Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of these materials, however, present major barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium, Pseudomonas putida , to funnel these oxygenated compounds into a single exemplary chemical product, either β-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products.

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Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p -coumaric acid ( p CA) and ferulic acid (FA) individually and in combination ( p CA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L p CA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of p CA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in p CA ​+ ​FA TALE-derived strains but did not affect growth in p CA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

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Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking, yet has implications for improved yield from plants, algae and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites-including carbohydrates, lipids, amino acids, pigments, cofactors, nucleic acids and polysaccharides-in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light:dark cycles was developed and applied. A custom photobioreactor and multiplatform mass spectrometry workflow enabled metabolite profiling every 30-120 min across a 24-h diurnal sinusoidal LD ('sinLD') cycle peaking at 1600 lmol photons m À2 sec À1 . We report widespread oscillations across the sinLD cycle with 90%, 94% and 40% of the identified polar/semi-polar, non-polar and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation and cell division phases of growth. During the lag phase, amino acids and nucleic acids accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially relevant strain design.