Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes.

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability 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 in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes , we have developed a gene disruption system in . The pT18-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene () from the genome of the wild-type strain, producing the Δ strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ strain as a parent strain and as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ and Δ 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 a no-carbon source. Moreover, the Δ strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for metabolism of PET. 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 studies, including biochemical and crystal structure analyses, few studies have been reported. Here, we developed a targeted gene disruption system in , which was then applied for constructing Δ and Δ strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in , while PETase and MHETase play essential roles in its PET assimilation.