Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells

Bayer, Thomas; Pfaff, Lara; Branson, Yannick; Becker, Aileen; Wu, Shuke; Bornscheuer, Uwe T.; Wei, Ren

doi:10.1016/j.isci.2022.104326

Cited by 19 publications

(32 citation statements)

References 74 publications

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“…During the preparation of this manuscript, the first example of a CAR that exhibits activity on TPA was reported. 58 This report demonstrated activity of mmCAR (from Mycobacterium marinum ) on TPA in resting E. coli whole cells, with no other CARs tested. Intrigued by that result, we performed our own analysis on mmCAR, albeit in an in vitro setting.…”

Section: Resultsmentioning

confidence: 78%

Section: Productsmentioning

confidence: 80%

“…1.2.1.30) may be able to catalyze the reduction of carboxylates like TPA to its corresponding dialdehyde, terephthalaldehyde (TPAL). [34][35][36] CARs are multidomain and polyspecific enzymes that perform the desirable 2ereduction of carboxylic acids to aldehydes at a cost of ATP and NADPH. 37 Given the broad substrate scope of both CAR and ω-TA families, some precedent for this type of cascade exists for certain aliphatic or heterocyclic chemistries.…”

Section: Introductionmentioning

confidence: 99%

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Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

Gopal

Dickey

Butler

et al. 2022

Preprint

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To better incentivize the collection of plastic wastes, new chemical transformations must be developed that add value to plastic deconstruction products. Polyethylene terephthalate (PET) is a common plastic whose deconstruction through chemical or biological means has received much attention. However, a limited number of alternative products have been formed from PET deconstruction, and only a small share could serve as building blocks for alternative materials or therapeutics. Here, we demonstrate the production of useful mono-amine and diamine building blocks from known PET deconstruction products. We achieve this by designing one-pot biocatalytic transformations that are informed by the substrate specificity of an ω-transaminase and diverse carboxylic acid reductases (CAR) towards PET deconstruction products. We first establish that an ω-transaminase fromChromobacterium violaceum(cvTA) can efficiently catalyze amine transfer to potential PET-derived aldehydes to form the mono-amine para-(aminomethyl)benzoic acid (pAMBA) or the diamine para-xylylenediamine (pXYL). We then identified CAR orthologs that could perform the bifunctional reduction of TPA to terephthalaldehyde (TPAL) or the reduction of mono-(2-hydroxyethyl) terephthalic acid (MHET) to its corresponding aldehyde. After characterizing 17 CARs in vitro, we show that the CAR fromSegniliparus rotundus(srCAR) had the highest observed activity on TPA. Given these newly elucidated substrate specificity results, we designed modular enzyme cascades based on coupling srCAR and cvTA in one-pot with enzymatic co-factor regeneration. When we supply TPA, we achieve a 69 ± 1% yield of pXYL, which is useful as a building block for materials. When we instead supply MHET and subsequently perform base-catalyzed ester hydrolysis, we achieve 70 ± 8% yield of pAMBA, which is useful for therapeutic applications and as a pharmaceutical building block. This work expands the breadth of products derived from PET deconstruction and lays the groundwork for eventual valorization of waste PET to higher-value chemicals and materials.

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Section: Resultsmentioning

confidence: 78%

Section: Productsmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 99%

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Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

Gopal

Dickey

Butler

et al. 2022

Preprint

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“…By using several rounds of mutagenesis, this transcription factor was optimized to recognize TPA at 10 μM concentration. While this manuscript was under review, yet another study showed that the conversion of TPA to an aldehyde leads to bioluminescence suitable for online monitoring and detection of TPA in living cells (Bayer et al, 2022 ).…”

Section: Smart Strategies For the Identification Of Plastic‐active En...mentioning

confidence: 99%

Microbial enzymes will offer limited solutions to the global plastic pollution crisis

Chow

Pérez-García

Dierkes

et al. 2022

Microbial Biotechnology

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Global economies depend on the use of fossil‐fuel‐based polymers with 360–400 million metric tons of synthetic polymers being produced per year. Unfortunately, an estimated 60% of the global production is disposed into the environment. Within this framework, microbiologists have tried to identify plastic‐active enzymes over the past decade. Until now, this research has largely failed to deliver functional biocatalysts acting on the commodity polymers such as polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC), ether‐based polyurethane (PUR), polyamide (PA), polystyrene (PS) and synthetic rubber (SR). However, few enzymes are known to act on low‐density and low‐crystalline (amorphous) polyethylene terephthalate (PET) and ester‐based PUR. These above‐mentioned polymers represent >95% of all synthetic plastics produced. Therefore, the main challenge microbiologists are currently facing is in finding polymer‐active enzymes targeting the majority of fossil‐fuel‐based plastics. However, identifying plastic‐active enzymes either to implement them in biotechnological processes or to understand their potential role in nature is an emerging research field. The application of these enzymes is still in its infancy. Here, we summarize the current knowledge on microbial plastic‐active enzymes, their global distribution and potential impact on plastic degradation in industrial processes and nature. We further outline major challenges in finding novel plastic‐active enzymes, optimizing known ones by synthetic approaches and problems arising through falsely annotated and unfiltered use of database entries. Finally, we highlight potential biotechnological applications and possible re‐ and upcycling concepts using microorganisms.

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“…Furthermore, the luciferase LuxAB from Photorhabdus luminescens was employed to establish a TPA sensitive reporter system ( 24 ). The authors of this study combined a carboxylic acid reductase (CAR) and the luciferase LuxAB to generate a luminescent readout in E. coli , which semiquantitatively correlated with TPA concentration and was suitable for high-throughput screening.…”

Section: Introductionmentioning

confidence: 99%