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References (2)

Title : Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to beta-ketoadipic acid by Pseudomonas putida KT2440 - Werner_2021_Metab.Eng_67_250
Author(s) : Werner AZ , Clare R , Mand TD , Pardo I , Ramirez KJ , Haugen SJ , Bratti F , Dexter GN , Elmore JR , Huenemann JD , Peabody GLt , Johnson CW , Rorrer NA , Salvachua D , Guss AM , Beckham GT
Ref : Metab Eng , 67 :250 , 2021
Abstract : 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, beta-ketoadipic acid (betaKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L betaKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to betaKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.
ESTHER : Werner_2021_Metab.Eng_67_250
PubMedSearch : Werner_2021_Metab.Eng_67_250
PubMedID: 34265401
Gene_locus related to this paper: idesa-mheth , idesa-peth

Title : Characterization and engineering of a two-enzyme system for plastics depolymerization - Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
Author(s) : Knott BC , Erickson E , Allen MD , Gado JE , Graham R , Kearns FL , Pardo I , Topuzlu E , Anderson JJ , Austin HP , Dominick G , Johnson CW , Rorrer NA , Szostkiewicz CJ , Copie V , Payne CM , Woodcock HL , Donohoe BS , Beckham GT , McGeehan JE
Ref : Proc Natl Acad Sci U S A , 117 :25476 , 2020
Abstract : Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 A resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.
ESTHER : Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
PubMedSearch : Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
PubMedID: 32989159
Gene_locus related to this paper: idesa-mheth