Metabolism

P450-driven plastic-degrading synthetic bacteria

Done by :Tuqa Abuatiq

Soo-Jin Yeom , 1,2,3,* Thien-Kim Le , 1 and Chul-Ho Yun 1,4,5,*, (2021).

Outline:
 Introduction : Plastic and Cytochromes P450 .

 Method: How can synthetic bacteria solve plastic accumulation

and contamination problems.

 Results

 Conclusions

 References
 Introduction

▪ Plastic contamination currently threatens a wide variety of ecosystems and its scale
of 350–400 million metric tons each year and approximately 80% of the plastic
accumulates in landfills or the natural environment.
▪ Plastic pollution causes harmful environment effects, but the development of
biocatalysts can solve them.
▪ avoiding plastic contamination problems through biocatalysts, including enzymes and
microorganisms because of their consequent degradation.
Introduction
▪ Bacteria play important roles in the elimination of plastics  Cytochrome P450 is a heme-thiolate protein
by using the P450 enzyme. found in most living organisms and has over
300,000 known genes.
▪ Synthetic bacteria are emerging interdisciplinary
technologies that can solve plastic issues by applying
 cytochromes (cyt) prosthetic group, called a
degrading enzymes that contribute to the breakdown of
plastics. heme group, has an iron atom that accepts and
donates electrons.
▪ Cascading enzymatic systems that are started via in-chain
hydroxylation and catalyzed by a hypothetical P450 enzyme
with an engineered active site can depolymerize
polyethylene into small degradation molecules.
Introduction
▪ Plastic types according to chemical structures:
• Plastics are produced through two main processes:
- Plastics with a carbon–carbon backbone: polyethylene polymerization and polycondensation. Polymers like
(PE), polypropylene (PP), polyvinyl chloride (PVC), and PE, PP, PVC, PET, PU, and PS have unique
polystyrene (PS). properties and structures based on their basic
monomers such as ethylene and propylene.

• PE, PP, and PS are polymers with rigid C-H bonds

that are highly resistant to degradation.

- Plastics with heteroatoms in the main chain:

terephthalate (PET) and a typical polyurethane (PU). • PET and PU have functional groups with ester and
amide bonds that are accessible to hydrolytic enzymes.
 Introduction
▪ Plastic biodegradation begins with oxidation of PE, with C-H
hydroxylation occurring primarily at terminal and in-chain
positions. The primary C–H bond’s dissociation energy
(101.1 kcal/mol) is higher than that of the secondary C–H
bond (98.6 kcal/mol) in alkanes, the tertiary C-H bond in a
few chain branches in PE has the lowest bond dissociation
energy (96.5 kcal/mol), making the C-H hydroxylation at the
terminal position (ꞷ) unfavorable.

ꞷ
▪ Hypothetical hydroxylases are needed for aerobic
biodegradation of PE, with AlkB alkane hydroxylase enzymes
being the only known ones. Cytochrome P450, acting as an
PE
oxygenase, hydroxylates PE intermediates with different
positions.
 Introduction
 P450s can hydroxylate linear alkanes, alcohols,
and fatty acids of various chain lengths.
▪ most P450s require NAD(P)H-driven redox
partners to function in vivo.
▪ A family of P450 enzymes (CYP152) has been
discovered to act as peroxygenases using H2O2
as an oxidant, rather than NAD(P)H-driven redox
partner systems. These enzymes hydroxylate
fatty acids mainly at the α position, with
potential biotechnological applications in
developing P450s as industrial biocatalysts.
 Some enzymes, such as AlkB, laccase, manganese
peroxidase, cutinase, and PETase, are reported to be
involved in plastic biodegradation, information on their
biochemical mechanisms for plastic degradation is
limited.
 Alkane monooxygenase (AlkB), a recombinant
enzyme from Pseudomonas sp., has been reported to
degrade PE. AlkB is a membrane protein and requires
redox partners.
 CYP153 family enzymes, along with AlkB, are
important for aerobic alkane degradation in oil-polluted
environments, often found in bacteria lacking AlkB.
 Method
▪ AlkB degrades PE, potentially involving P450 enzymes ▪ Wild types of CYP102A1 and CYP102A3 enzymes
in the degradation process. hydroxylate medium-chain fatty acids but have no
hydroxylation activities towards n-alkanes. Directed
▪ P450s initiate PE degradation through carbon evolution has been used to construct mutants with
hydroxylation at different alkane positions. high hydroxylation activities.
▪ P450 belong to the monooxygenases superfamily and ▪ WT P450s, such as CYP505E3 and CYP505D6, are
are useful biocatalysts involved in various compound active with fatty acids and alkanes, catalyzing
reactions. hydroxylation reactions to produce corresponding
products.
▪ P450s require redox partners to obtain electrons for
monooxygenation reactions and they have ▪ CYP52A3 and CYP52A4 are enzymes that catalyze
peroxygenase activity like H2O2. the hydroxylation of alkanes and fatty acids, while
CYP505A30 exhibits hydroxylation activities towards
fatty acids.
 Method
 Selected CYP enzymes involved in the hydroxylation of linear alkanes
 Method
P450s require two electrons for catalysis, which are
provided by NAD(P)H, via one or two reduction partners.

• P450 transfers oxygen atoms into substrates,

reducing heme-Fe (III) to Fe (II) and releasing water
molecules, requiring a single redox partner protein.

• The redox partner carries FAD (flavin adenine

dinucleotide) and FMN (flavin mononucleotide )
cofactors.

• (C) P450 systems require two redox partners, the

iron–sulfur protein ferredoxin (Fdx), and a ferredoxin
reductase (FdR) containing an FAD or FMN cofactor
 Method
P450s require two electrons for catalysis, which are
provided by hydrogen peroxide (H2O2).

• P450s use hydrogen peroxide as an electron

source to drive catalysis in the peroxide
shunt. The P450 peroxygenase does not
need any of the redox partners, molecular
oxygen, or NAD(P)H.
Method
 PE composed of long, linear alkanes with few
chain branches. These can be metabolized in
bacteria by P450 (CYP), (AlkB), Adh, BVMO,
Aldh and UPO including terminal, subterminal,
biterminal oxidation.
 P450s hydroxylate 2-alkanol at the
subterminal, followed by secondary alcohol
dehydrogenase (Adh), Baeyer–Villiger
monooxygenase (BVMO), and esterase.
 In-chain hydroxylation produces alkanol and
alkanoic acid, which can be further oxidized to
aldehyde and alkanoic acid.
 The final products of degradation are fatty
acids, with varying chain lengths, which can
be used as metabolic intermediates for living
organisms via the β-oxidation pathway.
▪ C12-C18 fatty acids being common substrates for
P450s.
▪ Some hydroxy fatty acids can be oxidized for
various products, including shorter alkanoic acids,
hydroxyl fatty acids, and dicarboxylic acids.
▪ CYP152 enzymes, convert fatty acids into
terminal alkenes and shorter-alkyl-chain fatty
acids through oxidative decarboxylation and
hydroxy fatty acids through α- and β-
hydroxylation.
▪ The most efficient PE decomposition pathway
involves in-chain hydroxylation with highly active
P450s triggering random PE decomposition.
Method
 The process of alkane degradation involves the
formation of ketones, followed by secondary
hydroxylation.
 In which it forms an ester group through Baeyer-
Villiger monooxygenase (BVMO).
 These final products, including fatty
acids, are used in metabolic pathways
and energy production for living
organisms, including the β-oxidation
pathway and tricarboxylic acid cycle.

 Esterase then cleaves the ester bond, resulting in

alkanols and acetic acids.
 Method
▪ Hydroxylation reactions at terminal and subterminal
positions are not efficient in breaking down very long
hydrocarbon chains. Highly active P450s with random
in-chain hydroxylase activity should trigger random PE
decomposition.
▪ Engineered P450s are ideal for PE biodegradation
due to their open and flat active sites, This allows new
P450s in PE-degrading bacteria to perform in-chain
hydroxylation.
▪ CYP1A enzymes have large active sites, allowing
five-ring polycyclic molecules to access them based
on substrate size recognition.
▪ Unspecific peroxygenases (UPOs) are potential
hydroxylating enzymes, they are fungi-secreted
enzymes with similar alkane hydroxylation activities to
P450s.
▪ UPOs display peroxygenase activities supported by
H2O2 without the need for external redox cofactors in
hydroxylation of linear, branched, and cyclic alkanes.
▪ UPOs' heterologous functional expression is a major
problem, while AlkB's complexity makes it difficult to
apply to enzyme engineering and synthetic bacteria.
▪ So P450s are more suitable for PE-degrading
synthetic bacteria than UPOs and AlkB enzymes.
 Results
 Schematic diagram of
proposed polyethylene
(PE)-degrading synthetic
bacteria with:
• Hydroxylase

• Alcohol dehydrogenase (Adh).

• Baeyer–Villiger monooxygenase
(BVMO).

• Esterase.
Results
▪ Biofilms form due to PE's high surface
hydrophobicity and microorganisms secrete
exopolysaccharides for strong adhesion.
▪ the microorganisms of the biofilms secrete
extracellular enzymes, which catalyze the
depolymerization of the polymer chain into
oligomers, dimers, or monomers.
 The P450-driven cascade enzymatic reaction for
PE degradation involves microorganisms growing
in harsh environments.
 Synthetic artificial bacteria are able to assimilate
limited nutrients in extreme environments. such as
E. coli, which can significantly assimilate methanol
using new enzymes and a modified metabolic
pathway.
 The biodegradation of PE involves four steps:
biodeterioration, biofragmentation,
assimilation, and mineralization.
 Biofragmentation is a process that generates
lower-molecular-weight compounds, which are
then transported into the cytoplasm of a
microbe, forming and releasing into the
environment.
 Bulk PE uptake is challenging due to its size
limit (~500 Dalton), but enzymes can access it
directly on the bacterial surface, and
membrane-bound metabolons can be
regulated through interactions or scaffolding
proteins.
 Results
 TF-based genetic biosensors are being developed for
strain evolution and enzyme engineering in high-
throughput screening platforms.
 These biosensors evaluate the operational range of
target metabolites needed for a significant change in
output signal.
 The output signals in the genetic circuit can be
reporter genes like fluorescent proteins as Green
fluorescent protein GFP or luciferases, which detect
target molecules.
▪ Genetically encoded biosensors using ligand-
inducible TF have untapped potential for screening
novel enzymes or enhancing enzyme evolution for
desired activity.
▪ TFs have been reported as potential biosensors for
PE biodegradation, potentially aiding in enzyme
engineering and screening natural PE-eating.
 using transcription factor-based genetic biosensors
for screening novel enzymes to enhance the
enzyme activity for efficient enzymatic degradation
of synthetic polymers.
 Results
 Applying synthetic biology-based genetic biosensors.

(A) The small products from the polyethylene (PE)

depolymerization pathway in synthetic or natural bacteria
can be observed by a PE monomer-detecting biosensor
 Results

(B) The combination of a genetic biosensor with high-

throughput fluorescence-activated cell sorting (FACS) for
rational protein engineering and direction evolution is
necessary to improve the related enzyme’s activity and
stability, which will enhance the enzymatic degradation
efficiency.
 Conclusion
 hypothetical P450 enzymes with an engineered active
site might be the most important candidates as trigger
enzymes for PE degradation.
 Synthetic biology tools can create artificial PE-eating
synthetic bacteria harboring a PE biodegradation
synthetic pathway with engineered P450, Adh, BVMO,
and esterase or other necessary enzymes.
 Rational protein engineering and directed evolution via
synthetic biology-based biosensors are necessary to
improve the related enzymes’ activity and stability
▪ Engineered biocatalysts could be applied to other
plastics such as PS and PP, and small
depolymerization products could be used as
feedstock.
▪ If synthetic microbial cell factories are possible, they
will not only contribute to the disposal of plastic
wastes but also establish an improved upcycling
utilization of plastics.
 References
Soo-Jin Yeom , 1,2,3,* Thien-Kim Le , 1 and Chul-Ho Yun 1,4,5,*, (2021).

P450-driven plastic-degrading synthetic bacteria.

School of Biological Sciences and Technology, Chonnam National

University.

https://doi.org/10.1016/j.tibtech.2021.06.003

Trends in Biotechnology
Thank you for your attention
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