Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis

The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages.

First molecular report of causative agent of otomycosis due to Aspergillus luchuensis

Otomycosis is a fungal infection of the external auditory canal caused mainly by the genus Aspergillus. Aspergillus luchuensis, an industrially important fungus, is a member of Aspergillus section Nigri. In this report, we present a case of otomycosis due to Aspergillus luchuensis in a 43-year-old female patient. We performed a partial PCR-sequencing of β-tubulin and calmodulin genes to identify the isolate to the species level. Further, we determined the in vitro susceptibility of the isolate to nystatin, clotrimazole and itraconazole according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 protocol. Accordingly, the minimum inhibitory concentrations of clotrimazole, nystatin and itraconazole were 0.25μg/mL, 0.5μg/mL and 1μg/mL, respectively. This is the first report of clinically relevant isolation of Aspergillus luchuensis identified by a molecular technique as a causative agent of otomycosis.

Chromosome-Level Genome Sequence of the Black Koji Fungus Aspergillus luchuensis RIB2601

Aspergillus luchuensis is used for the production of awamori and shochu, which are traditional Japanese distilled alcoholic beverages. Here, we determined the chromosome-level genome sequence of A. luchuensis RIB2601.

Making Traditional Japanese Distilled Liquor, Shochu and Awamori, and the Contribution of White and Black Koji Fungi

The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called “honkaku shochu.” It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a unique temperature control method to ensure that these three important elements, which greatly affect the taste of the produced liquor, are balanced without any excess or deficiency. This review describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan and whose market is expected to expand worldwide, with special attention paid to the koji fungi cultivation step. Furthermore, we describe the history of the koji fungi used today in the production of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also report the latest research progress on this topic.

Mikroskopisko sēņu izmantošana sadzīves notekūdeņu attīrīšanā no farmaceitiski aktīvajām vielām

Promocijas darbā “Mikroskopisko sēņu izmantošana sadzīves notekūdeņu attīrīšanā no farmaceitiski aktīvajām vielām” veikta mikroskopisko sēņu izpēte un pilnveidota alternatīva metode notekūdeņu attīrīšanai no farmaceitiski aktīvajām vielām. Klasiskās notekūdeņu attīrīšanas metodes ne vienmēr var attīrīt sadzīves notekūdeņus no farmaceitiski aktīvo vielu piesārņojuma. Šī iemesla dēļ apkārtējā vidē var nonākt farmaceitiski aktīvās vielas un radīt apdraudējumu dzīvajiem organismiem. Lai novērstu farmaceitiski aktīvo vielu nokļūšanu apkārtējā vidē no sadzīves notekūdeņiem, nepieciešams meklēt jaunas un alternatīvas tehnoloģijas, lai uzlabotu gan jau esošās metodes, gan izstrādātu jaunas notekūdeņu attīrīšanas metodes. Kā viena no metodēm šī mērķa sasniegšanai var tikt izmantota bioloģiskā metode ar mikroskopiskajām sēnēm. Promocijas darba pētījums ir iedalīts divās galvenajās daļās. Pirmajā daļā veikti laboratorijas mēroga eksperimenti, lai noskaidrotu atbilstošākās mikroskopiskās sēnes notekūdeņu attīrīšanai no farmaceitiski aktīvajām vielām. Promocijas darba gaitā noskaidrots, ka mikroskopisko sēņu augstākā efektivitāte farmaceitisko vielu attīrīšanā no notekūdeņiem tika sasniegta, ja sēnes tiek izmantotas atsevišķi, nevis savstarpēji kombinētas. Noskaidrots, ka Trametes veriocolor un Aspergillus luchuensis ir vispiemērotākās mikroskopiskās sēnes notekūdeņu attīrīšanai no farmaceitiskajām vielām, tāpēc promocijas darba otrajā daļā izmantotas šīs mikroskopiskās sēnes. Promocijas darba otrajā daļā izstrādāts bioreaktors, kurā pārbaudīta izvēlēto mikroskopisko sēņu efektivitāte farmaceitiski aktīvo vielu attīrīšanā no sadzīves notekūdeņiem. Darba gaitā pārbaudīta bioaugmentācija kā iespējamā metode, lai uzlabotu mikroskopisko sēņu efektivitāti farmaceitiski aktīvo vielu attīrīšanā no sadzīves notekūdeņiem, un mikroskopisko sēņu spēja attīrīt notekūdeņus no fosfora un slāpekļa savienojumiem. Noskaidrots, ka mikroskopiskās sēnes spēj samazināt kopējo fosfora daudzumu sadzīves notekūdeņos. Iegūtie rezultāti parādīja arī to, ka mikroskopiskās sēnes spēj sadzīves notekūdeņus attīrīt no farmaceitiski aktīvo vielu piesārņojuma un bioaugmentācijas izmantošana var kalpot kā efektīva metode, lai uzturētu nepārtrauktu bioreaktora darbību ar mikroskopiskajām sēnēm.

Development of an itraconazole resistance gene as a dominant selectable marker for transformation in Aspergillus oryzae and Aspergillus luchuensis

ABSTRACT There are only a few combinations of antifungal drugs with known resistance marker genes in the Aspergillus species; therefore, the transformation of their wild-type strains is limited. In this study, to develop the novel dominant selectable marker for itraconazole, a fungal cell membrane synthesis inhibitor, we focused on Aspergillus luchuensis cyp51A (Alcyp51A), which encodes a 14-α-sterol demethylase related to the steroid synthesis pathway. We found that the G52R mutation in AlCyp51A and the replacement of the native promoter with a high-expression promoter contributed to itraconazole resistance in Aspergillus oryzae, designated as itraconazole resistant gene (itrA). The random integration in the A. luchuensis genome of the itrA marker cassette gene also allowed for transformation using itraconazole. Therefore, we succeed in developing a novel itraconazole resistance marker as a dominant selectable marker for transformation in A. oryzae and A. luchuensis.

Safety assessment of purine nucleosidase from Aspergillus luchuensis

Purine nucleosidase (EC 3.2.2.1) catalyzes the N-riboside hydrolysis of purine nucleosides to D-ribose and a purine base. This enzyme may be used in the production of beer and other alcoholic beverages to reduce the purine content of these products. Purine nucleosidase was obtained from Aspergillus luchuensis naturally occurring in grain sources. The safety profile of purine nucleosidase is not well documented in the scientific literature, and a series of toxicological studies were undertaken to investigate the safety of its use in food production. Purine nucleosidase from A. luchuensis was non-mutagenic and non-clastogenic in a standard Ames test and in vitro mammalian chromosome aberration assay. Administration of purine nucleosidase in a 90-day subchronic toxicity study in Sprague-Dawley rats did not elicit adverse findings on any hematology, clinical chemistry, urinalysis, organ weight, or histopathological parameter at doses up to 1700 mg total organic solids (TOS)/kg body weight/day, the highest dose tested. The results suggest purine nucleosidase to lack systemic toxic effect. The no-observed-adverse-effect level was concluded to be 1700 mg TOS/kg body weight/day. The results of the toxicology studies support the safety of purine nucleosidase from a non-genetically modified strain of A. luchuensis when used in food production.