Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Lin, Paul P.; Mi, Luo; Morioka, Amy H.; Yoshino, Kouki Matthew; Konishi, S.; Xu, Sharon C.; Papanek, Beth; Riley, Lauren A.; Guss, Adam M.; Liao, James C.

doi:10.1016/j.ymben.2015.07.001

Cited by 152 publications

(93 citation statements)

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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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confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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“…However, several investigators routinely add bicarbonate, a dissolved form of CO 2 , into the medium to promote C. thermocellum growth (6,(10)(11)(12), implying its ability to use CO 2 . This observation raises the questions of how CO 2 plays a role in promoting C. thermocellum growth (13), and whether CO 2 is incorporated into the metabolism.…”

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CO ₂ -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Xiong

Lin

Magnusson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2 . This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13 C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13 C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO 2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 2 fixation via the reductive C1 metabolic pathway in C. thermocellum. These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO 2 as well.Clostridium thermocellum | pyruvate:ferredoxin oxidoreducase | formate | 13 C-isotopic tracing | one-carbon metabolism T he gram-positive Clostridium thermocellum is a thermophilic and strict anaerobic bacterium. It has gained a great amount of interest due to its cellulolytic abilities. By taking advantage of an extracellular cellulase system called the cellulosome (1), C. thermocellum can depolymerize cellulose into soluble oligosaccharides. The latter are further transported into the cells and fermented through its glycolytic pathway to pyruvate, the precursor to an array of fermentation products (e.g., H 2 , formate, lactate, acetate, ethanol, secreted amino acids) (2, 3). This capability makes C. thermocellum an attractive candidate for consolidated bioprocessing of lignocellulosic biomass, a process configuration that directly converts plant biomass into biofuels and chemicals without separate additions of enzymes (4). To date, there have been numerous works describing the molecular and genetic details of the cellulolytic system and fermentation pathways in C. thermocellum (5-9), whereas other metabolic characteristics of this species have not been adequately understood.Currently, few details regarding inorganic carbon utilization are known for C. thermocellum. However, several investigators routinely add bicarbonate, a dissolved form of CO 2 , into the medium to promote C. thermocellum growth (6, 10-12), implying its ability to use CO 2 . Thi...

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“…Fermentation data from a set of C. thermocellum strains with single deletions of each of the five annotated pfor s points to pfor4 being responsible for isobutanol production. Given the interest in producing isobutanol either for use as a biofuel or as a feedstock chemical [36]; the knowledge that pfor4 is necessary for isobutanol production could be beneficial to further improve its production from cellulosic substrates.…”

Section: Resultsmentioning

confidence: 99%

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

Hon

Holwerda

Worthen

et al. 2018

Biotechnol Biofuels

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BackgroundClostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA, for ethanol production.ResultsHere, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum. The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes (adhA, nfnA, nfnB, and adhEG544D) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production.ConclusionsHere, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1245-2) contains supplementary material, which is available to authorized users.

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Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Cited by 152 publications

References 26 publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

CO ₂ -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

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Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Cited by 152 publications

References 26 publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

CO 2 -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

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CO ₂ -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum