Ustiloxin B

Ustiloxin A (UA) and ustiloxin B (UB), two major mycotoxins produced by the pathogen of rice false smut (RFS) during rice cultivation, have attracted increasing attentions due to their potential health risks. However, limited data are available about their occurrence and fate in paddy fields and contamination profiles in rice. In this study, a field study was performed to investigate the occurrence and translocation of UA and UB in RFS-occurred paddies. For the first time to our knowledge, we reported a ubiquitous occurrence of the two ustiloxins in the paddy water (range: 0.01-3.46 μg/L for UA and <0.02-1.15 μg/L for UB) and brown rice (range: 0.09-154.08 μg/kg for UA and <0.09-23.57 μg/kg for UB). A significant positive correlation was observed between ustiloxin levels in paddy water and brown rice (rs = 0.48-0.79, p < 0.01). The occurrence of ustiloxin uptake in water-rice system was also evidenced by the rice exposure experiment, suggesting paddy water might be an important source for ustiloxin accumulation in rice. These results suggested that the contamination of ustiloxins in rice might occur widely, which was supported by the significantly high detection frequencies of UA (96.6%) and UB (62.4%) in polished rice (149 samples) from Hubei Province, China. The total concentrations of ustiloxins in the polished rice samples collected from Hubei Province ranged from <20.7 ng/kg (LOD) to 55.1 μg/kg (dry weight). Further studies are needed to evaluate the potential risks of ustiloxin exposure in the environment and humans.

Ustiloxin B is a ribosomally synthesized and post-translationally modified peptide (RiPP) first reported in Ascomycetes. Its biosynthetic pathway was recently identified in the filamentous fungus Aspergillus flavus. The precursor protein of ustiloxin B, UstA, has a signal peptide to the endoplasmic reticulum at its N-terminal and a subsequent tandemly highly repeated segment cleaved at Lys-Arg dipeptides by Kex2 protease; such proteins are called Kex2-processed repeat proteins (KEPs). RiPP biosynthetic pathways using KEPs as precursor proteins are widely distributed in the Fungi kingdom, with high diversity of precursor protein sequences. UstA in A. flavus has a 16-fold tandemly repeated segment containing the core peptide Tyr-Ala-Ile-Gly, which forms the ustiloxin B backbone structure, but it is unknown why such a costly-to-maintain highly repeated sequence is retained. Here, we replaced ustA, the gene encoding the ustiloxin B precursor protein, with synthetic genes encoding 1-, 3-, 5-, 7-, and 11-fold tandem-repeat segments in A. flavus, to investigate the relationship between the repeat number and ustiloxin B production. Ustiloxin B production increased quadratically with increasing repeat number in ustA variants, although it dropped in a previously constructed ustA variant that had a substituted synthetic gene encoding a 16-fold repeat segment probably because of the presence of the many rare codons in the sequence. We also examined the transcript levels of substituted synthetic genes in ustA variants, and surprisingly we found that the transcript levels of the synthetic genes increased linearly with increasing repeat number. This result implies that an unknown mechanism stabilizes ustA transcripts via the highly repeated structure in a feedback manner. We also constructed a transformant without the intron in native ustA, but no effect of intron removal was observed on either ustiloxin B production or the precursor gene transcript level. The costly-to-maintain highly repeated sequence in KEPs probably serves the purpose of maintaining stable transcripts and thus increasing the amount of substrate.

Ustiloxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) first reported in Ascomycetes. Originally identified as metabolites of the rice pathogenic fungus Ustilaginoidea virens, they were recently identified among the metabolites of the mold Aspergillus flavus, along with their corresponding biosynthetic gene cluster. Ustilaginoidea virens produces ustiloxins A and B, whereas A. flavus produces only ustiloxin B. Correspondingly, in U. virens, the ustiloxin precursor peptide, from which the compound backbone is cleaved and cyclized, contains the core peptides Tyr(Y)-Val(V)-Ile(I)-Gly(G) and Tyr(Y)-Ala(A)-Ile(I)-Gly(G) for ustiloxins A and B, respectively, whereas that of A. flavus contains only the YAIG motif for ustiloxin B. In this study, the gene that encodes the precursor peptide in A. flavus, ustA, was replaced with synthetic genes encoding the core peptides YVIG or FAIG, to investigate their compatibility with the ustiloxin biosynthetic machinery. We also examined the importance of the hydroxyl group on the aromatic ring of Tyr for cyclization of the YAIG core peptide. Against our expectation, the ustA variant possessing YVIG core peptides did not produce a detectable amount of ustiloxin A, even though the ustiloxin biosynthetic gene clusters of A. flavus and U. virens both contain 13 homologous genes. We confirmed that the lack of ustiloxin A production was not due to lack or insufficient expression of the substituted synthetic gene. This result, along with the differences between the primary sequences of UstYa and UstYb in A. flavus and U. virens, suggests that the ustiloxin biosynthetic machinery is optimized for the native core peptide sequences. The synthetic FAIG-encoding ustA did not yield any compounds specific to the FAIG core peptide, suggesting that the hydroxyl group on the aromatic ring of Tyr in the core peptide is indispensable for cyclization of the core peptide, even though it is not structurally involved in the cyclization.