Ritsuko Kodaira

Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae.

Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.

Purification and characterization of chitosanase and exo-β-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.

Isolation of sake yeast strains possessing various levels of succinate- and/or malate-producing abilities by gene disruption or mutation

Succinate and malate are the main taste components produced by yeast during sake (Japanese alcohol beverage) fermentation. Sake yeast strains possessing various organic acid productivities were isolated by gene disruption. Sake fermented using the aconitase gene (ACO1) disruptant contained a two-fold higher concentration of malate and a two-fold lower concentration of succinate than that made using the wild-type strain K901. The fumarate reductase gene (OSM1) disruptant produced sake containing a 1.5-fold higher concentration of succinate as compared to the wild-type, whereas the alpha-ketoglutarate dehydrogenase gene (KGD1) and fumarase gene (FUMI) disruptants gave lower succinate concentrations. The Deltakgd1 disruptant exhibited lower succinate productivity in the earlier part of the sake fermentation, while the Deltafum1 disruptant showed lower succinate productivity later in the fermentation, indicating that succinate is mainly produced by an oxidative pathway of the TCA cycle in the early phase of sake fermentation and by a reductive pathway in the later phases. Sake yeasts with low succinate productivity and/or high malate productivity was bred by isolating mutants unable to assimilate glycerol as a carbon source. Low malate-producing yeasts were also obtained from phenyl succinate-resistant mutants. The mutation of one of these mutant strains with low succinate productivity was found to occur in the KGD1 gene. These strains possessing various succinate- and/or malate-producing abilities are promising for the production of sake with distinctive tastes.

Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101

Abstract A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101. The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0–30%). The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetyl-glucosamine oligomers and colloidal chitin. The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined. B. gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues. Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain. The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases.

Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells.

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn(2+), Co(2+) and Cu(2+) ions, as well as by SH-blocking reagents. The K(m) values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic Bacterium Aeromonas hydrophila Strain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A β-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-β-D-glucosaminide and pNP-N-acetyl-β-D-galactosaminide. A gene coding for the purified β-N-acetylglucosaminidase was isolated. The ORF identified is 2,661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial β-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.

The bacterium Burkholderia gladioli strain CHB101 produces two different kinds of chitinases belonging to families 18 and 19 of the glycosyl hydrolases

Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.

Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has an ability for production of scopoletin. In this cell culture, scopoletin is taken up from culture medium and accumulated in vacuoles after conversion to scopolin when cells are treated with 2,4-dichlorophenoxyacetic acid (2,4-D) (Taguchi et al. (2000)). To clarify the effect of 2,4-D on tobacco cells, its interaction with several other plant hormones was investigated. Other auxins also stimulated the uptake in the same manner as 2,4-D did, although higher concentrations were required than that of 2,4-D. When p-chlorophenoxyisobutyric acid (PCIB), an antiauxin, was added to the cell culture before 2,4-D, it inhibited 2,4-D-stimulated scopoletin uptake. This result suggests that the stimulation of scopoletin uptake was one of the auxin effects on tobacco cells. Among other classes of plant hormones that were tested, only salicylic acid stimulated the uptake. When these hormones were added to the cell cultures before 2,4-D, methyl jasmonate and kinetin reduced scopoletin uptake. These results suggest that this scopoletin uptake by tobacco cells is regulated by the interaction between different plant hormones.

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from the white rot basidiomycete Irpex lacteus.

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.