In this report, Shinella zoogloeoides NN6 was discovered to produce two rare sugar producing enzymes, D-allulose 3-epimerase (DAE) and L-rhamnose isomerase (LRhI), when cultured with L-rhamnose as the sole carbon source. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified DAE and LRhI revealed that the molecular masses of the monomeric subunits are 37 and 43 kDa, respectively, whereas gel filtration analysis showed that purified DAE and LRhI are 148 and 162 kDa, respectively, indicating that both enzymes form tetramers. The activity of DAE was the highest at 80°C in acetate buffer (pH 6.5) with Co²⁺, whereas LRhI exhibited maximum activity at 60°C in glycine–NaOH buffer (pH 9.0) with Mn²⁺. A co-immobilized biocatalyst was constructed using DAE (3.2 U) and LRhI (40 U). Activity profile analysis of this co-immobilized biocatalyst revealed that DAE activity was highest at 80°C in acetate buffer (pH 5.5), whereas the highest activity for LRhI was observed at 55°C in sodium phosphate buffer (pH 7.0). D-Allose was produced from 2% (w/w) D-fructose via D-allulose at 60°C and pH 9.0 in a one-pot reaction, providing a mixture of D-glucose, D-fructose, D-allulose and D-allose at a ratio of 1.3:62.7:23.6:12.4. This is the first report describing one-pot D-allose production using LRhI and DAE expressed in a single microorganism.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

The His-Asp phosphorelay signal transduction from histidine kinase (HK) to the response regulator (RR) is an important mechanism for adaptation to environmental changes. Aspergillus nidulans expresses 15 different HKs, which may be involved in different types of adaptations. As reactive oxygen species (ROS) are a key signal of environmental changes, some HKs might be involved in ROS generation through transcriptional regulation. Previously, we identified 3 HK (NikA, FphA, and HysA) deletion strains that showed increased ROS production during growth. We also showed that the phosphorylation function of HysA is involved in ROS generation. Here, we investigated the role of HysA in ROS production in A. nidulans cells. HysA protein was detected in both the cytosol and mitochondria by biological fractionation of the vegetative cell lysate of A. nidulans. The subcellular localization analysis by expressing the the HysA-GFP fusion protein along with MitoTracker Red staining did not clearly reveal mitochondrial localization of HysA at the conidiophore during asexual development. However, mitochondrial ROS in hysA mutant strains were detected by MitoSOX Red staining, and their excess levels possibly caused morphological changes during asexual development.

Musty or moldy off-odor in sake severely reduces its quality. Such off-odor is caused by 2,4,6-trichloroanisole (TCA), a compound that is produced by Aspergillus oryzae during sake production by O-methylating the precursor 2,4,6-trichlorophenol (TCP). TCP suppresses the growth of fungi, including A. oryzae, although TCA does not. Therefore, strains that are unable to convert TCP to TCA should be sensitive to TCP in the medium. Nevertheless, A. oryzae with a disrupted O-methyltransferase gene (ΔomtT) grew in a medium containing TCP. In agar medium, we observed no growth difference between the ΔomtT strain and a non-disrupted transformant; however, a significant growth delay was observed with the ΔomtT strain grown in liquid medium containing 0.5 µg/mL of TCP. This strain was more sensitive to low concentrations of TCP, suggesting that omtT contributes to the conversion (detoxification) of TCP in liquid culture. We generated A. oryzae RIB 40 mutants by ultraviolet irradiation and then cultured them in liquid medium containing TCP to obtain strains that did not produce moldy odor. The slow-growing strains were cultured in agar plates and then used to make koji with added TCP. We obtained three strains with lower TCA-producing ability and with sufficient hydrolase activities for sake brewing.

Screening for new sake yeasts can expand the sensory diversity of sake, due to their production of metabolites that characterize sake’s aroma and taste. In this study, mud from tidal flats in the Ariake Sea was screened for Saccharomyces cerevisiae strains with ethanol productivity suitable for sake brewing, and the brewing characteristics of isolated strains were evaluated. Five strains (H1-1, H1-2, H1-3, H3-1, and H3-2) classified as S. cerevisiae were isolated. Karyotype analysis by pulsed-field gel electrophoresis showed that five isolated strains were closely related to sake yeast strains (K7, K701, K9, K901, and Y52) instead of laboratory yeast strain. Results of small-scale brewing tests including sake yeast strains K701, K901, and Y52 showed that the five isolated strains have fermentation activity comparable to sake yeast strains. Principal component analysis (PCA) revealed that the five isolated strains produce higher levels of ethyl caproate and lower levels of acidic compounds than sake yeasts. In addition, isolated strains H3-1 and H3-2 produce higher levels of isoamyl acetate and lower levels of acetic acid than other isolated strains. Consequently, five S. cerevisiae strains that have high fermentation activity and differ from common sake yeast strains in terms of brewing characteristics were successfully isolated from the Ariake Sea.

Nitric oxide (NO) functions in cell protection or cell death, depending on its concentration. Therefore, regulation of the intracellular concentrations of NO by its degradation systems is important for cellular functions. One of the NO degrading enzymes, flavohemoglobin (FHb), which has NO dioxygenase (NOD) activity, is a promising target for antibiotics, based on the finding that FHb-deficient pathogens exhibited reduced host toxicity. Here, we developed a high-throughput method to measure the NOD activity. Our newly developed method could contribute to the screening of potential antibiotics with NOD inhibitory activity.