Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum

Deng, Yu; Olson, Daniel G.; Jing-lun, Zhou; Herring, Christopher D.; Shaw, Aubie; Lynd, Lee R.

doi:10.1016/j.ymben.2012.11.006

Cited by 78 publications

(100 citation statements)

References 33 publications

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“…PEP synthase seems to be an unlikely candidate for PEP-to-pyruvate conversion due to its low expression at the transcript (14) and protein level (15). The remaining option is conversion via PEPCK, which was present at a high level of activity, and we suspect that the majority of PEP is converted to OAA via this enzyme (7).…”

Section: Discussionmentioning

confidence: 97%

Atypical Glycolysis in Clostridium thermocellum

Jing-lun

Olson

Argyros³

et al. 2013

Appl Environ Microbiol

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197

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dCofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PP i )-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PP i in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PP i -dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PP i must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PP i generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.

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Section: Discussionmentioning

confidence: 97%

Atypical Glycolysis in Clostridium thermocellum

Jing-lun

Olson

Argyros³

et al. 2013

Appl Environ Microbiol

Self Cite

197

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“…Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3)(4)(5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6)(7)(8), as well as secreted amino acids (9,10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.…”

mentioning

confidence: 99%

“…A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10,11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes.…”

mentioning

confidence: 99%

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Taillefer

Rydzak

Levin

et al. 2015

Appl Environ Microbiol

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Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a k cat of 45.8 s ؊1 or 14.9 s ؊1 , respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a k cat of 520.8 s ؊1 . The malic enzyme used NADP ؉ as a cofactor along with NH 4 ؉ and Mn 2؉ as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a K i of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH 4 ؉ based on the characterization of the malate dehydrogenase and malic enzyme.C lostridium thermocellum is a Gram-positive, anaerobic thermophile capable of reaching one of the highest growth rates on crystalline cellulose (1, 2). Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3-5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6-8), as well as secreted amino acids (9, 10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10, 11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes. In contrast to most clostridia and other fermentative ethanol and/or H 2 -producing organisms (7), C. thermocellum ATCC 27405 (GenBank accession number NC_009012.1) does not encode a pyruvate kinase, which catalyzes PE...

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“…Also, another strain with heterologous pyruvate kinase activity whose malic enzyme gene and a part of its malate dehydrogenase genes were deleted showed over 3-fold higher ethanol yield than the wild-type strain. Overall, their novel mutant strains achieved 3-fold higher ethanol yield, increased carbon recovery, increased formate production, increased ethanol tolerance, and decreased amino acid secretion relative to the parent strain (Deng et al 2013;Ragauskas et al, 2014). These findings confirmed that the malate shunt could be used to convert phosphoenol pyruvate to pyruvate.…”

Section: Clostridium Thermocellummentioning

confidence: 98%

“…In their work, the Δldh Δpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain (DSM1313 Δhpt Δspo0A), whereas its biomass accumulation was comparable to Δpta. Deng et al (2013) engineered the ‗‗malate shunt'' pathway, including phosphoenolpyruvate carboxykinase, NADH-linked malate dehydrogenase, and NADP-dependent malic enzymes. The engineering included expression of the pyruvate kinase gene from T. saccharolyticum, mutation of the phosphoenolpyruvate carboxykinase and deletion of malic enzyme gene in C. thermocellum.…”

Section: Clostridium Thermocellummentioning

confidence: 99%

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

2015

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HIGHLIGHTS Various CBP strategies have been discussed. Recently, lignocellulosic biomass as the most abundant renewable resource has been widely considered for bioalcohols production. However, the complex structure of lignocelluloses requires a multi-step process which is costly and time consuming. Although, several bioprocessing approaches have been developed for pretreatment, saccharification and fermentation, bioalcohols production from lignocelluloses is still limited because of the economic infeasibility of these technologies. This cost constraint could be overcome by designing and constructing robust cellulolytic and bioalcohols producing microbes and by using them in a consolidated bioprocessing (CBP) system. This paper comprehensively reviews potentials, recent advances and challenges faced in CBP systems for efficient bioalcohols (ethanol and butanol) production from lignocellulosic and starchy biomass. The CBP strategies include using native single strains with cellulytic and alcohol production activities, microbial co-cultures containing both cellulytic and ethanologenic microorganisms, and genetic engineering of cellulytic microorganisms to be alcohol-producing or alcohol producing microorganisms to be cellulytic. Moreover, high-throughput techniques, such as metagenomics, metatranscriptomics, next generation sequencing and synthetic biology developed to explore novel microorganisms and powerful enzymes with high activity, thermostability and pH stability are also discussed. Currently, the CBP technology is in its infant stage, and ideal microorganisms and/or conditions at industrial scale are yet to be introduced. So, it is essential to bring into attention all barriers faced and take advantage of all the experiences gained to achieve a high-yield and low-cost CBP process.

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Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum

Cited by 78 publications

References 33 publications

Atypical Glycolysis in Clostridium thermocellum

Atypical Glycolysis in Clostridium thermocellum

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

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