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In this study, we investigated a deleterious mutation in the β-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial β-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose levels during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

In this study, we developed an efficient gene targeting system for the osmophilic fungus Aspergillus chevalieri, which is commonly used in the production of a dried bonito, katsuobushi. Specifically, we utilized the clustered regularly interspaced short palindromic repeats/Cas9 system to disrupt the ATP sulfurylase encoding sC gene. This results in methionine auxotroph and selenate-resistance. Additionally, we disrupted the DNA ligase IV encoding ligD gene, which is required for nonhomologous end joining. Using the sC marker and selenate-resistance as a selection pressure, we were able to rescue the sC marker and generate a ΔligD ΔsC strain. We determined that the gene targeting efficiency of the ΔligD ΔsC strain was significantly higher than that of the parental ΔsC strain, which indicates that this strain provides efficient genetic recombination for the genetic analysis of A. chevalieri., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Escherichia coli strain, whose gene is one of the subunits of succinate dehydrogenase (sdhA), and gene of the transcriptional repressor of isocitrate lyase (iclR) were disrupted, accumulated 6.6 times as much intracellular succinate as the wild-type MG1655 strain in aerobic growth, but succinate was not found in the culture medium. E. coli citT gene that encodes a citrate transporter was cloned under the control of the lacI promoter in pBR322-based plasmid and the above strain was transformed. This transformant, grown under aerobic condition in M9-tryptone medium with citrate, accumulated succinate in the medium while no succinate was found in the medium without citrate. CitT was active as a succinate transporter for 168 h by changing the culture medium or for 24 h in fed-batch culture. This study suggests that the CitT transporter functions as a succinate exporter in E. coli for succinate production in the presence of citrate., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1 -based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii . The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S-23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.

The influence of carbon sources on bacterial community structure in the gut of the wood-feeding higher termite Nasutitermes takasagoensis was investigated. 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analyses revealed that the bacterial community structure changed markedly depending on feed components at the phylum level. Spirochaetes was predominant in the clone libraries from wood- and wood powder-fed termites, whereas Bacteroidetes was the largest group in the libraries from xylan-, cellobiose-, and glucose-fed termites, and Firmicutes was predominant in the library from xylose-fed termites. In addition, clones belonging to the phylum Termite Group I (TG1) were found in the library from xylose-fed termites. Our results indicate that the symbiotic relationship between termite and gut microorganisms is not very strong or stable over a short time, and that termite gut microbial community structures vary depending on components of the feeds.

Seven-week old female rats fed restricted foods including the fish oils Docosahesaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) and perilla oil with food intake decreased by 50%, had increases of fracture force and bone mineral density (BMD) and decreases in levels of Deoxypiridinoline (Dpd) and Calcium (Ca) in the urine, compared with those of rats with osteoporosis due to restricted soy bean oil food intake. Therefore, the fish oils DHA and EPA and perilla oil depressed excretion of urinary Ca and inhibited osteoporosis due to restricted food intake.

Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.

Independent use of K(2) and D(3) and simultaneous application of K(2) and D(3) inhibited the development of osteoporosis caused by PD food intake. The ALP activity of urine as a marker of bone formation osteoporosis did not rise in rats fed PD foods containing D(3), K(2) or both together. Body and womb weights fell in rats fed PD foods with D(3) K(2) and both D(3), K(2). Osteoporosis caused by PD food intake found to be very similar to type II osteoporosis in respects of inhibition by D(3) and K(2) and rising urinary ALP activity.

Galactinol, 1-O-(alpha-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material. UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots. Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source. Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves. The product was identified as galactinol by the retention times of HPLC, alpha-galactosidase digestion, and NMR spectrometry.

Rats fed on a restricted, semi-purified diet containing a 50%-reduced level of carbohydrate and oil, but normal levels of protein, minerals and vitamins, exhibited osteoporosis. However, rats fed on this restricted diet, but containing sugar cane wax, did not exhibit this bone disease. Sugar cane wax, containing a long-chain carbohydrate with an OH radical, prevented the development of osteoporosis via a non-estrogenic mechanism.

Genomic DNA encoding a beta-D-glucosidase (EC 3.2.1.21), which has beta-D-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this beta-D-glucosidase from Bifidobacterium breve clb had 46% identity with that of beta-glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more beta-D-glucosidase activity than that from Bifidobacterium breve clb. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme.