Transcriptomic adaptations of Clostridium thermocellum strains evolved to grow on xylose
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ABSTRACT:
ORGANISM(S): Acetivibrio thermocellus DSM 1313
PROVIDER: GSE276070 | GEO | 2025/08/31
REPOSITORIES: GEO
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Project description:To broaden microbial cell factories´ substrate scope towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by detailed knowledge of metabolism remain scarce, especially for non-model organisms. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous pathway remained unresolved. Here, we elucidated the xylose metabolism and established a foundation for further engineering followed by ALE. By de-repressing native glycolysis, we unlocked the route for xylose-derived carbon and obtained a strain with a substantially reduced lag phase on xylose. We then enhanced the pentose phosphate pathway in two lag-shortened strains and allowed ALE to fine-tune the rewired metabolism. Following the metabolism tuning, we employed multi-level analysis that provided unique insights into the parallel paths of bacterial adaptation to the non-native carbon source.
2024-03-06 | PXD047537 | Pride
Project description:Clostridium acetobutylicum was grown in a batch-culture with minimal medium containing glucose and xylose as substrate. Diauxie growth was observed after glucose was consumed. Following the organism grows on xylose. Transcriptional analysis was done to pursue the cellular processes during the switch from growth on glucose to growth on xylose. We compared DNA-Microarray data from cells grown during the exponential phase on glucose (A), with cells growing during the start of diauxie growth lag (B), during the end of diauxie growth lag (C) and during exponential growth on xylose (D). We used cells grown in a continuous culture with glucose as substrate as common reference for the samples A-D.
2010-09-30 | E-MEXP-2613 | biostudies-arrayexpress
Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
2017-10-03 | GSE104491 | GEO
Project description:<p><strong>BACKGROUND:</strong> Converting carbon dioxide (CO2) into value-added chemicals using engineered cyanobacteria is a promising strategy to tackle the global warming and energy shortage issues. However, most cyanobacteria are autotrophic and use CO2 as a sole carbon source, which makes it hard to compete with heterotrophic hosts in either growth or productivity. One strategy to overcome this bottleneck is to introduce sugar utilization pathways to enable photomixotrophic growth with CO2 and sugar (e.g., glucose and xylose). Advances in engineering mixotrophic cyanobacteria have been obtained, while a systematic interrogation of these engineered strains is missing. This work aimed to fill the gap at omics level.</p><p><strong>RESULTS:</strong> We first constructed two engineered <em>Synechococcus elongatus</em> YQ2-gal and YQ3-xyl capable of utilizing glucose and xylose, respectively. To investigate the metabolic mechanism, transcriptomic and metabolomic analysis were then performed in the engineered photomixotrophic strains YQ2-gal and YQ3-xyl. Transcriptome and metabolome of wild-type <em>S. elongatus</em> were set as baselines. Increased abundance of metabolites in glycolysis or pentose phosphate pathway indicated that efficient sugar utilization significantly enhanced carbon flux in <em>S. elongatus</em> as expected. However, carbon flux was redirected in strain YQ2-gal as more flowed into fatty acids biosynthesis but less into amino acids. In strain YQ3-xyl, more carbon flux was directed into synthesis of sucrose, glucosamine and acetaldehyde, while less into fatty acids and amino acids. Moreover, photosynthesis and bicarbonate transport could be affected by upregulated genes, while nitrogen transport and assimilation were regulated by less transcript abundance of related genes in strain YQ3-xyl with utilization of xylose.</p><p><strong>CONCLUSIONS:</strong> Our work identified metabolic mechanism in engineered <em>S. elongatus</em> during photomixotrophic growth, where regulations of fatty acids metabolism, photosynthesis, bicarbonate transport, nitrogen assimilation and transport are dependent on different sugar utilization. Since photomixotrophic cyanobacteria is regarded as a promising cell factory for bioproduction, this comprehensive understanding of metabolic mechanism of engineered <em>S. elongatus</em> during photomixotrophic growth would shed light on the engineering of more efficient and controllable bioproduction systems based on this potential chassis.</p>
2022-03-21 | MTBLS2825 | MetaboLights
Project description:Pseudomonas alloputida KT2440 (previously misclassified as P. putida KT2440 based on 16S rRNA gene homology) has emerged as an ideal host strain for plan t biomass valorization. However, P. alloputida KT2440 is unable to natively utilize abundant pentose sugars (e.g., xylose and arabinose) in hydrolysate streams, which may account for up to 25% of lignocellulosic biomass. In the last decades, microbes have been engineered to utilize the pentose sugars. However, most of the engineered strains were either slow-growing or displayed phenotypes that could not be replicated. In this work, we successfully isolated five Pseudomonas species with the native capability to utilize glucose, xylose and p-coumarate as a sole carbon source. These isolates were in two clusters; one set of isolates (M2 and M5) and the second set of isolates (BP6 and BP7) showed 85.6% and 96.2% ANI, respectively, to P. alloputida KT24440. BP8 showed 84.6% ANI to P. putida KT2440 and does not belong to any neighboring type strains indicating a new species. Notably, the isolates showed robust growth solely on xylose and higher growth rates (m, 0.36-0.49 h-1) when compared to only known xylose-utilizing Pseudomonas taiwanenesis VLB120 (m, 0.28 h-1) as a control. Unexpectedly, among five isolates, M2 and M5 grew solely on arabinose as well. Comprehensive analysis of genomics, transcriptomics and proteomics revealed the isolates utilize xylose and arabinose via Weimberg pathway (xylD-xylX-xylA) and oxidative pathway (araD-araX-araA), respectively. Furthermore, a preliminary result demonstrated the production of flaviolin solely on xylose and arabinose in the isolate, showing noteworthy potential to be an alternative host for lignocellulosic feedstocks into valuable products. This is the first report on isolating Pseudomonas strains natively capable of utilizing all of the major carbon sources in lignocellulosic biomass, and leading to higher consumption of available substrates and therefore maximizing the product yield.
2023-05-10 | PXD031549 | Pride
Project description:Gas-fermenting acetogens, such as Clostridium autoethanogenum, have emerged as promising biocatalysts capable of converting CO and CO2-containing gases into fuels and chemicals relevant for a circular economy. However, functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in C. autoethanogenum. In this study, we deleted one of the targets – CLAU_0471 (proposed amino acid permease) – with high mutation occurrence in ALE isolates and extensively characterised autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterized two previously reverse-engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g. low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3-butanediol production while RE1 performed similar to the best-performing previously characterised ALE isolate LAbrini. The three reverse-engineered strains showed similarities in proteome expression and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype-phenotype relationships for a better understanding of the metabolism of an industrially-relevant acetogen.
2025-08-12 | PXD062027 | Pride
Project description:Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomyces cerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. Despite the sterility of this hybrid strain, we were able to develop novel methods to genetically characterize its xylose utilization phenotype, using bulk segregant analysis in conjunction with high-throughput sequencing. We found that its growth in xylose is governed by at least two genetic loci: one of the loci maps to a known xylose-pathway gene, a novel allele of the aldo-keto reductase gene GRE3, while a second locus maps to an allele of APJ1, a chaperonin gene not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a non-genetically-tractable hybrid strain that contains a complex, polygenic trait, and it identifies new avenues for metabolic engineering as well as for construction of non-genetically modified xylose-fermenting strains.
2012-07-19 | GSE35528 | GEO
Project description:To understand the mechanisms of enhanced xylose metabolism of XUSE, we reported transcriptome analysis of XUS (rationally engineered strain) and XUSE (evolved XUS strain) grown on xylose by performing RNA-sequencing. Compared with XUS, XUSE showed significantly different gene expression landscape with 463 up-regulated and 675 down-regulated genes (>2-fold, p-value<0.05). While the genes involved in non-oxidative pentose phosphate pathway, such as TKL1, TKL2 and TAL1, were down-regulated in XUSE, it shows the decreased transcriptional patterns of a number of genes encoding TCA cycle and respiratory enzymes. Also, the genes involved in the cellular metal ion regulation and sugar transporters showed significantly changed expression levels in XUSE. This study shows the gene expression landscape of XUSE revealing engineering strategies for the enhanced xylose metabolism in S. cerevisiae.
2018-12-26 | GSE116076 | GEO
Project description:Efficient assimilation of renewable feedstocks is the cornerstone for achieving sustainable and economical microbial production of commodity chemicals. Unfortunately, most renewables are foreign to the cellular metabolism of classical industrial workhorses, resulting in unsatisfactory biomanufacturing performance. Here, Corynebacterium glutamicum was systematically engineered for rapid non-natural xylose metabolism and the underlying adaptations were elucidated by combining metabolic engineering, adaptive laboratory evolution and systems biology techniques. A plasmid-free, stable and efficient xylose-utilizing chassis strain, named CGS15, was reconstructed with both a rapid specific growth rate of 0.341 h-1 on xylose and an excellent co-utilization of glucose and xylose at a ratio of about 2:1. For the first time, we revealed a novel xylose regulatory mechanism by the endogenous transcription factor IpsA with global regulatory effects on C. glutamicum core carbon and energy metabolism. The coordination between the heterologous xylose metabolism and the endogenous carbon/energy metabolism both endowed cells with accelerated growth and released carbon catabolite repression. Finally, this chassis demonstrated great promise for lignocellulosic biorefinery applications by producing 97.1 g/L of the platform compound succinate from corn stalk hydrolysate with an average productivity of 8.09 g/L/h. This work provides an elegant paradigm to understand and engineer the metabolism of renewable substrates for sustainable biomanufacturing.
2022-10-03 | GSE184402 | GEO
Project description:Xylose induced effects on metabolism and gene expression during anaerobic growth of an engineered Saccharomyces cerevisiae on mixed glucose-xylose medium were quantified. Gene expression of S. cerevisiae harbouring an XR-XDH pathway for xylose utilisation was analysed from early cultivation when mainly glucose was metabolised, to times when xylose was co-consumed in the presence of low glucose concentrations, and finally, to glucose depletion and solely xylose being consumed. Cultivations on glucose as a sole carbon source were used as a control. Genome-scale dynamic flux balance analysis models were developed and simulated to analyse the metabolic dynamics of S. cerevisiae in the cultivations. Model simulations quantitatively estimated xylose dependent dynamics of fluxes and challenges to the metabolic network utilisation. Increased relative xylose utilisation was predicted to induce two-directionality of glycolytic flux and a redox challenge already at low glucose concentrations. Xylose effects on gene expression were observed also when glucose was still abundant. Remarkably, xylose was observed to specifically delay the glucose-dependent repression of particular genes in mixed glucose-xylose cultures compared to glucose cultures. The delay occurred during similar metabolic flux activities in the both cultures. Xylose is abundantly present together with glucose in lignocellulosic streams that would be available for the valorisation to biochemicals or biofuels. Yeast S. cerevisiae has superior characteristics for a host of the bioconversion except that it strongly prefers glucose and the co-consumption of xylose is yet a challenge. Further, since xylose is not a natural substrate of S. cerevisiae, the regulatory response it induces in an engineered yeast strain cannot be expected to have evolved for its utilisation. Dynamic cultivation experiments on mixed glucose-xylose medium having glucose cultures as control integrated with mathematical modelling allowed to resolve specific effects of xylose on the gene expression and metabolism of engineered S. cerevisiae in the presence of varying amounts of glucose.
2016-05-24 | GSE69966 | GEO