Combining chemistry and protein engineering for new-to-nature biocatalysis

Miller, David C.; Athavale, Soumitra V.; Arnold, Frances H.

doi:10.1038/s44160-021-00008-x

Cited by 130 publications

(83 citation statements)

References 59 publications

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“…[100] In particular, the green and sustainable credentials of biocatalysis are beyond any doubt. [4,9,11,[51][52][53][54][55] The exquisite precision of biocatalysts (in terms of chemo-, regio-, and stereoselectivity), combined with their biodegradability, non-toxicity and capability for catalyzing a broad palette of reactions under very mild reaction conditions, makes them extremely powerful. [101] Nowadays, the combination of these advantages with the rapid implementation of enzymatic processes (when needed, as in the SARS-CoV-2 example) at industrial level, gives an exciting view of the future for biocatalysis as a core technology to reach sustainability and efficiency in chemical processes.…”

Section: Rolling Out Biocatalysis Globally At All Levelsmentioning

confidence: 99%

“…With the growing attention to sustainability, the application of biocatalysis has been increasing in both research and industrial areas as its power is more widely appreciated. [11] It has even been recently stated that we are in a Golden Age of Biocatalysis. [12] Advanced DNA sequencing of microbial (meta)genomes and enzyme engineering through directed evolution have enabled the rapid identification of new robust enzyme activities and optimization for industrial applications.…”

Section: Introductionmentioning

confidence: 99%

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Biocatalysis as Key to Sustainable Industrial Chemistry

Alcántara

Marı́a²,

Littlechild

et al. 2022

ChemSusChem

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The role and power of biocatalysis in sustainable chemistry has been continuously brought forward step by step to its present outstanding position. The problem‐solving capabilities of biocatalysis have been realized by numerous substantial achievements in biology, chemistry and engineering. Advances and breakthroughs in the life sciences and interdisciplinary cooperation with chemistry have clearly accelerated the implementation of biocatalytic synthesis in modern chemistry. Resource‐efficient biocatalytic manufacturing processes have already provided numerous benefits to sustainable chemistry as well as customer‐centric value creation in the pharmaceutical, food, flavor, fragrance, vitamin, agrochemical, polymer, specialty, and fine chemical industries. Biocatalysis can make significant contributions not only to manufacturing processes, but also to the design of completely new value‐creation chains. Biocatalysis can now be considered as a key enabling technology to implement sustainable chemistry.

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Section: Rolling Out Biocatalysis Globally At All Levelsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biocatalysis as Key to Sustainable Industrial Chemistry

Alcántara

Marı́a²,

Littlechild

et al. 2022

ChemSusChem

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“…The evolutionary improvements demonstrated here make them as efficient as the previously known metal-containing phosphotriesterases, suggesting that not only accidental low-efficieny promiscuous catalyst exist, but instead alternative enzymatic strategies are available in a given metagenome, with the potential to be truly proficient and ready to be recruited for a survival advantage or for their biocatalytic application. Natural repertoires thus hold a variety of solutions for catalytic challenges: even if not explored thus far, they are available to contribute new-to-nature reactions 42 or mechanisms.…”

Section: Implications and Conclusionmentioning

confidence: 99%

Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase

Fernández

Klein

Kamiński

et al. 2022

Preprint

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The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators. Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the α/β-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105 members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91's phosphotriesterase activity was increased ≈ 400-fold to a kcat/KM of ≈ 106 M-1s-1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in Nature, resulting in alternative reagents with a novel catalytic machinery.

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“…It is important to know all of the enzymes in the natural biosynthetic route to a metabolite and any missing enzymes need to be identified (Caputi et al, 2018). While natural pathway enzymes provide within their substrate scope facile synthetic access to metabolites or metabolite-like molecules, more diversified and non-natural metabolite-like compounds can become accessible by broadening of the substrate by enzyme engineering, by introducing new catalytic activities with non-natural substrates and by developing new chemical transformations by combining chemistry and protein engineering (Miller et al, 2022). The transformations which can effectively be performed by either a classical chemical reaction, a chemocatalytic or a biocatalytic reaction can enable the combination of the two worlds of classical organic synthesis and biocatalysis (Wohlgemuth, 2007;Roberts et al, 2010;Rudroff et al, 2018;Bering et al, 2022).…”

Section: Integration Of Biocatalytic Reactionsmentioning

confidence: 99%

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

Wohlgemuth

Littlechild²

2022

Front. Bioeng. Biotechnol.

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The biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules.

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Combining chemistry and protein engineering for new-to-nature biocatalysis

Cited by 130 publications

References 59 publications

Biocatalysis as Key to Sustainable Industrial Chemistry

Biocatalysis as Key to Sustainable Industrial Chemistry

Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

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