Gene_locus Report for: idesa-mheth

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.

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 pT18mobsacB-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 deltapyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the deltapyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both deltapetase and deltamhetase 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 deltapetase 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 deltapetase and deltamhetase 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.

In 2016, one of the two enzymes involved in the polyethylene terephthalate (PET) degradation pathway of Ideonella sakaiensis 201-F6, MHETase, was found to exhibit a strong ability to degrade the PET monomer mono-(2-hydroxyethyl)terephthalate (MHET) at room temperature, converting it back into the precursors used in PET production. MHETase engineering to improve efficiency is an active field that suffers from an incomplete characterization of its reaction mechanism. In this paper, we analyze the reaction mechanism of MHETase using umbrella sampling molecular dynamics simulations at the B3LYP/MM level of theory. The combination of a high theoretical level and extensive sampling generated a very robust computational prediction. We found that MHETase catalyzed the conversion of MHET in two steps, with a rate-limiting step activation barrier of deltaG = 19.35 +/- 0.15 kcal.mol-1 (from the weighted-histogram analysis). Our calculations are in line with the hypothesis that a transient tetrahedral intermediate mediates the reaction mechanism in each step, which is quite common in the serine hydrolase class. The energy of the first tetrahedral intermediate was similar to that of the reactant state, while the tetrahedral intermediate of the deacylation step was observed to lie closer to the rate-limiting transition state. Nevertheless, both determined tetrahedral states were found to be transient, with activation barriers close to 2.0 kcal.mol-1 relative to the product state of the acylation and deacylation steps, corresponding to a half-life of about 3 ps at 303.15 K.

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.

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 pT18mobsacB-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 deltapyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the deltapyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both deltapetase and deltamhetase 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 deltapetase 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 deltapetase and deltamhetase 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.

Poly (ethylene terephthalate) (PET) is one of the most widely used synthetic polyesters but also a main cause of plastic pollution. Since the chemical degradation of PET would be uneconomical and rather burdensome, considerable efforts have been devoted to exploring enzymatic processes for the disposal of PET waste. Many PET hydrolyzing enzymes have been consecutively reported in recent decades, some of which demonstrate excellent potential for industrial applications. This review sets out to summarize the investigation status of Is PETase, a cutinase-like enzyme from I. sakaiensis possessing ability to degrade the crystalline PET, and to gain further insight into the structure-function relationship of Is PETase. Benefiting from the continuing identification of novel cutinase-like proteins and growing availability of the engineered Is PETase, we may anticipate future developments in this type of enzyme would generate suitable biocatalyst for industrial use.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary.

Accumulation of plastics in the oceans presents a major threat to diverse ecosystems. The introduction of biodegradable plastics into the market aims to alleviate the ecological burden caused by recalcitrant plastics. Poly (butylene adipate-co-terephthalate) (PBAT) is a biodegradable commercial plastic that can be biodegraded similarly to polyethylene terephthalate (PET) by PETase-like enzymes and MHETases. The role of MHETases is to hydrolyze the intermediate degradation product of PET, mono-2-hydroxyethyl terephthalate (MHET) to its monomers. We recently identified a homolog of the MHETase of the PET-degrading bacterium Ideonella sakaiensis, Mle046, from a marine microbial consortium. In this consortium, Mle046 was highly expressed when a PBAT-based blend film (PF) was supplied as the sole carbon source. In this study, we recombinantly expressed and biochemically characterized Mle046 under different conditions. Mle046 degrades MHET but also 4-(4-hydroxybutoxycarbonyl) benzoic acid (Bte), the intermediate of PF degradation. Mle046 is a mesophilic enzyme adapted to marine conditions, which rapidly degrades MHET to terephthalate and ethylene glycol at temperatures between 20 and 40 degreesC. Mle046 degradation rates were similar for Bte and MHET. Despite its mesophilic tendency, Mle046 retains a considerable amount of activity at temperatures ranging from 10 to 60 degreesC. In addition, Mle046 is active at a range of pH values from 6.5 to 9. These characteristics make Mle046 a promising candidate for biotechnological applications related to plastic recycling.

In 2016, one of the two enzymes involved in the polyethylene terephthalate (PET) degradation pathway of Ideonella sakaiensis 201-F6, MHETase, was found to exhibit a strong ability to degrade the PET monomer mono-(2-hydroxyethyl)terephthalate (MHET) at room temperature, converting it back into the precursors used in PET production. MHETase engineering to improve efficiency is an active field that suffers from an incomplete characterization of its reaction mechanism. In this paper, we analyze the reaction mechanism of MHETase using umbrella sampling molecular dynamics simulations at the B3LYP/MM level of theory. The combination of a high theoretical level and extensive sampling generated a very robust computational prediction. We found that MHETase catalyzed the conversion of MHET in two steps, with a rate-limiting step activation barrier of deltaG = 19.35 +/- 0.15 kcal.mol-1 (from the weighted-histogram analysis). Our calculations are in line with the hypothesis that a transient tetrahedral intermediate mediates the reaction mechanism in each step, which is quite common in the serine hydrolase class. The energy of the first tetrahedral intermediate was similar to that of the reactant state, while the tetrahedral intermediate of the deacylation step was observed to lie closer to the rate-limiting transition state. Nevertheless, both determined tetrahedral states were found to be transient, with activation barriers close to 2.0 kcal.mol-1 relative to the product state of the acylation and deacylation steps, corresponding to a half-life of about 3 ps at 303.15 K.

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2(II)A3(II)B(II)A1(II) from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, beta-ketoadipic acid (betaKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L betaKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to betaKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.

Few reports have described the biological degradation or utilization of poly(ethylene terephthalate) (PET) to support microbial growth. We screened environmental samples from a PET bottle recycling site and identified the microbial consortium no. 46, which degraded amorphous PET at ambient temperature; thereafter, we isolated the resident Ideonella sakaiensis 201-F6 strain responsible for the degradation. We further identified two hydrolytic enzymes from I. sakaiensis, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase), which synergistically converted PET into its monomeric building blocks. Here, we provide original methods of microbial screening and isolation of PET degrading microbe(s). These novel approaches can be adapted for exploring microorganisms that degrade PET and other plastics. Furthermore, our enzyme assay protocols to characterize PETase and MHETase can be applied to evaluate new enzymes that target PET and its hydrolysates.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 A resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

The degradation of synthetic polymers by marine microorganisms is not as well understood as the degradation of plastics in soil and compost. Here, we use metagenomics, metatranscriptomics and metaproteomics to study the biodegradation of an aromatic-aliphatic copolyester blend by a marine microbial enrichment culture. The culture can use the plastic film as the sole carbon source, reaching maximum conversion to CO(2) and biomass in around 15 days. The consortium degrades the polymer synergistically, with different degradation steps being performed by different community members. We identify six putative PETase-like enzymes and four putative MHETase-like enzymes, with the potential to degrade aliphatic-aromatic polymers and their degradation products, respectively. Our results show that, although there are multiple genes and organisms with the potential to perform each degradation step, only a few are active during biodegradation.

Monohydroxyethyl terephthalate (MHET) hydrolase (MHETase) is an enzyme known to be involved in the final degradation step of poly(ethylene terephthalate) (PET) by hydrolyzing MHET into terephthalic acid and ethylene glycol in Ideonella sakaiensis. Here, we report the extracellular production of MHETase in an active form with a proper folding. Based on the structural observations and biochemical experiments, we reveal that MHETase also functions as exo-PETase by hydrolyzing the synthesized PET pentamer. We further present that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme. We also develop a MHETase R411K/S416A/F424I variant with a higher BHET activity, and the variant exhibits an enhanced degradation activity against the PET film. Based on these results, we propose that MHETase plays several roles in the biodegradation of PET using the BHETase and exo-PETase activities as well as the MHET hydrolysis function

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70degC. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETase(S242T) and IsPETase(N246D), with higher enzymatic activity at both 25 and 37degC compared with IsPETase(WT). We also developed the IsPETase(S121E/D186H/S242T/N246D) variant by integrating the S242T and N246D mutations into the previously reported IsPETase(S121E/D186H/R208A) variant. At the 37degC incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETase(WT) that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETase(WT).

The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus alpha/beta-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic alpha/beta-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.

Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymatic degradation of PET can be a promising solution. Although PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degradation activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degradation activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized beta6-beta7 connecting loop and extended subsite IIc, had a Tm value that was increased by 8.81 C and PET degradation activity was enhanced by 14-fold at 40 C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure determination of the variants, and high thermal stability was further confirmed by a heat-inactivation experiment. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegradation of PET under mild conditions

Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.