The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Production of the natural iron chelator deferriferrichrysin from Aspergillus oryzae and evaluation as a novel food-grade antioxidant.

BACKGROUND: Deferriferrichrysin (Dfcy) is a siderophore found in foods fermented by Aspergillus oryzae and is a promising candidate for an antioxidant food additive because of its high binding constant toward iron. However, the Dfcy concentration is typically low in foods and cultures. RESULTS: We optimised culture conditions to improve Dfcy production to 2800 mg L(-1) from 22.5 mg L(-1) under typical conditions. Then, we evaluated the potential of Dfcy as a food additive by measuring its safety, stability, and antioxidant activity. Dfcy was sufficiently stable that over 90% remained after pasteurisation at 63 °C for 30 min at pH 3-11, or after sterilisation at 120 °C for 4 min at pH 4-6. Dfcy showed high antioxidant activity in an oil-in-water model, where inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assays. Dfcy decreased PV and TBARS by 83% and 75%, respectively. Antioxidant activity of Dfcy was equal to or higher than that of the synthetic chelator EDTA. CONCLUSION: Our study provides the first practical method for production of Dfcy. Dfcy can be a novel food-grade antioxidant and the first natural alternative to the synthesised iron chelator EDTA. © 2015 Society of Chemical Industry.

Aspergillus oryzae FaeA is responsible for the release of ferulic acid, a precursor of off-odor 4-vinylguaiacol in sake brewing.

4-Vinylguaiacol (4-VG) is one of the most common off-flavors found in sake. 4-VG is produced from its precursor, ferulic acid, which is a component of the cell wall of the rice endosperm. The release of ferulic acid in sake brewing is thought to be mediated by feruloyl esterase produced by either Aspergillus oryzae or Saccharomyces cerevisiae. To investigate the effect of FaeA, a feruloyl esterase produced by A. oryzae, its loss-of-function strain was produced by genome co-editing. The feruloyl esterase activity of the faeA-deficient strain was drastically reduced. Sake was fermented using koji with S. cerevisiae strain G046, which can convert ferulic acid to 4-VG. Fermented sake was analyzed by measuring the 4-VG content and sensory evaluation. 4-VG content was reduced to approximately 10% of that of sake fermented with control koji. Sensory evaluation revealed that 4-VG was almost undetectable. Our findings showed that disruption of faeA in A. oryzae is a promising strategy to reduce 4-VG off-flavors in sake.

Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.

Over-expression of rate-limiting enzymes to improve alkaloid productivity.

Benzylisoquinoline alkaloids are one of the most important groups of secondary metabolites and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the productivity of these alkaloids, we investigated the effects of putative rate-limiting step enzymes in alkaloid biosynthesis. We constructed several over-expression vectors for biosynthetic enzymes and introduced them into cultured California poppy, a model isoquinoline alkaloid-producing plant. HPLC/LC-MS analysis of transgenic cells revealed that these enzymes varied in their ability to increase alkaloid production. We describe the use of a rate-limiting step gene to improve alkaloid productivity.