Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display

Cribari, Mario A.; Unger, Maxwell J.; Unarta, Ilona C.; Ogorek, Ashley N.; Huang, Xuhui; Martell, Jeffrey D.

doi:10.1021/jacs.3c08291

Cited by 12 publications

(3 citation statements)

References 41 publications

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“…Recently, this mutation was independently identified using a different library background and selection platform, namely a high-throughput yeast surface display platform, following the screening of 10 7 LCC mutants. [58] Calibiri et al [58] observed a similar PET degradation rate for the constructed ICCG-H218Y, where 80% PET film was degraded after 18 h. Furthermore, Caribari et al…”

Section: Discussionmentioning

confidence: 82%

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation

Orr,

Niv,

Barakat

et al. 2024

Biotechnology Journal

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Enzyme‐mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial‐scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC—leaf compost cutinase) has resulted in the isolation of a variant harboring four‐point mutations (LCC‐ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error‐prone genetic libraries based on LCC‐ICCG for enhanced activity toward consumer‐grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC‐ICCG (named here LCC‐ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC‐ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer‐grade PET.

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Section: Discussionmentioning

confidence: 82%

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation

Orr,

Niv,

Barakat

et al. 2024

Biotechnology Journal

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“…Cutinase has solvent-oriented Lys residues (Lys194 at LCC and Lys199 at TfCut2), so there is space for Phe at the same position as Phe233 of PET2. In addition, the activity of LCC-ICCG was increased by H218Y mutation . On the other hand, PET2-7M has a different loop structure because the length is one amino acid longer than cutinase, the side chain of Ile203 interferes with Phe233, and the angle of Trp199 is unsuitable for PET degradation.…”

Section: Discussionmentioning

confidence: 99%

Direct Screening of PET Hydrolase Activity in Culture Medium Based on Turbidity Reduction

Ogura,

Hashino,

Nakamura

2024

ACS Omega

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The development of an efficient screening method for the activity of PET-degrading enzymes represents a significant technological advance in the field of enzyme research, with the potential to facilitate the advancement of enzymes for PET recycling. By examining the stable conditions of PET suspension and enzyme production conditions, we developed a method to quantify PET-degrading enzyme activity in E. coli culture medium using turbidity reduction as an indicator. High PET concentration or ionic strength caused aggregation of PET, and the best condition for activity detection was 0.5 mg mL–1 PET in 50 mM sodium phosphate pH 7.0. Preculture of E. coli increased the purity of enzyme secreted in medium. To evaluate the screening method, 720 colonies of the PET2-7M-H229X-F233X mutant library were analyzed and three candidates of high-activity mutants were obtained. The thermostability of the mutants could also be easily measured by measuring the residual activity after heat treatment. The H229T-F233M mutant showed 3.4 times higher degradation rate against PET film than the template enzyme at the initial time. The molecular dynamics simulation implied that the F233M mutation makes space for making an α helix and that the H229T mutation resolved the steric hindrance with Trp199. These mutations were speculated to change the angle of the Trp199 side chain of PET2 to an angle similar to that of the Trp185 of IsPETase, making it suitable for PET binding to the active center. Screening of activity using PET suspensions is compatible with robotic automation and is expected to be useful for validating computationally predicted mutations.

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“…2022 , Xu et al. 2023 , Cribari et al. 2023 ), hence these may be used as starting points for novel methods to address the limitations within directed evolution.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Improving plastic degrading enzymes via directed evolution

Joho,

Vongsouthi,

Gomez

et al. 2024

Protein Engineering, Design and Selection

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Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

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Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display

Cited by 12 publications

References 41 publications

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation

Direct Screening of PET Hydrolase Activity in Culture Medium Based on Turbidity Reduction

Improving plastic degrading enzymes via directed evolution

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