Search

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 b-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products. [ABSTRACT FROM AUTHOR]

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2 II A3 II B II A1 II from Comamonas and tpaK from Rhodococcus jostii , (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis , and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET. • Enabled terephthalate (TPA) and bis(2-hydroxyethyl)TPA (BHET) catabolism in P. putida • Engineered conversion of BHET to β-ketoadipate (βKA), a performance-advantaged bioproduct • Achieved a titer of 15.1 g βKA/L at a 76% molar yield from commercial BHET • Demonstrated βKA production from glycolyzed poly(ethylene TPA) (PET) [ABSTRACT FROM AUTHOR]

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters. • Engineered Acinetobacter baylyi ADP1 to grow on terephthalic acid (TPA). • Developed a sensitive and specific biosensor to detect intracellular TPA in vivo. • Identified a native TPA transporter using gene amplification and evolution. • Generated improved transporter variants for use in TPA uptake in heterologous hosts. [ABSTRACT FROM AUTHOR]

Total length (LT) and standard length (LS)(mm) were compared to weight MB(g) in the Spottail Shiner, Notropis hudsonius (Clinton 1824), in the Western Basin of Lake Erie. Length and weight relationship (n = 529), lengthfrequency distribution, and sex ratio were evaluated for tributary habitats and compared to coastal habitats. The total population had a positive correlation between log normalized LT and MB (y = 3.0902x-5.2289, R² = 0.995) and a positive correlation between LS and MB (y = 3.1397x-5.0501, R² = 0.996). Male Spottail Shiner had a positive correlation between log normalized LT and MB (y = 3.0984x-5.2465, R² = 0.995,) and a significant positive correlation between log normalized standard length and body weight (y = 3.1551x-5.0775, R² = 0.996). Female Spottail Shiner had a positive correlation between log normalized LT and MB (y = 3.078x-5.2034, R² = 0.993) and a positive correlation between log normalized LS and MB (y = 3.1338x-5.0393, R² = 0.996). Males and females were not significantly different in LT or LS (F-test = 1.020, df = 260, 267, p = 0.437). Sex ratio was 1:1. Spottail Shiner exhibit indeterminate growth and did not exhibit gender influenced growth patterns. Tributary individuals were significantly smaller than coastal individuals (F-test = 0.346, df = 65, 202, p = < 0.001). Male age I individuals ranged from 12-54 mm, age II individuals ranged from 57-99 mm, and age III individuals ranged from 99-132 mm. Female Age I individuals ranged from 12-48 mm, age II individuals ranged from 53-102 mm, and age III individuals ranged from 102-129 mm. [ABSTRACT FROM AUTHOR]