Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

Abstract

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.

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

“…Infinity with a Zorbax Eclipse Plus-C18 column with a guard column installed. The methods for detection of TPAL, 4HMB, 4HMBA, BDM, and pXYL used were described in Gopal et al 14 3 | RESULTS…”

“…12,13 We recently identified carboxylic acid reductase enzymes that can efficiently generate the versatile dialdehyde terephthalaldehyde (TPAL) from TPA. 14 Our first step was to report the use of these enzymes in vitro, in part because it is unclear if these steps would function well in live cells given the potential instability and reactivity of TPAL. 15 However, the use of live cells to convert TPAL to useful chemical building blocks could provide a more cost effective and efficient biosynthetic process compared with the use of purified enzymes.…”

“…Here, we envision utilizing the dialdehyde functionality of TPAL to access potential new opportunities in biosynthetic pathways, including asymmetrically functionalized products. Aldehyde-derived biosynthetic targets have ranged broadly, 19 including diamine polymer building blocks, 14,20 hydroxylated non-standard amino acids, [21][22][23][24][25] nitro alcohols, 26 and pharmaceutical mono-amine precursors. [27][28][29] Despite the biosynthetic possibilities available from aldehydes, their redox instability in cellular environments due to endogenous oxidoreductase activity remains a key challenge.…”

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by resting cells ofE. coliRARE strains

“…Aliphatic aldehydes generated from fermentation or fatty acid synthesis have been used to produce fuels and commodities based on alcohols (Akhtar et al, 2013; Sheppard et al, 2014) or alkanes (Kallio et al, 2014; Sheppard et al, 2016). Exemplary newer aldehyde-derived biosynthetic targets that have applications in the pharmaceutical and materials industries are tetrahydroisoquinoline alkaloid plant natural products (Pyne et al, 2020), primary or heterocyclic mono-amine precursors to small molecule pharmaceuticals (Citoler et al, 2019; France et al, 2016; Hepworth et al, 2017), diamine polymer building blocks (Fedorchuk et al, 2020; Gopal et al, 2022), hydroxylated non-standard amino acids (Doyon et al, 2022; Ellis et al, 2022; Kumar et al, 2021; Xu et al, 2020, 2019), and β-lactone antibiotics (Schaffer et al, 2017; Scott et al, 2017). Additionally, opportunities to blend biological and abiological synthesis methods to access additional chemical functional groups are emerging, such as the recent chemoenzymatic synthesis of nitriles (Horvat et al, 2022; Winkler et al, 2022).…”

Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products

“…The ωTAs are pyridoxal 5’-phosphate (PLP)-dependent enzymes that have previously been applied to reversibly catalyze amination by transferring an amine group from an amine donor to the substrate aldehyde or ketone through a pyridoxamine 5’-phosphate (PMP) intermediate (Höhne and Bornscheuer, 2009; Ngo et al, 2022; Rocha et al, 2019). Recently, we reported bioprospecting of diverse CAR enzymes and their coupling to the well-known ω-TA from Chromobacter violaceum (cvTA) to build a one-pot cascade for highly selective and efficient conversion of deconstruction product from polyethylene terephthalate (PET) to mono-and diamines (Gopal et al, 2023).…”