Mutsumi Watanabe

Thiamine‐regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch‐like domain in the 5′‐UTR

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5′‐untranslated region (5′‐UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate‐binding riboswitch‐like domain that has been quite recently found in the 5′‐UTR of thiA.

Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859

5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.

Isolation of sake yeast mutants resistant to isoamyl monofluoroacetate to improve isoamyl acetate productivity

Abstract The growth of sake yeast was significantly inhibited by isoamyl monofluoroacetate. This inhibition was caused by free monofluoroacetic acid, a highly toxic compound, formed from isoamyl monofluoroacetate by yeast esterase. Yeast mutants with low esterase activity were expected to become resistant to isoamyl monofluoroacetate. Fifty mutants resistant to this ester were isolated from haploid strains derived from sake yeast. Two strains which had less than 15% of the original activity of esterase were obtained. These 2 strains could be used for brewing sake like their parental strain, and produce sake which contained 1.5 to 2.0 times higher amounts of isoamyl acetate and isobutyl acetate and had an excellent fruity flavor.

Different missense mutations in PDR1 and PDR3 genes from clotrimazole-resistant sake yeast are responsible for pleiotropic drug resistance and improved fermentative activity.

Series of gain-of-function missense mutations in PDR1 and PDR3 that encode transcription factors are known to increase the expression of ABC transporters that extrude various kinds of drugs out of a cell, leading to pleiotropic drug resistance (PDR). Previously, the PDR1-H176 (L309S) allele from a clotrimazole (CTZ)-resistant sake yeast mutant has been cloned and found to be sufficient to confer PDR and improved fermentative activity on the arginase-deficient (car1/car1) sake yeast HL163. In this study, another PDR1 and three PDR3 a!leles were cloned from other CTZ-resistant mutants. These alleles contain different missense mutations, PDR1-2 (M308I), PDR3-C21 (L950S), PDR3-C231 (G948D), and PDR3-11 (G957D). The gene recombinants containing each of the mutations found in CTZ-resistant mutants were constructed using the wild-type sake yeast K1001. The gene recombination was designed to generate only a nucleotide replacement without any other DNA changes such as insertion of external DNA fragments. The resultant recombinants that were heterozygous for each of the mutations showed various degrees of drug resistance that were comparable to those of the CTZ-resistant mutants. The recombinants, except one introduced with PDR1-H176, also fermented sake mash faster and produced higher amounts of alcohol than K1001 did. These results demonstrated that PDR1-2, PDR3-C21, PDR3-C231, and PDR3-11 are sufficient to confer PDR and improved fermentative activity on K1001, and suggested that previous results in which fermentative activity of HL163 was significantly improved by PDR1-H176 must have depended on its genetic background, probably arginase deficiency.

Characterization of a PDR1 mutant allele from a clotrimazole-resistant sake yeast mutant with improved fermentative activity

Clotrimazole-resistant mutants from various sake yeasts show improved fermentative activity in sake mash while retaining their parental advantages for sake making. These mutants also exhibit pleiotropic drug resistance (PDR) phenotypes. To investigate the relationship between the improvement of fermentative activity and PDR phenotypes, a PDR1 mutant allele (pdr1-h176) encoding a transcription factor was cloned from a clotrimazole-resistant mutant, HL176 (MATa/MATalpha), using PCR amplification. The nucleotide sequences of pdr1-h176 and its wild allele were determined. The mutant allele contained a missense point mutation (L309S) that can confer a PDR phenotype on yeast. This amino acid substitution is located in the conserved motif II in the inhibitory domain of Pdr1p, and is very close to the cluster of three mutation points (P298A, K302Q, and M308I) described by Carvajal et al. (Mol. Gen. Genet., 256, 406-415, 1997) in laboratory strains. A PDR1 wild allele of HL163, the parent strain of HL176, was replaced by pdr1-h176 using gene recombination at the homologous site. The resultant transformants (PDR1/pdr1-h176) showed the same PDR phenotype as HL176, and they fermented sake mash efficiently even in the final fermentation stage, while HL163 did not. The amino acid substitution (L309S) in pdr1-h176 was considered to be sufficient to improve the fermentative activity of sake yeast, in addition to conferring the PDR phenotype.

Properties of sake yeast mutants resistant to isoamyl monochloroacetate

Abstract Isoamyl monochloroacetate ( i -AmOCIAc) inhibited the growth of sake yeast. AOF10, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate ( i -AmOAc), also showed resistance to i -AmOClAc. Monochloroacetic acid formed by an esterase through the hydrolysis of i -AmOClAc was more effective at glycolysis inhibition than i -AmOClAc. Forty five mutants resistant to i -AmOClAc were isolated from a parental haploid strain (HL-69) derived from sake yeast. Three had less than 20% of the original activity of the esterase. All mutants fermented sake mash as well as the parent strain, HL-69, and accumulated 1.3 to 1.7-fold the levels of i -AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by HL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i -AmOAc and 1.3-fold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (1.6-fold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band ( EST2 ) were closely related to the activity of the esterase toward i -AmOAc.

Synthesis of 5-methyluridine by a thermophile, Bacillus stearothermophilus JTS 859

Many thermophiles, which can grow at 65°C, were examined as to their ability to produce 5-methyluridine from inosine and thymine (5-methyluracil) in the presence of phosphate and cells as enzyme sources. Bacillus stearothermophilus JTS 859 was selected as a strain that synthesized 5-methyluridine efficiently. The reaction is supposed to be carried out by a combination of a thermostable purine nucleoside phosphorylase and a thermostable pyrimidine nucleoside phosphorylase. Their half-lives were 7200 hr and 400 hr at 63°C, and 148 hr and 14 hr at 70°C, respectively.

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of deltapdr5 and deltasnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity - 1) x 1443] of the deltapdr5 and deltasnq2 strains rose faster than that of HL69. On the other hand, a deltayor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the deltayor1 slowed down compared with that of CTZ21. A deltayor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the deltapdr5 and deltasnq2 strains of CTZ21. In contrast, the deltayor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.

Optimal culture conditions of Bacillus stearothermophilus JTS 859 for the production of a thermostable enzyme to synthesize 5-methyluridine

Abstract The optimal culture conditions of a thermophile, Bacillus stearothermophilus JTS 859, were examined for the production of a thermostable enzyme to synthesize 5-methyluridine from inosine and thymine in the presence of phosphate by the ribosyl transfer reaction. Elevation of the agitation velocity and the addition of inosine to the medium were effective in improving the production of the enzyme. By optimizing the culture conditions, the production amounts of cells and enzyme per volume of medium were increased by 4.6 and 10.1 times, respectively, compared to the initial results.