A PROCESS AND SYSTEM FOR ENHANCED BIODEGRADATION OF PLASTIC WASTE USING GENETICALLY ENGINEERED MICROORGANISMS

Abstract

The present invention relates to the development of genetically engineered microorganisms designed for the efficient biodegradation of plastic waste. This innovative approach addresses the growing environmental challenge posed by plastic pollution by utilizing microbial strains, such as Escherichia coli and Pseudomonas putida, genetically modified to express plastic-degrading enzymes like PETase and MHETase. These enzymes accelerate the breakdown of synthetic plastics, including polyethylene (PE) and polyethylene terephthalate (PET), into environmentally benign byproducts such as carbon dioxide and water. The microorganisms are further optimized through directed evolution and metabolic engineering to enhance enzyme activity, stability, and byproduct utilization. This technology offers a scalable, eco-friendly solution for plastic waste management, applicable in industrial waste treatment plants, landfills, and polluted natural environments. The invention promises significant advancements in reducing plastic pollution and promoting sustainable environmental practices.

Specification

Description:[0017].The following description provides specific details of certain aspects of the disclosure illustrated in the drawings to provide a thorough understanding of those aspects. It should be recognized, however, that the present disclosure can be reflected in additional aspects and the disclosure may be practiced without some of the details in the following description.
[0018].The various aspects including the example aspects are now described more fully with reference to the accompanying drawings, in which the various aspects of the disclosure are shown. The disclosure may, however, be embodied in different forms and should not be construed as limited to the aspects set forth herein. Rather, these aspects are provided so that this disclosure is thorough and complete, and fully conveys the scope of the disclosure to those skilled in the art. In the drawings, the sizes of components may be exaggerated for clarity.
[0019].It is understood that when an element or layer is referred to as being "on," "connected to," or "coupled to" another element or layer, it can be directly on, connected to, or coupled to the other element or layer or intervening elements or layers that may be present. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
[0020].The subject matter of example aspects, as disclosed herein, is described with specificity to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. Rather, the inventor/inventors have contemplated that the claimed subject matter might also be embodied in other ways, to include different features or combinations of features similar to the ones described in this document, in conjunction with other technologies.
[0021].The present invention introduces genetically engineered microorganisms specifically designed for the enhanced biodegradation of plastic waste. The innovation addresses the critical environmental issue of plastic pollution by leveraging advanced genetic engineering techniques to improve the natural plastic-degrading capabilities of microbial strains such as Escherichia coli and Pseudomonas putida.
[0022].These microorganisms are engineered to express key enzymes, notably PETase and MHETase, which are known for their ability to break down polyethylene terephthalate (PET) into simpler molecules. PETase catalyzes the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET), which is further broken down by MHETase into terephthalic acid (TPA) and ethylene glycol. To enhance the degradation of polyethylene (PE) and other types of synthetic plastics, additional enzymes are incorporated based on their effectiveness in breaking down long-chain polymers.
[0023].The genetic modification process begins with the selection and optimization of target genes responsible for plastic degradation. These genes are either synthesized or extracted from organisms naturally producing the required enzymes. Using codon optimization, the DNA sequences are tailored for high expression levels in the chosen host microorganisms. The optimized genes are then inserted into plasmid vectors equipped with strong promoters, such as the T7 promoter, to drive robust and inducible gene expression. Selectable markers and terminator sequences ensure successful integration and controlled transcription within the host cells.
[0024].Transformation of the host microorganisms is achieved through methods such as heat shock or electroporation, which facilitate the uptake of plasmid DNA. Following transformation, the microorganisms are cultured in selective media to confirm the presence of the target genes. Verification is performed using molecular techniques like PCR, sequencing, and protein expression assays, ensuring the correct insertion and functionality of the genes.
[0025].To maximize the biodegradative efficiency, enzyme activity is further optimized through directed evolution and site-directed mutagenesis. These techniques introduce beneficial mutations that enhance enzyme stability and catalytic efficiency under various environmental conditions. The engineered microorganisms are capable of thriving in diverse environments, including industrial waste facilities, landfills, and natural habitats polluted with plastic waste.
[0026].The metabolic pathways of the host microorganisms are also engineered to process degradation byproducts effectively. By introducing or enhancing pathways that convert intermediates into non-toxic compounds such as carbon dioxide and water, the overall biodegradation process is made more efficient and environmentally benign. This metabolic engineering ensures that the microorganisms can sustain their activity and prevent the accumulation of harmful byproducts.
[0027].Moreover, the invention incorporates strategies to improve the interaction between plastic substrates and microbial enzymes. Techniques such as surface modification of plastics through UV irradiation or mild acid treatment increase surface roughness, enhancing enzyme binding and degradation efficiency. Additionally, cell-surface display technology is employed to anchor the enzymes to the outer membrane of the microorganisms, bringing the degradation machinery in close proximity to the plastic waste.
[0028].The scalability of this technology is a crucial aspect of its application. Engineered microorganisms are tested and optimized in laboratory settings before being scaled up for use in industrial bioreactors. These bioreactors are designed to maintain optimal conditions for plastic degradation, including controlled temperature, pH, and oxygen levels. Continuous monitoring of the degradation process ensures high efficiency and sustainability.
[0029].The genetic modification process begins with the identification and selection of target genes responsible for plastic degradation. Key enzymes, such as PETase and MHETase, are identified for their ability to hydrolyze PET into simpler molecules. PETase catalyzes the breakdown of PET into mono(2-hydroxyethyl) terephthalate (MHET), which is further hydrolyzed by MHETase into terephthalic acid (TPA) and ethylene glycol. To extend the degradation capability to polyethylene (PE) and other synthetic plastics, additional enzymes such as alkane hydroxylase (AlkB) and polystyrene dioxygenase are also incorporated into the microbial strains.
[0030].The selected genes are either synthesized de novo or extracted from naturally occurring organisms that produce these enzymes. Codon optimization is performed to tailor the DNA sequences for high expression levels in the chosen host microorganisms, such as Escherichia coli and Pseudomonas putida. The optimized genes are then cloned into plasmid vectors containing strong, inducible promoters (e.g., the T7 promoter) to drive robust gene expression. These plasmids also include selectable markers, such as antibiotic resistance genes, to ensure the successful selection and maintenance of the transformed cells.
[0031].Transformation of the host microorganisms is achieved through techniques such as heat shock or electroporation, which facilitate the uptake of the plasmid DNA by temporarily permeabilizing the cell membrane. Following transformation, the microorganisms are cultured in selective media containing antibiotics to ensure that only the cells harboring the plasmid with the target genes survive. Verification of successful transformation and gene expression is carried out using molecular techniques such as polymerase chain reaction (PCR), DNA sequencing, and protein expression assays (e.g., SDS-PAGE and Western blotting).
[0032].To further enhance the biodegradative efficiency of the engineered microorganisms, the activity of the plastic-degrading enzymes is optimized through directed evolution and site-directed mutagenesis. Directed evolution involves the iterative process of inducing mutations in the enzyme genes, selecting variants with improved activity, and further refining these variants. Site-directed mutagenesis is used to introduce specific changes to the enzyme's active sites, enhancing their catalytic efficiency and stability under diverse environmental conditions.
[0033].The engineered microorganisms are designed to thrive in a variety of environmental settings, including industrial waste treatment facilities, landfills, and polluted natural environments. To ensure their efficacy across different conditions, the enzymes are engineered to maintain high activity and stability over a broad range of temperatures and pH levels. For instance, PETase and MHETase are optimized to function efficiently at elevated temperatures (up to 60°C) and across a pH range of 4 to 9.
[0034].In addition to enhancing enzyme activity, the metabolic pathways of the host microorganisms are engineered to efficiently process the degradation byproducts. For example, the pathways responsible for metabolizing TPA and ethylene glycol, the primary byproducts of PET degradation, are optimized to convert these compounds into carbon dioxide and water. This metabolic engineering ensures a complete and environmentally benign biodegradation process, preventing the accumulation of intermediate byproducts that could be harmful.
[0035].The interaction between the plastic substrates and the microbial enzymes is a critical factor in the degradation process. To improve this interaction, the plastic waste can be pre-treated to increase surface roughness, making it more accessible to enzyme binding. Methods such as UV irradiation, mild acid treatment, or surface coating with degradative compounds are employed to enhance the substrate's susceptibility to enzymatic action.
[0036].Moreover, cell-surface display technology is utilized to anchor the plastic-degrading enzymes directly onto the outer membrane of the microorganisms. This localization brings the enzymes in close proximity to the plastic waste, facilitating more efficient degradation. By displaying the enzymes on the cell surface, the microorganisms can effectively act on the plastic substrates without the need for extensive diffusion of the enzymes.
[0037].he scalability of this technology is a crucial aspect of its practical application. After successful laboratory optimization, the engineered microorganisms are scaled up for industrial use in bioreactors. These bioreactors are designed to maintain optimal conditions for plastic degradation, including controlled temperature, pH, and oxygen levels. Continuous monitoring of the degradation process is implemented through spectroscopic methods or chemical analysis, ensuring high efficiency and sustainability.
[0038].The bioreactor systems can be integrated into existing waste management infrastructure, such as industrial waste treatment plants or recycling facilities, providing a seamless transition to eco-friendly plastic waste processing. The engineered microorganisms can also be deployed in situ for environmental cleanup operations, targeting polluted natural habitats like oceans, rivers, and landfills.
[0039].This invention offers significant environmental and economic benefits. By providing a scalable and eco-friendly solution for plastic waste management, it reduces the environmental footprint associated with traditional methods such as landfilling and incineration. The biodegradation process converts plastic waste into harmless byproducts, mitigating the harmful impact of plastic pollution on ecosystems and human health.
[0040].Economically, the use of genetically engineered microorganisms for plastic degradation presents a cost-effective alternative to high-energy recycling or incineration techniques. The technology's scalability ensures that it can be deployed on a large scale, addressing the global challenge of plastic waste pollution.
[0041].The invention of genetically engineered microorganisms for the enhanced biodegradation of plastic waste marks a significant breakthrough in addressing the global challenge of plastic pollution. By leveraging advanced genetic engineering techniques, this technology optimizes the natural plastic-degrading capabilities of microbial strains such as Escherichia coli and Pseudomonas putida. The integration of key enzymes like PETase and MHETase into these microorganisms accelerates the breakdown of synthetic plastics, including polyethylene (PE) and polyethylene terephthalate (PET), into environmentally benign byproducts such as carbon dioxide and water.
[0042].This innovative approach not only improves the efficiency and speed of plastic degradation but also ensures that the process is environmentally safe. By enhancing the stability and activity of plastic-degrading enzymes through directed evolution and metabolic engineering, the engineered microorganisms can thrive under diverse environmental conditions, making them suitable for various waste management scenarios. The ability to scale up the technology for industrial applications, such as in bioreactors and waste treatment facilities, further underscores its practicality and economic viability.
[0043].The invention provides a sustainable and eco-friendly alternative to traditional plastic waste management methods, which often involve landfilling, incineration, or mechanical recycling, all of which have significant environmental drawbacks. By converting plastic waste into harmless byproducts, this technology reduces the environmental footprint and mitigates the adverse impacts of plastic pollution on ecosystems and human health.
[0044].Moreover, the cost-effectiveness of using genetically engineered microorganisms for plastic degradation presents a promising solution for large-scale implementation. The potential applications of this technology extend beyond waste treatment facilities to include environmental cleanup operations in polluted natural habitats such as oceans, rivers, and landfills.
[0045].In conclusion, the development of genetically engineered microorganisms for enhanced plastic biodegradation represents a pivotal advancement in biotechnology and environmental sustainability. It offers a robust, scalable, and eco-friendly solution to the growing problem of plastic waste, contributing to a cleaner and more sustainable future. This invention not only addresses a critical environmental issue but also sets the stage for future innovations in waste management and bioremediation technologies. , Claims:1.A genetically engineered microorganism for the biodegradation of plastic waste, wherein the microorganism comprises:
a) One or more plastic-degrading enzymes, selected from the group consisting of PETase and MHETase, introduced into a host microbial strain;
b) Modifications to metabolic pathways to convert plastic degradation byproducts into environmentally benign compounds such as carbon dioxide and water.
2.The genetically engineered microorganism as claimed in claim 1, wherein the host microbial strain is selected from the group consisting of Escherichia coli and Pseudomonas putida. The plastic waste comprises polyethylene (PE) and polyethylene terephthalate (PET).
3.The genetically engineered microorganism as claimed in claim 1, further comprising genetic modifications to enhance enzyme activity and stability at higher temperatures and a broader range of environmental conditions. The plastic-degrading enzymes are optimized through directed evolution and site-directed mutagenesis to improve their catalytic efficiency.
4.The genetically engineered microorganism as claimed in claim 1, wherein the microorganism is capable of degrading a wide range of plastic materials, including low-density polyethylene (LDPE), high-density polyethylene (HDPE), and PET.
5.The genetically engineered microorganism as claimed in claim 1, wherein the enzyme expression is controlled by strong inducible promoters and multiple gene copies to ensure high levels of enzyme production.
6.A method for biodegrading plastic waste, comprising:
a) Exposing the plastic waste to the genetically engineered microorganism under conditions that promote the degradation of the plastic waste into environmentally benign by-products.
7.The method as claimed in claim 6, wherein the plastic waste is pre-treated to enhance surface roughness and accessibility for enzyme binding.
8.The method as claimed in claim 6, wherein the genetically engineered microorganism is applied in industrial waste treatment plants, landfills, or polluted natural environments.

Documents