Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

Tolonen, Andrew C.; Zuroff, Trevor R.; Ramya, Mohandass; Boutard, Magali; Cerisy, Tristan; Curtis, Wayne R.

doi:10.1128/aem.00619-15

Cited by 21 publications

(13 citation statements)

References 43 publications

(36 reference statements)

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“…The targeted intron was PCR amplified and inserted between the NdeI and BsrGI sites of pQint (15). Targeted pQint plasmids were transferred into C. phytofermentans by conjugation with Escherichia coli under anaerobic conditions (15,36,37). Colonies on GS2 plates containing the 40 g ml Ϫ1 erythromycin were picked, and the genomic intron insertion was confirmed by PCR and sequencing using primers in Table S3.…”

Section: Methodsmentioning

confidence: 99%

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

et al. 2019

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The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose. IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans. We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

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Section: Methodsmentioning

confidence: 99%

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

et al. 2019

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“…Duplicate cultures were sampled in mid-log phase or after 2 days (RAC) or 3 days (stover). Fermentation products were quantified by HPLC43.…”

Section: Methodsmentioning

confidence: 99%

Global repositioning of transcription start sites in a plant-fermenting bacterium

et al. 2016

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Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5′ mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.

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“…Other organisms such as Clostridium and Pseudomonas can be used to produce alternative biochemicals (Ragauskas et al ., 2014; Tolonen et al ., 2015; Ramos et al ., 2016; Sanford et al ., 2016). …”

Section: Lignocellulosic Facility Process Descriptionmentioning

confidence: 99%

“…The saccharification is followed by simultaneous fermentation of xylose and glucose to ethanol using a genetically modified strain of brewer's yeast (Saccharomyces cerevisiae). Other organisms such as Clostridium and Pseudomonas can be used to produce alternative biochemicals (Ragauskas et al, 2014;Tolonen et al, 2015;Ramos et al, 2016;Sanford et al, 2016).…”

Section: Lignocellulosic Facility Process Descriptionmentioning

confidence: 99%

Biofuels 2020: Biorefineries based on lignocellulosic materials

Valdivia

Galán-González

Briones

et al. 2016

Microbial Biotechnology

201

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SummaryThe production of liquid biofuels to blend with gasoline is of worldwide importance to secure the energy supply while reducing the use of fossil fuels, supporting the development of rural technology with knowledge‐based jobs and mitigating greenhouse gas emissions. Today, engineering for plant construction is accessible and new processes using agricultural residues and municipal solid wastes have reached a good degree of maturity and high conversion yields (almost 90% of polysaccharides are converted into monosaccharides ready for fermentation). For the complete success of the 2G technology, it is still necessary to overcome a number of limitations that prevent a first‐of‐a‐kind plant from operating at nominal capacity. We also claim that the triumph of 2G technology requires the development of favourable logistics to guarantee biomass supply and make all actors (farmers, investors, industrial entrepreneurs, government, others) aware that success relies on agreement advances. The growth of ethanol production for 2020 seems to be secured with a number of 2G plants, but public/private investments are still necessary to enable 2G technology to move on ahead from its very early stages to a more mature consolidated technology.

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Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

Cited by 21 publications

References 43 publications

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

Global repositioning of transcription start sites in a plant-fermenting bacterium

Biofuels 2020: Biorefineries based on lignocellulosic materials

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