Pyruvate Formate-Lyase Enables Efficient Growth of <i>Escherichia coli</i> on Acetate and Formate

Zelcbuch, Lior; Lindner, Steffen N.; Zegman, Yonatan; Slutskin, Ilya Vainberg; Antonovsky, Niv; Gleizer, Shmuel; Milo, Ron; Bar‐Even, Arren

doi:10.1021/acs.biochem.6b00184

Cited by 59 publications

(52 citation statements)

References 28 publications

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“…3 and 5). We provided multiple lines of evidence showing that the fixed CO 2 was channeled to the C1 metabolism through PFL, which could operate reversibly (21,30) and contribute to the biosynthesis of essential amino acids, including serine and methionine. In addition, the reductive C1 pathway mediated by PFL may play a critical role in nucleotide biosynthesis (reconstructed purine biosynthesis pathway in C. thermocellum shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…Supplementation of formate into the MAP strain increased isobutanol concentration by 7%, compared to MAP without formate in the medium (Supporting Information, Figure S2B). This phenomenon can be explained by the conversion of acetate and formate into pyruvate by reversible pflB-pflA reactions (Zelcbuch et al, 2016). Further introduction of cofactor regeneration in the MAP strain did not produce any positive results.…”

Section: Introduction Of Additional Nadph Regeneration System and Amentioning

confidence: 93%

“…Additional 5 mM of formate was included in the medium for fdh to increase NADPH regeneration. This phenomenon can be explained by the conversion of acetate and formate into pyruvate by reversible pflB-pflA reactions (Zelcbuch et al, 2016). Supplementation of formate into the MAP strain increased isobutanol concentration by 7%, compared to MAP without formate in the medium (Supporting Information, Figure S2B).…”

Section: Introduction Of Additional Nadph Regeneration System and Amentioning

confidence: 96%

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl‐CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Song

Seo

Jeon

et al. 2018

Biotech & Bioengineering

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Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.

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“…However, it is unclear if an organism could use only pyruvate formate-lyase to assimilate acetate and formate into pyruvate in defined medium. The only relevant study we found in literature did not test the effect of deleting the pyruvate-flavodoxin oxidoreductaselike ydbK (Nakayama et al, 2013) in their E. coli strain to exclude its contribution to pyruvate production (Zelcbuch et al, 2016). Moreover, threonine can be converted into pyruvate via serine (Figure 3).…”

Section: Thiamine Biosynthesis and Metabolismmentioning

confidence: 99%

Refined experimental annotation reveals conserved corrinoid autotrophy in chloroform-respiring Dehalobacter isolates

et al. 2016

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Two novel chlorinated alkane-respiring Dehalobacter restrictus strains CF and DCA were isolated from the same enrichment culture, ACT-3, and characterized. The closed genomes of these highly similar sister strains were previously assembled from metagenomic sequence data and annotated. The isolation of the strains enabled experimental verification of predicted annotations, particularly focusing on irregularities or predicted gaps in central metabolic pathways and cofactor biosynthesis. Similar to D. restrictus strain PER-K23, strains CF and DCA require arginine, histidine and threonine for growth, although the corresponding biosynthesis pathways are predicted to be functional. Using strain CF to experimentally verify annotations, we determined that the predicted defective serine biosynthesis pathway can be rescued with a promiscuous serine hydroxymethyltransferase. Strain CF grew without added thiamine although the thiamine biosynthesis pathway is predicted to be absent; intracellular thiamine diphosphate, the cofactor of carboxylases in central metabolism, was not detected in cell extracts. Thus, strain CF may use amino acids to replenish central metabolites, portending entangled metabolite exchanges in ACT-3. Consistent with annotation, strain CF possesses a functional corrinoid biosynthesis pathway, demonstrated by increasing corrinoid content during growth and guided cobalamin biosynthesis in corrinoid-free medium. Chloroform toxicity to corrinoid-producing methanogens and acetogens may drive the conservation of corrinoid autotrophy in Dehalobacter strains. Heme detection in strain CF cell extracts suggests the 'archaeal' heme biosynthesis pathway also functions in anaerobic Firmicutes. This study reinforces the importance of incorporating enzyme promiscuity and cofactor availability in genome-scale functional predictions and identifies essential nutrient interdependencies in anaerobic dechlorinating microbial communities.

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Pyruvate Formate-Lyase Enables Efficient Growth of Escherichia coli on Acetate and Formate

Cited by 59 publications

References 28 publications

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

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

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl‐CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Refined experimental annotation reveals conserved corrinoid autotrophy in chloroform-respiring Dehalobacter isolates

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Pyruvate Formate-Lyase Enables Efficient Growth of Escherichia coli on Acetate and Formate

Cited by 59 publications

References 28 publications

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

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

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl‐CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Refined experimental annotation reveals conserved corrinoid autotrophy in chloroform-respiring Dehalobacter isolates

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

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