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Biotechnology & Applied Microbiology
Polyethylene terephthalate (PET) is a widely used plastic that is polymerized by terephthalic acid (TPA) and ethylene glycol (EG).
- Polyethylene terephthalate
- Biodegradation
- Hydrolases
- Chassis
1. Introduction
Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in people’s lives [1]. It is polymerized by terephthalic acid (TPA) and ethylene glycol (EG) through ester bonds [2]. Since PET was first used to produce disposable plastic bottles in the 20th century, it has been welcomed worldwide and has become an indispensable part of people’s lives [3]. As PET is highly resistant to natural degradation, the recycling of PET has been encouraged [4]. At present, the main methods for managing PET waste include landfilling, incineration, as well as physical and chemical recycling [5,6]. These methods usually cause secondary pollution to the environment and consume huge amounts of energy, which is not economical or environmentally friendly. Due to the improper recycling strategies and the strong mechanical properties of plastic products, serious environmental problems, such as soil pollution and the disturbance of marine ecosystems, have occurred [7]. Therefore, PET biodegradation has attracted attention as an environmentally friendly alternative, requiring milder temperatures and lower energy consumption than other recycling methods [8,9]. Additionally, the degradation monomers can easily be recycled, with the hope of converting PET into high value chemicals.
In 1977, several commercial lipases and an esterase were reported to hydrolyze various kinds of polyesters [10]. Since then, many PET hydrolases, such as lipases, cutinases and esterases, have been discovered and characterized by various microorganisms [1,11]. In 2016, Ideonella sakaiensis 201-F6 was isolated from a waste recycling station [12]. It was found to produce PET hydrolase (PETase) and monohydroxyethyl terephthalate (MHET) hydrolase (MHETase), which can degrade PET into intermediate products at 30 °C. Then, the structures of the two enzymes were analyzed and a series of effective enzyme modifications were carried out [13,14,15,16,17], efficiently improving the activity and stability of the two enzymes. The discovery and modification of PETase and MHETase has provided an important basis for the degradation of PET waste under ambient temperatures.
Synthetic biology and metabolic engineering strategies have been applied to the biodegradation and bioconversion of PET waste, especially in the modification of PET hydrolases, optimization of microbial chassis, and reconstruction of degradation pathways. At present, some bacteria, fungi, and marine microalgae have been reported as being good microbial chassis for PET biodegradation. The whole-cell biocatalysts have been able to achieve the initial degradation of PET. The bioconversion pathways of TPA and EG have been identified. Some microorganisms have been engineered to produce high value chemicals from PET monomers, which is an important development direction for PET upcycling. Based on these current advances, developing enhanced microbial chassis and constructing artificial microbial consortia to couple the biodegradation of PET by secreted PET hydrolases with the bioconversion of high value chemicals from monomers is a promising method to realize the circular economy of PET waste.
2. PET Biodegradation
During PET biodegradation, the microorganisms first adhere onto the surface of PET films and then secrete extracellular PET hydrolases, which bind to the PET films and initiate the biodegradation process [18,19]. PET hydrolases act on the ester bond of PET, hydrolyzing it into TPA and EG and generating incomplete hydrolysis products, such as MHET and Bis-(2-hydroxyethyl) terephthalate (BHET). In I. sakaiensis 201-F6, MHET can be further hydrolyzed into TPA and EG under the action of MHETase [12]. It was reported that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme [20]. PET hydrolases can further hydrolyze BHET to produce MHET, TPA, and EG [12]. The products TPA and EG can be used by different microorganisms and be further metabolized into the tricarboxylic acid cycle (TCA cycle) [21,22,23,24,25,26,27,28,29]. Additionally, these intermediate and final products of PET biodegradation have been identified as competitive inhibitors of PET hydrolases [30,31].
2.1. Engineered PET Hydrolases
The hydrolases, including lipases [31,32,33,34], cutinases [35,36,37,38,39,40,41,42], esterases [43,44,45,46], PETase [12] and MHETase [12], that can degrade PET have been identified. Among them, lipases have the lowest hydrolysis activity of PET mainly because their catalytic centers are covered by lid structures, which limits the hydrolases’ contact and catalysis with the substrate PET. Cutinases always have a strong PET hydrolysis ability due to their large substrate binding pockets without lid structures, which is conducive to the combination of PET with their active centers. However, cutinases usually degrade PET at high temperatures (50–70 °C), while PETase and MHETase can efficiently and specifically hydrolyze PET at 30 °C [12]. The discovery of PETase and MHETase is helpful in achieving the high efficiency biodegradation of PET at ambient temperatures. At present, the structures of these two enzymes have been studied extensively, and more high activity hydrolases variants have appeared.
The PET hydrolases identified in nature always have poor stability, low activity, and low expression levels, which limit their large-scale industrial application. A series of strategies that could enhance the catalytic activity of PET hydrolases have been proposed [13].
One strategy is to engineer the binding pocket, which can improve the specificity of the PET hydrolases and increase the effective adsorption of enzymes and substrates [15,47,48,49]. Our laboratory previously focused on six key amino acids near the binding of PETase to the substrate and conducted site-directed mutations. The R61A, L88F, and I179F mutants were successfully screened, and the enzyme activity increased 1.4-fold, 2.1-fold, and 2.5-fold, respectively, in comparison to wild-type PETase [50]. Silva et al. [51] modified the cutinase from Thermobifida fusca_0883 by site-directed mutagenesis and constructed a single mutation Ile218Ala and a double mutation Q132A/T101A, which expanded the catalytic space and improved the efficiency of the PET biodegradation. Chen et al. [52] identified the unique amino acids S214 and I218 through the structural analysis of PETase and noted that they are associated with W185 wobbling and β6-β7 loop flexibility. This research is helpful in designing PETase mutants that increase the flexibility of the substrate binding pocket.
Some studies focused on using enzyme engineering strategies to improve the stability of the PET hydrolases to improve PET biodegradation efficiency [53,54]. Methods such as adding Ca2+ or Mg2+ [38,55], introducing a disulfide bond and salt bridge [56,57], and glycosylation have all been proven to improve the stability of PET hydrolases. Researchers added disulfide bonds to improve the thermal stability of leaf-branch compost cutinase (LCC) and performed site-directed mutations on hot amino acids near the substrate binding to obtain the combined mutation F243I/D238C/S283C/Y127G (ICCG) [53]. Finally, 90% of shredded PET plastic bottles were degraded at 72 °C for 10 h, which is by far the most efficient PET hydrolase [53].
Additionally, increasing the substrate accessibility for PET hydrolases by engineering the PET hydrolases has also been widely studied [58,59,60]. It is reported that the fusion expression of Thc_Cut1 from Thermobifida cellulosilytica and hydrophobins (HFB4 and HFB7) from Trichoderma reesei can increase the hydrolysis effect of PET by more than 16 times, while a mixture of the enzyme and the hydrophobins led to only a 4-fold increase at most [61].
The intermediate and final products of PET biodegradation, such as Bis-(2-hydroxyethyl) terephthalate (BHET), monohydroxyethyl terephthalate (MHET), TPA, and EG, are all competitive inhibitors of the PET hydrolases [30]. Therefore, the mixtures of hydrolases that act synergistically or protein engineering strategies that reduce the interaction between the enzymes and products are effective methods for solving the inhibition [62,63,64].
2.2. Engineered PET Biodegradation Chassis
Most of the microorganisms identified that are capable of secreting PET hydrolases are non-model microorganisms and they are difficult to genetically engineer due to their complex genetic background. In addition, the expression level of the PET hydrolases from wild strains is insufficient to satisfy the demand for large-scale degradation. Therefore, it is necessary to develop recombinant expression systems using model microorganisms to express the PET hydrolases efficiently. PET is a high molecular polymer that is polymerized from TPA and EG and cannot enter cells, so in vitro enzymatic degradation of PET has been studied extensively. Owing to the purification and preparation process of PET hydrolases being time-consuming and cost-intensive, the efficient expression PET hydrolases extracellularly for practical applications is necessary [76,77].
At present, some microbial chassis such as bacteria, fungi, and marine microalgae have been applied to the secretion and expression of PET hydrolases, which have been studied and proven to be promising chassis to degrade PET. Several whole-cell biocatalysts have been designed to degrade PET, which are able to not only avoid the complicated steps of enzyme purification but also be reused in multi-step reactions, in comparison to the free enzyme-based approach [78,79]. Additionally, the difficulty of the reduced activity of the enzymes, or even enzymes being inactivated, under the influence of environmental factors has been solved. The following is a summary of several microbial chassis that are suitable for PET biodegradation.
2.2.1. Bacteria
Escherichia coli
E. coli is an important model microorganism for the production of recombinant proteins due to its clear genetic background, simple growth conditions, and its advantages in high density cultivation [89]. In recent years, with the continuous discovery of PET hydrolases, more and more enzymes have achieved the heterologous expression in E. coli [12,14,16,50,53,69]. PET hydrolases heterologously expressed in E. coli have been summarized [76] and it is helpful in further analyzing the crystal structures of these enzymes and explore the degradation mechanism for PET.
Recent studies have shown that engineered E. coli can be used as a whole-cell biocatalyst for PET biodegradation. Selecting the optimal signal peptide is a common strategy used to improve the section of heterologous PET hydrolases. A study tested the effects of Sec-dependent and SRP-dependent signal peptides from E. coli in secreting PETase, and successfully produced 6.2 mg/L PETase by fusing SPLamB and PETase [80]. Some other research improved the expression titer and enzymatic activity by modifying the signal peptide. An evolved signal peptide PelB (G58A) obtained by random mutagenesis was successfully used to express heterologous PETase in E. coli and enabled up to 1.7-fold higher PETase secretion [81]. An enhancer of signal peptides B1 (MERACVAV) was studied to mediate the excretion of PETase, and finally, the excretion efficiency of PETase mediated by B1PelB demonstrated a 62-fold increase over that of PelB [82].
Bacillus subtilis
Gram-positive B. subtilis has the advantages of high secretion capacity, fast growth, and the lack of an outer membrane, and it is regarded as an excellent microbial chassis for secreting heterologous proteins compared to E. coli, which usually forms an inclusion body [90,91]. Additionally, B. subtilis has a strong resistance to harsh environments and it has been used to secrete proteins that can degrade many pollutants, which is why it is considered to be a promising microbial chassis for biodegradation [92,93].
In terms of PET biodegradation, B. subtilis has been engineered to secrete PET hydrolases. It is reported that PETase was successfully secreted into the medium by B. subtilis 168 under the direction of its native signal peptide (SPPETase). SPPETase is predicted to be a twin-arginine signal peptide, and the inactivation of twin-arginine translocation (Tat) complexes improved the secretion amount of PETase 3.8-fold [83]. Another two PET hydrolases (BhrPETase and LCC) were also expressed in B. subtilis, and the expression titer of BhrPETase and LCC reached 0.66 g/L and 0.89 g/L in an engineered chaperone-overexpression of B. subtilis, respectively [42]. Additionally, the combinations of signal peptides and promoters were optimized to promote the expression of PETase in B. subtilis WB600, and the combination of the signal peptide SPamy and the weak promoter P43 was proved to be best [84].
Thermophilic Bacteria
Most of the hydrolases capable of degrading PET, including lipases, cutinases and esterases, have higher enzymatic activity at higher temperatures, while the optimal growth temperature of most model microorganisms that can produce heterologous PET hydrolases is usually 30–40 °C. Whole-cell biocatalyst is not compatible with some PET hydrolases that are only functional at high temperatures [94]. Therefore, a thermophilic expression system is necessary to improve the efficiency of PET biodegradation [36,95]. Most thermophilic microorganisms are usually difficult to genetically engineer except for Clostridium thermocellum, which has a mature genetic manipulation platform [94]. C. thermocellum has been engineered for lignocellulose bioconversion [96] and biofuel production [97], which is why it is regarded as a potential microbial chassis for the biodegradation of PET.
LCC has been successfully obtained from an engineered C. thermocellum. This engineered whole-cell biocatalyst realized a high level expression of LCC and more than 60% of a commercial PET film was converted into soluble monomers at 60 °C after 14 days [85]. This thermophilic whole-cell degradation system has the advantage of simultaneous enzyme production and PET degradation compared to only using free enzymes, which is why it is a promising strategy to degrade PET using other high temperature hydrolases [98,99]. In addition to the thermophilic whole-cell degradation system, an alkali-tolerant whole-cell catalytic system has also been reported [100,101,102].
2.2.2. Fungi
In addition to bacteria, the potential of some yeasts, including Pichia pastoris and Yarrowia lipolytica, being used in PET biodegradation has been studied. P. pastoris, with a great secretion expression system and scalable fermentation capability, has become a common strain for protein production in industrial applications. Researchers have expressed BurPL (H344S/F348I) and PETase in P. pastoris and E. coli and noted that both enzymes produced from P. pastoris showed higher activity than that expressed in E. coli because of the protein half-life protection mechanism of P. pastoris [52,103]. A whole-cell biocatalyst was developed by displaying PETase on the surface of P. pastoris and the enzymatic activity of PETase increased 36-fold towards a highly crystalline PET in comparison to that of purified PETase [86]. Additionally, this whole-cell biocatalyst can be reused seven times without obvious activity loss, which is helpful in developing other whole-cell biocatalysts for PET biodegradation [86]. Considering the ability of P. pastoris to perform N-linked glycosylation, some researchers studied the effects of glycosylation on the LCC expressed in P. pastoris and found that the kinetic stability and activity of LCC were both improved [35]. Y. lipolytica is also a great microbial chassis for bioremediation [104]. Researchers isolated Y. lipolytica IMUFRJ 50682 with the ability to convert PET into MHET and verified that the PET monomers may act as inducers in the process of lipase production [105], which showed that Y. lipolytica is a potential microbial chassis for PET biodegradation. Other research expressed PETase in Y. lipolytica Po1f with a signal peptide from lipase and confirmed that the engineered strain could hydrolyze BHET and PET powder into the monomers [106]. Surface display systems and whole-cell biocatalysts provide novel ideas and strategies for achieving the high efficiency expression of PET hydrolases and promoting PET biodegradation [77,107,108]. Yeasts, together with efficient genetic tools, have been used as great microbial chassis for biodegradation and bioconversion [109].
2.2.3. Marine Microalgae
At present, the existing native and engineered microbial chassis that are capable of producing PET hydrolases are usually difficult to adapt to the complexity of the marine environment and produce much PET waste. Recently, some marine microalgae have been used as chassis for PET biodegradation [110]. A photosynthetic microalga Phaeodactylum tricornutum has been reported as being engineered as a chassis capable of secreting a PETase mutant into the culture medium, and the recombinant PETase was able to efficiently degrade different substrates, including PET films, poly (ethylene terephthalateco-1,4-cylclohexylenedimethylene terephthalate) (PETG) film, and shredded PET, at 30 °C or even at mesophilic temperatures (21 °C) [87]. Additionally, Chlamydomonas reinhardtii, the green algae, was also successfully engineered to produce PETase with degrading activity, and the chemical and morphological changes appeared on the PET films after 4 weeks of culture [88]. As environmentally friendly chassis for the biodegradation of PET waste in a saltwater-based environment, marine microalgae have the potential for future biotechnological applications in the degradation of PET polluted seawater [87].
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms10010039
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