Simultaneous xylose and glucose utilization by an E. coli crp mutant
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Escherichia Coli (ncbitaxon:562)
SUBMITTER: 
Xin Wang
PROVIDER: MSV000092810 | MassIVE |
SECONDARY ACCESSION(S): PXD045049
REPOSITORIES: MassIVE
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Project description:Background Lignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results A new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance. Conclusions Our results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.
2014-09-08 | GSE57553 | GEO
Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment and enzymatic hydrolysis of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and eventually the production rate and yield. Acetate is one of the major inhibitors liberated from hemicelluloses during dilute acid pretreatment. An understanding of the toxic effects of acetate on the fermentation microorganism is critical to improving biofuel yields in the process. In addition, the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results: We have observed previously that some pretreatment inhibitors affect growth and performance in Zymomonas mobilis 8b differently when different sugars (e.g. glucose or xylose) are used as substrate. To investigate this phenomenon at the cellular level, microarray technology was used to investigate the acetate stress responses of Z. mobilis 8b when using single carbon sources of glucose or xylose, and mixed sugars of glucose and xylose. We designed a microarray based on the most up-to-date genome annotation for both coding sequences and intergenic regions. In the presence of acetate, 8b still can utilize all the glucose (though xylose utilization was inhibited) with similar ethanol yield although the growth, final biomass, and ethanol production rate were reduced. The presence of acetate caused genes related to biosynthesis, flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Our result indicates that Z. mobilis utilized different mechanism for xylose utilization compared to that of glucose, with even more dramatic results than those caused by treatment of the culture with the inhibitor acetate. Our study also suggests that redox imbalance caused by stressful conditions may trigger a metabolic reaction that leads to the accumulation of toxic intermediates such as xylitol, but Z. mobilis appears to be capable of managing its carbon and energy metabolism through the control of individual reactions to overcome the inhibition caused by stressful conditions. Several target gene candidates based on transcriptomic result have been selected for genetic manipulation and a TonB-dependent receptor knockout mutant was confirmed to have advantage on acetate tolerance. Conclusions: We have gained insights into the molecular responses of the model ethanologenic bacterium Z. mobilis to the inhibitor acetate when grown in different sugar sources. These insights will facilitate future metabolic modeling studies and help further strain metabolic engineering efforts for better xylose utilization and acetate tolerance. Two series of microarray studies using total RNA extracted from Zymomonas mobilis subsp mobilis 8b (an xylose-utilizing recombinant) were carried out to investigate the effect of carbon source and acetate on Z. mobilis. One study compared the acetate effect in either glucose or xylose at exponential phase and another study investigated the acetate effect in mixed sugar of glucose and xylose at three growth phases of exponential, transition, and stationary. Tthree biological replicates were used for each condition.
2014-09-08 | E-GEOD-57553 | biostudies-arrayexpress
Project description:Though highly efficient at fermenting hexose sugars, the ethanologenic yeast Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomasses is the five carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars. Here we combined classical candidate gene approach with systems biology to develop xylose-utilising S. cerevisiae strains. The introduction of an exogenous xylose isomerase (XYLA) and an additional copy of the endogenous xylulokinase gene (XKS1) results in the significant improvement of xylose consumption. Microarray studies reveal that the introduction of XYLA and XKS1 results in the dramatic transcriptional remodelling of the cell under both glucose and xylose conditions. To further investigate the cellular processes impacted by the introduction of XYLA and XKS1, using genome-wide chemical and synthetic lethal screens we identified greater than 40 deletion mutants that impact xylose utilization. We identified four genes, ALP1, ISC1, RPL20B and BUD21, that when individually deleted allow S. cerevisiae to utilize xylose as the sole carbon source. When these mutants are combined with XYLA and XKS1, it results in strains with significant improvement in xylose consumption. We have demonstrated that systems biology techniques combined with candidate gene approaches can successfully lead to novel genetic strategies for the improvement of xylose utilization
2012-07-15 | GSE27325 | GEO
Project description:Though highly efficient at fermenting hexose sugars, the ethanologenic yeast Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomasses is the five carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars. Here we combined classical candidate gene approach with systems biology to develop xylose-utilising S. cerevisiae strains. The introduction of an exogenous xylose isomerase (XYLA) and an additional copy of the endogenous xylulokinase gene (XKS1) results in the significant improvement of xylose consumption. Microarray studies reveal that the introduction of XYLA and XKS1 results in the dramatic transcriptional remodelling of the cell under both glucose and xylose conditions. To further investigate the cellular processes impacted by the introduction of XYLA and XKS1, using genome-wide chemical and synthetic lethal screens we identified greater than 40 deletion mutants that impact xylose utilization. We identified four genes, ALP1, ISC1, RPL20B and BUD21, that when individually deleted allow S. cerevisiae to utilize xylose as the sole carbon source. When these mutants are combined with XYLA and XKS1, it results in strains with significant improvement in xylose consumption. We have demonstrated that systems biology techniques combined with candidate gene approaches can successfully lead to novel genetic strategies for the improvement of xylose utilization For this experiment samples (2 reference and 2 test samples), consisting of at least 3 biological reps were hybridized. Cy3 one-color labelling was used for each sample.
2012-07-14 | E-GEOD-27325 | biostudies-arrayexpress
Project description:Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).
2023-09-07 | PXD041554 | Pride
Project description:Efficient utilization of lignocellulosic biomass-derived sugars is essential to improve the economics of biorefinery. While Pseudomonas putida is a promising microbial host, its usage is limited because this strain cannot utilize xylose or galactose as a sole carbon source. To address this issue, we heterologously introduced a xylose utilizing gene (xylD) from Caulobacter crescentus and a galactose operon (galETKM) from E. coli MG1655. To improve the utilization further, we evolved the engineered strains in minimal medium conditions. After the evolution, they acquired better fitnesses on the non-native sugars. To understand transcriptional changes after the evolution, the transcriptomes of few evolved isolates were analyzed.
2021-07-15 | GSE155767 | GEO
Project description:Plant biomass holds tremendous potential as a renewable feedstock in the production of biofuels and biochemicals. The effective co-utilization of the main components cellulose and hemicellulose in plant lignocellulose is critical to the economic viability of lignocellulosic biorefineries. Here, we found that the thermophilic cellulolytic fungi Myceliophthora thermophila can utilize cellulose and hemicellulose synchronously. To investigate the underlying molecular mechanisms, we firstly checked the soluble carbohydrate of the culture using plant biomass (corncob) as sole carbon source and revealed the presence of various oligosaccharides including cellodextrin and xylodextrin, both intracellularly and extracellularly in the cultures, in addition to glucose or xylose. Based on these, intracellular oligosaccharide metabolism was proposed and confirmed by identification of cellodextrin and xylodextrin metabolism pathway. Furthermore, sugar consumption assay showed that contrasting with synchronous utilization of mixed cello-/xylo-dextrin, the inhibition effect of glucose for the metabolism of xylose and cello-/xylo-dextrin exists in this fungus, suggesting Carbon Catabolite Repression (CCR) is largely avoided at the form of oligosaccharides. Transporter MtCDT-2 showing preference to xylobiose and the tolerance of cellobiose inhibition also helps to bypass metabolic inhibition. Finally, the expression of cellulase and hemicellulase genes, were found orthogonal induction by cellobiose/Avicel and xylobiose/xylan, which conferred the ability of the strain to synchronously utilize cellulose and hemicellulose. Taken together, the orthogonal oligosaccharide catabolic pathway in this fungus establishes the molecular basis for the synchronous utilization of cellulose and hemicellulose, which sheds new light on understanding the plant biomass degradation by fungi and provides alternative paradigm for development of lignocellulose biorefinery such as consolidated bioprocessing in the future.
2024-02-28 | GSE222371 | GEO
Project description:Background: Bacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into biofuels by the anaerobic bacterium Clostridium acetobutylicum is a promising alternative energy source to fossil fuels. Understanding the complex metabolic pathways it employs and how they are regulated will contribute to improved biofuel production. Recently, it has been demonstrated that xylose is not appreciably fermented in the presence of arabinose, suggesting a hierarchy of pentose utilization in this organism. Our goal is to uncover if transcriptional regulation contributes to this hierarchy. Results: Growth and sugar consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. RNA-Seq revealed there was a large overlap in differentially regulated genes after addition of arabinose or glucose, suggesting a common mechanism of regulation. A putative ORF was identified that may be important for transcriptional regulation in response to the nutritional state of the cells. Conclusions: Decreased xylose consumption, increased acetate production, and transcription of the phosphoketolase gene (CA_C1343) revealed a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway after addition of arabinose. Together, these results substantiate the claim that arabinose is utilized preferentially over xylose in C. acetobutylicum. Furthermore, they demonstrate that this phenomenon is modulated in part at the transcriptional level, and they provide valuable insight into potential mechanisms for altering pentose utilization to modulate fermentation products for biofuel production.
2017-12-20 | GSE107804 | GEO
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, Gram-positive anaerobe, which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2 and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to co-utilize glucose and xylose, make this bacterium an attractive candidate for microbial bioenergy production. Glycolytic pathways and an ABC-type sugar transporter were significantly up-regulated during growth on glucose and xylose, indicating that C. saccharolyticus co-ferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks represents a highly desirable feature of a lignocellulose-utilizing, biofuel-producing bacterium. Keywords: substrate response
2008-04-15 | GSE11153 | GEO
Project description:Transcriptional landscape of MyceliopTranscriptional profiling of Myceliophthora thermophila on galactose and metabolic engineering for improved galactose utilization thora thermophila responding to soluble starch and the role of regulator AmyR on polysaccharide degradation Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for cost-effective production of biofuels and commodity chemicals. Galactose is the next most abundant sugar in many hemicelluloses and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fugus Myceliophthora thermophila has been used as cell factory to produce biochemicals directly from renewable polysaccharides. This study determined the transcriptomic profiles of M. thermophila responding to galactose and identified the gene involved in the oxido-reductive pathway for galactose degradation, of which hexokinase might be the rate-limiting enzyme in this thermophilic fungus. Galactokinase was necessary for high induction of galactose transporter and disruption of galK resulted in decreased galactose utilization. Both of galactokinase deletion and sugar transporter overexpression could further activate the oxido-reductive pathway and led to improved galactose utilization. In addition, disruption of the Leloir pathway brought about reduced tolerance to cell wall perturbation, oxidative stress and high osmolality. In order to accelerate galactose consumption, the third galactose-degradation pathway- De Ley-Doudoroff pathway was successfully integrated into M. thermophila and the consumption rate of galactose was increased by 57%. This study will be benefit for the rational design of fungal strains to produce biofuels and biochemical from plant biomass, especially, marine plant biomass with the abundant of galactose, such as red seaweed and molasses.
2021-02-05 | GSE165516 | GEO