The four classes of PHA synthases
1.1. Environmental problems caused by petroleum-based plastics
The last half century has witnessed the development of synthetic plastics from petroleum resources, and more than 300 million tons of synthetic plastics are annually produced at present. The recently increased consumption of petroleum resources has led to environmental problems such as a depletion of the resources as well as a global warming due to a marked increase of atmospheric CO2 level. In addition to these problems, wasted plastics used in short-term applications may cause the environmental damage to a wide variety of wild animals including terrestrial, aquatic animals and birds. Furthermore, it has been suggested that even the wasted plastics in the form of small particles potentially induce the alteration of pelagic ecosystems . Therefore, the development of environmentally sound alternatives, such as bioplastics, to conventional petroleum-based plastics is urgently desired to sustain the environment [2-4].
Bioplastics include biodegradable and bio-based plastics (Figure 1) [5, 6]. The former are produced from renewable or petroleum resources via biological or chemical processes, and degraded by enzymes and microorganisms in natural environment. The latter are synthesized from renewable resources via biological or chemical processes, and some of them show non-biodegradability although bio-based plastics are generally biodegradable. Poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES) and poly(butylene succinate) (PBS) are synthesized from petroleum resources via chemical processes, but they show an excellent biodegradability. Currently, cost-effective processes for the production of succinic acid and 1,4-butanediol, raw materials of PBS, from biomass resources are being developed. Meanwhile, poly(ethylene) (PE) and poly(propylene) (PP) are chemically synthesized from their monomers derived from biological sources, but they are not biodegradable. Poly(hydroxyalkanoate)s (PHAs) and poly(lactide) (PLA) show an excellent biodegradability, and are produced from renewable resources via biological and chemical processes, respectively. Thus, the bio-based bioplastics having biodegradability, such as PHAs and PLA, are the most favorable bioplastics to avoid the above-mentioned problems associated with the use of petrochemical-based synthetic plastics.
PHAs are the only bioplastics completely synthesized from renewable resources by a wide variety of microorganisms in soil, active sludge, marine and extreme environments [7, 8]. In the cells, PHAs form amorphous granules and is degraded by intracellular PHA depolymerases (i-PHA depolymerases) produced by the PHA-accumulating bacterium itself. In contrast, after PHAs are extracted from the cells, PHAs are converted to semicrystalline form and is degraded by extracellular PHA depolymerases (e-PHA depolymerases) secreted from microorganisms in natural environments, such as soil, active sludge, fresh water, and seawater [9, 10].
Many bacteria can synthesize various types of PHAs containing 3-, 4-, and 5-hydroxyalkanoate units, and over 150 different hydroxyalkanoates other than 3-hydroxybutyrate have been reported as constitutive units of PHAs . PHAs consisting of short-chain hydroxyalkanoates (SCL-HAs; 3–5 carbon atoms) or medium-chain hydroxyalkanoates (MCL-HAs; 6–14 carbon atoms) have been detected. The former are thermoplastic in nature, whereas, the latter are elastomeric in nature. The physical and mechanical properties of PHAs can be regulated by varying monomer composition in order to gain properties comparable to petrochemical-based thermoplastics that have been used for various applications in industry, medicine, pharmacy, agriculture, and electronics . Accordingly, PHAs have attracted industrial interest as bio-based, biodegradable, biocompatible, and versatile thermoplastics [13, 14].
PLA is representative bio-based plastics with good processability and transparency that are used in packaging, containers, stationary, etc. . In addition, medical and agricultural uses of the material have been investigated because of their biocompatibility and biodegradability . PLAs are produced from renewable biomass through a chemo-bioprocess consisting of fermentative production of lactic acid (LA) and chemical polymerization. LA is spontaneously polymerized by refluxing, but the molecular mass of yielded polymer tends to be low . There are several methods for synthesizing high-molecular-mass PLAs: condensation, chain elongation, and ring-opening polymerization of cyclic lactides . Currently, the major industrial method to produce PLAs is ring-opening polymerization which is catalyzed by heavy metal catalysts, typically tin [18, 19]. However, the trace residues of the heavy metal catalyst are unfavorable for certain applications, in particular, medical and food applications. Thus, replacement of the heavy metal catalyst with a safe and environmentally acceptable alternative is an important issue. For this purpose, enzymes are attractive targets because they are natural non-harmful catalysts that can drive the reactions under mild conditions. In addition, highly specific enzymatic reactions may be capable of synthesizing polymers with fine structure from crude materials, which would reduce the cost of preparing the starting substances. This could be an advantage over chemical polymerization of LA or lactides, since the chemoprocess requires extremely pure monomers (contamination of carbonic acids is known to inhibit polymerization), along with anhydrous and high temperature conditions to proceed.
In such a situation, LA-polymerizing enzyme (LPE) functioning in replacement of metal catalysts should enable the biosynthesis of PLA, even though it is enormously challenging both in terms of research and industrial implementation. The best solution could be the development of a PLA-producing microorganism introduced with LPE gene, but this has not been reported so far. In 2008, Taguchi et al. nonetheless successfully obtained encouraging results by developing a recombinant
1.3. Toward an enhanced sustainable production
Three main issues have hindered widespread use of PHAs: (1) the high production cost compared to petroleum-based polymers with similar properties; (2) the inability to produce high-performance PHAs in substantial amounts; and (3) the difficulty in controlling the life cycle of PHAs, i.e., the control of their biodegradability and their effective chemical recycling.
To solve the former two issues, we have focused on the genetic engineering of PHAs metabolism, which will lead to the cost-effective biological production of PHAs and the improvement of their properties, such as molecular mass and monomer composition. In particular, protein engineering of PHA synthase can improve both PHA production efficiency and the properties of the generated polymer because PHA synthase plays a central role in PHA biosynthesis . Here we would like to highlight the current special topic on the biosynthesis of new PHA polymers incorporating unusual monomer units such as LA by PHA synthase engineering. Further, gene cloning and expression in plants has created new possibilities of using photosynthesis to convert atmospheric CO2 directly into PHA, in hopes of reducing production cost in the future.
In addition, to solve the latter issue, we have also focused on the engineering of PHB depolymerases. PHB is the most common form of PHAs. In natural environment, the microbial and enzymatic degradation of PHB is an important first step in the PHB recycling process. However, PHB degradation depends on the surrounding conditions and proceeds on the order of a few months in anaerobic sewage or a few years in seawater . Such PHB degradation process is undesirable from the standpoint of the efficient use of biomass resources. To overcome this issue, chemical recycling using spent PHB materials as recyclable monomer-concentrated resources is rapidly gaining importance due to its high degradation rate . In addition, as chemical recycling is cost-efficient and has low CO2 emissions, it has great potential as a low-cost and environmentally compatible process. PHB monomerization, the first step in chemical recycling, is currently carried out via a thermal decomposition process. However, this chemical recycling method presents some drawbacks, such as racemization of the decomposed products, high reaction temperature, and contamination with residual metal catalysts [28-31]. As one of the solutions, the development of alternative PHB monomerization methods that use such enzymes as PHB depolymerases is highly awaited because those methods do not produce undesirable byproducts, have high enantio- and regioselectivities, and can be performed at moderate temperatures [32, 33]. Moreover, as the efficient use of biocatalysts requires suitable enzymes with high activity and stability under process conditions, the desired substrate selectivity, and high enantioselectivity, the improvement of PHB depolymerases is expected to result in the construction of an effective PHB chemical recycling system. In this chapter we will also provide some case studies on protein engineering of PHB depolymerase based on domain structure-based and random mutagenesis approaches.
2. Protein engineering of PHA synthases
2.1. Biochemical properties and engineering concepts of PHA synthases
PHA synthases catalyze the polymerization reaction of hydroxyalkanoate (HA) to polymer PHA. The monomer substrates of PHA synthase are mainly 3HA-CoAs with various side-chain lengths, and only
|I||PhaC||C3 - C5|
C3 – C7
|II||PhaC||C6 – C14|
C3 – C12
|III||PhaC - PhaE||C3 – C5|
|IV||PhaC - PhaR|
Type I and type II PHA synthases consist of single subunits (PhaC). Type I PHA synthases, represented by
The lack of a suitable structural model for any PHA synthase has limited attempts to improve the activity and to alter the substrate specificity of these enzymes in “irrational” manners, such as random mutagenesis and gene shuffling [36, 37]. Generally, natural diversity provides us with attractive starting materials for artificial evolution as it represents functionalized sequence spaces to some extent. A tremendous population (over 60 species) of randomly screened PHA producing bacteria suggests that attractive prototype enzymes for molecular breeding would exist. Among them, enzyme evolution approach has been applied to the following type I and type II PHA synthases derived from some bacteria.
2.2. Activity improvement and substrate specificity alteration of PHA synthases
2.2.1. Application to type I PHA synthases
A pioneering study that established methods for protein engineering PHA synthase initiated in 2001 using the best-studied enzyme, the
As a next case, screened beneficial mutation, Gly4Asp (G4D), exhibited higher levels of protein accumulation and PHB production compared to the recombinant
Junction site for interconnection of heterogeneous enzymes based on the predicted secondary structures allowed chimeragenesis of the PHA synthase from
2.2.2. Application to type II PHA synthases
Contrasted with the type I PHA synthases, type II PHA synthases typically have substrate specificity towards MCL-3HA-CoA substrates but relatively poor substrate specificity towards SCL-3HA-CoA substrates like 3HB-CoA. An exception to this is the type II PHA synthase of
The findings obtained in these studies for the type II PHA synthase would be very useful for evaluating a similar evolution strategy to the other types of PHA synthases based on the amino acid sequence alignment of the PHA synthases. For example, position 481 in PhaC1 PHA synthase from
The other two beneficial positions, Glu130  and Ser477 , were also identified through the
2.3. Engineering of lactate-polymerizing enzyme (LPE) from PHA synthases
The pioneering work on the exploration of LA-polymerizing activity by PHA synthases was reported by Valentin et al. . In that attempt, the PLA biosynthesis was carried out by monitoring the activity of PHA synthases towards synthetic LA-CoAs (
In this context, Taguchi et al. formally reported the first prototype LPE in the year 2008 as a PHA synthase with an acquired LA-polymerizing activity through
The finding that PhaC1Ps(STQK) could polymerize LA was a demonstration of evolutionary engineering as a powerful tool for the generation of biocatalysts with desired properties. By demonstrating the
In a subsequent study, based on the improved activity of a point mutation at position 420 (F420S) of a type I PHA synthase (PhaCRe) from
Regarding the reports on LPE, the following several studies have been published [63, 64]. Currently, the best-studied PHA synthase from
3. Protein engineering of PHB depolymerase
3.1. Biochemical and genetic properties of PHB depolymerases
A number of PHA depolymerases have been purified from diverse PHA-degrading microorganisms and characterized [9, 10, 12]. As described earlier, depending on the substrates and localization of PHA depolymerases, PHA depolymerases are grouped generally into four families: PHA depolymerases degrading the native intracellular granules (i-PHAMCL depolymerases and i-PHASCL (i-PHB) depolymerases) and PHA depolymerases degrading the denatured extracellular PHA granules (e-PHAMCL depolymerases and e-PHASCL (e-PHB) depolymerases). To date, the genes of about 30 PHA depolymerases with experimentally verified PHA depolymerase activity have been identified. On the basis of their sequence similarity, the PHA Depolymerase Engineering Database has been established as a tool for systematic analysis of PHA depolymerase family .
Among the PHA depolymerases, multi-domain e-PHB depolymerases have been extensively examined . The multi-domain e-PHB depolymerases generally have a domain structure consisting of a catalytic domain (CD) at N-terminus, a substrate-binding domain (SBD) at C-terminus, and a linker region connecting the two domains, while e-PHB depolymerases from
From a biological viewpoint, the structure-function relationship of maluti-domain e-PHB depolymerases has been studied extensively, and several mutants were designed to analyze the function of each domain, in particular, SBD. Using a truncated multi-domain e-PHB depolymerases, Behrends et al., Nojiri and Saito, and our group revealed that the C-terminal domain is essential for PHB-specific binding [74-76]. Further, Nojiri and Saito genetically prepared many mutants of PhaZRpiT1 in various forms such as inversions, chimeras, and fusion to extra linker domains, and demonstrated that its SBD organization also influences the PHB degradation but not water-soluble substrates. Doi and co-workers prepared fusion proteins of SBDs of several PHB depolymerases with glutathione-S-transferase [77-81], and demonstrated specific interactions based on molecular recognition between SBD and polyester surface.
3.2. Effects of chemical and solid-state structures and surface properties of PHAs on enzymatic degradation
Chemical structures of PHAs have influence on their enzymatic hydrolysis by multi-domain e-PHB depolymerases. Various types of PHAs including racemic PHA [82-89] and 3HA oligomers [90, 91], PHAs with different main- and side-chain lengths (Kasuya et al., 1997) , and random copolymers of (
To investigate the influence of the chemical structure or surface properties of polymer on enzymatic adsorption at nano-level sensitivity, several studies using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) have been performed. Yamashita et al. investigated the PhaZRpiT1 adsorption to the film surface of several polymers including polyethylene, polystyrene and PHA using the QCM technique, and found that the enzyme showed adsorption specificity for PHA [100-102]. In addition, AFM analysis of PhaZRpiT1 on polyester surface has revealed that small ridges are formed around the enzyme molecule due to movement of some polyester chains at the adsorption area, suggesting that a strong chemical interaction exists between the enzyme and the polyester chains [102, 103]. Furthermore, AFM analysis of interaction between PHB single crystal and a hydrolytic-activity-disrupted PhaZRpiT1 mutant has demonstrated that its SBD disturbs the molecular packing of PHB polymer chains, resulting in fragmentation of the PHB single crystal . Taking these findings into consideration, the specific adsorption of PHB depolymerase to the PHB surface probably involves both the adsorption of the enzyme to the surface and the non-hydrolytic disruption of the substrate to promote PHB degradation. Recently, we have developed the AFM technique by using an AFM tip modified with SBD protein to evaluate the interaction between the SBD molecule and the PHB surface at the molecular level. Through this, it has been shown that the adsorption force of one SBD molecule to the PHB surface is approximately 100 pN [105, 106].
3.3. Analysis of polymer binding ability of e-PHB depolymerase using directed evolution technique
The structural aspects of an enzyme generally provide crucial information about the interaction between the enzyme and its ligand. Some researchers have reported the tertiary structures of polymer-degrading enzymes, such as glycoside hydrolases and single-domain e-PHB depolymerases, and proposed an interaction model between the enzymes and the polymer surfaces [68, 107-109]. However, because of the paucity of information about the 3D structures of multi-domain e-PHB depolymerases, there are few insights into which and how amino acid residues in their SBD are involved in the enzyme adsorption to PHB surface.
Directed evolution is a useful and powerful tool to explore, manipulate, and optimize the properties of an enzyme as no information on the tertiary structure of the enzyme is required and new and unexpected beneficial mutations can be discovered [110-112]. Random mutagenesis via error-prone PCR (epPCR) and DNA recombination are widely used approaches to generate a large mutant pool and screen for the desired characteristics [113, 114]. Using those approaches, many enzymes with improved substrate specificity, catalytic activity, thermostability, or solubility were obtained . Further, analysis of the effects of mutations could also provide useful information for the improvement of enzyme function.
To improve e-PHB depolymerases, it is important to understand the mechanisms underlying its adsorption and hydrolysis, such as which and how amino acid residues participate in the catalytic process. To clarify this issue, we have investigated the interaction between PhaZRpiT1 and PHB surface by a combination of PCR random mutagenesis targeted to only SBD and an
Nevertheless, because only little knowledge was obtained on the biochemistry and kinetics of the purified mutant enzymes, the roles of these amino acids (Ser410, Tyr412, Val415, Tyr428, Ser432, Leu441, Tyr443, Ser445, Ala448, Tyr455, and Val457) and their contributions to the enzymatic activity remain poorly understood, resulting in little information to develop e-PHB depolymerases. Among these positions, Leu441, Tyr443, and Ser445 were predicted to form a β-sheet structure to orient in the same direction as shown in Figure 6(B). As polymer-degrading enzymes generally align their amino acid residues in a plane to interact with polymer surfaces, these three residues in PhaZRpiT1 may interact directly with the PHB surface. Since the hydropathy indices of such mutations as L441H (replacement of Leu441 with His), Y443H (replacement of Tyr443 with His), and S445C (replacement of Ser445 with Cys) dramatically changed among the mutations at these positions, their PHB-binding and -degrading properties were examined in detail . Functional analyses of the purified L441H, Y443H, and S445C enzymes indicated that these mutations had no influence on their structures and their ability to cleave the ester bond, while their PHB-degrading activity differed from that of the wild type. Kinetic analysis of PHB degradation by the mutants suggested that the hydrophobic residues at these positions are important for the enzyme adsorption to the PHB surface, and may more effectively disrupt the PHB surface to enhance the hydrolysis of PHB polymer chains than the wild-type enzyme. Further, surface plasmon resonance (SPR) analysis revealed that these substitutions mentioned above altered the association phase rather than the dissociation phase in the enzyme adsorption to the polymer surface.
Recently, Hisano et al. determined the crystal structure of PhaZPfu and proposed that hydrophobic residues, including Tyr, Leu, Ile, and Val, contribute to adsorption to the PHB surface, and that hydrophilic residues (Ser and Asn) located around the mouth of the enzyme crevice may also contribute to the affinity of the enzyme for PHB . Jendrossek group determined PhaZ7Ple crystal structure and demonstrated that the enzyme was enriched in hydrophobic amino acids including eight tyrosine residues . All tyrosine residues (Tyr103, Tyr105, Tyr172, Tyr173, Tyr189, Tyr190, Tyr203, and Tyr204), which are located at the surface of PhaZ7Ple but are far from the active site (Ser136), were changed to alanine or serine and the substitution effects were examined . It turned out that mutation of Tyr105, Tyr189 or Tyr190 resulted in reduced PHB-degrading activity and in occurrence of a lag phase of the depolymerase reaction, indicating that these residues are possibly involved in the enzyme adsorption. Similar results have been obtained for the e-PHAMCL depolymerase of
3.4. Improvement in SBD function of PhaZRpiT1
The above-mentioned findings imply that PHB binding ability of PhaZRpiT1 can be improved by substituting a hydrophilic residue with a hydrophobic one at the positions of 441, 443 and 445. Tyr at position 443 was targeted for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues .
Table 2 shows the hydrophobicity, the potential for β-sheet formation, and the volume of 20 common amino acid residues [122-124]. In this table, the properties of the original amino acid residue are colored blue and the desirable characteristics of the amino acid residues are colored orange, respectively. In the design of a mutant enzyme with an amino acid substitution at this position, the following factors were taken into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. Analysis of the purified Y443F enzyme indicated that the mutation had no influence on the structure and the ester bond cleavage activity, while this mutant had higher PHB degradation activity than the wild type. Thus, this finding supports our previous assumption and indicates the importance of highly hydrophobic residues at these positions for PHB degradation.
3.5. Cell surface display system for protein engineering of PhaZRpiT1
Cell surface display is a valuable technique for the expression of peptides or proteins on the surface of bacteria and yeasts by fusion with the appropriate anchoring motifs . Therefore, the cell surface display of functional and useful peptides and proteins, such as enzymes, receptors, and antigens, has become an increasingly used strategy in various applications, including whole-cell biocatalysts and bioabsorbents, live vaccine development, antibody production, and peptide library screening. In addition, this method is very useful for enzyme library screening because the displayed protein is accessible to the external environment and thus, is able to interact with substrates easily, allowing the screening of large libraries .
A variety of surface anchoring motifs, including outer membrane proteins, lipoproteins, autotransporters, subunits of surface appendages, and S-layer proteins, have been employed to achieve the display systems [125, 127, 128]. We used the OprI anchoring motif for the functional display of PhaZRpiT1 on
3.6. Protein engineering of CD region of PhaZRpiT1 using cell surface display system
In contrast to SBD, there is little knowledge on the CD of PhaZRpiT1, such that which and how amino acid residues in the CD contribute to the enzymatic activity remain poorly understood, and this has resulted in the lack of information for the improvement of the CD function of PhaZRpiT1. The CD of PhaZRpiT1 was targeted for the directed evolution, employing random mutagenesis and DNA recombination to enhance its ester bond cleavage ability (Figure 7) . The mutant genes generated from these reactions were expressed as surface-displayed enzymes, and the mutant enzymes were screened through a high-throughput system using pNPC4, a water-soluble substrate. As a result, clones displaying mutant enzymes with a 4- to 8-fold increase in pNPC4 hydrolysis activity were obtained in comparison with those displaying the wild type. This result was roughly consistent with the results of pNPC4 hydrolysis using purified enzymes with the unfused and undisplayed forms, concluding that the current screening system is feasible and effective for the search of improved enzymes.
As the aliphatic part in pNPC4 is similar to the monomer unit in PHB polymer chain and pNPC4 is generally used as a model substrate, changes in pNPCn hydrolysis rates by the purified mutant enzymes as a function of the chain length of the aliphatic part in
As opposed to pNPCn hydrolysis by the N285D and N285Y mutant enzymes, their PHB degradation rates were slower than that of the wild-type enzyme, indicating that these mutations are unfavorable for PHB degradation. The kinetics of PHB degradation demonstrated that the N285D and N285Y mutations lowered the hydrolysis activity for the PHB polymer chain compared to the wild-type enzyme despite retention of the binding activity for the PHB polymer surface.
3.7. Proposed models of the active site in e-PHB depolymerases
The correct orientation of a PHB polymer chain to the active site is necessary to realize effective PHB degradation by e-PHB depolymerase. Hisano et al. have determined the crystal structures of PhaZPfu-3HB trimer complex as well as PhaZPfu enzyme alone . In the PhaZPfu-3HB trimer complex, 3HB trimer binds to the crevice with its carbonyl terminus towards the catalytic residues (Figure 8(A)). From the structural insight gained from PhaZPfu, they proposed the mechanism of action of PhaZPfu. Figure 8(B) shows the location of the catalytic residues and the interaction between PHB polymer chain and the residues in the subsite of the active site of PhaZPfu. In their model, Ser39 participates in the nucleophilic attack of the carbonyl carbon of a PHB chain, resulting in the formation of a covalent acyl-enzyme intermediate followed by the hydrolysis by an activated water molecule. The nucleophilicity of the hydroxyl group of Ser39 is enhanced by the His155-Asp121 hydrogen bonding system.
For PhaZRpiT1, Bachmann and Seebach proposed that this enzyme has four subsites (2, 1, -1, and -2) in its active site, in which three of the subsites must be occupied by (
This review describes the development of PHA synthases to synthesize the wide variety of custom-made bioplastics as well as PHB depolymerase with higher activity for PHB adsorption or pNPCn hydrolysis.
Bioplastics present a multitude of benefits as substitutes for conventional petroleum-based plastics. Among them, PHAs are one of the desirable alternatives to petrochemical-derived polymers because PHAs are produced directly from renewable resources completely by biological process and can be renewed over a relatively-short time. However, three main issues have hindered widespread use: the high production cost compared to petroleum-based polymers with similar properties; the inability to produce high-performance PHA polymers in substantial amounts; and the difficulty in controlling the life cycle of PHA polymers, i.e., the control of their biodegradability and their efficient recycling. Thus, with the development in recombinant DNA technology and high-throughput screening techniques, protein engineering methods and applications on the improvement of processes of bioplastic production as well as bioplastic degradation are becoming increasingly important and widespread.
The enzyme modification by protein engineering is an increasingly important scientific field. The well-known methods of rational design and directed evolution as well as new techniques including computational design, catalytic antibodies and mRNA display will be crucial for de novo design of enzymes. With recent advances in recombinant DNA technology tools including omics technologies and high-throughput screening facilities, improved methods for protein engineering will be available for easy modification or improvement of more enzymes for further specific applications.
Against such backgrounds, directed evolution of enzymes involved in PHA biosynthesis as well as metabolic engineering approaches of bacterial hosts will become the driving force to establish bioprocesses for the controlled production of PHAs with desired monomer compositions. In addition, systems-level analysis of metabolic, signaling, and regulatory networks is also making it possible to comprehensively understand global biological processes taking place in PHA-accumulating strains. The resultant knowledge will provide new targets and strategies for the improvement of PHA production, including tailor-made PHAs with desired monomer compositions and molecular masses.
Furthermore, from the viewpoint of preserving the ecosystem, bioplastics are most beneficial when they can be actually biodegraded. In order to achieve it, it is vital to elucidate the biodegradation mechanism of bioplastics and engineer their depolymerases. By contrast to PHA synthases, there have been very few protein engineering studies of PHA depolymerases using directed evolutionally methods, resulting in the less information about the improvement of PHA biodegradability as well as PHA depolymerases so far. In addition, as one of the recent trends in green polymer chemistry,
Here, we present the recent approaches of protein engineering with potential for a total recycle system of bioplastics via combination of biological production with biological degradation. In the future, custom-made prominent enzymes generated via evolutionary engineering will be utilized extensively to create high-performance bioplastics from renewable resources in various organisms and applied to effective and eco-friendly chemical recycling of bioplastics.