Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however its non-biodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium
Ideonella sakaiensis
201-F6 strain was isolated and the enzymes involved in PET-digestion, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase), were identified. Despite the great potentials of
I. sakaiensis
in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes
in vivo
, we have developed a gene disruption system in
I. sakaiensis
. The pT18
mobsacB
-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into
I. sakaiensis
cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (
pyrF
) from the genome of the wild-type strain, producing the Δ
pyrF
strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ
pyrF
strain as a parent strain, and
pyrF
as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ
petase
and Δ
mhetase
strains on terephthalic acid (TPA, one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on no carbon source. Moreover, the Δ
petase
strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for
I. sakaiensis
metabolism of PET.
IMPORTANCE
The poly(ethylene terephthalate) (PET)-degrading bacterium
Ideonella sakaiensis
possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention as they have potential to be used for bioconversion of PET. Compared to many
in vitro
studies including the biochemical and crystal structure analyses, few
in vivo
studies have been reported. Here, we developed a targeted gene disruption system in
I. sakaiensis
, which was then applied for constructing Δ
petase
and Δ
mhetase
strains. Growth of these disruptants revealed that PETase is a sole enzyme responsible for PET degradation in
I. sakaiensis
, while PETase and MHETase play essential roles in its PET assimilation.
Development of a targeted gene disruption system in the PET-degrading bacterium
Ideonella sakaiensis
and its applications to PETase and MHETase genes