Engineered microorganisms for the deconstruction of polymers
Inventors
BECKHAM, Gregg Tyler • JAYAKODY, Thelhawadigedara Lahiru Niroshan • GUSS, Adam Michael • MAND, Thomas David • Johnson, Christopher W. • PARDO MENDOZA, Isabel • ZIMONT WERNER, Allison Jean
Assignees
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Publication Number
US-12371718-B2
Publication Date
2025-07-29
Expiration Date
Abstract
Disclosed herein are methods and compositions for catalytic glycolysis to deconstruct PET to bis(2-hydroxyethyl) terephthalate (BHET). For BHET conversion to terephthalate and ethylene glycol, we engineer Pseudomonas putida KT2440 with PETase and MHETase enzymes from Ideonella sakaiensis. We further engineer P. putida to convert terephthalate to a performance-advantaged bioproduct, β-ketoadipic acid, and for improved utilization of ethylene glycol, a byproduct of BHET catabolism. In a bioreactor, we produce 15.1±0.6 g/L of β-ketoadipic acid (βKA) from BHET at 76±3% molar yield. Lastly, we demonstrate 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.
Core Innovation
The invention describes engineered Pseudomonas putida KT2440 strains in which Ideonella sakaiensis PETase and Ideonella sakaiensis MHETase are co-expressed and secreted in a SecB-compatible native/native secretion system. Each enzyme is provided with a secretion signal peptide to enable secretion, and the strains are configured to metabolize bis(2-hydroxyethyl) terephthalate (BHET) into BHET deconstruction products.
The invention further introduces heterologous terephthalic acid (TPA) transport and catabolism to convert PET-derived deconstruction products toward downstream beta-ketoadipic acid (βKA/β-ketoadipate). It includes introduction of tpaK and related tph/tpa genes from Comamonas sp. E6 and Rhodococcus jostii RHA1, and deletion of pcaIJ to accumulate βKA. The engineered strains are also configured for improved ethylene glycol utilization via gclDEFG:PP_3749 with gclR deletion.
The invention demonstrates production of βKA in bioreactors and includes conversion of catalytically depolymerized PET to βKA. The document reports quantitative βKA performance and includes evidence of PET degradation selectivity, including improved BHET to TPA conversion rate.
Claims Coverage
The independent claim covers a genetically modified Pseudomonas sp. organism engineered to metabolize BHET by expressing secreted Ideonella sakaiensis PETase and Ideonella sakaiensis MHETase with respective secretion signal peptides to produce BHET deconstruction products. The dependent claims refine this core by adding sequence-identity constraints, codon optimization, genome incorporation, specified BHET deconstruction product components, and additional condition constraints including minimal salt medium.
Secreted Ideonella sakaiensis PETase and MHETase for BHET metabolism
A genetically modified Pseudomonas sp. organism comprising a nucleic acid sequence encoding a functional Ideonella sakaiensis PETase with a first secretion signal peptide and a nucleic acid sequence encoding a functional Ideonella sakaiensis MHETase with a second secretion signal peptide, wherein the genetically modified organism metabolizes BHET to produce BHET deconstruction products.
Secretion signal peptides with sequence-identity constraints
The genetically modified organism defined by first and second secretion signal peptides that each have greater than 90% sequence identity to SEQ ID NO: 17 and SEQ ID NO: 19, respectively.
Codon optimization of both enzyme-encoding nucleic acids
The genetically modified organism in which both nucleic acid sequences are codon optimized.
Genome incorporation of both nucleic acid sequences
The genetically modified organism defined by having both specified nucleic acid sequences incorporated into its genome.
Specified BHET deconstruction product components
The genetically modified organism produces BHET deconstruction products including at least one selected from mono-(2-hydroxyethyl) terephthalate, terephthalate, ethylene glycol, β-ketoadipate, or muconate.
Contacting in minimal salt medium
The method is carried out by performing the contacting step in minimal salt medium.
Across the claim set provided, the core inventive concept is BHET-to-BHET deconstruction product metabolism enabled by secreted Ideonella sakaiensis PETase and MHETase in a genetically modified Pseudomonas sp. The refinements require specific secretion signal peptides, codon optimization, genome incorporation, and specify candidate deconstruction products, with additional narrowing to minimal salt medium in a dependent method context.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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