Protein and the Planet 2: Directed Evolution of Proteins

Following on from our more general blog, “Protein and the Planet” where we reviewed various examples of how protein biotechnology is having an impact as solutions to Climate Change, in this blog Sam Bloor focuses on the directed evolution of proteins. In particular, he comments on how engineered enzymes are being used to produce biofuels and pharmaceutical intermediates more efficiently and how the structural biology capabilities we have here at Peak Proteins (a Sygnature Discovery business) can be used to facilitate a directed evolution project.

Introduction

Climate change is an ever-present societal issue at the forefront of modern media. The United Nations (UN), signed the Paris Agreement in 2016 providing a global treaty for green initiatives to limit the increase in average global temperature to no more than 2.0 ^oC. It was also noted at the time that in increase in average global temperature above 1.5 ^oC would have catastrophic effects on the environment¹. The production of high value chemicals, such as biofuels² and pharmaceutical intermediate compounds³, through biotechnology rather than energy intensive chemical reactions has been identified as one of the most important routes to a sustainable green economy^4,5. These chemicals can be produced by a specific type of proteins known as enzymes.

Limitations of Enzymes at Industrial Scale

Enzymes are biological catalysts which lower the activation energy required for a specific reaction to occur⁶, they are typically proteins but there are examples of RNA molecules acting as biocatalysts. Enzymes can provide regio- and stereo-specificity to a reaction, are often utilised in ambient conditions and do not require toxic solvents for use. Taken together these attributes provide a sustainable route to a wide range of high value chemicals. However, enzymes can have limitations at an industrial scale such as poor thermostability, yields or turnover rates.

Directed Evolution as a Solution

Directed evolution, pioneered by Nobel laureate Frances Arnold, has enabled the rapid evolution of enzymes to overcome many of these industrial limitations. Directed evolution, like natural selection, is survival of the fittest by means of enhanced enzyme function: increased yields of products, enhanced thermostability, the unlocking of previously not possible in biology chemical reactions and the repurposing enzymes for different biochemical reactions^7,8,9.

Structural Biology to Support Directed Evolution

Whilst not a prerequisite for directed evolution, structural information for a given enzyme can rapidly advance the evolution process and reduce the overall cost of a directed evolution workflow. If a scientist, such as yourself, has obtained a crystal structure of a protein of interest, they/you can target residues contained within the active site which are most likely to influence activity. This technique known as saturation mutagenesis produces a limited mutant library which contains every possible residue at a specific residue position. Saturation mutagenesis is therefore an extension of the rationale design method previously employed for protein engineering.

Peak Proteins has a dedicated team of structural biologists with a wealth of experience in determining structures by crystallography, cryo-EM and NMR. If you are about to embark on a directed evolution workflow to enhance your targets abilities for industrial biotechnology, you should consider using Peak Proteins to give you a head start by obtaining accurate structural information to inform your screening process. Get in touch to find out more info@peakproteins.com

References

1. UNFCCC. United Nations Framework Convention on Climate Change: Paris Agreement (ed. United Nations) 1-27. (United Nations, Paris, 2016)
2. Wang, C. et al. Metabolic engineering of Escherichia coli for alpha-farnesene production. Metabolic Engineering. 13. 648-655 (2011).
3. Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nature Chemical Biology. 5. 568-574. (2009).
4.Panoutsou, C. et al. Advanced biofuels to decarbonise European transport by 2030: Markets, challenges, and policies that impact their successful market uptake. Energy Strategy Reviews. 34. 23. (2021).
5. Thomas, S. M., DiCosimo, R. & Nagarajan, A. Biocatalysis: applications and potentials for the chemical industry. Trends in Biotechnology. 20. 238-242. (2002).
6.Bell, E. L. et al. Biocatalysis. Nature Reviews: Methods Primer 2021. 1. (2021).
7. Li, G. Y., Wang, J. B. & Reetz, M. T. Biocatalysts for the pharmaceutical industry created by structure-guided directed evolution of stereoselective enzymes. Bioinorganic & Medicinal Chemistry. 26. 1241-1251. (2018).
8. Burke, A. J. et al. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature. 570. 219-223. (2019).
9. Heath, R. S. et al. An engineered alcohol oxidase for the oxidation of primary alcohols. Chembiochem. 20. 276-281. (2019).