Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.
Project description:Xylose-utilizing yeasts with tolerances to fermentation inhibitors (such as weak organic acids) and high temperature are needed for cost-effective simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic materials. We constructed a novel xylose-assimilating Saccharomyces cerevisiae strain with improved fermentation performance under heat and acid co-stress using the genome shuffling technique. Two xylose-utilizing diploid yeasts with different genetic backgrounds were used as the parental strains for genome shuffling. The hybrid strain Hyb-8 showed significantly higher xylose fermentation ability than both parental strains (Sun049T-Z and Sun224T-K) under co-stress conditions of heat and acids. To screen for genes that might be important for fermentation under heat and acid co-stress, a transcriptomic analysis of hybrid strain Hyb-8 and its parental strains was performed.
Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.
Project description:High-temperature fermentation of the Bacillus subtilis isolated from the black part of maotai Daqu. Studying on the gene expression profile using microarray for analyzing the connection between metabolites and the maotai flavor substances. 84 differential expressed genes were obtained, including 40 up-regulated genes and 44 down-regulated genes.The differentially expressed genes involved in the metabolic pathways were just only KBL (glycine C - acetyltransferase) and ripA (bifunctional 3, 4 - dihydroxy - 4-2 - butanone phosphate synthase), up-regulated 2.9 and 2.9 times respectively, and their catalytic reaction prodction of aminobutyric acid and dihydroxy ethyl ketone phosphate, respectively. They may be further derived into alcohol and ketone flavoring substances. However, a large number of differential expressed genes was related to sporulation, such as ybaN (polysaccharide deacetylase) and rapA (aspartic acid phosphatase), they were up-regulated 17.5 times and down-regulated 112.5 times. YbaN is closely related to the formation of spore cortex and high temperature group spore cortex obvious thickening by TEM. RapA is signaling molecules to restrain spore formation, its lower expression can promote the sporulation in group A. Formation and release of peptidoglycan and the DPA (2, 6 - Pyridinedicarboxylic acid) of spore cortex during theseveral rounds of low temperature to high temperature circulation fermentation may be the main source of furan and pyranand nitrogen heterocyclic compounds in maotai flavor substances . In this paper, the formation of high-temperature fermentation Bacillus subtilis spores is closely related to the generation of maotai flavor substances.
Project description:High-temperature fermentation of the Bacillus subtilis isolated from the black part of maotai Daqu. Studying on the gene expression profile using microarray for analyzing the connection between metabolites and the maotai flavor substances. 84 differential expressed genes were obtained, including 40 up-regulated genes and 44 down-regulated genes.The differentially expressed genes involved in the metabolic pathways were just only KBL (glycine C - acetyltransferase) and ripA (bifunctional 3, 4 - dihydroxy - 4-2 - butanone phosphate synthase), up-regulated 2.9 and 2.9 times respectively, and their catalytic reaction prodction of aminobutyric acid and dihydroxy ethyl ketone phosphate, respectively. They may be further derived into alcohol and ketone flavoring substances. However, a large number of differential expressed genes was related to sporulation, such as ybaN (polysaccharide deacetylase) and rapA (aspartic acid phosphatase), they were up-regulated 17.5 times and down-regulated 112.5 times. YbaN is closely related to the formation of spore cortex and high temperature group spore cortex obvious thickening by TEM. RapA is signaling molecules to restrain spore formation, its lower expression can promote the sporulation in group A. Formation and release of peptidoglycan and the DPA (2, 6 - Pyridinedicarboxylic acid) of spore cortex during theseveral rounds of low temperature to high temperature circulation fermentation may be the main source of furan and pyranand nitrogen heterocyclic compounds in maotai flavor substances . In this paper, the formation of high-temperature fermentation Bacillus subtilis spores is closely related to the generation of maotai flavor substances. There are total of eight samples. It divided two groups, set as group A (High temperature fermentation) and B (normal temperature fermentation, continuous 37C). There are four replicates for each group.