Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers

Introduction:
Plastics, like synthetic polymers, are ubiquitous in our everyday lives; they’re
strong, flexible and cost effective to produce. But the thing is, they are hard to
decompose in nature, so they accumulate in the environment and cause severe
environmental problems. Conventional methods for cutting down plastic garbage,
such as recycling and chemical treatments, are energy intensive and not very
effective. As such, people have begun having second thoughts about using
microbes to degrade (nature-works) plastics naturally. Advances in microbial
biotechnology and strain development have now enabled optimization. This
capability of some microorganisms such as bacteria and fungi to decompose plastic
materials via biocatalysis has been referred to as microbial and enzymatic
degradation of synthetic plastics. Due to their strong chemical bonds and complex
structures, the artificial plastics like PE [81,82], PET [83], PP [84] and PS exhibit
extremely high resistance against degradation. However, in the presence of
appropriate conditions acidity and microbiological flora some microorganisms
continue to stick on plastic surface and form biofilms which allow enzymes to start
slow degradation. Enzymes Hydrolases such as esterases, lipases and cutinases
including PET‐specific enzymes like PETase, MHETase are the key players in this
mechanism.
These enzymes have an action of breaking the chemical bonding that binds the
plastic polymers together to cause them to be broken down to smaller molecules or
monomers. As an example, PET-degrading enzymes break down the ester bonds to
liberate ethylene glycol and terephthalic acid which are absorbed by the microbial
cells and are metabolized. On the other hand, because they are free of functional
groups that can be easily attacked by an enzyme, plastics such as PE degrade much
more slowly and often require previous oxidation brought about by exposure to
heat, sunlight or chemicals. Generally, microbial and enzymatic degradation is a
viable alternative to conventional methods of managing plastic wastes. The
knowledge of these biological processes has provided valuable knowledge on
improving biodegradation of plastics despite the low natural degradation rates.
This information is the base for the development of genetically modified
microorganisms and engineered enzymes with improved stability, efficiency and
substrate range for long-term solutions to reduce plastic pollution
Plastics, like synthetic polymers, are ubiquitous in our everyday lives; they’re
strong, flexible and cost effective to produce. But the thing is, they are hard to
decompose in nature, so they accumulate in the environment and cause severe
environmental problems. Conventional methods for cutting down plastic garbage,
such as recycling and chemical treatments, are energy intensive and not very
effective. As such, people have begun having second thoughts about using
microbes to degrade (nature-works) plastics naturally. Advances in microbial
biotechnology and strain development have now enabled optimization. This
capability of some microorganisms such as bacteria and fungi to decompose plastic
materials via biocatalysis has been referred to as microbial and enzymatic
degradation of synthetic plastics. Due to their strong chemical bonds and complex
structures, the artificial plastics like PE [81,82], PET [83], PP [84] and PS exhibit
extremely high resistance against degradation. However, in the presence of
appropriate conditions acidity and microbiological flora some microorganisms
continue to stick on plastic surface and form biofilms which allow enzymes to start
slow degradation. Enzymes Hydrolases such as esterases, lipases and cutinases
including PET‐specific enzymes like PETase, MHETase are the key players in this
mechanism.
These enzymes have an action of breaking the chemical bonding that binds the
plastic polymers together to cause them to be broken down to smaller molecules or
monomers. As an example, PET-degrading enzymes break down the ester bonds to
liberate ethylene glycol and terephthalic acid which are absorbed by the microbial
cells and are metabolized. On the other hand, because they are free of functional
groups that can be easily attacked by an enzyme, plastics such as PE degrade much
more slowly and often require previous oxidation brought about by exposure to
heat, sunlight or chemicals. Generally, microbial and enzymatic degradation is a
viable alternative to conventional methods of managing plastic wastes. The
knowledge of these biological processes has provided valuable knowledge on
improving biodegradation of plastics despite the low natural degradation rates.
This information is the base for the development of genetically modified
microorganisms and engineered enzymes with improved stability, efficiency and
substrate range for long-term solutions to reduce plastic pollution
This image is taken from research article Biolgical degradation of Microplastics
presenting how PET plastic breaks down through microbial degradation. Plastic is
initially weakened by sunlight and weathering into micro plastics, which are then
inhabited by bacteria such as Ideonella sakaiensis which produce PETase and
MHETase enzymes. These enzymes break down the plastic into tiny monomers
which enter the cell and are ultimately degraded into CO2 and water.

Enzymes such as PETase and cutinases are important for the degradation of
polymers such as poly ethylene terephthalate (PET), but their natural counterparts
are often poorly active and stable. With the use of protein engineering methods,
such as site-directed mutagenesis and rational design, these enzymes have been
modified to increase their catalytic efficiency, thermal stability, and capacity to act
on a broader spectrum of substrates. In addition, engineering of suitable host
strains for expression and secretion of these enzymes has further increased
degradation performance. These developments indicate that engineering of
enzymes and host strains are valid measures of enhancing microbial degradation of
plastics and developing sustainable solutions to plastic waste management.
Scientists have successfully engineered well-studied bacteria like Escherichia coli,
as well as naturally hardy organisms like Pseudomonas, to produce enzymes that
are capable of breaking down plastics, including PETase, MHETase and cutinases.
These modifications assist the bacteria in breaking down plastics into smaller
components and utilize these products in a better way. In addition to the research
on single bacterial species, scientists are also engineering engineered microbial
consortia (populations of various microorganisms) that collaborate. In such
systems, every microorganism has a certain role to play. For example, some
degrade the plastic and others break down products. This cooperative approach
enhances the overall efficiency and ensures better utilization of available resources.

This image is taken from research article Biolgical degradation of Microplastics

elaborating how the bacterium Ideonella sakaiensis breaks down PET plastic into
smaller and sustainable molecules using its two enzymes PETase and MHETase.

Ensuring enough enzyme activity and stability in the real environment conditions is one of the greatest
challenges in genetic modification of bacteria to be used in synthetic polymers degradation. The majority
of plastic-degrading enzymes such as PETase or cutinases can be produced in engineered bacteria but in
the laboratory setting these enzymes are inefficient in non-controlled environments. Temperature
variations, pH variations, presence of the inhibitors, and poor accessibility of the polymer surfaces are
factors that are capable of playing a big role towards reducing the rate of degradation. Moreover, most
synthetic polymers are crystalline and hydrophobic and extremely difficult to bind and degrade by
enzymes, despite being genetically optimized. The compatibility of host strain and metabolic burden is
the other critical problem. The alien gene arriving to bacterial hosts can disrupt the normal cellular
activities, retard growth or reduce survival. When plastic-degrading enzymes are expressed on high
levels, they can result in a metabolic cost and, therefore, a degradation of the fitness of the engineered
bacteria. In addition, additional metabolic pathway engineering would be required to conduct efficient
uptake and metabolism of degradation by-products, e.g. terephthalic acid or ethylene glycol, which
complicates the design of strains, in addition to posing the risk of unintended metabolic imbalances.
There is also a significant barrier to practice in bio safety, scalability and regulatory complications.
Horizontal gene transfer, environmental imbalance and environmental impact in the long run are the
issues linked with the environmental release of genetically modified bacteria. Safe operation and genetic
stability on an industrial level are also not easy to assure and contain. Moreover, the procedure of the
actualization of the laboratory-designed systems to perform successfully in the natural or industrial
setup requires the optimization of the process, the cost-effective production, and the compliance with
the rigid rules and regulations. These issues are critical to deal with to put genetic engineering strategies
at the center of credible and viable solutions to the problem of plastic waste degradation.

Genetically modified bacteria, therefore, provide a good and environmentally friendly solution to the
increasing menace of synthetic polymer pollution. These microorganisms have the ability to degrade
plastics more effectively than natural systems by enhancing the bacterial enzymes and metabolic
pathways. Studies have indicated that to make this method effective in a practical scenario, the systems
should be secure, scalable, and be able to operate effectively on a large scale. Despite the fact that some
challenges like biosafety and massive application are still there, advancement in genetic engineering and
biotechnology continues to reduce these challenges. All in all, the engineered bacteria have high
chances of emerging as a powerful and a sustainable means of handling the synthetic polymer waste in
the future.

Applications:

1. Heterologous Expression of Degradation Enzymes:

Esterase and cutinase expression in genetically modified bacteria has emerged as
one of the promising methods of degrading synthetic polymers like polylactic acid
(PLA), polybutylene succinate (PBS), and polybutylene adipate terephthalate
(PBAT). These plastics have ester bonds which can be cleavaged easily with
hydrolytic enzymes. Transposable genes of esterases or cutinases of naturally
plastic-degrading microorganisms can be transferred into fast-growing bacterial
hosts such as Escherichia coli or Bacillus species, leading to a significant
enhancement in the overall level of polymer degradation. These enzymes then
target the ester bonds within the polymer chains to break large, bulky polymers
down into smaller oligomers or monomers that the microbial systems find easier to
deal with.
These enzymes, especially cutinases have proven to be the most effective since
they can directly impact on the surface of solid polymer without prior chemical or
physical processing. Cutinases that are expressed and engineered in heterologous
systems tend to be more thermally stable and have a more extensive substrate
range, allowing efficient degradation of biodegradable plastics like PLA, PBS and
PBAT at comparatively mild conditions. Esterases also facilitate the process by
dismantling the smaller fragments of the polymers that are produced in the early
stages of degradation. The combination of esterases and cutinases enables
genetically modified bacteria to not only depolymerize synthetic polyesters, but
also to use the products of the depolymerization process and proves that they can
be useful in sustainable plastic waste management and bio-recycling strategies.

2. Protein & Enzyme Engineering:

Protein and enzyme engineering is a relevant solution to increasing the capacity of

microorganisms to degrade plastic materials by enhancing the activity and stability
of enzymes. A case in point is the creation of engineered PETase where specific
mutations in the active site of the enzyme are made to enhance its binding to
polyethylene terephthalate (PET). These changes make the enzyme more active to
attach to the plastic and cleave the ester bonds in the polymer more effectively.
Therefore, the engineered PETase is highly degradable in a shorter time and can be
active in a wider temperature and environmental range in comparison with the
natural form. This points to the potential of enzyme engineering to overcome the
limitations of indigenous plastic-degrading enzymes and enable more efficient and
sustainable biological plastic recycling.
This is an image taken in Wikipedia which demonstrates that protein engineering
enhances enzymes in bio catalysis in three primary approaches namely directed
evolution, semi-rational design and rational design.3. CRISPR-Based Genome
Editing:

CRISPR-based genome editing is state-of-the-art technology that allows scientists

to accurately manipulate the DNA of microbes to increase their capacity to break
down plastics. Under this approach, individual genes in plastic degradation can be
introduced, eliminated or optimized in the microbial genome. Plastic-degrading
pathways can be directly targeted and optimized using tools such as PlastiCRISPR,
and the process is more efficient and controlled than other methods of genetic
engineering. CRISPR can be used to alter the major genes of microbes to increase
the numbers of plastic-degrading enzymes, or to enable the enzymes to be more
effective within the cell. This does not only accelerate the degradation of plastic
polymers but also enables the microbes to utilize the smaller molecules as sources
of energy. Altogether, CRISPR-based technologies such as PlastiCRISPR offer an
accurate and promising means of producing some engineered microorganisms to
recycle plastics and clean the environment sustainably.

This picture is borrowed in a research article PlastiCRISPR: Genome Editing-

Based plastic Waste Managements describing how plastic waste can be recycled
with the help of CRISPR-Cas9.
Metabolic Engineering and Synthetic Biology: Metabolic engineering and
synthetic biology are processes that redesign the inner workings of microbes to
enhance the way they process things (including plastics). Changing the mechanism
of metabolism of a microbe, scientists can not only help it to decompose plastic
polymers more effectively but also transform the smaller products into valuable
products, such as biofuels or bioplastics. Synthetic biology goes one step further
than that, and constructs entirely new pathways or combines genes from other
organisms to create "custom-made" microbes with increased plastic-degrading
capabilities. Combined these strategies enable scientists to come up with bacteria
capable of cleaning plastic waste as well as generating useful byproducts, thus
plastic recycling will be more sustainable and efficient.

Microbial consortia engineering:

Microbial consortia engineering entails development of communities of various

bacteria each of which has been specially engineered to perform a specific step in
plastic degradation. Rather than relying on any one of the microbes to consume all
of the components, each member specialises in specific polymers or fragments. An
example is that one bacterium may be programmed to break down PET into small
pieces and another bacterium will be programmed to pick them up and transform
them into some useful products such as biofuels or biodegradable materials. This
teamwork, which is comparable to the natural ecosystem, enables the system to
perform in a more efficient and quicker way than any single strain would have
acted alone. It is also more flexible and scalable. Through the integration of
microbes with complementary capacities, researchers will be able to address a
wider scope of synthetic plastics including PLA, PBS or PBAT. The developed
consortia is able to treat mixed plastic wastes which guarantees complete
degradation. Moreover, this approach not only will assist in reducing plastic
pollution but it will also aid in a circular bio economy, converting waste into
resources in a sustainable and environmentally friendly manner, by converting the
breakdown products into something valuable.