ENGINEERING E. COLI FOR THE VALORIZATION OF PLASTIC AND BIOMASS DERIVED DECONSTRUCTION PRODUCTS

Abstract

Biocatalysis has become a vital aspect of modern organic synthesis. By harnessing nature’s catalyst, enzymes, synthetic chemistry unlocked a greener technology capable of countless applications from bench scale to industrial production. As global environmental challenges intensify and the transition toward a carbon-neutral economy accelerates, biocatalysis continues to gain recognition for its potential to drive environmentally responsible innovation. This transition to a sustainable economy hinge on the ability for our waste products to be integrated into a more circular economy. These waste streams, such as plastic and lignocellulose waste, offer a valuable and untapped chemical feedstock. Enzymes such as PET hydrolases and cutinases have been engineered to depolymerize polyethylene terephthalate (PET) into its monomeric components under mild conditions, facilitating closed-loop recycling and offering the potential for further downstream biotransformations. Similarly, lignocellulosic biomass can be deconstructed into fermentable sugars and diverse aromatic building blocks. Through the integration of biocatalytic pathways, these waste streams can be redirected into the production value-added aromatic aldehydes. These aldehyde containing compounds can be utilized in the production of bio-based polymers, flavors and fragrances as well as specialty chemicals, aligning with circular economy principles and reducing reliance on fossil resources. By coupling waste deconstruction with enzymatic functionalization, these biocatalyst platforms enable sustainable routes to generate diverse products from renewable and waste derived feedstocks. In this thesis, I present a microbial platform for the valorization of plastic and lignin waste with engineered bacterial that enables the production of aromatic aldehydes and their derivative compounds. As an initial stage in development, we engineered strains of Escherichia coli capable of stabilizing aromatic aldehydes in both growing and resting cells. As aromatic aldehydes are rapidly reduced to alcohols in fermentation processes, we investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain capable of stabilizing the PET derived dialdehyde, TPAL. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over previously engineered strains after 24 h. Additionally, colleagues and I identified and addressed unexpected aldehyde oxidation on a collection of aromatic aldehydes, including many that originate from biomass degradation, with bio-catalytically relevant resting cell conditions. By performing combinatorial inactivation of up to 22 candidate alcohol dehydrogenases, aldo-keto reductases and aldehyde dehydrogenase genes in the E. coli genome we were able to engineer enhanced aldehyde stabilizing strains capable of retaining over 70% of the initial aldehyde concentration after 4 h of 11 out of the 17 tested compounds. With engineered strains now capable of stabilizing aldehydes, we then aimed to generate a whole cell platform for PET valorization. PET is an exemplary plastic whose deconstruction by chemical and enzymatic methods has been well explored. However, efforts to transform PET deconstruction products by live cells have been slow given a bottleneck in terephthalic acid (TPA) uptake. Here, we investigated two strategies to enhance deconstruction product uptake in E. coli without heterologous expression of a TPA transporter, with our ultimate goal being upcycling transformations to aryl aldehydes and amines. Through cellular expression of modifying enzymes, we identified that apparent TPA uptake sharply increases at pH values < 6 with maximum conversion occurring at pH 5.2 . Surprisingly, the alternative deconstruction products bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET) exhibit far greater apparent uptake at neutral pH. We exploited this finding along with strain engineering to design chemoenzymatic routes that convert real PET wastes to target dialdehydes and diamines at high yields. Our work has important ramifications for PET biovalorization and catabolism, and it adds new perspectives on why a two-enzyme PETase/MHETase system may have evolved. Finally, we aimed to biosynthesize aldehydes from biomass-derived alcohols using alcohol oxidases (AOs). The use of AOs in biocatalysis is still modest, despite their advantageous capability to produce valuable aldehydes via oxidation of the respective alcohols without the need for costly external cofactors. These flavin-based redox enzymes have been used in the development of various industrial processes and products primarily for the production of various industrially useful carbonyl compounds. Here, we characterized 52 diverse putative AOs through the use of a sequence similarity network to explore the specificity and activity of the AO enzyme class. We identified four novel AOs that exhibited high activity on a broad range of aromatic alcohols including the conversion of vanillyl alcohol and piperonyl alcohol to fragrance compounds vanillin and piperonal. We also show the use of an AO in coupled biocatalytic pathways in engineered E. coli to enable full conversion of aromatic alcohol substrates to high-value amines and non-standard amino acids. Overall, our work reveals novel AOs in an enzyme family whose members could play key roles in the biosynthesis of flavor and fragrance products and presents a simple whole cell platform for aldehyde production that requires no additional cofactor supplementation. In summary, this thesis focuses on the biosynthesis and retention of aromatic aldehydes to help enable a cost-effective platform for the upcycling of plastic and biomass derived feedstocks.