Bio-upcycling of even and uneven medium-chain-length diols and dicarboxylates to polyhydroxyalkanoates using engineered Pseudomonas putida.

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous ß-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native ß-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous ß-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.

Biotechnological Plastic Degradation and Valorization Using Systems Metabolic Engineering.

Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to ß-ketoadipic acid by Pseudomonas putida KT2440.

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 tphA2IIA3IIBIIA1II 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, ß-ketoadipic acid (ßKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L ßKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to ßKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.

Seamless and orthogonal expression of genetic parts in polyhydroxyalkanoate (PHA)-producing bacterial chassis for plastic bio-upcycling applications.

Polyhydroxyalkanoate (PHA)-producing bacteria represent a powerful synthetic biology chassis for waste bioconversion and bio-upcycling where PHAs can be produced as the final products. In this study, we present a seamless plasmid construction for orthogonal expression of recombinant PET hydrolase (PETase) in model PHA-producing bacteria P. putida and C. necator. To this end, this study described seamless cloning and expression methods utilizing SureVector (SV) system for generating pSV-Ortho-PHA (pSVOP) expression platform in bioengineered P. putida and C. necator. Genetic parts specifically Trc promoter, pBBR1 origin of replication, anchoring proteins and signal sequences were utilized for the transformation of pSVOP-based plasmid in electrocompetent cells and orthogonal expression of PETase in both P. putida and C. necator. Validation steps in confirming functional expression of PETase activity in corresponding PETase-expressing strains were also described to demonstrate seamless and detailed methods in establishing bioengineered P. putida and C. necator as whole-cell biocatalysts tailored for plastic bio-upcycling.•Seamless plasmid construction for orthogonal expression in PHA-producing bacteria.•Step-by-step guide for high-efficiency generation of electrotransformants of P. putida and C. necator.•Adaptable methods for rapid strain development (Design, Build, Test and Learn) for whole-cell biocatalysis.

Current biotechnologies on depolymerization of polyethylene terephthalate (PET) and repolymerization of reclaimed monomers from PET for bio-upcycling: A critical review.

The production of polyethylene terephthalate (PET) has drastically increased in the past half-century, reaching 30 million tons every year. The accumulation of this recalcitrant waste now threatens diverse ecosystems. Despite efforts to recycle PET wastes, its rate of recycling remains limited, as the current PET downcycling is mostly unremunerative. To address this problem, PET bio-upcycling, which integrates microbial depolymerization of PET followed by repolymerization of PET-derived monomers into value-added products, has been suggested. This article critically reviews current understanding of microbial PET hydrolysis, the metabolic mechanisms involved in PET degradation, PET hydrolases, and their genetic improvement. Furthermore, this review includes the use of meta-omics approaches to search PET-degrading microbiomes, microbes, and putative hydrolases. The current development of biosynthetic technologies to convert PET-derived materials into value-added products is also comprehensively discussed. The integration of various depolymerization and repolymerization biotechnologies enhances the prospects of a circular economy using waste PET.

Bioconversion of Plastic Waste Based on Mass Full Carbon Backbone Polymeric Materials to Value-Added Polyhydroxyalkanoates (PHAs).

This review article will discuss the ways in which various polymeric materials, such as polyethylene (PE), polypropylene (PP), polystyrene (PS), and poly(ethylene terephthalate) (PET) can potentially be used to produce bioplastics, such as polyhydroxyalkanoates (PHAs) through microbial cultivation. We will present up-to-date information regarding notable microbial strains that are actively used in the biodegradation of polyolefins. We will also review some of the metabolic pathways involved in the process of plastic depolymerization and discuss challenges relevant to the valorization of plastic waste. The aim of this review is also to showcase the importance of methods, including oxidative degradation and microbial-based processes, that are currently being used in the fields of microbiology and biotechnology to limit the environmental burden of waste plastics. It is our hope that this article will contribute to the concept of bio-upcycling plastic waste to value-added products via microbial routes for a more sustainable future.

Engineering Microbes to Bio-Upcycle Polyethylene Terephthalate.

Polyethylene terephthalate (PET) is globally the largest produced aromatic polyester with an annual production exceeding 50 million metric tons. PET can be mechanically and chemically recycled; however, the extra costs in chemical recycling are not justified when converting PET back to the original polymer, which leads to less than 30% of PET produced annually to be recycled. Hence, waste PET massively contributes to plastic pollution and damaging the terrestrial and aquatic ecosystems. The global energy and environmental concerns with PET highlight a clear need for technologies in PET "upcycling," the creation of higher-value products from reclaimed PET. Several microbes that degrade PET and corresponding PET hydrolase enzymes have been successfully identified. The characterization and engineering of these enzymes to selectively depolymerize PET into original monomers such as terephthalic acid and ethylene glycol have been successful. Synthetic microbiology and metabolic engineering approaches enable the development of efficient microbial cell factories to convert PET-derived monomers into value-added products. In this mini-review, we present the recent progress of engineering microbes to produce higher-value chemical building blocks from waste PET using a wholly biological and a hybrid chemocatalytic-biological strategy. We also highlight the potent metabolic pathways to bio-upcycle PET into high-value biotransformed molecules. The new synthetic microbes will help establish the circular materials economy, alleviate the adverse energy and environmental impacts of PET, and provide market incentives for PET reclamation.

Upcycling of hydrolyzed PET by microbial conversion to a fatty acid derivative.

The enzymatic degradation of polyethylene terephthalate (PET) results in a hydrolysate consisting almost exclusively of its two monomers, ethylene glycol and terephthalate. To biologically valorize the PET hydrolysate, microbial upcycling into high-value products is proposed. Fatty acid derivatives hydroxyalkanoyloxy alkanoates (HAAs) represent such valuable target molecules. HAAs exhibit surface-active properties and can be exploited in the catalytical conversion to drop-in biofuels as well as in the polymerization to bio-based poly(amide urethane). This chapter presents the genetic engineering methods of pseudomonads for the metabolization of PET monomers and the biosynthesis of HAAs with detailed protocols concerning product purification.