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  • Primer
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Biocatalysis

Nature Reviews Methods Primers volume 1, Article number: 46 (2021) Cite this article

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Abstract

Biocatalysis has become an important aspect of modern organic synthesis, both in academia and across the chemical and pharmaceutical industries. Its success has been largely due to a rapid expansion of the range of chemical reactions accessible, made possible by advanced tools for enzyme discovery coupled with high-throughput laboratory evolution techniques for biocatalyst optimization. A wide range of tailor-made enzymes with high efficiencies and selectivities can now be produced quickly and on a gram to kilogram scale, with dedicated databases and search tools aimed at making these biocatalysts accessible to a broader scientific community. This Primer discusses the current state-of-the-art methodology in the field, including route design, enzyme discovery, protein engineering and the implementation of biocatalysis in industry. We highlight recent advances, such as de novo design and directed evolution, and discuss parameters that make a good reproducible biocatalytic process for industry. The general concepts will be illustrated by recent examples of applications in academia and industry, including the development of multistep enzyme cascades.

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Fig. 1: Examples of different products synthesized using biocatalysis.
Fig. 2: Sources of biocatalysts.
Fig. 3: Directed evolution cycle.
Fig. 4: Computational approaches to enzyme design.
Fig. 5: Design and engineering of biocatalysts using non-canonical amino acids.
Fig. 6: Biocatalytic cascades and their development.
Fig. 7: Beneficial properties of an excellent biocatalyst under industrial process conditions.
Fig. 8: Industrial examples of biocatalytic cascades.

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Acknowledgements

The authors are grateful for funding from the European Research Council (ERC) (S.L.F., 788231; A.P.G., 757991; S.O., 679001; N.J.T., 742987), the Engineering and Physical Sciences Research Council (EPSRC) (S.L.F. and N.J.T., EP/S005226/1), the Biotechnology and Biological Sciences Research Council (BBSRC) (S.L.F. and N.J.T., BB/M027791/1, BB/M028836/1; A.P.G., BB/M027023/1), the Spanish Ministry of Economy and Competitiveness (MINECO) (S.O., PGC2018-102192-B-I00), Generalitat de Catalunya (S.O., SGR 2017 1707) and the University of Manchester (Presidential Fellowship to S.L.L.).

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Authors and Affiliations

  1. School of Chemistry and MIB, The University of Manchester, Manchester, UK

    Elizabeth L. Bell, William Finnigan, Anthony P. Green, Lorna J. Hepworth, Sarah L. Lovelock, Nicholas J. Turner & Sabine L. Flitsch

  2. Pfizer Worldwide Research and Development, Groton, CT, USA

    Scott P. France

  3. Compound Synthesis and Management, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

    Martin A. Hayes & Elvira Romero

  4. Department of Chemistry, The University of British Columbia, Vancouver, British Columbia, Canada

    Haruka Niikura & Katherine S. Ryan

  5. CompBioLab Group, Institut de Quimica Computacional i Catalisi, Departament de Química, Universitat de Girona, Girona, Spain

    Sílvia Osuna

  6. ICREA, Barcelona, Spain

    Sílvia Osuna

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  1. Elizabeth L. Bell
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Contributions

Introduction (S.L.F., H.N. and K.S.R.); Experimentation (E.L.B., A.P.G., H.N., S.O., K.S.R., N.J.T., S.L.L., L.J.H. and W.F.); Results (M.A.H. and E.R.); Applications (S.P.F., M.A.H. and E.R.); Reproducibility and data deposition (W.F. and L.J.H.); Limitations and optimizations (W.F., L.J.H., M.A.H. and E.R.); Outlook (S.L.F.); overview of Primer (S.L.F.). All authors contributed equally to planning and revision of the manuscript as described. Please note that co-authors have been listed in alphabetical order.

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Correspondence to Sabine L. Flitsch.

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Nature Reviews Methods Primers thanks L. Betancor, A. Fryszkowska and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

BioCatNet: https://www.biocatnet.de/

Basic Local Alignment Search Tool : https://blast.ncbi.nlm.nih.gov/Blast.cgi

BRENDA: https://www.brenda-enzymes.org/

CAVER: http://www.caver.cz/

KEGG: https://www.genome.jp/kegg/

InterPro: https://www.ebi.ac.uk/interpro/

Pfam: https://pfam.xfam.org/

PrenDB: http://prendb.pharmazie.uni-marburg.de/prendb/home/

Protein Data Bank: https://www.rcsb.org/

RetroBioCat: https://retrobiocat.com/

Revenant: https://revenant.inf.pucp.edu.pe/

Rosetta: https://www.rosettacommons.org/

UniProt: https://www.uniprot.org/

Glossary

Enzyme cascade

Within the biocatalysis community this term is used broadly for concurrent, multienzyme one-pot biocatalytic reactions as well as reactions in which components are added sequentially or process steps are telescoped.

Metagenomic libraries

Genomic libraries constructed by the direct cloning of the large fragments of the environmental DNA into an appropriate vector, transformed into the host bacteria.

C(sp 3)–H functionalization

A type of reaction in which a C–H bond, in which the carbon is sp3 hybridized, is cleaved and a new C–X bond is formed (where X is usually carbon, oxygen, nitrogen or a halide).

Diels–Alderases

Enzymes that catalyse a [4 + 2] cycloaddition reaction between a conjugated diene and a substituted alkene forming a cyclohexene derivative.

Rates of catalysis

The rates by which substrates are converted into products in catalytic reactions.

Saturation mutagenesis

A method that allows the randomization of a target codon or set of codons in a gene.

Iterative combinatorial active site testing

A method that allows the generation of DNA libraries where active site positions are randomized in pairs

Error-prone PCR

A PCR (polymerase chain reaction) that is run under reaction conditions that introduce random mutations into the target DNA sequence.

Gene shuffling

A method that allows for the generation of chimeric libraries of genes.

DNA shuffling

A method that allows the recombination of beneficial mutations in a directed evolution experiment.

High performance liquid chromatography

An analytical technique that allows for the rapid separation and quantification of compound mixtures using pressurized liquid solvent passed through chromatographic columns.

Nonsense codon

A codon within the genetic code that does not encode an amino acid but is recognized as a stop codon in transcription and translation of DNA.

Regioselectivity

The property that favours bond formation or breaking at a particular atom over all other possible atoms in a molecule.

Enzyme operational stability

Retention of enzyme activity when the enzyme is in use.

Evolvability

Capacity of an enzyme to acquire beneficial properties or functions through genetic modification.

Design of experiments

A statistical approach to analyse the influence of various factors in a system to predict the optimal operating conditions.

BLAST

(Basic Local Alignment Search Tool). A tool that compares nucleotide or protein sequences of interest (most commonly to sequences within a database), and finds regions of statistically significant similarity.

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Bell, E.L., Finnigan, W., France, S.P. et al. Biocatalysis. Nat Rev Methods Primers 1, 46 (2021). https://doi.org/10.1038/s43586-021-00044-z

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