Machine learning-aided engineering of hydrolases for PET depolymerization

Lu, Hong-Qian; Diaz, Daniel J.; Czarnecki, Natalie J.; Zhu, Congzhi; Kim, Wantae; Shroff, Raghav; Acosta, Daniel J.; Alexander, Bradley R.; Cole, Hannah; Zhang, Yan Jessie; Lynd, Nathaniel A.; Ellington, Andrew D.; Alper, Hal S.

doi:10.1038/s41586-022-04599-z

Cited by 645 publications

(673 citation statements)

References 34 publications

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“…Although we discovered that a few PETase variants with more than two mutation sites have stable docking conformations [ 32 , 54 ], the probability of finding these beneficial variants was low due to the difficulty in computationally and experimentally searching in a high-dimensional protein sequence space. Alternatively, structure-based, machine learning was recently used to predict potential mutations of PETase [ 55 ]. In addition to the number of mutation sites, increasing PETase thermal stability is another factor to consider for activity, as demonstrated by the successful example of PETase with three mutated residues that stabilized the β6–β7 connecting loop [ 32 ].…”

Section: Discussionmentioning

confidence: 99%

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

et al. 2022

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The accumulation of polyethylene terephthalate (PET) seriously harms the environment because of its high resistance to degradation. The recent discovery of the bacteria-secreted biodegradation enzyme, PETase, sheds light on PET recycling; however, the degradation efficiency is far from practical use. Here, in silico alanine scanning mutagenesis (ASM) and site-saturation mutagenesis (SSM) were employed to construct the protein sequence space from binding energy of the PETase–PET interaction to identify the number and position of mutation sites and their appropriate side-chain properties that could improve the PETase–PET interaction. The binding mechanisms of the potential PETase variant were investigated through atomistic molecular dynamics simulations. The results show that up to two mutation sites of PETase are preferable for use in protein engineering to enhance the PETase activity, and the proper side chain property depends on the mutation sites. The predicted variants agree well with prior experimental studies. Particularly, the PETase variants with S238C or Q119F could be a potential candidate for improving PETase. Our combination of in silico ASM and SSM could serve as an alternative protocol for protein engineering because of its simplicity and reliability. In addition, our findings could lead to PETase improvement, offering an important contribution towards a sustainable future.

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Section: Discussionmentioning

confidence: 99%

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

et al. 2022

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“…Enzymatic PET hydrolysis by native, non-engineered enzymes typically occurs on the order of days to weeks and is severely impaired by the semi-crystalline structure of commercial PET, which consists of amorphous regions interspersed with crystalline regions that are highly refractory to enzymatic attack [ 1 , 17 , 18 ]. PET hydrolases are frequently engineered to function near the glass transition temperature (T g ) of PET at ≥ 70 °C where amorphous regions become more accessible, with the latest generation of enzymes capable of degrading PET samples at elevated temperatures within as little as 10 h [ 19 – 21 ].…”

Section: Introductionmentioning

confidence: 99%

“…The PET monomers TA and EG can be used as metabolic feedstocks for biochemical production pathways in engineered microbes. Recent examples include the engineering of bacterial strains capable of transforming TA and/or EG into value-added products such as β-ketoadipic acid, glycolic acid, and the bioplastics polyhydroxyalkanoate (PHA) and poly(amide urethane) [ 21 – 25 ].…”

Section: Introductionmentioning

confidence: 99%

“…TA and EG can be extracted and processed into new virgin PET ( A ); used as feedstocks for engineered microbes and production of value-added bioproducts ( B ); or used as feedstocks for engineered microbes producing PET hydrolases in a continuous bioprocessing-like one-pot system ( C ). Path 2-A was explored by Tournier et al [ 19 ] and Lu et al [ 21 ]; path 1-B was explored by Kenny et al [ 22 , 94 ], Werner et al [ 24 ] and Kim et al; [ 25 ] path 2-B was described by Tiso et al [ 23 ] and Lu et al [ 21 ]; path 3-C is what I. sakaiensis perfoms natively [ 26 ] and was explored in this study. Additionally, several recent studies described whole-cell biocatalysts displaying or secreting PET hydrolases ( 3 ) without attempting downstream extraction or microbial utilization of released monomers.…”

Section: Introductionmentioning

confidence: 99%

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Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

2022

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Background Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. Results P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Conclusion Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

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“…Protein engineering is a growing area of research in which scientists use a variety of methods to design new proteins that can perform certain functions. For instance, enzymes that can biodegrade plastics, materials inspired by spider silk, or antibodies to neutralize viruses ( Lu et al, 2022 ; Shan et al, 2022 ).…”

mentioning

confidence: 99%