Biocatalytic reduction of carboxylic acids

Napora‐Wijata, Kamila; Strohmeier, Gernot A.; Winkler, Margit

doi:10.1002/biot.201400012

Cited by 96 publications

(82 citation statements)

References 81 publications

(87 reference statements)

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“…The acetaldehyde:ferredoxin oxidoreductase has been shown to only catalyze the reduction of the undissociated acetic acid (pKa ϭ 4.7) rather than the dissociated acetate (64,73). At an intracellular pH of 6 and an intracellular acetate concentration of 0.1 M, as assumed in the example above, the intracellular concentration of acetic acid is 5 mM and thus in the order of the reported K m of 6 mM acetic acid for the acetaldehyde:ferredoxin oxidoreductase (64).…”

Section: H 2 Activationmentioning

confidence: 96%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

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Most acetogens can reduce CO 2 with H 2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO 2 and H 2 . How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H 2 /CO 2 -grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD ؉ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H 2 /CO 2 -grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H 2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO 2 and H 2 . IMPORTANCEEthanol formation from syngas (H 2 , CO, and CO 2 ) and from H 2 and CO 2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H 2 and CO 2 , considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H 2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

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Section: H 2 Activationmentioning

confidence: 96%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

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“…A homolog of Car Ni from Mycobacterium marinum was also demonstrated to have activity on straight-chain aliphatic acids ranging from C 6 to C 18 (26). A recent review describes a larger number of carboxylic acid reductases that could be harnessed for biosynthesis of a variety of aldehydes (27). The general stoichiometry for reactions catalyzed by carboxylic acid reductases is as follows (where "e Ϫ " represents a reducing equivalent):…”

Section: Engineering Aldehyde Biosynthetic Reactions and Pathwaysmentioning

confidence: 99%

Microbial Engineering for Aldehyde Synthesis

Kunjapur

Prather

2015

Appl Environ Microbiol

143

129

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Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity.

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“…A mild and selective alternative to chemical reduction protocols [1–3] is the enzymatic reduction of carboxylic acids. Carboxylate reductase (CAR) enzymes exhibit a broad substrate tolerance for the conversion of organic acids to the respective aldehydes 4. However, only few CAR enzyme sequences have been elucidated5, 6, 7, 8, 9, 10, 11, 12 and are available for biocatalysis to date, although Nature provides a great versatility of organisms with the capability to catalyze this reaction.…”

Section: Introductionmentioning

confidence: 99%

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

et al. 2016

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The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a T m of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.

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Biocatalytic reduction of carboxylic acids

Cited by 96 publications

References 81 publications

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Microbial Engineering for Aldehyde Synthesis

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

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Biocatalytic reduction of carboxylic acids

Cited by 96 publications

References 81 publications

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Microbial Engineering for Aldehyde Synthesis

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

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Product

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Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation