Biotechnology Information (NCBI). To study the phosphate dissolving mechanisms of the cloned gene, we analyzed the changes of the pH value, the soluble phosphate content and the production of organic acids in the insoluble phosphate liquid medium inoculated with the clones harboring the phosphate-dissolving gene. RESULTS: A cDNA library of A. niger H1 was successfully constructed. Titer tests showed that the content of constructed A. niger H1 cDNA library reached 5.65 x 10(6) cfu/mL, in which the percentage of recombinant clones was 99.15%. We screened 61 clones with phosphate-dissolving activities on the solid medium with insoluble phosphate. The corresponding gene in one of these clones was identified. The full length cDNA of clone H-54 was 839 bp, encoding a predicted protein with 180 amino acid residues. The expression of phosphate-dissolving gene in E. coli enhanced organic acids secretion and improved the phosphate solubilizing activity. Formic acid and acetic acid were found in 12 h, and malic acid and alpha-ketoglutarate were secreted in 24 h. The clone H-54 decreased the pH value of medium from 6.32 to 3.93 and released soluble phosphate up to 0.105 mg/mL in 36 h. CONCLUSION: We had obtained a phosphate-dissolving gene designated psgA from Aspergillus niger H1.