Evolutionary engineering of Geobacillus thermoleovorans for growth on adipic acid and 1,4-butanediol

The growing plastic pollution crisis urges for innovative recycling solutions. Among promising approaches, enzymatic hydrolysis and microbial upcycling of plastic hydrolysates offers an option to valorize mixed plastic wastes especially those containing polyesters. For efficient enzymatic hydrolysis of polyesters elevated temperatures near their glass transition temperature (typically 70–80 °C) are required, necessitating thermophilic microbial chassis for consolidated bioprocessing (CBP) that can utilize released plastic monomers at high temperatures. In this study, we engineered Geobacillus thermoleovorans through adaptive laboratory evolution (ALE) for robust growth on adipic acid (AA) and 1,4-butanediol (BDO), two relevant monomers for example derived from the biodegradable polymer PBAT. Both evolved strains were characterized for substrate consumption and growth, genomic mutations as well as gene expression levels when grown on the respective substrate compared to glucose. This revealed key insights into the underlying catabolic pathways. For BDO, an alcohol dehydrogenase (pgap_001044) and an aldehyde dehydrogenase (pgap_001082) were identified to be likely responsible for its oxidative degradation. AA uptake appears to be mediated by a dicarboxylate transporter (pgap_003270), followed by CoA-activation and β-oxidation involving several upregulated CoA-family dehydrogenases. A deletion in pgap_003191 was linked to the constitutive expression of itself and the downstream gene pgap_003192, which likely encodes the CoA-transferase initiating AA catabolism. To demonstrate the applicability of these strains in plastic upcycling, they were co-cultivated with PBAT as sole carbon source in combination with the cutinase HiC for PBAT hydrolysis. This resulted in growth and the consumption of released AA and BDO. Given the potential to purify the released and accumulating terephthalate (TA) by acid precipitation, this approach highlights the feasibility of selective monomer valorization in future bioprocesses. Additional ALE enabled co-utilization of AA and BDO by a single strain and improved AA consumption in the initial AA-evolved strain at lower substrate concentrations, underscoring the strains' adaptability and their high potential for future plastic upcycling applications.