Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum

Biswas, Ranjita; Prabhu, Sandeep; Lynd, Lee R.; Guss, Adam M.

doi:10.1371/journal.pone.0086389

Cited by 65 publications

(37 citation statements)

References 19 publications

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“…S1 in the supplemental material). Thus, the enhanced growth of the ET strain at a high ethanol concentration is accompanied by reduced growth under standard conditions, supporting the idea that the ET strain has altered physiology, with results similar to those seen with C. thermocellum adapted to 5% ethanol (26,27). However, we observed no morphological differences between ET and WT colonies grown on GS2 agar plates or cells grown in liquid GS2 medium (see Fig.…”

Section: Phytofermentans Ethanol Tolerance and Productionsupporting

confidence: 87%

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

Tolonen

Zuroff

Ramya

et al. 2015

Appl Environ Microbiol

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cNovel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.

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Section: Phytofermentans Ethanol Tolerance and Productionsupporting

confidence: 87%

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

Tolonen

Zuroff

Ramya

et al. 2015

Appl Environ Microbiol

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“…However, wild-type C. thermocellum is limited by its low ethanol yield and titer, producing acetate, lactate, H 2 , formate, free amino acids, and other compounds as additional fermentation products [2, 3]. Recent development of genetic tools for C. thermocellum [4–9] has enabled the construction of numerous targeted mutants, eliminating acetate, lactate, formate, and H 2 production [4, 7, 10–14]. One of the mutations, C. thermocellum ΔhydG , eliminated a hydrogenase maturase protein involved in assembly of the [FeFe]-active site of three of the four hydrogenases in C. thermocellum .…”

Section: Introductionmentioning

confidence: 99%

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

Biswas

Wilson

Giannone

et al. 2017

Biotechnol Biofuels

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Background Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate of ΔhydG decreased substantially compared to wild type.ResultsAddition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ∆hydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ΔhydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG. Clostridium thermocellum ΔhydGΔech had a higher growth rate than ΔhydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate.ConclusionsSulfur metabolism is perturbed in C. thermocellum ΔhydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0684-x) contains supplementary material, which is available to authorized users.

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“…To date, the majority of C. thermocellum genetic engineering studies have focused on improving fuel production (24)(25)(26)(27)(28)(29)(30), but little genetic work has targeted understanding regulatory pathways. Here, we combine use of gene deletions with transcriptomics and DNA binding assays to gain insights into the regulatory networks of the three LacI transcription factors in the genome of C. thermocellum DSM1313.…”

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confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ␤-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5-to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and ␣-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters. IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase 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.

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Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum

Cited by 65 publications

References 19 publications

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

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

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

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