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KJC335’s transcriptomic responses to xylose versus cellobiose

ABSTRACT: Clostridium (Ruminiclostridium) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335’s transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log2 fold change (FC) > |1| and 106 highly DEGs (log2 FC > |2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD, were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes (tktAB) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation-reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose. KJC355 unevolved

ORGANISM(S): Acetivibrio thermocellus

PROVIDER: GSE137509 | GEO | 2019/09/17

REPOSITORIES: GEO

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Project description:Background (Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium producing the most extensive and intricate cellulosomal system known in nature. Recently, an elaborate architecture of B. cellulosolvens cellulosomal system was revealed from its genome sequence analysis, and first evidence on the interactions between its structural and enzymatic components were detected in vitro. Yet, the cellulolytic potential of the bacterium in carbohydrate deconstruction may reside only within complete high-molecular weight protein complexes, which are secreted from the bacterium. Results The current proteome-wide work reveals patterns of protein expression of various cellulosomal components, and explores the differential expression signature upon bacterial growth two carbon sources, either cellobiose or microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome fraction revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing enzymes, in addition to free enzymatic subunits. All these components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family memebers were represented among 102 carbohydrate-degrading enzymes, including the most abundant GH48 exoglucanase. Specific cellulosomal components were associatred with different carbon sources, in cellulosomal fractions of different molecular weights. Overall, microcrystalline cellulose-derived cellulosomes showed higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by C. thermocellum purified cellulosomes. Conclusions The current research demonstrates the involvement of key cellulosomal factors participating in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies of various carbon sourcesis revealed. Thus, a novel components reservoir of degradation machineries that may serve for subsequent cellulosomal research, as a pool for designing new cellulolytic cocktails for biotechnological purposes.

Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a Î²-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum. Four strains of C. thermocellum DSM1313 including a parental strain (Îhpt) and three others with deletion in lacI transcription factors were characterised by RNA-seq. The strains were grown in 1L bioreactors, cells (from 50 ml samples) were harvested at mid exponential, late exponential and 30 min after acid production halted during the transition to stationary phase. The growth studies were performed in triplicate and thirty six samples were analyzed by RNA-sequencing.

Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a β-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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.

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KJC335’s transcriptomic responses to xylose versus cellobiose

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