Using ethylene glycol from polyethylene terephthalate biodegradation to fuel Escherichia coli metabolism and produce value-added compounds

Funding Information: Although P. denitrificans is the only organism where this pathway has been fully characterized, Schada von Borzyskowski and colleagues have implemented the BHA cycle into P. putida KT2440 with the goal of assessing whether the implementation of this cycle would permit better growth than the natural EG oxidative pathway (Schada von Borzyskowski et al., 2023). In their work, a P. putida strain unable to assimilate EG through their native glycerate pathway \u2013 due to a deletion of the glyoxylate carboligase gene (gcl) \u2013 and expressing the BHA cycle enzymes was subjected to adaptive laboratory evolution (ALE). The evolved strain was able to grow 35% faster on 20 mM EG with a 20% higher biomass yield when compared to another P. putida strain evolved to grow on EG with the glycerate pathway (Li et al., 2019; Schada von Borzyskowski et al., 2023). Interestingly, before application in P. putida, the authors implemented part of this pathway in E. coli SIJ488 (Schada von Borzyskowski et al., 2023). The expression of BhcC (BHA aldolase), BhcB (BHA dehydratase) and BhcD (iminosuccinate reductase), a linear metabolic module that transforms glyoxylate and glycine into aspartate, was able to support biomass formation in an E. coli \u0394gcl after ALE. The deletion of the gcl gene means that this strain is not able to grow on glycolate and glycine, unless the BHA proteins BhcCBD are used. After prolonged incubation in M9 medium with glycolate and glycine, spontaneous mutants capable of growing on both carbon sources were obtained. These mutants add acquired point mutations in genes encoding for enzymes of the TCA cycle, suggesting the need for slight adaptations in the native metabolic network to support growth using the BHA cycle (Schada von Borzyskowski et al., 2023). Thus, it would be interesting to, in the future, connect this strain to EG as feedstock.Overall, these cyclic pathways (Fig. 6B\u2013D) are complex, and any kinetic or stoichiometric imbalance has the potential to trigger the collapse of GA assimilation. Also, since they rely on the recycling of a metabolite (G3P and E4P) that can be diverted into other metabolic reactions, it might be very difficult to implement these pathways in vivo with a significant production yield. Particularly, this was observed in the case of the Ara5P-GAA pathway, where the extremely low acetyl-CoA yields obtained in vivo from EG suggest the requirement for a co-substrate, such as glycerol, to replenish G3P and support cellular growth (Wagner et al., 2023). Overall, further research is essential to enhance the applicability of GA-assimilating circular pathways within industrial context. Conversely, the adoption of a linear GA assimilating pathway like the SACA pathway may offer greater efficacy due to its simplicity and its independence from an acceptor molecule, and further reports are highly expected.Thus, the selection of E. coli strains for metabolic engineering tasks must be carefully considered, taking into account their metabolic diversity and the final aim. Furthermore, understanding the genetic basis of strain-specific adaptations and elucidating the regulatory mechanisms and possible bottlenecks governing EG metabolism are essential for developing robust chassis across different E. coli strains. Systems biology methodologies such as evolutionary algorithms and simulation methods using genome-scale metabolic models may also offer valuable insights into the complex genetic and metabolic networks underlying these strain-specific responses (Monk et al., 2013), aiding in the rational design of optimized microbial cell factories. Targeted genetic interventions can redirect the metabolism of E. coli to use EG as the main carbon source (Orth et al., 2010, 2011). Furthermore, almost all reports reviewed here indicate that when EG or GA are used as sole carbon sources, there is often a need to supplement with additional carbon sources like glycerol (Wagner et al., 2023), amino acid mixtures like CSM (Panda et al., 2021, 2023), or even complex media (Fraz\u00E3o et al., 2023; Lu et al., 2019; Pandit et al., 2021; Yan et al., 2024) to support adequate microbial growth and more efficient EG assimilation. Moving forward, a thorough investigation of the metabolic pathways and regulatory mechanisms of E. coli during EG and GA assimilation is crucial. These discoveries may help create chassis strains and fermentation conditions that are more economically feasible and efficient.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. Grant (grant number 2020.07984.BD). Funding Information: This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. Grant (grant number 2020.07984.BD). Publisher Copyright: © 2024