Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production

Moura, Matthew; Pertusi, Dante A.; Lenzini, Stephen; Bhan, Namita; Broadbelt, Linda J.; Tyo, Keith E.J.

doi:10.1002/bit.25860

Cited by 36 publications

(36 citation statements)

References 40 publications

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“…Controls with all potential by‐products showed that the precipitate was pyrophosphate. Consistent with the observations from the Prather group with Ni CAR,11 these results indicate the inhibition of Nc CAR by the co‐product pyrophosphate. An experiment including pyrophosphatase showed that the concentration of 1b can indeed be increased‐two fold in vitro (Supporting Information, Figured S4).…”

Section: Resultssupporting

confidence: 90%

“…The hit with highest scores was a hypothetical protein with NCBI accession code XP_955820.1. On the sequence level, this protein showed low identities with the original search templates Ni CAR, Mm CAR, Sr CAR and At CAR (17.3%, 17.5%, 18.5% and 22.5%, respectively), and also low identities with CARs that have been published during this study: Mn CAR,9 Tv CAR,10 Ms CAR,11 Nb CAR11 and Sb CAR12 (16.4%, 26.1%, 17.7%, 17.3% and 22.4%, respectively). A codon optimized synthetic gene coding for XP_955820.1 from N. crassa OR74A was expressed with an N ‐terminal TEV cleavable HIS‐tag from the multiple cloning site 2 of the pETDUET1 vector.…”

Section: Resultsmentioning

confidence: 67%

“…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. [4,13–18] The reduction of salicylic acid,19 benzoic acid and derivatives20 as well as cinnamic acid and derivatives21 has been observed in the ascomycete Neurospora crassa ( Nc ).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…Of specific interest were amine nucleophiles that would lead to amide bond formation from acid and amine,an important challenge in organic chemistry. [8] Thea ctivated substrate is then attacked by the thiol of the enzyme-bound PPant group,generating athioester intermediate that is transferred to the reduction domain. CARs are composed of three distinct protein domains:t he adenylation domain, which itself is divided into ac ore-domain and subdomain, [7] the peptidyl carrier protein with the bound 4'-phosphopantetheine (PPant) prosthetic group,and areductase domain.…”

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confidence: 99%

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

et al. 2017

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Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

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“…Previous studies have demonstrated that substrates with an amine group at the α-position are scarcely accepted by carboxylic acid reductases [48], suggesting that production of ( S )-2-aminobutanol may be further boosted by optimizing the substrate binding site of the carboxylic acid reductase.…”

Section: Discussionmentioning

confidence: 99%

Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae

et al. 2017

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Background Saccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker’s yeast.ResultsWe first designed a two-step heterologous pathway, starting with the endogenous amino acid l-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional l-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional l-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol.ConclusionsOur results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0667-z) contains supplementary material, which is available to authorized users.

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Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production

Cited by 36 publications

References 40 publications

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

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

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae

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