Biology subjects

Dickey, R. M.

Publications and source records attributed to Dickey, R. M..

4 recordsLinked to original sources

bioRxiv2025

Robust cellular transformations of PET deconstruction products by import of glycol esters

Efforts to transform polyethylene terephthalate (PET) deconstruction products using live cells have been limited by terephthalic acid (TPA) uptake. Here, we used an intracellular carboxylate reduction assay to show that apparent TPA uptake in E. coli cells that lack a dedicated TPA transporter sharply increases between pH 5-6. Furthermore, we discovered that glycol ester deconstruction products, mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), surprisingly each result in rapid pH-independent uptake. We exploited glycol ester uptake along with deletion of 22 cellular oxidoreductases to design intracellular hydrolysis routes for synthesis of upcycled reduction products from BHET at >90% yields, and from real PET wastes after tandem catalytic glycolysis and cell-based valorization at >80% combined yields. Our work has important ramifications for PET utilization by cells and adds new perspectives on the evolution of the PETase/MHETase system.

bioengineering↗

bioRxiv2024

Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids in engineered bacteria

Applications of genetic code expansion in live cells are widespread and continually emerging, yet they have been limited by their reliance on the supplementation of non-standard amino acids (nsAAs) to cell culturing media. While advances in cell-free biocatalysis are improving nsAA synthesis cost and sustainability, such processes remain reliant on multi-step processes of product isolation followed by supplementation to engineered cells. Here, we report the design of a modular and genetically encoded system that combines the steps of biosynthesis of diverse phenylalanine derivatives, which are the most frequently used family of nsAAs for genetic code expansion, and their site-specific incorporation within target proteins using a single engineered bacterial host. Unlike previous demonstrations in which individual nsAAs were targeted for biosynthesis and site-specific incorporation, our system serves as a platform that exhibits broad substrate specificity towards commercially ubiquitous, achiral building blocks of aryl aldehydes or carboxylic acids. We demonstrate that this modular system enables high fidelity biosynthesis and incorporation of nsAAs for multiple industrially relevant nsAAs, such as the incorporation of 4,4-L-biphenylalanine within proteins after supplementation with biphenylaldehyde and the incorporation of 4-azido-L-phenylalanine within proteins after supplementation with 4-azido-benzoic acid. Finally, we show that the combination of nsAA biosynthesis and incorporation steps can extend the chemical reach of the intrinsic biological containment strategy of synthetic auxotrophy from nsAAs to low-cost and achiral building blocks. We anticipate that our system will aid industrial-scale manufacturing of proteins that contain nsAAs and democratize access to expensive or commercially unavailable chemistries for labs that lack separations or synthesis expertise.

synthetic biology↗

bioRxiv2023

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

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild-type strain MG1655 and an engineered strain for reduced aromatic aldehyde rection (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24h. We applied this new strain for the production of para-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared to RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

synthetic biology↗

bioRxiv2022

Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

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 {omega}-transaminase and diverse carboxylic acid reductases (CAR) towards PET deconstruction products. We first establish that an {omega}-transaminase from Chromobacterium 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 from Segniliparus 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/520786v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1bb7386org.highwire.dtl.DTLVardef@19d8c55org.highwire.dtl.DTLVardef@68569eorg.highwire.dtl.DTLVardef@ed398_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗