Metagenomic ene-reductases for the bioreduction of sterically challenging enones

Dobrijevic, Dragana; Benhamou, Laure; Aliev, Abil E.; Méndez-Sánchez, Daniel; Dawson, Natalie L.; Baud, Damien; Tappertzhofen, Nadine; Moody, Thomas S.; Orengo, Christine A.; Hailes, Helen C.; Ward, John M.

doi:10.1039/c9ra06088j

Cited by 20 publications

(26 citation statements)

References 39 publications

(58 reference statements)

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“…DNA was extracted from a domestic drain metagenome and sequenced using the Illumina MiSeq platform 52. Predicted protein sequences from the drain metagenome were previously functionally annotated by scanning the sequences against Pfam28.0 libraries of domain families (pfam.xfam.org).…”

Section: Methodsmentioning

confidence: 99%

The identification and use of robust transaminases from a domestic drain metagenome

et al. 2019

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Section: Methodsmentioning

confidence: 99%

The identification and use of robust transaminases from a domestic drain metagenome

et al. 2019

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“…To further promote an off-the-shelve synthetic use of ene reductases, which can reduce the time and cost of the implementation of a biocatalytic step into a process significantly, we set out to expand the available biocatalytic toolbox [15]. In this context, not only the discovery and engineering of novel ene reductases is of great utility [33], but also a careful characterization of the new biocatalysts is needed as it may lead to the construction of a more targeted enzyme library associated with reduced screening time and costs.…”

Section: Isolated In 1932 Bymentioning

confidence: 99%

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

2020

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Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone.

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“…Identifying enzymes from metagenomic sources, where DNA is extracted from an environmental sample and analysed using high throughput sequencing technologies, has proved to be a valuable method. With the increasing availability and the decreasing cost of sequencing methods, there has been a rise in studies using functional metagenomics [9–11] …”

Section: Introductionmentioning

confidence: 99%

“…In recent work, we have utilised this approach by collecting metagenomic material from the human oral cavity and a domestic drain, sequencing the DNA and assembling the shorter reads into larger contiguous stretches of DNA (contigs), creating an in silico library. Using this library, open reading frames, operons and enzymes can be identified and this approach has been used to discover new transketolases, ene‐reductases and transaminases [9,10,12,13] . After using PCR to amplify identified DNA, the enzymes were cloned, and assayed for their functionality.…”

Section: Introductionmentioning

confidence: 99%

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Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

et al. 2021

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Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR‐17, when co‐expressed with a co‐factor recycling system produced the anti‐(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

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Metagenomic ene-reductases for the bioreduction of sterically challenging enones

Abstract: Exceptional organic solvent tolerant ene-reductases mined from a drain metagenome library are highly versatile catalysts for difficult enones.

Cited by 20 publications

References 39 publications

The identification and use of robust transaminases from a domestic drain metagenome

The identification and use of robust transaminases from a domestic drain metagenome

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

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