Dataset Information

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering Pseudomonas Putida.

ABSTRACT: Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

SUBMITTER: Liu P

PROVIDER: S-EPMC9569715 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering <i>Pseudomonas Putida</i>.

Liu Pan P Zheng Yi Y Yuan Yingbo Y Zhang Tong T Li Qingbin Q Liang Quanfeng Q Su Tianyuan T Qi Qingsheng Q

International journal of molecular sciences 20220920 19

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional <i>Pseudomonas putida</i> KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydr ...[more]

PMID: 36232310

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Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Project description:BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Project description:Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic polymer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We monitored several key physical and chemical properties, including bacterial growth and modification of the plastic surface, using scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of five strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consortium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) and ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.IMPORTANCE While several research groups are utilizing purified enzymes to break down postconsumer PET to the monomers TPA and ethylene glycol to produce new PET products, here, we present a group of five soil bacteria in culture that are able to partially degrade this polymer. To date, mixed Pseudomonas spp. and Bacillus spp. biodegradation of PET has not been described, and this work highlights the possibility of using bacterial consortia to biodegrade or potentially to biorecycle PET plastic waste.

Dataset Information

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering Pseudomonas Putida.

Publications

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering <i>Pseudomonas Putida</i>.

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