(67d) Leveraging multiplex genome engineering and metabolic pathway design to upcycle waste plastics and biomass via aldehyde transformations in live bacteria

The interconversion of chemical functional groups on building block molecules can broaden their properties and therefore their value as synthons and monomers for macromolecules. Carboxylic acids are an example class of molecules commonly found in the chemical or biological deconstruction products of waste plastic and biomass, and they serve as excellent targets for selective functional group interconversions. One of the most versatile classes of functional groups for the formation of C-C or C-N bonds are aldehydes, which can be biosynthesized from carboxylic acids via carboxylic acid reductase (CAR) enzymes. We recently exploited the breadth of CAR substrate specificity to design a route from terephthalic acid (TPA), a deconstruction product of polyethylene terephthalate plastic (PET), to a diamine via terephthalaldehyde (TPAL) using purified enzymes [1]. In our submission to the AIChE Futures Issue, we investigated whether we could conduct this biochemical transformation in an aerobic fermentation using engineered Escherichia coli [2]. Despite our prior efforts to limit the reduction of aldehydes to their corresponding alcohols in E. coli by performing gene deletions [3], to our surprise we found that TPAL was one of the only aldehydes that we have tested that remained subject to rapid reduction when added to cultures of our previously engineered strain. In this talk, I will present our efforts to chase down and eliminate the remaining aldehyde reductases responsible for the reduction of TPAL, which included one gene that had not previously been targeted in any aldehyde stabilization effort to our knowledge. I will then describe the various transformations that we could perform when supplementing TPAL to cultures of these cells in aerobic fermentation. These include amine products as well as a non-standard amino acid that we recently reported in another publication [4]. If time permits, I will also mention our surprising discovery that aldehyde oxidation, rather than reduction, is catalyzed by resting whole cell biocatalysts, which we have also addressed using genome engineering [5]. Finally, we can employ analogous strategies to obtain amines from biomass-derived carboxylic acids in addition to plastic-derived carboxylic acids [6]. Collectively, these strategies expand our design space for transformations that are challenging using traditional synthetic approaches and for which we are using low-cost biocatalysts and sustainable feedstocks.

[1] Gopal et al. ACS Catal. 2023 13 (7), 4778-4789.

[2] Dickey et al. AIChE J. 2023.

[3] Kunjapur et al. JACS. 2014 136 (33), 11644-11654.

[4] Jones et al. Commun. Biol. 2023.

[5] Butler et al. Metab. Eng. 2023 77, 294-305.

[6] Nain et al. BioRxiv. 2023.