Evidence of polyamines mediated 2-acetyl-1-pyrroline biosynthesis in aromatic rice rhizospheric fungal species Aspergillus niger

Abstract

Rhizosphere soil of aromatic rice inhabits different fungal species that produce many bioactive metabolites including 2-acetyl-1-pyrroline (2AP). The mechanism for the biosynthesis of 2AP in the fungal system is still elusive. Hence, the present study investigates the role of possible nitrogen (N) precursors such as some amino acids and polyamines as well as the enzymes involved in 2AP synthesis in the fungal species isolated from the rhizosphere of aromatic rice varieties. Three fungal isolates were found to synthesize 2AP (0.32-1.07 ppm) and maximum 2AP was synthesized by Aspergillus niger (1.07 ppm) isolated from rhizosphere of Dehradun Basmati (DB). To determine the N source for 2AP synthesis, various N sources such as proline, glutamate, ornithine putrescine, spermine, and spermidine were used in place of putrescine in the synthetic medium (Syn18). The results showed that maximum 2AP synthesis was found with putrescine (1.07 ppm) followed by spermidine (0.89 ppm) and spermine (0.84 ppm). Further, LC-QTOF-MS analysis revealed the mobilization of spermine and spermidine into the putrescine, indicating that putrescine is the key N source for 2AP synthesis. Moreover, higher enzyme activity of DAO, PAO, and ODC as well as higher content of methylglyoxal metabolite in the A. niger NFCCI 5060 as compared to A. niger NFCCI 4064 (control) suggests the prominent role of these enzymes in the synthesis of 2AP. In conclusion, this study showed evidence of the polyamines mediated 2AP biosynthesis in A. niger NFCCI 5060.

Keywords: 2-Acetyl-1-pyrroline; Aromatic rice; Aspergillus niger; Polyamines; Rhizosphere fungi.

Fig. 1

Effect of different N sources on the synthesis of 2AP by A. niger NFCCI 5060. The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 2

2AP synthesis by A. niger NFCCI 5060 under polyamine supplementation. A Medium with spermidine (Put + Spd). B Medium with spermine (Put + Spm). The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 3

Quantification of putrescine, spermidine and spermine by LC–MS in A. niger NFCCI 4064 (non 2AP-producing strain; control) and A. niger NFCCI 5060 (2AP-producing strain). A, C, E Spermidine (Spd) only as a nitrogen source. B, D, F Spermine (Spm) only as a nitrogen source. The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 4

LC–MS-based relative quantification of acetylspermidine and acetylspermine in A. niger NFCCI 4064 (non 2AP-producing strain; control) and A. niger NFCCI 5060 (2AP-producing strain). A, C With spermidine only. B, D With spermine only. The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 5

Enzyme assays of different enzymes involved in the 2AP biosynthetic pathway in A. niger NFCCI 4064 (non 2AP-producing strain; control) and A. niger NFCCI 5060 (2AP-producing strain), A ODC, B PAO, C DAO, and D BADH2. The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 6

Quantification of methylglyoxal inA. niger NFCCI 4064 (non 2AP-producing strain; control) and A. niger NFCCI 5060 (2AP-producing strain). The vertical bars indicate the standard deviation of the mean. The different letters indicate significant differences at P ≤ 0.05

Fig. 7

Proposed 2AP biosynthetic pathway in A. niger NFCCI 5060. (ODC, ornithine decarboxylase; SpdS, spermidine synthase; SMS, spermine synthase; SMOX, spermine oxidase; SAT1, spermidine/spermine-N1-acetyltransferase; PAO, polyamine oxidase; DAO, diamine oxidase)