Engineering Enzymes for Environmental Sustainability

Abstract

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO₂) and have been embedded into new efficient metabolic CO₂ fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Keywords: Biocatalysis; Directed Evolution; Enzyme Engineering; Sustainability.

Figure 1

Schematic overview of the topics covered in this review.

Figure 2

Directed Evolution. Directed evolution is comprised of three main steps: mutagenesis, selection of desired phenotypes, and isolation of the selected variants. Starting from the parent gene (input) the sequence is diversified e.g. by error prone PCR. Typically, the resulting DNA library is then transformed into bacteria for protein expression. Variants with desired properties can be identified (e.g. using a multi‐well plate assay) and isolated. The recovered genes serve as the input for the subsequent round of evolution. This cycle is iterated until the desired phenotypic activity is reached, typically resulting in accumulation of several mutations (output).

Figure 3

CO₂ capture and utilisation. A) Schematic active site depiction of a β‐carbonic anhydrase (CA). B) CA can be used as a catalyst in carbon capture and sequestration (CSS) technologies to extract CO₂ from flue gas as bicarbonate. CA also accelerates CO₂ release at low temperatures. C) CA from D. vulgaris (DVCA) was improved over nine rounds of directed evolution to increase enzyme tolerance to harsh process conditions. Overall, a 4×10⁶‐fold improvement in enzyme performance was achieved. D) The TaCo pathway is a synthetic metabolic pathway for fixation of CO₂, resulting in the formation of glycerate. This pathway can be interfaced with the CETCH cycle.[ ³¹ , ³² ] (Enzyme and cofactor diagram abbreviations: ACS=acetyl‐CoA synthetase, GCC=glycolyl‐CoA carboxylase, GCS=glycolyl‐CoA synthase, TCR=tartronyl‐CoA reductase, MePCC=M. extorquens propionyl‐CoA carboxylase, NADP=nicotinamide adenine dinucleotide phosphate). E) The ASAP pathway is a chemo‐enzymatic cascade reaction for production of starch. Following the chemical reduction of CO₂ to methanol, a 10‐enzyme cascade converts C1 compounds to starch. Enzymes highlighted in red were identified as bottlenecks in this pathway and subjected to engineering to improve cascade efficiency. The protein structure shows formolase (FLS), which is a computationally designed enzyme (PDB: 4QQ8) based on benzaldehyde lyase (BAL). Blue spheres indicate mutations introduced by computational design, red spheres indicate mutations discovered through mutagenesis and screening, and purple spheres indicate positions that were targeted by computation and mutagenesis. FLS catalysis is mediated by a TPP cofactor, shown in cyan. (Enzyme and cofactor diagram abbreviations: AGP=ADP‐glucose pyrophosphorylase, ADP=adenosine 5′‐diphosphate, AOX=alcohol oxidase, ATP=adenosine 5′‐triphosphate, BAL=benzaldehyde lyase, DAK=dihydroxyacetone kinase, FBA=Fructose‐bisphosphate aldolase, FBP=fructose‐bisphosphatase, FLS=formolase, PGI=phosphoglucose isomerase, PGM=phosphoglucomutase, SS=starch synthase, TPI=triose‐phosphate isomerase, Chemical compound abbreviations: ADPG=ADP glucose, DHAP=dihydroxyacetone phosphate, GAP=D‐glyceraldehyde 3‐phosphate, F‐1,6‐BP=D‐fructose‐1,6‐bisphosphate, F‐6‐P=D‐fructose‐6‐phosphate, G‐6‐P=glucose‐6‐phosphate, G‐1‐P=α‐D‐glucose‐1‐phosphate, P_i=inorganic phosphate, PP_i=pyrophosphate).

Figure 4

Degradation of pollutants. A) Zn‐dependent phosphotriesterases (PTE) are capable of hydrolysing paraoxon to p‐nitrophenol and diethyl‐phosphate. Despite being nearly diffusion controlled, PTE was further improved, giving rise to a 63‐fold increase in k _cat. B) The promiscuous hydrolase P91 shows phosphodiesterase activity towards paraoxon. P91 was further evolved towards a fluorescein‐based model substrate (FDDEP), increasing k _cat/K _M by 360‐fold. C) Rhodococcus rhodochrous haloalkane dehalogenase (DhaA) hydrolyses 1,2,3‐trichloropropane (TCP) to 2,3‐dichloropropane‐1‐ol. Rational engineering in combination with directed evolution yielded DhaA‐31, which displays a 16‐fold increased k _cat and a 30‐fold increase in enzyme efficiency. D) LinB dehalogenates β‐HCH to pentachlorocyclohexanol. The T _m of LinB was increased by 23 °C through incorporation of 12 mutations predicted by computation and rational design. (Diagram abbreviations: DhaA=R. rhodochrous haloalkane dehalogenase, FDDEP=fluorescein di(diethylphosphate), HCH=hexachlorocyclohexane, WT=wildtype, PTE=phosphotriesterases).

Figure 5

PET depolymerisation. A) Various enzymes capable of hydrolysing PET to MHET, TPA and EG have been discovered to date. B) PET deconstructing enzymes typically belong to the cutinase enzyme family, which are serine hydrolases and utilize a catalytic triad in conjunction with an oxy‐anion hole. C) LCC^ICCG is an engineered PETase derived from leaf‐branch compost cutinase. Four mutations (blue spheres) were introduced to increase thermostability (i.e. through installation of a disulphide bridge) (PDB: 7 W44 ^[75] ). D) HotPETase (model with docked PET substrate (cyan) based on PDB: 7QVH) was engineered for increased thermostability from the natural enzyme IsPETase through rational design (yellow & purple spheres) and directed evolution (blue spheres). A total of 24 mutations were introduced leading to a T _m increase of 34.5 °C. (Diagram abbreviations: PET=poly(ethylene terephthalate), MHET=mono‐(2‐hydroxyethyl) terephthalic acid, TPA=terephthalic acid, EG=ethylene glycol, LCC=Leaf‐branch compost cutinase).

Figure 6

Lignin as a chemical feedstock. A) Lignin can be depolymerised by RCF, yielding 4‐n‐propylguaiacol as a main product. VAO‐type oxidases convert 4‐n‐propylguaiacol to isoeugenol as well as 4‐(1‐hydroxypropyl)‐2‐methoxyphenol and 1‐(4‐hydroxy‐3‐methoxyphenyl)‐1‐propanone which emerge as side products via hydration of the methide intermediate. Isoeugenol is a versatile precursor for various fine chemicals and polymers. B) Eugenol oxidase (EUGO) was converted into an efficient catalyst for isoeugenol production, by introducing mutations that improve thermostability (blue spheres), chemo‐selectivity (S394 V‐EUGO5X: yellow sticks) and to protect the FAD cofactor (cyan sticks) by preventing adduct formation with the substrate (PROGO ‐ D151E & Q425S: purple sticks). The crystal structure of S394 V‐EUGO5X (PDB: 7YWU) shows the formation of a covalent adduct between the substrate (salmon) and FAD, which is absent in the PROGO crystal structure (PDB: 7YWV, substrate shown in green). (Diagram abbreviations: FAD= flavin adenine dinucleotide, RCF=reductive catalytic fractionation).