Physiological role of the EHL gene in sake yeast and its effects on quality of sake.

The EHL1/2/3 genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast Saccharomyces cerevisiae. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed ehl1/2/3 mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the EHL1/2/3 genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the EHL1/2/3 genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the ehl1/2/3 mutant was comparable with that of strain H3, but succinate production was decreased in the ehl1/2/3 mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the ehl1/2/3 mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the EHL1/2/3 genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.

Effect of spo0A, sigE, sigG, and sigK disruption on butanol production and spore formation in Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564).

Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564) is a butanol-producing strain suitable for application to butanol production from cellulosic materials by co-culture with cellulolytic and thermophilic species, such as Hungateiclostridium thermocellum (synonym: Clostridium thermocellum). The optimal temperature for butanol production by strain N1-4 is 30 °C, and the strain is sensitive to a high culture temperature of 37 °C. Given that spore formation is observed at high frequency when strain N1-4 is cultivated at 37 °C, we assumed in a previous study that the initiation of sporulation is related to a decrease in butanol production. Therefore, to investigate the relationship between butanol production and spore formation, we generated strain N1-4 isolates in which genes related to spore formation were disrupted. The sporulation-related gene disruptants of spo0A, sigE, sigG, and sigK lost the ability to produce heat-resistant spores, irrespective of the culture temperature. Among the gene disruptants produced, only the spo0A disruptant lost butanol-producing ability when cultivated at 30 °C. Interestingly, the sigE disruptant maintained butanol productivity similar to that observed at 30 °C, even when cultivated at 37 °C. In addition, the sigE disruptant successfully produced butanol from Avicel cellulose by co-culture with H. thermocellum at a fermentation temperature of 37 °C.

The bio3 mutation in sake yeast leads to changes in organic acid profiles and ester levels but not ethanol production.

Biotin is an essential coenzyme that is bound to carboxylases and participates in fatty acid synthesis. The fact that sake yeast exhibit biotin prototrophy while almost all other Saccharomyces cerevisiae strains exhibit biotin auxotrophy, implies that biotin prototrophy is an important factor in sake brewing. In this study, we inserted a stop codon into the biotin biosynthetic BIO3 gene (cording for 7,8-diamino-pelargonic acid aminotransferase) of a haploid sake yeast strain using the marker-removable plasmid pAUR135 and investigated the fermentation profile of the resulting bio3 mutant. Ethanol production was not altered when the bio3 mutant was cultured in Yeast Malt (YM) medium containing 10% glucose at 15 °C and 30 °C. Interestingly, ethanol production was also not changed during the sake brewing process. On the other hand, the levels of organic acids in the bio3 mutant were altered after culturing in YM medium and during sake brewing. In addition, ethyl hexanoate and isoamyl acetate levels decreased in the bio3 mutant during sake brewing. Metabolome analysis revealed that the decreased levels of fatty acids in the bio3 mutant were attributed to the decreased levels of ethyl hexanoate. Further, the transcription level of genes related to the synthesis of ethyl hexanoate and isoamyl acetate were significantly reduced. The findings indicated that although the decrease in biotin biosynthesis did not affect ethanol production, it did affect the synthesis of components such as organic acids and aromatic compounds. Biotin biosynthesis ability is thus a key factor in sake brewing.

Elucidation of the enzyme involved in 2,3,5-triphenyl tetrazolium chloride (TTC) staining activity and the relationship between TTC staining activity and fermentation profiles in Saccharomyces cerevisiae.

2,3,5-Triphenyl tetrazolium chloride (TTC) staining is a method to distinguish the mitochondrial activity of cells based on the color: colorless TTC turns red when under reducing conditions. Although the assay reflects the mitochondrial activity of cells, which enzyme(s) in the electron transport system contribute to TTC reduction has been unclear. TTC staining assays using gene disruptants related to the electron transport system in Saccharomyces cerevisiae revealed those disruptants related to electron transport from each electron donor to ubiquinone (red colonies) and disruptants that were related to ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase (white colonies). In addition, when the enzyme activities of ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase were measured using TTC as the electron acceptor, only ubiquinol-cytochrome c oxidoreductase showed TTC reduction activity, and the activity was enhanced by potassium cyanide, an inhibitor of cytochrome c oxidase. These results indicated that ubiquinol-cytochrome c oxidoreductase is involved in TTC reduction in S. cerevisiae. The fermentation profiles of BY4741UΔcor1 and BY4741UΔcox4, which exhibited no TTC staining activity, were almost identical to that of the parental strain BY4741U. However, cell growth and ethanol and succinate production of the ura3-mutated strain BY4741, which also exhibited no TTC staining activity, was altered compared to those of BY4741U, indicating that the fermentation profile varies among strains that show no TTC staining activity. The relationship between uracil metabolism and TTC staining activity was also determined based on metabolome analysis.

Isolation of ammonia gas-tolerant extremophilic bacteria and their application to the elimination of malodorous gas emitted from outdoor heat-treated toilets.

Ammonia gas-tolerant extremophilic bacteria capable of growing in atmospheres containing up to 4000 ppm of gaseous ammonia were isolated. These bacteria were capable of growing in nutrient broth containing high concentrations of ammonia water, with growth in medium augmented with 0.1-0.2% ammonia exceeding that in medium without ammonia. The minimal inhibitory concentration of ammonia in the medium was 0.5%. The isolated ammonia gas-tolerant bacterium was moderately alkaliphilic, with optimum growth was observed at pH 9. DNA sequence analysis of the 16SrRNA gene revealed that the isolated bacterium was Bacillus lentus. Furthermore, extremophilic bacteria cultured in a 1300 ppm ammonia gas atmosphere on agar medium containing no nitrogen sources were observed to use ammonia gas for growth. These bacteria were identified as Paenibacillus lentus and Bacillus altitudinis based on 16SrRNA gene sequence analysis. The deodorizing effect of ammonia odor by the isolated bacteria immobilized on sawdust was evaluated. The findings showed that forcing ammonia gas through a column containing B. altitudinis immobilized on sawdust reduced the concentration of ammonia gas by 30% compared to columns containing sawdust only. The isolated bacteria immobilized on wood sawdust lost the capacity deodorization after drying, but this function could be restored with increased moisture. The ammonia gas-tolerant extremophilic bacteria immobilized on sawdust show considerable potential for use in ameliorating malodors associated with outdoor heat-treated toilets.

Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

Potential use of methane fermentation digested slurry as a low-cost, environmentally-friendly nutrient for bioethanol production from crude glycerol by Klebsiella variicola TB-83D.

A methane fermentation digested slurry (MFDS) was evaluated as a substitute for the commercial nutrient, yeast extract (YE), in ethanol production from glycerol by Klebsiella variicola strain TB-83D. In pH-controlled fed-batch cultures, partial replacement of YE by MFDS did not reduce ethanol productivity significantly. However, non-sterilized MFDS had negative effects on glycerol fermentation by this strain. Although ethanol production decreased when YE was completely replaced by sterilized MFDS, the use of crude glycerol and sterilized MFDS achieved a yield of 14.6 g/L ethanol. This is the first study to report the use of MFDS as the sole nutrient for ethanol production from glycerol, which contributes to the development of a low-cost glycerol biorefinery derived from the biodiesel fuel industry.

Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C.

We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels.IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

Butanol production from alkali-pretreated rice straw by co-culture of Clostridium thermocellum and Clostridium saccharoperbutylacetonicum.

The co-culture of cellulolytic Clostridium thermocellum NBRC 103400 and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4 produced 5.5 g/L of butanol from 40 g/L of delignified rice straw pretreated with 1% (wt/vol) NaOH. The addition of cellulase (100 U/g biomass) in a co-culture system significantly increased butanol production to 6.9 g/L using 40 g/L of delignified rice straw. Compared to the control, this increase in butanol production was attributed to the enhancement of exoglucanase activity on lignocellulose degradation in experimental samples. The results showed that the co-culture system in conjunction with enhanced exoglucanase activity resulted in cost-effective butanol production from delignified rice straw.

Isolation of a high malic and low acetic acid-producing sake yeast Saccharomyces cerevisiae strain screened from respiratory inhibitor 2,4-dinitrophenol (DNP)-resistant strains.

We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+).

Butanol production from crystalline cellulose by cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4.

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.