ABSTRACT: Application of co-cultures of engineered Saccharomyces cerevisiae strains to reduce byproduct formation during anaerobic ethanol fermentation
Project description:Saccharomyces cerevisiae cannot metabolize cellobiose in nature. Here, S. cerevisiae was engineered to achieve cellobiose utilization by introducing both a cellodextrin transporter gene (cdt-1) and an intracellular β-glucosidase gene (gh1-1) from Neurospora crassa. We sequenced mRNA from anaerobic exponential cultures of engineered S. cerevisiae grown on cellobiose or glucose as a single carbon source in biological triplicate. Differences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induced mitochondrial activation and reduced amino acid biosynthesis under fermentation conditions.
Project description:Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol-production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h-1), an engineered PRK-RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK-RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate yields by 67% and 29%, respectively. Additional C-terminal fusion of a 19 amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15x cbbm strain. These modifications did not affect glycerol production at 0.05 h-1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h-1) anaerobic batch cultures than observed for the 15x cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2x cbbm strain. At 0.05 h-1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15x cbbm strain, without affecting glycerol yield. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol yield was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK-RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth-rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.
Project description:Thermotolerance development of robust Saccharomyces cerevisiae is necessary to enhance enzyme activity of cellulase, lower cooling costs, and reduce cell harm from the bad-distributed heat transfer in large-scale fermentation. The process-based studies of adaptive evolution have been well documented, but it remains unknown for the underlying molecular mechanism of the improved thermotolerance and the facilitated ethanol fermentability derived from adaptive evolution. Here, a robust thermotolerant S. cerevisiae Z100 was obtained with significantly improved ethanol fermentability under the stress of high temperature (50 oC) after 91 days’ adaptive evolution. RNA sequencing showed that adaptive evolution and its derived thermotolerance contributed to the unique gene transcriptional landscapes of the evolved strain. An interesting phenomenon was that the gene transcriptional signals of carbon metabolism were strengthened not at 50 oC but at 30 oC in S. cerevisiae Z100, and thus suggested that the improved thermotolerance led to the enhanced ethanol fermentability at 30 oC. The deeply repressed gene transcriptional expression indicated ribosome would be another key thermotolerant mechanism for the evolved strain. This study would provide a robust thermotolerant S. cerevisiae for bioethanol production and an important clue for future synthetic biology to thermotolerance engineering of fermentation strains.
Project description:High concenHigh concentration acetic acid in the fermentation medium represses cell growth, metabolism and fermentation efficiency of Saccharomyces cerevisiae, which is widely used for cellulosic ethanol production. Our previous study proved that supplementation of zinc sulfate in the fermentation medium improved cell growth and ethanol fermentation performance of S. cerevisiae under acetic acid stress condition. However, the molecular mechanisms is still unclear. To explore the underlying mechanism of zinc sulfate protection against acetic acid stress, transcriptomic and proteomic analysis were performed. The changed genes and proteins are related to carbon metabolism, amino acid biosynthesis, energy metabolism, vitamin biosynthesis and stress responses. In a total, 28 genes showed same expression in transcriptomic and proteomic data, indicating that zinc sulfate affects gene expression at posttranscriptional and posttranslational levels.tration acetic acid in the fermentation medium represses cell growth, metabolism and fermentation efficiency of Saccharomyces cerevisiae, which is widely used for cellulosic ethanol production. Our previous study proved that supplementation of zinc sulfate in the fermentation medium improved cell growth and ethanol fermentation performance of S. cerevisiae under acetic acid stress condition. However, the molecular mechanisms is still unclear. To explore the underlying mechanism of zinc sulfate protection against acetic acid stress, transcriptomic and proteomic analysis were performed. The changed genes and proteins are related to carbon metabolism, amino acid biosynthesis, energy metabolism, vitamin biosynthesis and stress responses. In a total, 28 genes showed same expression in transcriptomic and proteomic data, indicating that zinc sulfate affects gene expression at posttranscriptional and posttranslational levels.
Project description:Saccharomyces cerevisiae cannot metabolize cellobiose in nature. Here, S. cerevisiae was engineered to achieve cellobiose utilization by introducing both a cellodextrin transporter gene (cdt-1) and an intracellular β-glucosidase gene (gh1-1) from Neurospora crassa. We sequenced mRNA from anaerobic exponential cultures of engineered S. cerevisiae grown on cellobiose or glucose as a single carbon source in biological triplicate. Differences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induced mitochondrial activation and reduced amino acid biosynthesis under fermentation conditions. mRNA levels in cellobiose-grown and glucose-grown cells of engineered cellobiose-utilizing Saccharomyces cerevisiae were examined by deep sequencing, in triplicate, using Illumina Genome Analyzer-II. We sequenced 3 samples from cellobiose-grown cells and 3 samples from glucose-grown cells and identified differential expressions in the cellobiose versus glucose fermentations by using mRNA levels of glucose-grown cells as a reference.
Project description:The present work aimed to compare the transcriptome of three major ethanol-producer Saccharomyces cerevisiae strains in Brazil when fermenting sugarcane juice for fuel ethanol production. This was motivated by the reports presenting physiological and genomics differences among them, and by the attempt to identify genes that could be related to their fermentation capacity and adaptation for different industrial processes.
Project description:Industrial bioethanol production may involve a low pH environment,improving the tolerance of S. cerevisiae to a low pH environment caused by inorganic acids may be of industrial importance to control bacterial contamination, increase ethanol yield and reduce production cost. Through analysis the transcriptomic data of Saccharomyces cerevisiae with different ploidy under low pH stress, we hope to find the tolerance mechanism of Saccharomyces cerevisiae to low pH.
Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in pch2 and sir2 mutant strains of Saccharomyces cerevisiae.
Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in an SK1 x S88C F1 hybrid strain of Saccharomyces cerevisiae.
Project description:In our previous work, we showed the positive effect of the magnesium and the negative effect of the copper on yeast fermentation performance. The magnesium increases the ethanol yield and a faster glucose consumption by the yeast, on the other hand, the copper provides an opposite effect in yeast under fermentation condition. Therefore, from this contrasting effect we performed the gene-wide expression analysis in the industrial yeast Saccharomyces cerevisiae JP1 under fermentation condition in order to reveal the gene expression profile upon magnesium and copper supplementation.