To investigate an efficient system for degrading PET plastic,
we investigated the promotion of PET degradation by extracellular
expression of PETase.
Efficient PET degradation requires large amounts of PETase
and MHETase hydrolases to be expressed and secreted extracellularly, as
PET degradation depends on the secretion of PETase and MHETase.
Rhodococcus opacus degradation requires extracellular secretion of
hydrolases for effective degradation of PET plastics, facilitating PETase
isolation and purification. It reduces protein toxicity and the metabolic
burden on the chassis organisms while significantly increasing expression.
However, the extracellular secretion of PETase is species-specific.
Therefore, the appropriate signal peptide matching the chassis organisms
had to be found for optimal expression. Following a literature review, six
TAT pathway signal peptides were identified that regulate the
extracellular protein secretion of Rhodococcus opacus PD630.
To assess the ability of different signal peptides to promote
PETase secretion, different signal peptides linked to PETase were
transformed into pBSKR7756. The transformed bacteria were engineered to
secrete and express PETase, and SDS-PAGE was used to detect protein bands.
It allows us to screen for the signal peptide with the most robust PETase
secretion ability.
Three small peptides, called enhancers, with different
enhancement effects, were introduced to improve the secretion effect of
the signal peptide. These enhancers were fused and added to the front end
of the signal peptide to enhance the secretion and expression of exogenous
proteins within the Rhodococcus opacus expression system.SDS-PAGE was used
to identify protein bands to screen for enhancers with the most
significant potential to enhance PETase secretion.
However, Rhodococcus opacus has difficulty using the TPA and
EG produced by the degradation of PETase and MHETase. We need to convert
TPA into aromatic substrates that Rhodococcus opacus can use more
efficiently to achieve more resource-efficient plastic reuse. According to
literature research, Rhodococcus opacus metabolizes TPA via the
β-ketoadipic acid pathway to produce pyruvic acid, accumulating via the
tricarboxylic acid cycle to produce triacylglycerol. Crucially, TPA must
be converted to protocatechuic acid (PCA) by 1,2-dioxygenase. However,
Rhodococcus opacus lacks the necessary enzyme to perform this task. As a
result, with the literature review. We added the Tph operon from Comamonas
testosterone YZW-D to our design, allowing us to achieve this conversion
of TPA by 1,2 dioxygenase. It allows Rhodococcus opacus to degrade PET
plastic more rapidly. Coccidioides can accumulate and produce oil more
efficiently by rapidly using the TPA produced from the degradation of PET.
To produce microbial lipids from PET conversion, Rhodococcus
opacus containing PETase-MHETase and Rhodococcus opacus containing the Tph
operon were co-cultured in an oil-producing medium containing PET plastic.
Finally, oil production was detected.
Construction of the degradation and transformation system
The degradation and conversion system was designed to
biodegrade polyethylene terephthalate (PET) plastic into two monomers,
ethylene glycol (EG) and terephthalic acid (TPA), using Rhodococcus
Opacus. Rhodococcus Opacus then converted the TPA and EG produced by
degradation via the β-ketoadipic acid pathway to produce microbial lipids.
1、Amplify
ETase、MHETase、Tph operon(Tph A2、Tph A3、Tph A1、 Tph B and Tph K)
2、plasmid construction
The coding sequences: PETase, MHETase, Tph operon (Thp A2,
Tph A3,Tph B.and Tph K), signal peptide (1-6), EG promoter, and enhancer
(1-3) were cloned into the expression vector pBSKR7756 by genetic
recombination, and the recombinant pBSKR7756 plasmid was then transformed
into Rhodococcus Opacus PD6300.
