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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.

Aldehydes are attractive chemical targets given applications as end products in the flavors and fragrances industry and as intermediates due to their propensity for C-C bond formation. While biosynthetic routes to diverse aldehydes have been designed, a common challenge is the stability of these aldehydes in the presence of microbial hosts of engineered pathways. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation, in the presence ofEscherichia colistrains that were engineered to minimize aldehyde reduction. Of heightened interest to us were resting cell conditions as they offer numerous advantages for the bioconversion of toxic metabolites. Surprisingly, when diverse aldehydes are supplemented toE. coliRARE cells grown under aerobic conditions, they remain stabilized on the timescale of days, whereas when these same aldehydes are supplemented to resting cell preparations ofE. coliRARE that had been grown under the same conditions, we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in theE. coligenome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 hours after their addition. Given that our newly engineered strain exhibits Reduced Oxidation And Reduction of aromatic aldehydes, we dubbed it theE. coliROAR strain. Seeking to apply this new strain to resting cell biocatalysis, we compared the capability to synthesize the aldehyde furfural from 2-furoic acid via the carboxylic acid reductase enzyme from Nocardia iowensis. Here, we found that use of ROAR resting cells achieved 2-fold enhancement in furfural titer after 4 h and nearly 9-fold enhancement after 20 h as compared to resting cells of the RARE strain. Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

To better incentivize the collection of plastic wastes, 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 monoamine 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) toward PET deconstruction products. We first establish that an ω-transaminase from Chromobacterium violaceum (cvTA) can efficiently catalyze amine transfer to potential PET-derived aldehydes to form monoamine para-(aminomethyl)benzoic acid (pAMBA) or diamine para-xylylenediamine (pXYL). We then identified CAR orthologs that could perform the bifunctional reduction of terephthalic acid (TPA) to terephthalaldehyde 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 from Segniliparus rotundus (srCAR) had the highest observed activity on TPA. Given these elucidated substrate specificity results, we designed modular enzyme cascades based on coupling srCAR and cvTA in one pot with enzymatic cofactor regeneration. When we supply TPA, we achieve a 69 ± 1% yield of pXYL, which is useful as a building block for polymeric 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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Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 μM in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block.

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