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ABSTRACT: AIMS: To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid-producing engineered MG1363 strain under stress condition was investigated. METHODS AND RESULTS: We cloned carotenoid biosynthesis genes from yellow-pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid-producing Lactococcus lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C30 carotenoid 4,4'-diaponeurosporene. After exposure to 32-mmol l-1 H2 O2 , low pH (1.5, acidified with HCl), and 10% bile acid, the survival rates of the MG1363 strain harboring pRC were 18.7-, 6.8-, and 4.4-fold higher, respectively, than that of MG1363 strain harboring the empty vector pRH100. CONCLUSIONS: The expression of carotenoid biosynthesis genes from E. gilvus improves the multi-stress tolerance of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: First report of the improvement of multi-stress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production. © 2013 The Authors Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.
Journal of Applied Microbiology 03/2013; · 2.34 Impact Factor
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ABSTRACT: Haa1 is a transcriptional activator required for Saccharomyces cerevisiae adaptation to weak acids. Here we show that the constitutive HAA1-overexpressing strain acquired a higher level of acetic acid tolerance. Under conditions of acetic acid stress, the intracellular level of acetic acid was significantly lower in HAA1-overexpressing cells than in the wild-type cells.
Applied and environmental microbiology 09/2012; 78(22):8161-3. · 3.69 Impact Factor
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ABSTRACT: A massive screening was performed to identify an acetate-tolerant strain of Saccharomyces cerevisiae. We found that S. cerevisiae ATCC 38555 is acetate-tolerant, with a fermentation profile indicating that it has a high level of acetate adaptation. The global gene expression analysis indicated that AFT1- and HAA1-regulated genes are clearly up-regulated.
Journal of Bioscience and Bioengineering 07/2012; · 1.79 Impact Factor
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ABSTRACT: During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed.
We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance.
In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.
Microbial Cell Factories 04/2012; 11:40. · 3.55 Impact Factor
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ABSTRACT: The protective effects of live Lactobacillus paracasei NFRI 7415 on alcoholic liver disease were investigated. Male Fischer 344 rats were fed a control diet (CD), an ethanol diet (ED) (35.8% of total energy from ethanol), or an ethanol diet containing 20% live Lb. paracasei NFRI 7415 (10(7) cfu/g) (LD) for 10 weeks. The results indicated that live Lb. paracasei NFRI 7415 reduced the total cholesterol concentration of the plasma and liver in the rats fed the LD. The level of docosahexaenoic acid (DHA; 22:6n-3) in the plasma and liver of the LD group was higher than in the ED group. Chronic alcohol consumption decreased the level of n-3 fatty acid in the plasma and liver of the ED group. These results indicated that live Lb. paracasei NFRI 7415 can adjust the fatty acid composition of the plasma and liver, and that it is possible to decrease liver damage due to chronic alcohol intake.
Bioscience Biotechnology and Biochemistry 02/2012; 76(2):232-7. · 1.28 Impact Factor
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ABSTRACT: Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking.
Journal of Bioscience and Bioengineering 01/2012; 113(5):592-5. · 1.79 Impact Factor
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ABSTRACT: During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking.
International journal of food microbiology 01/2012; 152(1-2):40-3. · 3.01 Impact Factor
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ABSTRACT: We constructed a self-cloning diploid baker's yeast strain that overexpressed the transcription activator Msn2. It showed higher tolerance to freeze-thaw stress and higher intracellular trehalose level than observed in the wild-type strain. Overexpression of Msn2 also enhanced the fermentation ability of baker's yeast cells in frozen dough. Hence, Msn2-overexpressing baker's yeast should be useful in frozen-dough baking.
Bioscience Biotechnology and Biochemistry 01/2012; 76(3):624-7. · 1.28 Impact Factor
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The Journal of General and Applied Microbiology 01/2011; 57(5):303-7. · 0.98 Impact Factor
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ABSTRACT: High temperature and agitation are advantageous for ethanol production from insoluble feedstock when using the simultaneous saccharification and fermentation (SSF) technique. To construct an effective SSF system, a respiratory-deficient mutant was isolated from the thermotolerant yeast Candida glabrata. Our results suggest that this respiratory-deficient mutant has higher ethanol production abilities in SSF.
Journal of Bioscience and Bioengineering 08/2010; 110(2):176-9. · 1.79 Impact Factor
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ABSTRACT: During bread-making processes, yeast cells are exposed to multiple stresses. Air-drying stress is one of the most harmful stresses by generation of reactive oxygen species (ROS). Previously, we discovered that the novel N-acetyltransferase Mpr1/2 confers oxidative stress tolerance by reducing intracellular ROS level in Saccharomyces cerevisiae Sigma1278b strain. In this study, we revealed that Japanese industrial baker's yeast possesses one MPR gene. The nucleotide sequence of the MPR gene in industrial baker's yeast was identical to the MPR2 gene in Sigma1278b strain. Gene disruption analysis showed that the MPR2 gene in industrial baker's yeast is involved in air-drying stress tolerance by reducing the intracellular oxidation levels. We also found that expression of the Lys63Arg and Phe65Leu variants with enhanced enzymatic activity and stability, respectively, increased the fermentation ability of bread dough after exposure to air-drying stress compared with the wild-type Mpr1. In addition, our recent study showed that industrial baker's yeast cells accumulating proline exhibited enhanced freeze tolerance in bread dough. Proline accumulation also enhanced the fermentation ability after air-drying stress treatment in industrial baker's yeast. Hence, the antioxidant enzyme Mpr1/2 could be promising for breeding novel yeast strains that are tolerant to air-drying stress.
International journal of food microbiology 03/2010; 138(1-2):181-5. · 3.01 Impact Factor
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ABSTRACT: EOS1 is required for tolerance to oxidative stress in Saccharomyces cerevisiae; mutants are defective in the gene sensitive to hydrogen peroxide and tolerant to tunicamycin. To clarify the function of Eos1, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress the hydrogen peroxide-sensitive eos1 mutation. We identified one such gene, IZH2, which has previously been reported to be a Zap1-regulated gene. However, the EOS1 and IZH2 genes do not themselves appear to be functionally interchangeable. Double disruption of the EOS1 and IZH2 genes yielded a slow-growth phenotype, suggesting that the two proteins are involved in related cellular processes. DNA microarray analysis revealed decreased expression of Zap1-regulated genes in the eos1-deletion mutant (Deltaeos1). Thus, it is likely that Eos1 is involved in zinc homeostasis.
FEMS Yeast Research 01/2010; 10(3):259-69. · 2.40 Impact Factor
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ABSTRACT: Bacterial contamination causes yield reduction during ethanol production from molasses. To prevent contamination, construction of a fermentation system using acetate-tolerant yeast under an acetate-containing condition was attempted. Schizosaccharomyces pombe was screened as an acetate-tolerant strain. Bacterial contamination was significantly prevented by the combined use of Sc. pombe and acetate.
Journal of Bioscience and Bioengineering 10/2009; 108(3):216-9. · 1.79 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.
Applied and environmental microbiology 09/2009; 75(21):6706-11. · 3.69 Impact Factor
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ABSTRACT: A vanillin-tolerant strain of Saccharomyces cerevisiae was screened and its intracellular ergosterol levels were compared with several laboratory yeast strains to study the potential relationship between ergosterol content and vanillin tolerance. Saccharomyces cerevisiae NBRC1950 was selected as a vanillin-tolerant strain. Its ergosterol content was higher than those of the laboratory strains. The results of DNA microarray and quantitative reverse transcriptase-polymerase chain reaction analysis showed that five genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5, and ERG7) were upregulated in NBRC 1950 compared with strain X2180, suggesting that high expression of genes involved in ergosterol biosynthesis may cause high ergosterol content in strain NBRC 1950. The S. cerevisiae HX strain, which was a high-ergosterol-containing strain derived from X2180, was more tolerant to vanillin than the parental strain, suggesting that high ergosterol content may, in part, be responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin.
FEMS Microbiology Letters 08/2009; 299(1):95-9. · 2.04 Impact Factor
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ABSTRACT: During the fermentation of dough and the production of baker's yeast (Saccharomyces cerevisiae), cells are exposed to numerous environmental stresses (baking-associated stresses) such as freeze-thaw, high sugar concentrations, air-drying and oxidative stresses. Cellular macromolecules, including proteins, nucleic acids and membranes, are seriously damaged under stress conditions, leading to the inhibition of cell growth, cell viability and fermentation. To avoid lethal damage, yeast cells need to acquire a variety of stress-tolerant mechanisms, for example the induction of stress proteins, the accumulation of stress protectants, changes in membrane composition and repression of translation, and by regulating the corresponding gene expression via stress-triggered signal-transduction pathways. Trehalose and proline are considered to be critical stress protectants, as is glycerol. It is known that these molecules are effective for providing protection against various types of environmental stresses. Modifications of the metabolic pathways of trehalose and proline by self-cloning methods have significantly increased tolerance to baking-associated stresses. To clarify which genes are required for stress tolerance, both a comprehensive phenomics analysis and a functional genomics analysis were carried out under stress conditions that simulated those occurring during the commercial baking process. These analyses indicated that many genes are involved in stress tolerance in yeast. In particular, it was suggested that vacuolar H+-ATPase plays important roles in yeast cells under stress conditions.
Biotechnology and Applied Biochemistry 08/2009; 53(Pt 3):155-64. · 1.53 Impact Factor
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ABSTRACT: A spontaneous flocculation mutant (Cgflo1) was obtained from Candida glabrata. Flocculation of Cgflo1 was dependent on divalent cations such as Ca(2+) and Mg(2+) and was inhibited by galactose. Cgflo1 is believed to be a useful strain for bioethanol production based on its flocculation ability at higher temperatures.
Journal of Bioscience and Bioengineering 05/2009; 107(4):379-82. · 1.79 Impact Factor
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ABSTRACT: The survival, impacts on microbiota and gene transfer ability of genetically modified (GM) Lactococcus lactis containing ermAM gene in soil and water environments were investigated. After inoculation of GM L. lactis into environments, its DNA remained longer, and the microbial community was changed. No transfer of chromosomal ermAM was observed.
Journal of Bioscience and Bioengineering 04/2009; 107(3):339-43. · 1.79 Impact Factor
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ABSTRACT: Freezing tolerance is an important characteristic for baker's yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of -5 degrees C and -15 degrees C. We also found that the cell survival rates began to increase at 3h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.
Cryobiology 01/2009; 58(2):170-4. · 2.06 Impact Factor
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ABSTRACT: Changes in the gene expression of commercial baker's yeast during an air-drying process, which simulated dried yeast production, were analyzed. K-means clustering suggested that the genes involved in protein folding were transiently up-regulated at early stages, and that the genes involved in fatty acid metabolism were continuously up-regulated.
Journal of Bioscience and Bioengineering 11/2008; 106(4):405-8. · 1.79 Impact Factor
