RNAseq of engineered S. cerevisiae strains for isobutanol production
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ABSTRACT:
PROVIDER: PRJNA772701 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16481343_1.fastq.gz | Fastqsanger.gz | |||
| SRR16481343_2.fastq.gz | Fastqsanger.gz | |||
| SRR16481344_1.fastq.gz | Fastqsanger.gz | |||
| SRR16481344_2.fastq.gz | Fastqsanger.gz | |||
| SRR16481345_1.fastq.gz | Fastqsanger.gz |
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Project description:Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketoacid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses indicated that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2-null strain harboring a cytosolic localized isobutanol pathway outperformed the strain with the mitochondrial localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.
2021-10-22 | GSE186126 | GEO
Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways. The goal of the present study is to investigate the impact of specific growth rate on biomass-specific productivity, product yield, by-product formation and host strain physiology of an S. cerevisiae strain that was previously engineered for de novo production of resveratrol from glucose. To this end, (by)product formation, physiology and transcriptome were analysed in steady-state, glucose-limited chemostat cultures grown at different dilution rates.
2015-12-22 | E-GEOD-65942 | biostudies-arrayexpress
Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.
2015-12-22 | GSE65942 | GEO
Project description:Response of Saccharomyces cerevisiae engineered for xylose metabolism to changes in carbon source and aeration Keywords: ordered
2004-11-13 | GSE835 | GEO
Project description:Experiments to measure relative metabolite pools of wild type Synechococcus elongatus PCC 11801 and its recombinants producing succinate. The wild type and the engineered strains producing succinate were cultivated at 1% CO2 and their metabolome data was collected in three biological and three technical replicates (n=9). The study aims to find metabolomics changes between the wild type and the engineered to identify potential rate-limiting steps that be used as targets for improved production.
2020-01-03 | ST001298 | MetabolomicsWorkbench
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.
2023-05-17 | GSE221392 | GEO
Project description:Model-guided chassis strain design has the potential to accelerate cellfactory development. In this experiment genetic targets were identified in silico and implemented in vivo to design a yeast chassis strain for enhanced production of succinic, malic and fumaric acid. The phenotype of engineered chassis strains was further optimised through adaptive laboratory evolution. RNA-seq analysis of engineered yeast chassis strains, evolved strains and wild-type (CEN.PK background)was performed to determine the effect of engineered gene deletions and evolution on the transcriptome.
2021-07-01 | E-MTAB-9499 | biostudies-arrayexpress
Project description:RNAseq analysis of Hypomontagnella monticulosum under production and non-production conditions
2020-07-17 | E-MTAB-8948 | biostudies-arrayexpress
Project description:Chassis strain suitable for producing multiple compounds is a central concept in synthetic biology. Design of a chassis using computational, first-principle, models is particularly attractive due to the predictability and control it offers, including against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has not been put to experimental test. Here, we report two Saccharomyces cerevisiae chassis strains for dicarboxylic acid production based on genome-scale metabolic modelling. The chassis strain, harboring gene knockouts in serine biosynthesis and in pentose-phosphate pathway, is geared for higher flux towards three target products - succinate, fumarate and malate - but does not appreciably secrete any. Introducing modular product-specific mutations resulted in improved secretion of the corresponding acid as predicted by the model. Adaptive laboratory evolution of the chassis-derived producer cells further improved production for succinate and fumarate attesting to the evolutionary robustness of the underlying growth-product coupling. In the case of malate, which exhibited decreased production during evolution, the multi-omics analysis revealed flux bypass at peroxisomal malate dehydrogenase not accounted in the model. Transcriptomics, proteomics and metabolomics analysis showed overall concordance with the flux re-routing predicted by the model. Together, our results provide experimental evidence for model-based design of microbial chassis and have implications for computer-aided design of microbial cell factories.
2021-07-20 | PXD020611 | Pride
Project description:This work was primarily focused on removing the main bottleneck for isobutanol production which is the toxicity of isobutanol towards its host and the major reason for its low-level production. Recently, there have been many reports where people have tried various strategies including continuous in-situ product removal from the bioreactor which led to a significant increase in the final product titers. Clearly, it is the intrinsic tolerance levels of the host which decides the end product titers. Importantly, if the host microbe can originally tolerate higher concentrations of toxin (here isobutanol) then it is likely to have a better potential for further improvement of tolerance levels. In our work, we tried to enhance the isobutanol tolerance of wild type NZ9000 using continuous culture. We used the principle of adaptive laboratory evolution to enhance the natural tolerance ability (0.8% isobutanol) of the wild type strain. The strain was cultivated for more than 60 days (>250 generations), while increasing the selection pressure (here isobutanol) gradually in the feed. This finally led to the strain that showed exceptionally higher tolerance (4%) for isobutanol. Transcriptomic analysis also showed fluctuations in gene expression levels from a wide range of categories, precluding any attempt to reverse engineer this phenotype.
2020-07-08 | GSE107996 | GEO