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Genome-scale resources for Thermoanaerobacterium saccharolyticum.

Currie DH, Raman B, Gowen CM, Tschaplinski TJ, Land ML, Brown SD, Covalla SF, Klingeman DM, Yang ZK, Engle NL, Johnson CM, Rodriguez M, Shaw AJ, Kenealy WR, Lynd LR, Fong SS, Mielenz JR, Davison BH, Hogsett DA, Herring CD - BMC Syst Biol (2015)

Bottom Line: The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid.Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge.In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions.

View Article: PubMed Central - PubMed

Affiliation: Mascoma Corporation, 67 Etna Rd, 03766, Lebanon, NH, USA. Devin.H.Currie.GR@dartmouth.edu.

ABSTRACT

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation.

Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results.

Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

No MeSH data available.


Related in: MedlinePlus

Inhibitor shock. A) Plot showing the addition of HMF and furfural in culture supernatants and the temporary disruption of growth. B) Plot showing the levels of intracellular citric acid and hydroxymethylfurfurol, as well as the average of all other metabolites. C) A heat map of a hierarchical clustering of the concentration of all monitored intracellular metabolites over the course of the 4 hour experiment.
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Fig5: Inhibitor shock. A) Plot showing the addition of HMF and furfural in culture supernatants and the temporary disruption of growth. B) Plot showing the levels of intracellular citric acid and hydroxymethylfurfurol, as well as the average of all other metabolites. C) A heat map of a hierarchical clustering of the concentration of all monitored intracellular metabolites over the course of the 4 hour experiment.

Mentions: A total of 40 different metabolites were tracked over the time course of the experiment (Figure 5, Additional file 1: Figure S4, Additional file 1: Table S2). Almost all metabolite concentrations showed a marked decrease at the 15 minute time point post exposure to HMF and furfural, with the exception of hydroxymethylfurfurol and citric acid. Hydroxymethylfurfurol, presumably resulting from the reduction of HMF, increased steadily throughout the 4 hours that metabolites were tracked. HMF and furfural were almost entirely metabolized after 16 hours. It is notable that glucose-6-phosphate is among the many metabolites that decrease as the result of HMF and furfural addition. This suggests that the inhibition occurs very early in the glycolysis pathway, either at glucose transport or its phosphorylation, although additional experimentation will be required to confirm this hypothesis given the labile nature of glucose-6-phosphate.Figure 5


Genome-scale resources for Thermoanaerobacterium saccharolyticum.

Currie DH, Raman B, Gowen CM, Tschaplinski TJ, Land ML, Brown SD, Covalla SF, Klingeman DM, Yang ZK, Engle NL, Johnson CM, Rodriguez M, Shaw AJ, Kenealy WR, Lynd LR, Fong SS, Mielenz JR, Davison BH, Hogsett DA, Herring CD - BMC Syst Biol (2015)

Inhibitor shock. A) Plot showing the addition of HMF and furfural in culture supernatants and the temporary disruption of growth. B) Plot showing the levels of intracellular citric acid and hydroxymethylfurfurol, as well as the average of all other metabolites. C) A heat map of a hierarchical clustering of the concentration of all monitored intracellular metabolites over the course of the 4 hour experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4518999&req=5

Fig5: Inhibitor shock. A) Plot showing the addition of HMF and furfural in culture supernatants and the temporary disruption of growth. B) Plot showing the levels of intracellular citric acid and hydroxymethylfurfurol, as well as the average of all other metabolites. C) A heat map of a hierarchical clustering of the concentration of all monitored intracellular metabolites over the course of the 4 hour experiment.
Mentions: A total of 40 different metabolites were tracked over the time course of the experiment (Figure 5, Additional file 1: Figure S4, Additional file 1: Table S2). Almost all metabolite concentrations showed a marked decrease at the 15 minute time point post exposure to HMF and furfural, with the exception of hydroxymethylfurfurol and citric acid. Hydroxymethylfurfurol, presumably resulting from the reduction of HMF, increased steadily throughout the 4 hours that metabolites were tracked. HMF and furfural were almost entirely metabolized after 16 hours. It is notable that glucose-6-phosphate is among the many metabolites that decrease as the result of HMF and furfural addition. This suggests that the inhibition occurs very early in the glycolysis pathway, either at glucose transport or its phosphorylation, although additional experimentation will be required to confirm this hypothesis given the labile nature of glucose-6-phosphate.Figure 5

Bottom Line: The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid.Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge.In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions.

View Article: PubMed Central - PubMed

Affiliation: Mascoma Corporation, 67 Etna Rd, 03766, Lebanon, NH, USA. Devin.H.Currie.GR@dartmouth.edu.

ABSTRACT

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation.

Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results.

Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

No MeSH data available.


Related in: MedlinePlus