**Experimental Molecular Archeology: Reconstruction of Ancestral Mutants and Evolutionary History of Proteins as a New Approach in Protein Engineering**

Tomohisa Ogawa and Tsuyoshi Shirai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56255

## **1. Introduction**

using sensitive oligonucleotide microarray systems. Biochem Biophys Res Commun

[88] Horisawa K, Doi N, Takashima H, Yanagawa H. Application of quantitative realtime PCR for monitoring the process of enrichment of clones on *in vitro* protein selec‐

[89] Horisawa K, Doi N, Yanagawa H. Use of cDNA tiling arrays for identifying protein interactions selected by *in vitro* display technologies. PLoS ONE. 2008;3:e1646.

[90] Chinenov Y, Kerppola TK Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 2001;20:2438-2452.

[91] Campbell NA. Biology, 4th Ed. New York: The Benjamin/Cummings Publishing;

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110 Protein Engineering - Technology and Application

1996

tion. J Biochem 2005;137:121-124.

The diversity of life on Earth is the result of perpetual evolutionary processes beginning at life's origins; evolution is the fundamental development strategy of life. Today, studies of gene and protein sequences, including various genome-sequencing projects, provide insight into these evolutionary processes and events. However, the sequence data obtained is restricted to extant genes and proteins, with the exception of the rare fossil genome samples [1, 2], for example Neanderthal [3], archaic hominin in Siberia [4, 5], and ancient elephants such as mastodon and mammoth [6]. The fossil record, and genome sequences derived from it, has the potential to elucidate ancient, extinct forms of life, acting as missing links to fill evolution‐ ary gaps; however, the sequenced fossil genome is very limited, mainly due to the condition of samples and the challenges of preparing them. Discovering the forms of ancient organisms is one of the major purposes of paleontology, and is valuable in understanding of current life forms as these will be a reflection of their evolutionary history. However, the reconstruction of a living organism from fossils, which would be the ultimate paleontological methodology, is far beyond the currently available technologies, although there has recently been a report of the production of an artificial bacterial cell, using a chemically synthesized genome [7].

Meanwhile, for genes or the proteins they encode, it is already feasible to reconstruct their ancestral forms using phylogenetic trees constructed from sequence data; these techniques may well provide clues to the evolutionary history of certain extant genes and proteins with respect to their ancestors. Although phylogenetic analyses alone, or in combination with protein structure simulations, are useful to analyze structure-function relationships and evolutionary history [8], resurrected ancient recombinant proteins have the potential to

provide more direct observations. Production of ancestral or ancient proteins can be achieved comparably easily due to developments in molecular biology and protein engineering techniques, which allow nucleotide or amino acid sequences to be synthesized. Ancestral proteins can be tested in the laboratory using biochemical or biophysical methods, for their activity, stability, specificity, and even three-dimensional structure. Thus, ancestral sequence reconstruction (ASR) has proved a useful experimental tool for studying the diverse structure and function of proteins [9]. To date, such 'experimental molecular archeology' using ASR has been applied to several enzymes [10-24], including photo-reactive proteins [25-37], nuclear receptor and transmembrane proteins [38-48], lectins [49-52], viral proteins [53, 54], elongation factor [55-57], paralbmin [58], in addition to a number of peptides [59,60] (Table 1).

In early studies, ASR experiments using the technique of molecular phylogeny were based on basic site-directed mutagenesis and used to investigate the functional evolution of proteins, including the convergent evolution of lysozyme in ruminant stomach environments and the adaptation of enzymes to alkaline conditions [10-12]. However, if ancestral sequences have been determined, the most straightforward method is to reconstruct the full-length ancestral protein in the laboratory. No fundamental differences exist between ancestor reconstruction and standard site-directed mutagenesis, other than the number of amino acids residues requiring mutation, which, in the case of ancestor reconstruction, might be spread over the entire sequence. At present, ASR can be achieved using commercially available *de novo* synthetic genes. Thus, 'experimental molecular archeology' by ancestral protein reconstruc‐ tion using a combination of the technical developments in biochemistry, molecular biology, and bioinformatics can be exploited in both molecular evolutionary biology and protein engineering. In this chapter, we will provide an overview the experimental molecular arche‐ ology technique of ASR, and the case of ancestral fish galectins will be discussed in detail, based on our recent studies.

## **2. The early studies: Reconstruction of partial ancestors by site-directed mutagenesis**

The first studies exploiting the idea of ancestral protein reconstruction used site-direct mutagenesis, in which a small number of amino acids were substituted to produce the anticipated ancestral status. These studies include the reconstruction of a ribonuclease (RNase) of an extinct bovid ruminant [10, 11], and the lysozymes from a game-bird using ancestral lysozyme reconstructions predicted by the MP (Maximum Parsimony) method [12]. Benner and colleagues reconstructed RNase of an extinct bovid ruminant [10], by predicting four sequences of ancestral RNases from five closely related bovids including ox, swamp buffalo, river buffalo, nilgai, and the primitive artiodactyl using the MP method [61, 62]. The ancestor closest to the extant ox protein was selected from the four probable ancestors as the target of the experiment as it contained a mutation of amino acid residue 35, located close to Lys41, which is known to be important for catalysis. Three ancestral mutants of the ox RNase (A19S, L35M, and A19S/L35M) were examined for their kinetic properties and the thermal stabilities against tryptic digestion. However, no significant difference was found between the ancestral

#### Experimental Molecular Archeology: Reconstruction of Ancestral Mutants and Evolutionary History of Proteins as a New Approach in Protein Engineering http://dx.doi.org/10.5772/56255 113


ML: Maximum likelihood/bayesian, MP: Maximum Parsimony

provide more direct observations. Production of ancestral or ancient proteins can be achieved comparably easily due to developments in molecular biology and protein engineering techniques, which allow nucleotide or amino acid sequences to be synthesized. Ancestral proteins can be tested in the laboratory using biochemical or biophysical methods, for their activity, stability, specificity, and even three-dimensional structure. Thus, ancestral sequence reconstruction (ASR) has proved a useful experimental tool for studying the diverse structure and function of proteins [9]. To date, such 'experimental molecular archeology' using ASR has been applied to several enzymes [10-24], including photo-reactive proteins [25-37], nuclear receptor and transmembrane proteins [38-48], lectins [49-52], viral proteins [53, 54], elongation

factor [55-57], paralbmin [58], in addition to a number of peptides [59,60] (Table 1).

based on our recent studies.

112 Protein Engineering - Technology and Application

**mutagenesis**

In early studies, ASR experiments using the technique of molecular phylogeny were based on basic site-directed mutagenesis and used to investigate the functional evolution of proteins, including the convergent evolution of lysozyme in ruminant stomach environments and the adaptation of enzymes to alkaline conditions [10-12]. However, if ancestral sequences have been determined, the most straightforward method is to reconstruct the full-length ancestral protein in the laboratory. No fundamental differences exist between ancestor reconstruction and standard site-directed mutagenesis, other than the number of amino acids residues requiring mutation, which, in the case of ancestor reconstruction, might be spread over the entire sequence. At present, ASR can be achieved using commercially available *de novo* synthetic genes. Thus, 'experimental molecular archeology' by ancestral protein reconstruc‐ tion using a combination of the technical developments in biochemistry, molecular biology, and bioinformatics can be exploited in both molecular evolutionary biology and protein engineering. In this chapter, we will provide an overview the experimental molecular arche‐ ology technique of ASR, and the case of ancestral fish galectins will be discussed in detail,

**2. The early studies: Reconstruction of partial ancestors by site-directed**

The first studies exploiting the idea of ancestral protein reconstruction used site-direct mutagenesis, in which a small number of amino acids were substituted to produce the anticipated ancestral status. These studies include the reconstruction of a ribonuclease (RNase) of an extinct bovid ruminant [10, 11], and the lysozymes from a game-bird using ancestral lysozyme reconstructions predicted by the MP (Maximum Parsimony) method [12]. Benner and colleagues reconstructed RNase of an extinct bovid ruminant [10], by predicting four sequences of ancestral RNases from five closely related bovids including ox, swamp buffalo, river buffalo, nilgai, and the primitive artiodactyl using the MP method [61, 62]. The ancestor closest to the extant ox protein was selected from the four probable ancestors as the target of the experiment as it contained a mutation of amino acid residue 35, located close to Lys41, which is known to be important for catalysis. Three ancestral mutants of the ox RNase (A19S, L35M, and A19S/L35M) were examined for their kinetic properties and the thermal stabilities against tryptic digestion. However, no significant difference was found between the ancestral

**Table 1.** The experimental molecular archeology analysis using ancestral proteins

mutants and the modern ox RNase. The results suggested that these amino acid substitutions were evolutionarily neutral, although this conclusion is limited to the extent of the examined properties [11].

Malcolm et al. succeeded in identifying a non-neutral evolutionary pathway of game-bird lysozymes using ancestral lysozyme reconstructions predicted by the MP method [12]. Seven mutations in game-bird lysozyme proteins included combinations of residues Thr40, Ile55, and Ser91, which were anticipated to be Ser40, Val55 and Thr91, respectively, in ancestral molecules. The mutants were synthesized as possible intermediates in the evolutionary pathway of bird lysozyme and comparative molecular properties and crystal structures of these revealed that the thermostabilities of the proteins were correlated with the bulkiness of their side chains. The T40S mutant increased its thermostability by more than 3°C, allowing the conclusion that this mutation was non-neutral effect of natural selection.

Yamagishi and colleagues used ancestral protein reconstruction [14-16] to obtain direct evidence for the hypothesis that the common ancestor of all organisms was hyper-thermophilic [63]. Because the catalytic activities of 3-isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase (ICDH) are similar to one another and their three-dimensional structures conserved, these proteins are diverged from an ancient common ancestor [64], of which sequence was inferred from a phylogenetic tree constructed from IPMDH and ICDH sequences from various species, including the thermophile (*Thermus thermophilus*) and the extreme thermophile (*Sulfolobus* sp. strain 7). Five of the seven ancestral mutants, in which substituted amino acids were located close to the substrate and cofactor-binding sites, demonstrated higher thermostability than wild type IPMDH from *Sulfolobus* sp. strain 7. These findings were taken to support the hypothesis of a hyperthermophile common ancestor. Moreover, the successful thermostabilization of ICDH [17] and Glycyl-tRNA synthetase [22] by ASR has been reported. Thus, the incorporation of ancestral residues into a modern protein can be used not only to test evolutionary hypotheses, but also as a powerful protein engineer‐ ing technique for protein thermostabilization.

Recently, Whittington and Moerland reported that ASR analysis of parvalbumins (PVs) was able to identify the set of substitutions most likely to have caused a significant shift in PV function during the evolution of *Antarctic notothenioids* in the frigid waters of the Southern Ocean [58]. The results suggest that the current thermal phenotype of Antarctic PVs can be recapitulated by only two amino acid substitutions, namely, K8N and K26N.

These studies were performed by introducing a limited number of mutations into extant proteins, or by carefully selecting ancestors that were separated from an extant protein by only few substitutions. However, such ancestral reconstruction by site-directed mutagenesis appears to be incomplete, as the possibility that sites remaining in a non-ancestral state may significantly affect the molecular property of interest, cannot be ruled out. Although it is difficult and expensive to introduce many mutations into sites widely distributed over gene sequences by site-directed mutagenesis, *de novo* gene synthesis is now available, allowing preparation of ancestral proteins. Therefore, the majority of recent ASR studies have been conducted using full-length or partial ancestral sequence reconstruction, including substitu‐ tion of corresponding sites in target proteins.

## **3. Methods for ancestral sequence prediction**

mutants and the modern ox RNase. The results suggested that these amino acid substitutions were evolutionarily neutral, although this conclusion is limited to the extent of the examined

Malcolm et al. succeeded in identifying a non-neutral evolutionary pathway of game-bird lysozymes using ancestral lysozyme reconstructions predicted by the MP method [12]. Seven mutations in game-bird lysozyme proteins included combinations of residues Thr40, Ile55, and Ser91, which were anticipated to be Ser40, Val55 and Thr91, respectively, in ancestral molecules. The mutants were synthesized as possible intermediates in the evolutionary pathway of bird lysozyme and comparative molecular properties and crystal structures of these revealed that the thermostabilities of the proteins were correlated with the bulkiness of their side chains. The T40S mutant increased its thermostability by more than 3°C, allowing

Yamagishi and colleagues used ancestral protein reconstruction [14-16] to obtain direct evidence for the hypothesis that the common ancestor of all organisms was hyper-thermophilic [63]. Because the catalytic activities of 3-isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase (ICDH) are similar to one another and their three-dimensional structures conserved, these proteins are diverged from an ancient common ancestor [64], of which sequence was inferred from a phylogenetic tree constructed from IPMDH and ICDH sequences from various species, including the thermophile (*Thermus thermophilus*) and the extreme thermophile (*Sulfolobus* sp. strain 7). Five of the seven ancestral mutants, in which substituted amino acids were located close to the substrate and cofactor-binding sites, demonstrated higher thermostability than wild type IPMDH from *Sulfolobus* sp. strain 7. These findings were taken to support the hypothesis of a hyperthermophile common ancestor. Moreover, the successful thermostabilization of ICDH [17] and Glycyl-tRNA synthetase [22] by ASR has been reported. Thus, the incorporation of ancestral residues into a modern protein can be used not only to test evolutionary hypotheses, but also as a powerful protein engineer‐

Recently, Whittington and Moerland reported that ASR analysis of parvalbumins (PVs) was able to identify the set of substitutions most likely to have caused a significant shift in PV function during the evolution of *Antarctic notothenioids* in the frigid waters of the Southern Ocean [58]. The results suggest that the current thermal phenotype of Antarctic PVs can be

These studies were performed by introducing a limited number of mutations into extant proteins, or by carefully selecting ancestors that were separated from an extant protein by only few substitutions. However, such ancestral reconstruction by site-directed mutagenesis appears to be incomplete, as the possibility that sites remaining in a non-ancestral state may significantly affect the molecular property of interest, cannot be ruled out. Although it is difficult and expensive to introduce many mutations into sites widely distributed over gene sequences by site-directed mutagenesis, *de novo* gene synthesis is now available, allowing preparation of ancestral proteins. Therefore, the majority of recent ASR studies have been conducted using full-length or partial ancestral sequence reconstruction, including substitu‐

recapitulated by only two amino acid substitutions, namely, K8N and K26N.

the conclusion that this mutation was non-neutral effect of natural selection.

ing technique for protein thermostabilization.

tion of corresponding sites in target proteins.

properties [11].

114 Protein Engineering - Technology and Application

How can we determine the sequences of ancestral proteins or genes? In most cases, since the ancestral genes do not currently exist, the ancestral sequences need to be estimated and reconstructed mainly *in silico* (using a computer). Ancestral sequences are calculated using computational methods originally developed for molecular phylogeny construction. Some of these methods, such as maximum parsimony (MP) and maximum likelihood (ML), have an integral procedure of ancestral sequence inference at each node of the phylogenetic tree under construction [65, 66]. The MP method assumes that a phylogenetic tree with minimum substitutions is the most likely. This method assigns a possible nucleotide/amino acid for each site at every node of a phylogenetic tree to evaluate the minimum substitution number. Because of this assumption of parsimony, the MP method tends to underestimate the number of substitutions if a branch is relatively long. The method is also fragile if the evolutionary rate varies among branches.

By contrast, the ML method, which does not require this assumption, is currently more widely used. This method evaluates the posterior probability of a nucleotide/amino acid res‐ idue at each node of a phylogenetic tree, based on empirical Bayesian statistics, using the provided sequences and a substitution probability matrix as inputs (observations). There‐ fore, results can be significantly affected by the choice of input sequences and the choice of substitution probability matrix; the probability of a reconstructed sequence at a node might be low when the node is connected to the provided sequences through longer and/or more intervening branches. The ML method is popular in the field, largely owing to the presence of the excellent software package PAML [67]. Several other software applications have been also developed for this purpose, such as FastML [68], ANSESCON [69], and GASP [70]. With the exception of GASP which partly employs the MP method to enable ancestral state prediction at gapped sites in a sequence alignment, these applications are based on the ML method. In many cases, ancestral sequences cannot be unambiguously determined, and sev‐ eral amino acids might be assigned to a residue site with almost equal probabilities. To avoid false conclusions as a result of such ambiguity, the accuracy of reconstructed ancestral sequence is critical for such studies. However, it is often difficult to obtain a complete, high‐ ly accurate sequence, as molecular evolution is believed to be a highly stochastic process and there is no guarantee that ancestral sequences can be identified without errors. Even if each residue of a protein made up of 100 residues, is identified with posterior probability of 0.99 (ie. 99% are expected to be correct), the probability that the sequence as a whole is accu‐ rate is only ~0.37 (i.e., 0.99100). In many actual cases, site probabilities are likely to be much lower. This is a major problem in ancestor reconstruction studies, and considerable efforts have been made to avoid incorrect conclusions due to imperfect reconstruction.

Williams et al reported the assessment of the accuracy of ancestral protein reconstruction by MP, ML and Bayesian inference (BI) methods [71]. Their results indicated that MP and ML methods, which reconstruct "best guess" amino acids at each position, overestimate thermo‐ stability, while the BI method, which sometimes chooses less-probable residues from the posterior probability distribution, does not. ML and MP tend to eliminate variants at a posi‐ tion that are slightly detrimental to structural stability, simply because such detrimental var‐ iants are less frequent. Thus, Williams et al caution that ancestral reconstruction studies require greater care to come to credible conclusions regarding functional evolution [71]. Thornton and colleagues also examined simulation-based experiments, under both simpli‐ fied and empirically derived conditions, to compare the accuracy of ASR carried out using ML and Bayesian approaches [72]. They showed that incorporating phylogenetic uncertain‐ ty by integrating over topologies very rarely changes the inferred ancestral state and does not improve the accuracy of the reconstructed ancestral sequence, suggesting that ML can produce accurate ASRs, even in the face of phylogenetic uncertainty, and using Bayesian in‐ tegration to incorporate the uncertainty is neither necessary nor beneficial [72].

In the case for experimental molecular archeology using ASR, the effects of equally probable residues at unreliable sites have been tested by site-directed mutagenesis to confirm directly that molecular properties are not largely affected by these. Indeed, in the case of ancestral congerin genes, the single mutant Con-anc'-N28K, in which the suspicious site was replaced with the alternate suggested amino acid was reconstructed in addition to the ancestral con‐ gerin (Con-anc', the last common ancestor of ConI and ConII) inferred from the phylogeny of extant galectins using the ML method based on DNA sequences [51]. Nucleotide sequen‐ ces were retrieved from the DDBJ database [73] , and the ancestral sequence were inferred using the PAML program [67]. The alignment of amino acid sequences of the extant galec‐ tins was first prepared using the XCED program [74], and an alignment of the correspond‐ ing nucleotide sequences was made in accordance with the amino acid sequence alignment. Tree topology was based on the amino acid sequences of extant proteins using the neighborjoining (NJ) method. PAML was applied to the phylogeny and alignment to infer the ances‐ tral sequences. The F1X4 matrix was used as the codon substitution model with the universal codon table. The free *dN*/*dS* ratio with M8 (beta & omega) model was adapted [75]. The reproduction rate of each Con-anc' amino acid residue was also calculated from the re‐ constructed sequences, with the exclusion of one extant gene in each case, in order to identi‐ fy highly unstable sites depending on the choice of extant genes. The results indicated that the average reproduction rate over the sequence was 0.98. The average site posterior proba‐ bility in the sequence of Con-anc' was 0.81. Seventy-two of 135 sites (53%) had a posterior probability > 0.9. By contrast, 11 sites were found to have posterior probabilities < 0.5. Only one residue, Asn28 of Con-anc', was reproduced with a distinguishably low rate of 0.286, with a suggested alternative amino acid of Lys. Therefore, the single mutant Con-anc'-N28K was also reconstructed. Several reconstruction tests demonstrated that the ancestral se‐ quence had constantly converged into that of Con-anc', and the expected shift by adding a newly found extent sequence was reduced to 1.4% (s.d. 3.2%).

In the case of alcohol dehydrogenase (Adh) ancestral mutants reported by Thomson et al., the posterior probability of the sequence predicted by the ML method was found to be low at three sites. Amino acid residues 168, 211 and 236 of Adh had two (Met and Arg), three (Lys, Arg and Thr), and two (Asp and Asn) equally probable candidates as the ancestral res‐ idues, respectively. Therefore, all possible combinations (2 x 3 x 2 = 12) of the candidates at the ambiguous sites were reproduced, and their kinetic properties assessed [13]. The results confirmed with consistency among the alternative mutants that acetaldehyde metabolism was the original function of Adh, that ancestral yeast could not consume ethanol, and that the function of ethanol metabolism was most likely acquired in the linage of the Adh2 locus after gene duplication.

## **4. Reconstruction of full-length ancestral proteins: Selective adaptive evolution of Conger eel galectins**

require greater care to come to credible conclusions regarding functional evolution [71]. Thornton and colleagues also examined simulation-based experiments, under both simpli‐ fied and empirically derived conditions, to compare the accuracy of ASR carried out using ML and Bayesian approaches [72]. They showed that incorporating phylogenetic uncertain‐ ty by integrating over topologies very rarely changes the inferred ancestral state and does not improve the accuracy of the reconstructed ancestral sequence, suggesting that ML can produce accurate ASRs, even in the face of phylogenetic uncertainty, and using Bayesian in‐

In the case for experimental molecular archeology using ASR, the effects of equally probable residues at unreliable sites have been tested by site-directed mutagenesis to confirm directly that molecular properties are not largely affected by these. Indeed, in the case of ancestral congerin genes, the single mutant Con-anc'-N28K, in which the suspicious site was replaced with the alternate suggested amino acid was reconstructed in addition to the ancestral con‐ gerin (Con-anc', the last common ancestor of ConI and ConII) inferred from the phylogeny of extant galectins using the ML method based on DNA sequences [51]. Nucleotide sequen‐ ces were retrieved from the DDBJ database [73] , and the ancestral sequence were inferred using the PAML program [67]. The alignment of amino acid sequences of the extant galec‐ tins was first prepared using the XCED program [74], and an alignment of the correspond‐ ing nucleotide sequences was made in accordance with the amino acid sequence alignment. Tree topology was based on the amino acid sequences of extant proteins using the neighborjoining (NJ) method. PAML was applied to the phylogeny and alignment to infer the ances‐ tral sequences. The F1X4 matrix was used as the codon substitution model with the universal codon table. The free *dN*/*dS* ratio with M8 (beta & omega) model was adapted [75]. The reproduction rate of each Con-anc' amino acid residue was also calculated from the re‐ constructed sequences, with the exclusion of one extant gene in each case, in order to identi‐ fy highly unstable sites depending on the choice of extant genes. The results indicated that the average reproduction rate over the sequence was 0.98. The average site posterior proba‐ bility in the sequence of Con-anc' was 0.81. Seventy-two of 135 sites (53%) had a posterior probability > 0.9. By contrast, 11 sites were found to have posterior probabilities < 0.5. Only one residue, Asn28 of Con-anc', was reproduced with a distinguishably low rate of 0.286, with a suggested alternative amino acid of Lys. Therefore, the single mutant Con-anc'-N28K was also reconstructed. Several reconstruction tests demonstrated that the ancestral se‐ quence had constantly converged into that of Con-anc', and the expected shift by adding a

In the case of alcohol dehydrogenase (Adh) ancestral mutants reported by Thomson et al., the posterior probability of the sequence predicted by the ML method was found to be low at three sites. Amino acid residues 168, 211 and 236 of Adh had two (Met and Arg), three (Lys, Arg and Thr), and two (Asp and Asn) equally probable candidates as the ancestral res‐ idues, respectively. Therefore, all possible combinations (2 x 3 x 2 = 12) of the candidates at the ambiguous sites were reproduced, and their kinetic properties assessed [13]. The results confirmed with consistency among the alternative mutants that acetaldehyde metabolism was the original function of Adh, that ancestral yeast could not consume ethanol, and that the function of ethanol metabolism was most likely acquired in the linage of the Adh2 locus

tegration to incorporate the uncertainty is neither necessary nor beneficial [72].

116 Protein Engineering - Technology and Application

newly found extent sequence was reduced to 1.4% (s.d. 3.2%).

after gene duplication.

Conger eel galectins, termed Congerins I and II (Con I and Con II), function as biodefense molecules in the skin mucus and frontier organs including the epidermal club cells of the skin, wall of the oral cavity, pharynx, esophagus, and gills [76-79]. Con I and Con II are prototype galectins, composed of subunits containing 135 and 136 amino acids, respectively, and display 48% amino acid sequence identity [80]. While both Con I and Con II form 2-fold symmetric homodimers with 5- and 6-stranded β-sheets (termed a jellyroll motif), they have different stabilities and carbohydrate-binding specificities, although they do have the conserved carbohydrate recognition domain (CRD) common to other galectins [81-84]. Previous studies of Con I and Con II, based on molecular evolutionary and X-ray crystallography analyses, revealed that these proteins have evolved via accelerated substitutions under natural selection pressure [74-85].

To understand the rapid adaptive differentiation of congerins, experimental molecular archaeology analysis, using the reconstructed ancestral congerins, Con-anc and Con-anc', and their mutants has been conducted [49-51]. Since the ancestral sequences of congerin, Con-anc and Con-anc', were estimated from different phylogenetic trees, which were constructed from the varying numbers of extant genes available (eight for Con-anc, and sixteen for Con-anc') (Fig. 1A), the ancestral sequence Con-anc' showed a 27% discrepancy from the previously inferred sequence of Con-anc (Fig. 1B). Furthermore, as described in the 'Methods for Ancestral Sequence Prediction' section, the reproduction rate of each Con-anc' amino acid residue was examined for the reconstructed sequences, with one extant gene excluded for each estimation, in order to identify highly unstable sites. The result indicated that the average reproduction rate over the sequences were 0.98, and only one residue, Asn28 of Con-anc', was reproduced with a distinguishably low rate of 0.286, prompting verification of the results by the construc‐ tion of a single mutant Con-anc'-N28K. The revised ancestral congerins, Con-anc' or Con-anc'- N28K, were attached to the nodes of extant proteins with zero distance in the phylogeny constructed from amino acid sequences, indicating that the sequence was appropriate for that of an ancestor (Fig. 1A). On the other hand, the previously inferred Con-anc was attached midway on the ConI branch. Therefore, Con-anc' or Con-anc'-N28K are likely to be closer to the true common ancestor of ConI and ConII than Con-anc. The structures and molecular properties of congerins, as discussed below, also supported this conclusion.

Although Con-anc is an ancestral mutant located midway on the ConI branch and shares a higher sequence similarity with ConI (76%) than with ConII (61%), it showed unique carbo‐ hydrate-binding activity and properties, and more closely resembled ConII than ConI, in terms of thermostability and carbohydrate recognition specificity, with the exception of carbohy‐ drates containing α2, 3-sialyl galactose, for example GM3 and GD1a. The ancestral congerins, Con-anc' and Con-anc'-N28K, demonstrated similar carbohydrate binding activity and specificities to those of Con-anc [51]. These analyses of Con-anc suggested a functional evolutionary process for ConI, where it evolved from the ancestral congerin to increase its structural stability and sugar-binding activity. In the case of the ancestral congerin, Con-anc, the candidate amino acid residues responsible for the higher structural stability and carbohy‐ drate-binding activity of Con I were reduced to only 31 amino acid residues, from a total of 71 with apparent differences between Con I and Con II. These were mainly located in the N- and C-terminal and loop regions of the molecule, including the CRD [49, 50]. To identify the residues responsible for the properties of Con I, we next performed molecular evolution tracing analysis, by constructing pseudo-ancestral Con-anc proteins focused on the N-terminal, Cterminal, and some loop regions (loops 3, 5 and 6) [50].

<sup>(</sup>A) Phylogeny of extant and ancestral congerins. The tree is based on the amino acid sequences of extant galectins and ancestral congerins. The extant genes used for ancestral reconstruction and their accession codes are ConI (Con‐ *ger myriaster* congerin I, AB010276.1), ConII (*C. myriaster* congerin II, AB010277.1), *Anguilla japonica* galectin-1 (AJL1, AB098064.1), *Hippoglossus hippoglossus* galectin (AHA1, DQ993254.1), *Paralichthys olivaceus* galectin (PoGal, AF220550.1), *Tetraodon nigroviridis* galectin (TnGal, CR649222.2), *Danio rerio* galectin-like lectin lgals1l1 (DrGal1\_L1, BC164225.1), *D. rerio* galectin-like lectin Gal1-L2 (DrGal1\_L2, AY421704.1), *D. rerio* Galectin-like lectin lgals1l3 (DrGal1\_L3, BC165230.1), *Ictalurus punctatus* galectin (IpGal, CF261531), *Bos taurus* galectin-1 (BTG1, BC103156.1), *Homo sapiens* galectin-1 (HSG1, AK312161.1), *Mus musculus* galectin-1 (MMG1, BC099479.1), *Cricetulus* sp. galectin L-14 (CRG1, M96676.1), *Xenopus laevis* galectin-1 (XLG1, AF170341.1), and *H. sapiens* galectin-2 (HSG2, BC059782.1). The numbers associated with the branches are the percent reproductions of branches in 1000 bootstrap reconstruc‐ tions. This tree is rooted by using the fungus sequence of *Coprinopsis cinerea* galectin-1 (AF130360.1) as the out‐ group. The proteins indicated with asterisk were used for the inference of the previous ancestor (Con-anc). (B) Amino acid sequences of ancestral congerins, ConI, and ConII. Amino acids identical to that of the corresponding last ances‐ tor are represented by a dot.

**Figure 1.** Amino Acid Sequences and Structures of Ancestral Congerins

This is a protein engineering approach where a proportion of amino acid residues of an extant protein are substituted with those of an ancestor, to construct pseudo-ancestors, in order to reveal the residues determining functional differences between extant and ancestral proteins. These molecular evolutional approaches using pseudo-ancestors bridged from Con-anc to ConI successfully elucidated the regions of the protein relevant to the two adaptive features of ConI, thermostability and higher carbohydrate-binding activity [49]. Experimental molec‐ ular archeology analysis, using the reconstructed ancestral congerins, also revealed the process of ConII evolution, another extant galectin. ConII has evolved to enhance affinity for α2, 3 sialyl galactose, which is specifically present in pathogenic marine bacteria. The selection pressure to which Con II reacted was hypothesized to be a shift in carbohydrate affinity. The observed difference in α2, 3-sialyl galactose affinities between Con-anc and Con II support this hypothesis.

the candidate amino acid residues responsible for the higher structural stability and carbohy‐ drate-binding activity of Con I were reduced to only 31 amino acid residues, from a total of 71 with apparent differences between Con I and Con II. These were mainly located in the N- and C-terminal and loop regions of the molecule, including the CRD [49, 50]. To identify the residues responsible for the properties of Con I, we next performed molecular evolution tracing analysis, by constructing pseudo-ancestral Con-anc proteins focused on the N-terminal, C-

(A) Phylogeny of extant and ancestral congerins. The tree is based on the amino acid sequences of extant galectins and ancestral congerins. The extant genes used for ancestral reconstruction and their accession codes are ConI (Con‐ *ger myriaster* congerin I, AB010276.1), ConII (*C. myriaster* congerin II, AB010277.1), *Anguilla japonica* galectin-1 (AJL1, AB098064.1), *Hippoglossus hippoglossus* galectin (AHA1, DQ993254.1), *Paralichthys olivaceus* galectin (PoGal, AF220550.1), *Tetraodon nigroviridis* galectin (TnGal, CR649222.2), *Danio rerio* galectin-like lectin lgals1l1 (DrGal1\_L1, BC164225.1), *D. rerio* galectin-like lectin Gal1-L2 (DrGal1\_L2, AY421704.1), *D. rerio* Galectin-like lectin lgals1l3 (DrGal1\_L3, BC165230.1), *Ictalurus punctatus* galectin (IpGal, CF261531), *Bos taurus* galectin-1 (BTG1, BC103156.1), *Homo sapiens* galectin-1 (HSG1, AK312161.1), *Mus musculus* galectin-1 (MMG1, BC099479.1), *Cricetulus* sp. galectin L-14 (CRG1, M96676.1), *Xenopus laevis* galectin-1 (XLG1, AF170341.1), and *H. sapiens* galectin-2 (HSG2, BC059782.1). The numbers associated with the branches are the percent reproductions of branches in 1000 bootstrap reconstruc‐ tions. This tree is rooted by using the fungus sequence of *Coprinopsis cinerea* galectin-1 (AF130360.1) as the out‐ group. The proteins indicated with asterisk were used for the inference of the previous ancestor (Con-anc). (B) Amino acid sequences of ancestral congerins, ConI, and ConII. Amino acids identical to that of the corresponding last ances‐

terminal, and some loop regions (loops 3, 5 and 6) [50].

118 Protein Engineering - Technology and Application

tor are represented by a dot.

**Figure 1.** Amino Acid Sequences and Structures of Ancestral Congerins

The crystal structures of ancestral full-length proteins, Con-anc', Con-anc'-N28K and Con-anc, have been solved at 1.5, 1.6, and 2.0 Å resolutions, respectively [51]. Their three-dimensional (3D) structures clearly demonstrate that Con-anc' or Con-anc'-N28K are appropriate ancestors of extant congerins (Fig. 2). A notable difference between the structures of ConI and ConII is the swapping of S1 strands at the dimer interface, which is unique to ConI among known galectins, and should contribute to its higher stability [81]. The dimer interface of ancestral Con-anc' and Con-anc'-N28K resembled that of ConII, but lacking the strand-swap. This protein-fold is the prototype for dimeric galectins, and the congerin ancestor is expected to have ConII-like conformation. Conversely, Con-anc did display a strand-swapped structure, indicating it was more likely to be an intermediate from the ancestor to ConI, consistent with the results of phylogeny construction (Fig. 2). The differences in carbohydrate interactions between Con-anc' and the extant congerins were observed mainly at the A-face of galactose [51]. These modifications might be relevant to the observed differentiation of carbohydrate specificities between ConI and ConII; ConI prefers α1,4-fucosylated *N*-acetyl glucosamine, while ConII is adapted to bind α2,3-sialyl galactose-containing carbohydrates [49, 50]. Furthermore, structural or functional parameters, such as cytotoxic activity, thermostability of hemagglutination activity, urea and heat denaturation of the structures, and carbohydrate binding activities of the ancestral and extant congerins, were compared as a function of the evolutionary distances from Con-anc' or ConI [51]. Some of these molecular properties were found to be enhanced in both lineages of congerin, which was observed as a correlation with the evolutionary distance from Con-anc'. The dimer interface essential for these proteins to evoke divalent cross-linking activity was enhanced in both lineages as the number of interface H-bonds and dimer interface area increased in ConI and ConII. However, the lactose interface area and the number of lactose H-bonds showed a low correlation with *K*a for carbohydrates, implying that simply enhancing carbohydrate interaction was not likely to be a major selection pressure, and obtaining specificity was more significant for the function of congerins.

Taken together, the first full-length ancestral structures of congerin revealed that the dupli‐ cated genes have been differentiating under natural selection pressures for strengthening of the dimer structure and enhancement of the cytotoxic activity. However, the two genes did not react equally to selection pressure, with ConI reacting through protein-fold evolution to enhance its stability. The modification of the dimer interface in the ConII lineage was rather moderate.

**Figure 2. Structures of ConI, ConII, Con-anc and Con-anc'.** Con-anc', Con-anc, ConII, and ConI dimers are shown from top to bottom along their molecular phylogeny. The numbers on each branch are the numbers of substitutions. The β strands relevant to the strand-swap at the dimer interface are labeled for S1-S2, and S1′-S2′. Each protein is associated with a close-up of its dimer interface.

## **5. Reconstruction of ancestral proteins: thermal adaptation of proteins in thermophilic bacterium**

Ancestral mutant analysis has been performed to explore the thermal adaptation of proteins. Benner and colleagues reconstructed the ancestral elongation factor-Tu (EF-Tu) predicted using ML methodology, in order to infer the physical environment surrounding ancient organisms [55]. Because EFs play a crucial role in protein synthesis in cells, the thermostability of EFs shows a strong correlation with the optimal growth temperature of their host organisms. For example, the melting temperatures (*T* m) of EFs from *Escherichia coli* and *Thermus thermo‐ philus* (HB8) are 42.8°C and 76.7°C, respectively, and the optimal growth temperatures of their respective hosts are approximately 40°C and 74°C, respectively [86]. Thus, EFs are suitable for use in assessment of the ambient temperature at the time of ancient life. To predict the ancestral sequences of EFs, amino acid sequences of fifty EF-Tu proteins from various bacterial lineages were used to construct two kinds of molecular phylogenetic trees; one using the evolutionary distances calculated using the EF-Tu sequences and the second from distances calculated using ribosomal RNA sequences [87]. Both resulting ancestors had temperature profiles similar to that of the thermophilic EF of modern *Thermus aquaticus*, supporting the hypothesis that the common ancestor of all organisms is a hyperthermophile. Inclusion of additional microbial species into the analysis, and reconstruction of the ancestral EFs at various depths (evolution‐ ary distance from present time) in the phylogeny using the ML method [56, 57], demonstrated that ancestral EFs positioned closer to the root of the phylogenetic tree tended to have significantly higher thermostabilities.

enhance its stability. The modification of the dimer interface in the ConII lineage was rather

**Figure 2. Structures of ConI, ConII, Con-anc and Con-anc'.** Con-anc', Con-anc, ConII, and ConI dimers are shown from top to bottom along their molecular phylogeny. The numbers on each branch are the numbers of substitutions. The β strands relevant to the strand-swap at the dimer interface are labeled for S1-S2, and S1′-S2′. Each protein is

**5. Reconstruction of ancestral proteins: thermal adaptation of proteins in**

Ancestral mutant analysis has been performed to explore the thermal adaptation of proteins. Benner and colleagues reconstructed the ancestral elongation factor-Tu (EF-Tu) predicted using ML methodology, in order to infer the physical environment surrounding ancient organisms [55]. Because EFs play a crucial role in protein synthesis in cells, the thermostability of EFs shows a strong correlation with the optimal growth temperature of their host organisms. For example, the melting temperatures (*T* m) of EFs from *Escherichia coli* and *Thermus thermo‐ philus* (HB8) are 42.8°C and 76.7°C, respectively, and the optimal growth temperatures of their

associated with a close-up of its dimer interface.

**thermophilic bacterium**

moderate.

120 Protein Engineering - Technology and Application

Yamagishi and coworkers reported several ancestral proteins, including two metabolic enzymes; 3-isopropylmalate and dehydrogenase (IPMDH), which is involved in leucine biosynthesis, and isocitrate dehydrogenase (ICDH) involved in the TCA cycle. Ancestral amino acids were introduced into extant IPMDH sequences of the hyperthermophilic archaeon *Sulfolobus tokodaii,* the extremely thermophilic bacterium *Thermus thermophilus*, and the hyperthermophilic archaeon *Caldococcus noboribetus* [14-18].

More recently, Hobbs et al reported the reconstruction of several common Precambrian ancestors of the core metabolic enzyme LeuB, 3-isopropylmalate dehydrogenase, estimated from various *Bacillus* species, in addition to the 3D structure of the last common ancestor at 2.9 Å resolution [19]. Their data indicated that the last common ancestor of LeuB was thermo‐ philic, suggesting that the origin of thermophily in the *Bucillus* genus was ancient. Evolution‐ ary tracing analysis through the ancestors of LeuB also indicated that thermophily was not exclusively a primitive trait, and it could be readily gained as well as lost in evolutionary history [19].

Overall, these studies demonstrate that ancestral enzymes retained enzymatic activity and ac‐ quired enhanced thermostability over respective extant enzymes, and that introduction of an‐ cestral state amino acids into modern proteins frequently thermostabilizes them. This indicates that ancestral protein reconstruction can provide empirical access to the evolution of ancient phenotypes, and is useful as a strategy for thermostabilization protein engineering.

## **6. Reconstruction of ancestral proteins: Evolutionary history of nuclear receptors and visual pigment proteins**

Thornton and colleagues have reported seminal work using ancestral protein reconstructions of the nuclear receptors for steroid hormones to investigate evolution of their ligand specific‐ ities [38-47, 88, 89]. Vertebrates have six homologous nuclear receptors for steroid hormones; the estrogen receptors alpha and beta (ERα and ERβ), androgen receptor (AR), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR). As it is thought that these proteins evolved from a common ancestor through a series of gene dupli‐ cations [65], the reconstruction of their ancestral proteins is a useful tool for investigation of their evolution of ligand-specificity. Although GR and MR are close relatives, GR is activated only by the stress hormone cortisol in most vertebrates, while MR is activated by both aldosterone and cortisol [90, 91]. The amino acid substitutions responsible for the specificity of GR toward cortisol were identified by reconstruction studies of the common ancestor of GRs and MRs using ML methodology [38-47]. Thornton and colleagues also reconstructed the ancestral corticoid receptor (AncCR), which corresponded to the protein predicted to be formed at the duplication event between GR and MR genes. Functional analysis showed that AncCR could be activated by both aldosterone and cortisol, suggesting that GR of vertebrates had lost aldosterone specificity during the evolutionary process. Furthermore, site-direct mutagenesis and X-ray crystallographic studies of AncCR revealed that amino acid substitu‐ tions at S106P and L111Q were key for the specificity shift of GR [38, 39]. AncCR is the first complete domain ancestor (ligand-binding domain only), for which 3D structure was deter‐ mined. Ancestral mutant analysis of the NR5 nuclear orphan receptors, including steroido‐ genic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) was also reported [41]. The structurefunction relationships of the SF-1/LRH-1 subfamily and their evolutionary ligand-binding shift, where the characteristic phospholipid binding ability of the SF-1/LRH-1 subfamily was subsequently reduced and lost in the lineage leading to the rodent LRH-1, due to specific amino acid replacements, were elucidated [41].

Reconstruction of visual pigment proteins, including rhodopsin and green fluorescent protein (GFP)-like proteins, has been also conducted. Chang et al reconstructed an ancestral archosaur rhodopsin from thirty vertebrate species using the ML method and three different models; nucleotide-, amino acid-, and codon-based [25]. An ancestral protein can be reconstructed with each of these models and the inferred archosaur rhodopsin had the same amino acid sequences for all three, except for three amino acid sites (positions 213, 217, and 218), and all reconstructed ancestral proteins had four variants at the ambiguous sites (single mutants T213I, T217A, V218I, and the triple mutant of these) showed similar optical properties, with an apparent absorption maximum at 508–509 nm, slightly red-shifted from that of modern vertebrates (482– 507 nm). These data indicated that the alternative ancestral amino acids predicted by the different likelihood models showed similar functional characteristics. Dim-light and color vision in vertebrates are controlled by five visual pigments (RH1, RH2, SWS1, SWS2, and M/ LWS), each consisting of a protein moiety (opsin) and a covalently bound 11-cis-retinal (or 11 *cis*-3, 4-dehydroretinal) [91], with characteristic sensitivity to specific wavelengths of maximal absorptions (λmax) from 360 nm (UV) to 560 nm (red). How do the visual pigments achieve sensitivity to various wavelengths? Despite extensive mutagenesis analyses of visual pig‐ ments, the molecular mechanisms that modulate the variable λmax values observed in nature were not well understood until ancestral protein reconstruction analysis was applied to the question [92]. Yokoyama and colleagues successfully identified the molecular mechanism of the spectral tuning of visual pigments by generating 15 currently known pigment types using engineered ancestral pigments of SWS1, RH1, and red- and green-sensitive (M/LWS) pigments [26-28]. Kawamura and colleagues reported the reconstruction of ancestral mutants of four green visual pigments from zebrafish, namely, RH2-1, RH2-2, RH2-3 and RH2-4, with λmax values of 467, 476, 488, and 505 nm, respectively [29]. The ancestral pigments showed that spectral shifts occurred toward the shorter wavelength in evolutionary lineages [29]. Further‐ more, Yokoyama and colleagues demonstrated the molecular basis (structural elements) of the adaptation of rhodopsin for the dim-light (deep-sea) vision by ancestral reconstruction experiments using 11 ancestral pigments estimated by rhodopsin sequences of migratory fish from both the surface and deep ocean [30-32].

The great star coral *Montastrea cavernosa* has several green fluorescent protein (GFP)-like proteins, classified into four paralogous groups based on their emission spectra: cyan (emission maximum, 480–495 nm), short wavelength green (500–510 nm), long wavelength green (515– 525 nm), and red (575–585 nm) [93]. Matz and colleagues reconstructed the ancestral fluores‐ cent proteins corresponding to the root of each color group, and the common ancestor of the groups using the ML method [33-37]. The analyses of the fluorescence spectra using the ancestral proteins depicted the evolutionary process of the coral GFP-like proteins, in which the peak wavelength has shifted from green to red. Furthermore, they identified the amino acid substitutions responsible for the generation of recent cyan and red fluorescence proteins through site-direct mutagenesis studies of the ancestral green fluorescent protein as a template [35]. Thus, the engineering of ancestral molecules at various evolutionary stages, to recapitu‐ late the changes in their phenotypes over time, is an effective way to explore the molecular evolution and adaptation mechanisms of proteins, although the experimental demonstration of adaptive events at the molecular level is particularly challenging.

## **7. Concluding remarks**

cations [65], the reconstruction of their ancestral proteins is a useful tool for investigation of their evolution of ligand-specificity. Although GR and MR are close relatives, GR is activated only by the stress hormone cortisol in most vertebrates, while MR is activated by both aldosterone and cortisol [90, 91]. The amino acid substitutions responsible for the specificity of GR toward cortisol were identified by reconstruction studies of the common ancestor of GRs and MRs using ML methodology [38-47]. Thornton and colleagues also reconstructed the ancestral corticoid receptor (AncCR), which corresponded to the protein predicted to be formed at the duplication event between GR and MR genes. Functional analysis showed that AncCR could be activated by both aldosterone and cortisol, suggesting that GR of vertebrates had lost aldosterone specificity during the evolutionary process. Furthermore, site-direct mutagenesis and X-ray crystallographic studies of AncCR revealed that amino acid substitu‐ tions at S106P and L111Q were key for the specificity shift of GR [38, 39]. AncCR is the first complete domain ancestor (ligand-binding domain only), for which 3D structure was deter‐ mined. Ancestral mutant analysis of the NR5 nuclear orphan receptors, including steroido‐ genic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) was also reported [41]. The structurefunction relationships of the SF-1/LRH-1 subfamily and their evolutionary ligand-binding shift, where the characteristic phospholipid binding ability of the SF-1/LRH-1 subfamily was subsequently reduced and lost in the lineage leading to the rodent LRH-1, due to specific amino

Reconstruction of visual pigment proteins, including rhodopsin and green fluorescent protein (GFP)-like proteins, has been also conducted. Chang et al reconstructed an ancestral archosaur rhodopsin from thirty vertebrate species using the ML method and three different models; nucleotide-, amino acid-, and codon-based [25]. An ancestral protein can be reconstructed with each of these models and the inferred archosaur rhodopsin had the same amino acid sequences for all three, except for three amino acid sites (positions 213, 217, and 218), and all reconstructed ancestral proteins had four variants at the ambiguous sites (single mutants T213I, T217A, V218I, and the triple mutant of these) showed similar optical properties, with an apparent absorption maximum at 508–509 nm, slightly red-shifted from that of modern vertebrates (482– 507 nm). These data indicated that the alternative ancestral amino acids predicted by the different likelihood models showed similar functional characteristics. Dim-light and color vision in vertebrates are controlled by five visual pigments (RH1, RH2, SWS1, SWS2, and M/ LWS), each consisting of a protein moiety (opsin) and a covalently bound 11-cis-retinal (or 11 *cis*-3, 4-dehydroretinal) [91], with characteristic sensitivity to specific wavelengths of maximal absorptions (λmax) from 360 nm (UV) to 560 nm (red). How do the visual pigments achieve sensitivity to various wavelengths? Despite extensive mutagenesis analyses of visual pig‐ ments, the molecular mechanisms that modulate the variable λmax values observed in nature were not well understood until ancestral protein reconstruction analysis was applied to the question [92]. Yokoyama and colleagues successfully identified the molecular mechanism of the spectral tuning of visual pigments by generating 15 currently known pigment types using engineered ancestral pigments of SWS1, RH1, and red- and green-sensitive (M/LWS) pigments [26-28]. Kawamura and colleagues reported the reconstruction of ancestral mutants of four green visual pigments from zebrafish, namely, RH2-1, RH2-2, RH2-3 and RH2-4, with λmax values of 467, 476, 488, and 505 nm, respectively [29]. The ancestral pigments showed that

acid replacements, were elucidated [41].

122 Protein Engineering - Technology and Application

Experimental molecular archaeology using ASR is a new and potentially useful method not only for the study of molecular evolution, but also as a protein engineering technique. This method can provide us with experimental information about ancient genes and proteins, which cannot be obtained from fossil records or by simply constructing molecular phylogeny. However, as discussed above, ancestral sequences can have some issues with ambiguity, depending on the choice of evaluation method, evolutionary model, and sequences. Although inference methods such as MP, ML and BI can lead to errors in predicted ancestral sequences, resulting in potentially misleading estimates of the properties of the ancestral protein, experimental molecular archaeology using ASR could be a more reliable method as all possible ancestral mutants, in which ambiguous amino acid sites are replaced by equally probable candidates individually or in combination, are reproducible and the biological and physico‐ chemical properties and 3D structures of the molecules can be assessed. Indeed, when ancestral congerins were reconstructed based on insufficient sequence information lacking recently determined fish galectin genes, the ancestral Con-anc protein was shown to have a strandswapped structure resembling ConI, indicating that Con-anc was more likely to be an intermediate mutant of the ancestor to ConI, and that the revised Con-anc' or Con-anc'-N28K are more appropriate ancestors. Thus, the accuracy of ASR can be assessed by analysis of protein activities, stabilities, specificities, and even 3D structures in the laboratory using biochemical or biophysical methods.

Experimental molecular archaeology using ASR can be applied to more complex biological systems, such as heterologous subunit interactions and their evolution in molecular machines [48], host-viral interactions and their co-evolution [54, 94, 95], and proteome/structural proteome level analyses [96]. Furthermore, recent studies have indicated that ASR is applicable to not only to proteins, but also to nucleotides including ancestral rRNA [97] and transposons [95]. To understand the molecular strategies of evolution in nature and the structure-function relationships of proteins and nucleotides, it is important to learn more from 'nature' itself, and from its prodigious works and histories; proteins/nucleotides and their molecular evolution.

## **Author details**

Tomohisa Ogawa1 and Tsuyoshi Shirai2

1 Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Sendai, Japan

2 Nagahama Institute of Bio-Science and Technology, and Japan Science and Technology Agency, Bioinfomatic Research Division, Nagahama, Shiga, Japan

## **References**


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Experimental molecular archaeology using ASR can be applied to more complex biological systems, such as heterologous subunit interactions and their evolution in molecular machines [48], host-viral interactions and their co-evolution [54, 94, 95], and proteome/structural proteome level analyses [96]. Furthermore, recent studies have indicated that ASR is applicable to not only to proteins, but also to nucleotides including ancestral rRNA [97] and transposons [95]. To understand the molecular strategies of evolution in nature and the structure-function relationships of proteins and nucleotides, it is important to learn more from 'nature' itself, and from its prodigious works and histories; proteins/nucleotides and their molecular evolution.

1 Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University,

2 Nagahama Institute of Bio-Science and Technology, and Japan Science and Technology

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Tomohisa Ogawa1

Sendai, Japan

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## **Chapter 6**

## **Protein Engineering of Enzymes Involved in Bioplastic Metabolism**

Tomohiro Hiraishi and Seiichi Taguchi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55552

## **1. Introduction**

## **1.1. Environmental problems caused by petroleum-based plastics**

The last half century has witnessed the development of synthetic plastics from petroleum resources, andmore than300milliontonsof syntheticplastics are annuallyproducedatpresent. The recently increased consumption of petroleum resources has led to environmental prob‐ lems 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 includ‐ ingterrestrial,aquaticanimalsandbirds.Furthermore,ithasbeensuggestedthateventhewasted plastics in the form of small particles potentially induce the alteration of pelagic ecosystems [1]. Therefore, the development of environmentally sound alternatives, such as bioplastics, to conventional petroleum-based plastics is urgently desired to sustain the environment [2-4].

### **1.2. Bioplastics**

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 synthe‐ sized from renewable resources via biological or chemical processes, and some of them show non-biodegradability although bio-based plastics are generally biodegradable. Poly(εcaprolactone) (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(hy‐ droxyalkanoate)s (PHAs) and poly(lactide) (PLA) show an excellent biodegradability, and are produced from renewable resources via biological and chemical processes, respective‐ ly. 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.

#### T. Hiraishi and S. Taguchi

**Figure 1.** Bioplastics comprised of biodegradable and bio-based plastics.

## *1.2.1. PHAs*

Meanwhile, poly(ethylene) (PE) and poly(propylene) (PP) are chemically synthesized from their monomers derived from biological sources, but they are not biodegradable. Poly(hy‐ droxyalkanoate)s (PHAs) and poly(lactide) (PLA) show an excellent biodegradability, and are produced from renewable resources via biological and chemical processes, respective‐ ly. 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.

134 Protein Engineering - Technology and Application

T. Hiraishi and S. Taguchi

**Figure 1.** Bioplastics comprised of biodegradable and bio-based 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 semicrys‐ talline form and is degraded by extracellular PHA depolymerases (e-PHA depolymeras‐ es) 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 [11]. 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 [12]. Accordingly, PHAs have attracted industrial interest as biobased, biodegradable, biocompatible, and versatile thermoplastics [13, 14].

## *1.2.2. PLA*

PLA is representative bio-based plastics with good processability and transparency that are used in packaging, containers, stationary, etc. [15]. In addition, medical and agricultural uses of the material have been investigated because of their biocompatibility and biode‐ gradability [16]. PLAs are produced from renewable biomass through a chemo-biopro‐ cess 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 [17]. There are several methods for synthesizing high-molecular-mass PLAs: condensation, chain elongation, and ring-opening polymerization of cyclic lactides [15]. Currently, the major industrial method to produce PLAs is ring-opening polymeriza‐ tion 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 particu‐ lar, 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 develop‐ ment 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 *Escherichia coli* strain allowing the synthesis of LA-based polyesters by introducing the gene encoding engineered PHA synthase with acquired LApolymerizing activity [20, 21]. They thus achieved the one-step biosynthesis of a copolymer with 6 mol% of lactate and 94 mol% of 3-hydroxybutyrate units. This extremely important result represents a milestone towards the biological synthesis of PLA and confirms that the work is moving in the right direction, as mentioned in the section of 2.3. At present, the LA fraction in the copolyesters has been enriched up to 96 mol% [22], so the synthesis of homo‐ polymers of LA represents a major goal. To that end, the current microbial cell factory ought to be improved with further evolved LA-polymerizing enzymes (LPE) and metabolic engi‐ neering-based optimization [23, 24]. Matsumura et al. likewise reported the lipase PCcatalyzed polymerization of cyclic diester-D,L-lactide at a temperature of 80-130 °C to yield PLA with molecular masses of up to 12,600 [25].

#### **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 metab‐ olism, 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 [26]. 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 photo‐ synthesis 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 depoly‐ merases. 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 [13]. 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 monomerconcentrated resources is rapidly gaining importance due to its high degradation rate [27]. 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 condi‐ tions, 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**

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 develop‐ ment 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 *Escherichia coli* strain allowing the synthesis of LA-based polyesters by introducing the gene encoding engineered PHA synthase with acquired LApolymerizing activity [20, 21]. They thus achieved the one-step biosynthesis of a copolymer with 6 mol% of lactate and 94 mol% of 3-hydroxybutyrate units. This extremely important result represents a milestone towards the biological synthesis of PLA and confirms that the work is moving in the right direction, as mentioned in the section of 2.3. At present, the LA fraction in the copolyesters has been enriched up to 96 mol% [22], so the synthesis of homo‐ polymers of LA represents a major goal. To that end, the current microbial cell factory ought to be improved with further evolved LA-polymerizing enzymes (LPE) and metabolic engi‐ neering-based optimization [23, 24]. Matsumura et al. likewise reported the lipase PCcatalyzed polymerization of cyclic diester-D,L-lactide at a temperature of 80-130 °C to yield

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 metab‐ olism, 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 [26]. 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 photo‐ synthesis to convert atmospheric CO2 directly into PHA, in hopes of reducing production cost

In addition, to solve the latter issue, we have also focused on the engineering of PHB depoly‐ merases. 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 [13]. 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 monomerconcentrated resources is rapidly gaining importance due to its high degradation rate [27]. In addition, as chemical recycling is cost-efficient and has low CO2 emissions, it has great potential

PLA with molecular masses of up to 12,600 [25].

136 Protein Engineering - Technology and Application

**1.3. Toward an enhanced sustainable production**

in the future.

## **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 *R*-enantiomer HA-CoAs are accepted for polymerization by synthase [34]. Over 60 different PHA synthases have been classified into four types based on their substrate specificities and subunit compositions of enzymes (Table 1) [35].


**Table 1.** The four classes of PHA synthases

Type I and type II PHA synthases consist of single subunits (PhaC). Type I PHA synthases, represented by *Ralstonia eutropha* enzyme, mainly polymerize SCL-monomers (C3–C5), whereas type II PHA synthases, represented by *Pseudomonas oleovorans* enzyme, polymerize MCL-monomers (C6–C20). Type III PHA synthases, represented by *Allochromatium vinosum* enzyme, consist of two hetero-subunits (PhaC and PhaE). PhaC subunits of type III synthase are smaller than those of type I and II synthases, but possess catalytic residues. Like the type I synthases, these PHA synthases prefer to polymerize SCL-monomers (C3–C5). Type IV PHA synthases, represented by *Bacillus megaterium*, are similar to the type III PHA synthases with respect to possessing two subunits. However, unlike the PhaE of type III PHA synthases, a smaller protein designated as PhaR is required for full activity expression of type IV PhaC.

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 *R. eutropha* PHA synthase [38]. *In vitro* evolutionary program was firstly constructed by coupling an error-prone PCR-mediated point mutagenesis with the plate-based high-throughput screening method to generate mutants with acquired beneficial functions [38]. A mutant library of the *R. eutropha* PHA synthase gene was prepared by colony formation of transformant cells of *Escherichia coli*. It should be noted to meet a good correlation between the change in PHB accumulation resulting from the introduction of mutations into the *R. eutropha* PHA synthase gene and the change in the enzymatic activity of the mutants. To gain the mutants with increased activity, multi-step mutations, including an activity loss and an intragenic suppression-type activity reversion were attempted [39]. The mutant enzymes were once identified by primary mutation analysis, a secondary round of mutation was used to evolve these enzymes to proteins with better characteristics than the wild-type enzyme. As a result, through this intragenic suppression-type mutagenesis, an increased specific activity towards 3HB-CoA by 2.4-fold compared to the wild-type enzyme was acquired by a mutation of Phe420Ser (F420S) in a type I PHA synthase [39].

As a next case, screened beneficial mutation, Gly4Asp (G4D), exhibited higher levels of protein accumulation and PHB production compared to the recombinant *E. coli* strain harboring the wild-type PHA synthase [40]. As for intragenic suppression-type mutagenesis, second-site reversion is dependent or independent of primary mutation in the activity. Secondary mutations of F420S and G4D are the latter cases, being independent of primary mutation. Subsequently, site-specific saturation mutagenesis was also performed on the codon encoding the G4 residue of the *R. eutropha* PHA synthase and many substitutions resulted in much higher PHB content as well as higher molecular masses of the polymers [41].

*Aeromonas caviae* (*punctata*) PHA synthase is unique among type I PHA synthases since it can synthesize not only PHB homopolymer but also random copolyesters of 3HB and 3-hydrox‐ yhexanoate (3HHx). Kichise et al. performed the first successful *in vitro* molecular evolution experiments on PHA synthase from *A. caviae* by targeting to the limited region of the enzyme [42]. Two evolvants exhibited increased activity towards 3HB-CoA of 56% and 21%, respec‐ tively, compared to the wild-type enzyme by *in vitro* assays. These mutations led to enhanced accumulation (up to 6.5-fold higher than the wild-type enzyme) of P(3HB-*co*-3HHx) and increases in the 3HHx molar fraction (16-18 mol% compared to 10 mol% of the wild-type PHA synthase) in recombinant *E. coli* strains grown on dodecanoate. As an extended study, a combination of these two beneficial mutations (N149S/D171G) synergistically altered enzy‐ matic properties, leading to synthesis of PHA copolymers with enhanced 3HA fraction and increased molecular mass from in the recombinant *R. eutropha* [43]. In a separate study, *A. caviae* PHA synthase was engineered *in vivo* using the mutator strain *E. coli* which has a 5,000 fold higher mutation rate than wild-type *E. coli*, and mutants were again screened for enhanced PHB accumulation in recombinant *E. coli* [44]. Also, mutants synthesized PHAs with increased molecular mass, but in contrast to the previous study, the 3HHx fraction was only slightly different from wild-type composition.

Junction site for interconnection of heterogeneous enzymes based on the predicted secondary structures allowed chimeragenesis of the PHA synthase from *R. eutroph*a with the partner PHA synthase from *A. caviae*. Successfully obtained chimera-mutant exhibited improved activity increase and expanded substrate specificity compared to the original enzymes [45]. As for PHA synthases, directed evolution studies have thus progressed through advancements from random approach to much more systematic approaches such as chimera-genesis, recombina‐ tion and shuffling.

#### *2.2.2. Application to type II PHA synthases*

are smaller than those of type I and II synthases, but possess catalytic residues. Like the type I synthases, these PHA synthases prefer to polymerize SCL-monomers (C3–C5). Type IV PHA synthases, represented by *Bacillus megaterium*, are similar to the type III PHA synthases with respect to possessing two subunits. However, unlike the PhaE of type III PHA synthases, a smaller protein designated as PhaR is required for full activity expression of type IV PhaC. 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.

*2.2.1. Application to type I PHA synthases*

138 Protein Engineering - Technology and Application

**2.2. Activity improvement and substrate specificity alteration of PHA synthases**

was acquired by a mutation of Phe420Ser (F420S) in a type I PHA synthase [39].

PHB content as well as higher molecular masses of the polymers [41].

As a next case, screened beneficial mutation, Gly4Asp (G4D), exhibited higher levels of protein accumulation and PHB production compared to the recombinant *E. coli* strain harboring the wild-type PHA synthase [40]. As for intragenic suppression-type mutagenesis, second-site reversion is dependent or independent of primary mutation in the activity. Secondary mutations of F420S and G4D are the latter cases, being independent of primary mutation. Subsequently, site-specific saturation mutagenesis was also performed on the codon encoding the G4 residue of the *R. eutropha* PHA synthase and many substitutions resulted in much higher

*Aeromonas caviae* (*punctata*) PHA synthase is unique among type I PHA synthases since it can synthesize not only PHB homopolymer but also random copolyesters of 3HB and 3-hydrox‐

A pioneering study that established methods for protein engineering PHA synthase initiated in 2001 using the best-studied enzyme, the *R. eutropha* PHA synthase [38]. *In vitro* evolutionary program was firstly constructed by coupling an error-prone PCR-mediated point mutagenesis with the plate-based high-throughput screening method to generate mutants with acquired beneficial functions [38]. A mutant library of the *R. eutropha* PHA synthase gene was prepared by colony formation of transformant cells of *Escherichia coli*. It should be noted to meet a good correlation between the change in PHB accumulation resulting from the introduction of mutations into the *R. eutropha* PHA synthase gene and the change in the enzymatic activity of the mutants. To gain the mutants with increased activity, multi-step mutations, including an activity loss and an intragenic suppression-type activity reversion were attempted [39]. The mutant enzymes were once identified by primary mutation analysis, a secondary round of mutation was used to evolve these enzymes to proteins with better characteristics than the wild-type enzyme. As a result, through this intragenic suppression-type mutagenesis, an increased specific activity towards 3HB-CoA by 2.4-fold compared to the wild-type enzyme

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 *Pseudomonas* sp. 61-3 with significant substrate specificity towards the 3HB-CoA (Table 1). In the landmark study by Takase *et al*, the *in vitro* evolutionary technique was applied to the PhaC1 PHA synthase from *Pseudomonas* sp. 61-3 to increase the activity towards 3HB-CoA monomers [46]. Substitutions at two amino acid residues, Ser325 and Gln481 were found to dramatically effect the production of PHB homopolymer in recombinant *E. coli* with glucose as the carbon source. The codons for these amino acids were subjected to site-specific saturation mutagenesis and several individual substitutions were found that could dramatically increase the level of PHB production. These mutations were combined as double mutants to further increase the level of PHB production (340 - 400-fold higher than the wild-type enzyme) [46]. The changes in the *in vivo* produced P(3HB-*co*-3HA) copolymer molar compositions correlated well with the *in vitro* biochemical data of the substrate specificity and activity of the enzymes and represents one of the most well-rounded studies to date [47].

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 *Pseudomonas* sp. 61-3 was found to be one of the residues determining substrate specificity of the enzyme, as described above. Interestingly, the amino acid residues corre‐ sponding to the position of this enzyme are conserved within each type of PHA synthases; Ala for type I, Gln for type II, Gly for type III and Ser for type IV enzymes. Thus, the effects of mutating the highly conserved alanine (Ala510) of the *R. eutropha* PHA synthase (correspond‐ ing to the position 481 in *Pseudomonas* sp. 61-3 PhaC1) were analyzed via site-specific saturation mutagenesis. Mutations at Ala510 were found to affect the substrate specificity of the *R. eutropha* PHA synthase, allowing slightly higher 3HA incorporation compared to the wildtype PHA synthase in *R. eutropha* PHB-4 (PHA negative mutant) [48].

The other two beneficial positions, Glu130 [49] and Ser477 [50], were also identified through the *in vitro* evolution screening. As illustrated in Figure 2 (A) and (B), "mutation scrambling" among four beneficial positions (130, 325, 477, 481) for activity increase, change in substrate specificity, and regulation of polymer molecular mass would further create new superenzymes. Most recently, a possible mechanistic model for PHA polymerization has been proposed on the basis of the accumulated evolutionary studies [51]. Furthermore, the useful evolvants obtained through the systematic enzyme evolution have been supplied to other organisms including plants [52, 53]. The impacts of these reports prompted the other research groups to apply directed evolution to the individual PHA synthases of interests [54-56].

#### T. Hiraishi and S. Taguchi

**Figure 2.** Structural diversities in main-chain and side-chain of PHA back bone which can be recognized by natural and artificially evolved PHA synthases. Asterisks indicate the chiral center in monomer units of PHA.

## **2.3. Engineering of lactate-polymerizing enzyme (LPE) from PHA synthases**

synthase from *Pseudomonas* sp. 61-3 was found to be one of the residues determining substrate specificity of the enzyme, as described above. Interestingly, the amino acid residues corre‐ sponding to the position of this enzyme are conserved within each type of PHA synthases; Ala for type I, Gln for type II, Gly for type III and Ser for type IV enzymes. Thus, the effects of mutating the highly conserved alanine (Ala510) of the *R. eutropha* PHA synthase (correspond‐ ing to the position 481 in *Pseudomonas* sp. 61-3 PhaC1) were analyzed via site-specific saturation mutagenesis. Mutations at Ala510 were found to affect the substrate specificity of the *R. eutropha* PHA synthase, allowing slightly higher 3HA incorporation compared to the wild-

The other two beneficial positions, Glu130 [49] and Ser477 [50], were also identified through the *in vitro* evolution screening. As illustrated in Figure 2 (A) and (B), "mutation scrambling" among four beneficial positions (130, 325, 477, 481) for activity increase, change in substrate specificity, and regulation of polymer molecular mass would further create new superenzymes. Most recently, a possible mechanistic model for PHA polymerization has been proposed on the basis of the accumulated evolutionary studies [51]. Furthermore, the useful evolvants obtained through the systematic enzyme evolution have been supplied to other organisms including plants [52, 53]. The impacts of these reports prompted the other research groups to apply directed evolution to the individual PHA synthases of interests [54-56].

**Figure 2.** Structural diversities in main-chain and side-chain of PHA back bone which can be recognized by natural

and artificially evolved PHA synthases. Asterisks indicate the chiral center in monomer units of PHA.

4 (PHA negative mutant) [48].

type PHA synthase in *R. eutropha* PHB-

140 Protein Engineering - Technology and Application

T. Hiraishi and S. Taguchi

The pioneering work on the exploration of LA-polymerizing activity by PHA synthases was reported by Valentin et al. [57]. In that attempt, the PLA biosynthesis was carried out by monitoring the activity of PHA synthases towards synthetic LA-CoAs (*R* and *S* enantiomers). Several PHA synthases were evaluated for LA-polymerizing activities and a class III PHA synthase from *Allochromatium vinosum* exhibited a weak CoA releasing activity [57]. In a similar report, Yuan et al. reported in detail the activity of *A. vinosum* PHA synthase towards (*R*)-LA-CoA [58]. Unfortunately, in either case, polymerization was not observed/was negligible, suggesting that PHA synthase could hydrolyze CoA ester to release CoA but not progress from there with polymerization to form a polymer.

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 *in vitro* and *in vivo* experiments [20]. The first clue of LA-polymerizing activity was obtained through a waterorganic solvent two-phase *in vitro* system [20, 59]. The activity towards LA-CoA was tested in the absence or presence of 3HB-CoA using representative PHA synthases belonging to the four classes of natural PHA synthases together with three engineered (PhaC1Ps mutants) from *Pseudomonas* sp. 61-3. The engineered PHA synthases were two single mutants[Ser325Thr (ST) and Gln481Lys (QK)] and one double mutant carrying the two mutations (STQK). The two mutants were selected based on their improved activity and/or broader substrate specificity [36, 37]. The natural synthases and mutants did not form a clear-polymer with LA-CoA alone but did with 3HB-CoA. However, when LA-CoA was supplied together with 3HB-CoA, one mutant, PhaC1Ps(STQK) clearly exhibited a polymer-like precipitation. Subsequently, the analysis of the precipitant revealed that the precipitant consisted of 36 mol% of the LA unit. Therefore, this was the first report ever of a PHA synthase with ability to incorporate LA unit to form P(LA-*co*-3HB).

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 vitro* activity of PhaC1Ps(STQK) towards LA-CoA, it was presumed that heterologous expression of this LPE could result into an *in vivo* synthesis of LA-based poly‐ esters thus creating microbial factories for LA-based polyesters synthesis.

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 *R. eutropha* (Taguchi et al., 2002) [39], the same mutation was introduced into the ancestral LPE [PhaC1Ps(STQK) from *Pseudomonas* sp. 61-3] to create a triple mutant of LPE with S325T and Q481K along with a new mutation, F392S which corresponds to F420S of PhaCRe [60]. When the new further engineered LPE [PhaC1Ps (STQKFS)] was expressed in *E. coli*, a copolymer with 45 mol% LA and polymer content of 62 wt% was synthesized in comparison with P(26 mol% LA-*co*-3HB) obtained with the prototype LPE, PhaC1Ps(STQK) under aerobic culture conditions. Additionally, the cells harboring PhaC1Ps (STQKFS) synthesized P(LA-*co*-3HB) with 62 mol% LA with polymer content of 12 wt %. During the same study, saturation mutagenesis of LPE at the same site (position 392) yielded mutants that gave varying LA fractions in the copolymers however; F392S was superior to the other mutants in incorporating LA. This study demonstrated the effectiveness of enzyme engineering of the LPE towards two directions; there was improved LA incorporation and polymer yield improvement for both aerobic and anaerobic culture conditions [60]. Further‐ more, it will be interesting to note that copolymers incorporating 2-hydroxy acids (2HAs) such as 2-hydroxybutyrate [61] and glycolate (Matsumoto et al., 2011) [62] may lead to copolymers with novel properties (Figure 3). This expansion of PHA synthase to 2HAs-polymerizing enzymes has extensively prompted us to create further new enzymes with acquired activities toward new unusual monomer substrates, consequently create new polymers.

T. Hiraishi and S. Taguchi

**Figure 3.** Functional mapping of beneficial positions (A) and relationships among the residues related to enzymatic activity and substrate specificity of PHA synthase from *Pseudomonas* sp. 61-3 (B).

Regarding the reports on LPE, the following several studies have been published [63, 64]. Currently, the best-studied PHA synthase from *R. eutropha* has been successfully engi‐ neered to LPE by only single mutations at beneficial position corresponding to the position 481 of *Pseudomonas* sp. 61-3 PHA synthase [65]. This implies the functional compatibility between PHA members also for acquiring LPE activity. In the prospect, advanced types of LPE will be supplied by artificial evolution of the prototype LPE as well as exploration of natural PHA synthases with potentially possessing new substrate specificities such as LApolymerizing activity.

## **3. Protein engineering of PHB depolymerase**

engineering of the LPE towards two directions; there was improved LA incorporation and polymer yield improvement for both aerobic and anaerobic culture conditions [60]. Further‐ more, it will be interesting to note that copolymers incorporating 2-hydroxy acids (2HAs) such as 2-hydroxybutyrate [61] and glycolate (Matsumoto et al., 2011) [62] may lead to copolymers with novel properties (Figure 3). This expansion of PHA synthase to 2HAs-polymerizing enzymes has extensively prompted us to create further new enzymes with acquired activities

**Figure 3.** Functional mapping of beneficial positions (A) and relationships among the residues related to enzymatic

Regarding the reports on LPE, the following several studies have been published [63, 64]. Currently, the best-studied PHA synthase from *R. eutropha* has been successfully engi‐ neered to LPE by only single mutations at beneficial position corresponding to the position 481 of *Pseudomonas* sp. 61-3 PHA synthase [65]. This implies the functional compatibility between PHA members also for acquiring LPE activity. In the prospect, advanced types of LPE will be supplied by artificial evolution of the prototype LPE as well as exploration of

activity and substrate specificity of PHA synthase from *Pseudomonas* sp. 61-3 (B).

toward new unusual monomer substrates, consequently create new polymers.

142 Protein Engineering - Technology and Application

T. Hiraishi and S. Taguchi

#### **3.1. Biochemical and genetic properties of PHB depolymerases**

A number of PHA depolymerases have been purified from diverse PHA-degrading microor‐ ganisms 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 depoly‐ merases 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 [66].

Among the PHA depolymerases, multi-domain e-PHB depolymerases have been extensively examined [9]. 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 Cterminus, and a linker region connecting the two domains, while e-PHB depolymerases from *Penicillium funiculosum* (PhaZPfu) and PhaZ7 from *Paucimonas lemoignei* (PhaZ7Ple) have emerged as two exceptions (single-domain e-PHB depolymerases) [9, 67-69]. Genetic analysis also shows that e-PHB depolymerases contain a lipase box pentapeptide [Gly-X1-Ser-X2-Gly] as an active residue, indicating that these enzymes are one of the serine hydrolases. As an example, the domain structure of e-PHB depolymerase from *Ralstonia pickettii* T1 (PhaZRpiT1) is illustrated in Figure 4(A). Such domain structure has been found in many biopolymerdegrading enzymes, such as cellulase, xylanase, and chitinase, which are capable of hydro‐ lyzing water-insoluble polysaccharides [70-73]. The enzymatic degradation of PHB by the multi-domain e-PHB depolymerases is considered to proceed via a two-step reaction at the solid-liquid interface, as shown in Figure 4(B). The e-PHB depolymerase approaches and adheres to the PHB surface via SBD, followed by hydrolysis of the polymer chain by CD. Accordingly, it is considered that elucidation of the mechanisms of enzyme adsorption and enzymatic hydrolysis will contribute to the development of new PHB polymer materials with the desired environmental stability and biodegradability as well as the development of improved e-PHB depolymerases that can be used to effectively recycle PHB materials.

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

T. Hiraishi and S. Taguchi

**Figure 4.** A) Domain structure of e-PHB depolymerase from *Ralstonia pickettii* T1 (PhaZ*RpiT1*). (B) Schematic illustra‐ tion of the enzymatic degradation of PHA by e-PHB depolymerase.

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) [77], and random copolymers of (*R*)-3HB with various hydroxyalkanoate units [92-95] have been synthesized to examine their enzymatic degradation by a variety of e-PHB depolymerases. For instance, Abe et al. proposed a schematic model of the enzymatic cleavage of the PHA chain by PhaZRpiT1 (Figure 5), in which its active site can recognize at least three neighboring monomer units with a certain degree of difference in main-chain length [93]. Besides the chemical structure, the solid-state structure and surface properties of PHAs also influence the enzymatic hydrolysis. For example, the amorphous regions in PHA materials are preferentially hydrolyzed, followed by the hydrolysis of crystalline regions as a rate-limiting step in the enzymatic degradation process [96, 97]. Further, the enzymatic degradation rate of PHA materials decreases with increasing crystallinity, crystal size, and regularity of the chain packing state. In addition, Abe and co-workers demonstrated using proteinase K that the change in the surface properties of PLA film induced by end-capping with alkyl ester groups (carbon numbers 12 to 14) leads to a decrease in their enzymatic degradation rates [98, 99].

#### T. Hiraishi and S. Taguchi

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.

**Figure 4.** A) Domain structure of e-PHB depolymerase from *Ralstonia pickettii* T1 (PhaZ*RpiT1*). (B) Schematic illustra‐

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) [77], and random copolymers of (*R*)-3HB with various hydroxyalkanoate units [92-95] have been synthesized to examine their enzymatic degradation by a variety of e-PHB depolymerases. For instance, Abe et al. proposed a schematic model of the enzymatic cleavage of the PHA chain by PhaZRpiT1 (Figure 5), in which its active site can recognize at least three neighboring monomer units with a certain degree of difference in main-chain length [93]. Besides the chemical structure, the solid-state structure and surface properties of PHAs also influence the enzymatic hydrolysis. For example, the amorphous regions in PHA materials are preferentially hydrolyzed, followed by the hydrolysis of crystalline regions as a rate-limiting step in the enzymatic degradation process [96, 97]. Further, the enzymatic degradation rate of PHA materials decreases with increasing crystallinity, crystal size, and regularity of the chain

**3.2. Effects of chemical and solid-state structures and surface properties of PHAs on**

**enzymatic degradation**

T. Hiraishi and S. Taguchi

144 Protein Engineering - Technology and Application

tion of the enzymatic degradation of PHA by e-PHB depolymerase.

**Figure 5.** Schematic model of enzymatic cleavage of an ester bond in various sequences by PHB depolymerase.

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 microba‐ lance (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 [104]. 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 interac‐ tion 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 muta‐ genesis 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, thermosta‐ bility, or solubility were obtained [115]. 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 *in vivo* screening system as shown in Figure 6(A) [116]. In the analysis of recombinants showing low PHB-degrading activity, Ser410, Tyr412, Val415, Tyr428, Ser432, Leu441, Tyr443, Ser445, Ala448, Tyr455, and Val457 were replaced with other residues having hydropathy indices opposite to theirs at high frequency (Figure 6(B)). The results suggested that PhaZRpiT1 adsorbs to the PHB surface not only via the formation of hydrogen bonds between hydroxyl groups of Ser at these positions of the enzyme and carbonyl groups in the PHB polymer, but also via the hydrophobic interaction between hydrophobic residues at abovementioned positions and methyl groups in the PHB polymer.

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,

T. Hiraishi and S. Taguchi

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

**3.3. Analysis of polymer binding ability of e-PHB depolymerase using directed evolution**

The structural aspects of an enzyme generally provide crucial information about the interac‐ tion 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

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 muta‐ genesis 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, thermosta‐ bility, or solubility were obtained [115]. Further, analysis of the effects of mutations could also

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 *in vivo* screening system as shown in Figure 6(A) [116]. In the analysis of recombinants showing low PHB-degrading activity, Ser410, Tyr412, Val415, Tyr428, Ser432, Leu441, Tyr443, Ser445, Ala448, Tyr455, and Val457 were replaced with other residues having hydropathy indices opposite to theirs at high frequency (Figure 6(B)). The results suggested that PhaZRpiT1 adsorbs to the PHB surface not only via the formation of hydrogen bonds between hydroxyl groups of Ser at these positions of the enzyme and carbonyl groups in the PHB polymer, but also via the hydrophobic interaction between hydrophobic residues at above-

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,

residues in their SBD are involved in the enzyme adsorption to PHB surface.

provide useful information for the improvement of enzyme function.

mentioned positions and methyl groups in the PHB polymer.

100 pN [105, 106].

146 Protein Engineering - Technology and Application

**technique**

**Figure 6.** A) *In vivo* assay system for assessment of mutational effects of the substrate-binding domain of PhaZRpiT1 on PHB degradation. Schematic flow diagram of the system is illustrated. This system is composed of PCR-mediated ran‐ dom mutagenesis in the substrate-binding domain region of PhaZRpiT1 gene, preparation of mutant library, primary plate assay of PHB degradation (clear-zone formation), nucleotide sequencing and PHB degrading and adsorbing as‐ says of partially purified mutant enzymes. (B) Positions and frequencies of PCR-mediated single mutations in the re‐ gion coding for SBD of PhaZRpiT1, together with its predicted secondary structure.

these three residues in PhaZRpiT1 may interact directly with the PHB surface. Since the hydro‐ pathy indices of such mutations as L441H (replacement of Leu441 with His), Y443H (replace‐ ment 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 [117]. 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 hydro‐ phobic 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 [68]. Jendrossek group determined PhaZ7Ple crystal structure and demonstrated that the enzyme was enriched in hydrophobic amino acids including eight tyrosine residues [108]. 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 [118]. 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 *Pseudomonas fluorescens* GK13 by Jendrossek et al. [119]. They reported that several hydrophobic amino acids (Leu15, Val20, Ile26, Phe50, Phe63, Tyr143 and Val198) were identified to be involved in interaction between the enzyme and poly(3-hydroxyoctanoate) substrate surface. This finding was supported with the recent study by Ihssen et al. (2009) [120].

## **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 [121].

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 hydropho‐ bicity 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.


T. Hiraishi and S. Taguchi

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

Recently, Hisano et al. determined the crystal structure of PhaZPfu and proposed that hydro‐ phobic 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 [68]. Jendrossek group determined PhaZ7Ple crystal structure and demonstrated that the enzyme was enriched in hydrophobic amino acids including eight tyrosine residues [108]. 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 [118]. 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 *Pseudomonas fluorescens* GK13 by Jendrossek et al. [119]. They reported that several hydrophobic amino acids (Leu15, Val20, Ile26, Phe50, Phe63, Tyr143 and Val198) were identified to be involved in interaction between the enzyme and poly(3-hydroxyoctanoate) substrate surface. This finding

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

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 hydropho‐ bicity 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

in the enzyme adsorption to the polymer surface.

148 Protein Engineering - Technology and Application

was supported with the recent study by Ihssen et al. (2009) [120].

**3.4. Improvement in SBD function of PhaZRpiT1**

general naturally occurring amino acid residues [121].

hydrophobic residues at these positions for PHB degradation.

a J. Kyte and R. F. Doolittle, 1982 [122].

<sup>b</sup> P. Y. Chou and G. D. Fasman, 1978 [123]. Pb: potential for β-sheet formation ; h: former ; i: indifferent ; b: breaker.

c A. A. Zamyatnin, 1972 [124].

**Table 2.** Hydrophobicity, potential for β-sheet formation, and volume of amino acid residues

#### **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 [125]. 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 [126].

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 *Escherichia coli* cell surface [129]. The displayed enzyme retained its intrinsic characteristics, that is, hydrolytic activity for *p*-nitrophenyl butyrate (pNPC4) and the ability to adsorb to and degrade PHB, indicating that the engineered *E. coli* can be used in the form of a whole-cell biocatalyst by overcoming the uptake limitation of such substrates as insoluble PHB. These findings also indicate that the whole-cell catalyst is a promising and suitable tool to screen for mutant PhaZRpiT1 with enhanced catalytic activity.

## **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) [130]. 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 display‐ ing 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 *p*-nitrophenyl esters (pNPCn, n=2 to 6) can provide the information regarding the substrate recognition of the enzyme. The results of pNPCn hydrolysis by the mutants demonstrated that the elevation on their pNPCn hydrolysis activity for each pNPCn substrate occurred. DNA sequencing showed that eight improved mutant enzymes contained N285D or N285Y mutations. As beneficial mutations are accumulated and deleterious mutations are simultaneously removed from the improved mutants through DNA recombination procedures [131], the N285D and N285Y mutations found here are probably beneficial for pNPCn hydrolysis. Kinetic studies revealed that the increase in catalytic efficiency for pNPCn hydrolysis by the mutant enzymes is attributed to the high Vmax values.

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 demon‐ strated that the N285D and N285Y mutations lowered the hydrolysis activity for the PHB

T. Hiraishi and S. Taguchi

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 [126].

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 *Escherichia coli* cell surface [129]. The displayed enzyme retained its intrinsic characteristics, that is, hydrolytic activity for *p*-nitrophenyl butyrate (pNPC4) and the ability to adsorb to and degrade PHB, indicating that the engineered *E. coli* can be used in the form of a whole-cell biocatalyst by overcoming the uptake limitation of such substrates as insoluble PHB. These findings also indicate that the whole-cell catalyst is a promising and suitable tool to screen for mutant PhaZRpiT1 with enhanced catalytic activity.

**3.6. Protein engineering of CD region of PhaZRpiT1 using cell surface display system**

screening system is feasible and effective for the search of improved enzymes.

is attributed to the high Vmax values.

150 Protein Engineering - Technology and Application

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) [130]. 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 display‐ ing 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

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 *p*-nitrophenyl esters (pNPCn, n=2 to 6) can provide the information regarding the substrate recognition of the enzyme. The results of pNPCn hydrolysis by the mutants demonstrated that the elevation on their pNPCn hydrolysis activity for each pNPCn substrate occurred. DNA sequencing showed that eight improved mutant enzymes contained N285D or N285Y mutations. As beneficial mutations are accumulated and deleterious mutations are simultaneously removed from the improved mutants through DNA recombination procedures [131], the N285D and N285Y mutations found here are probably beneficial for pNPCn hydrolysis. Kinetic studies revealed that the increase in catalytic efficiency for pNPCn hydrolysis by the mutant enzymes

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 demon‐ strated that the N285D and N285Y mutations lowered the hydrolysis activity for the PHB

**Figure 7.** Directed evolution targeted at the catalytic domain (CD) of PhaZRpiT1 using the *in vivo* screening system in the cell surface display system. A schematic diagram of the mutational effects analysis is illustrated. This system consists of random mutagenesis by error-prone PCR in the CD of PhaZRpiT1, cell surface display of enzyme, high-throughput micro‐ plate screening via *p*-nitrophenyl butyrate (pNPC4) hydrolysis, staggered extension process (StEP), and nucleotide se‐ quencing.

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 [68]. 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.

#### T. Hiraishi and S. Taguchi

**Figure 8.** A) Molecular surface representation of PhaZPfu. 3HB trimer in the crevice is shown as a ball and stick model. The positions of catalytic triad residues (S39A, D121, and H155) (cyan), as well as residue S170 (color-coded according to molecular species) are indicated. (B) Proposed model of the active site in PhaZPfu by Hisano et al. (Hisano et al. 2006).

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 (*R*)-3-hydroxybutyrate (3HB) units for cleavage to occur at the center of the active site [90]. Homology modeling of PhaZRpiT1 using the SWISS-MODEL program based on the crystal structure of PhaZPfu (PDB accessionno.2d81A)wasperformedtospeculatethepossiblelocalizationofAsn285intheactive site. Figure 9(A) shows the homology modeling structure of PhaZRpiT1, in which the modeled residue range was positioned from 124 to 294. The residue Asn285 (color-coded according to molecular species)ofPhaZRpiT1 is locatedatthemouthofthe crevice andalso locatedimmediate‐ ly above His273, which corresponds to His155 in subsite -1 of PhaZPfu. However, Asn285 was positioned as if to cover the subsite -1 and to inhibit the substrate access. Taking the homology modeling results and the aforementioned information on the cleavage mechanism into consideration, we propose a simple schematic model for PhaZRpiT1, as shown in Figure 9(B). In this model, Ser139 participates in the nucleophilic attack of the carbonyl carbon of a PHB chain, anditsnucleophilicityisenhancedbytheHis273-Asp121hydrogenbondingsystem.Theresidue Asn285 is positioned relatively close to His273 located in subsite -1 as if to cover the subsite. The location of Asn285 in the subsite probably leads to the regulation of the recognition of sub‐ stratemolecules, such aspNPCn andPHBpolymer chain,possibly indirectly via conformation‐ al change. A similar situation has been described in lipases and PhaZ7Ple where activation via conformational change is required to uncover the active site [108, 109].

T. Hiraishi and S. Taguchi

T. Hiraishi and S. Taguchi

152 Protein Engineering - Technology and Application

**Figure 8.** A) Molecular surface representation of PhaZPfu. 3HB trimer in the crevice is shown as a ball and stick model. The positions of catalytic triad residues (S39A, D121, and H155) (cyan), as well as residue S170 (color-coded according to molecular species) are indicated. (B) Proposed model of the active site in PhaZPfu by Hisano et al. (Hisano et al. 2006).

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 (*R*)-3-hydroxybutyrate (3HB) units for cleavage to occur at the center of the active site [90]. Homology modeling of PhaZRpiT1 using the SWISS-MODEL program based on the crystal structure of PhaZPfu (PDB accessionno.2d81A)wasperformedtospeculatethepossiblelocalizationofAsn285intheactive site. Figure 9(A) shows the homology modeling structure of PhaZRpiT1, in which the modeled residue range was positioned from 124 to 294. The residue Asn285 (color-coded according to molecular species)ofPhaZRpiT1 is locatedatthemouthofthe crevice andalso locatedimmediate‐ ly above His273, which corresponds to His155 in subsite -1 of PhaZPfu. However, Asn285 was positioned as if to cover the subsite -1 and to inhibit the substrate access. Taking the homology modeling results and the aforementioned information on the cleavage mechanism into consideration, we propose a simple schematic model for PhaZRpiT1, as shown in Figure 9(B). In

**Figure 9.** A) Molecular surface representation of the homology model of PhaZRpiT1. The positions of catalytic triad resi‐ dues (S139, D214, and H273) (cyan), as well as residue N285 (color-coded according to molecular species) are indicat‐ ed. (B) Newly proposed schematic model of the active site in the CD of PhaZRpiT1.

## **4. Conclusion**

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 engi‐ neering 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 compo‐ sitions. 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, *in vitro* bioplastic synthesis using isolated bioplasticdegrading enzymes has been developed because of the close relationship between the substrate specificities of the enzymes for polymer degradation and polymer synthesis. *In vitro* enzymatic polymerization offers many advantages, including easier control of polymer structure and monomer reactivity than conventional chemical methods. Accordingly, novel bioplasticdegrading enzymes evolved by protein engineering are expected to become useful biocatalysts for the bioplastic production in the future.

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.

## **Author details**

**4. Conclusion**

154 Protein Engineering - Technology and Application

or pNPCn hydrolysis.

for further specific applications.

monomer compositions and molecular masses.

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

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 engi‐ neering 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

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 compo‐ sitions. 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

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, *in vitro* bioplastic synthesis using isolated bioplasticdegrading enzymes has been developed because of the close relationship between the substrate specificities of the enzymes for polymer degradation and polymer synthesis. *In vitro* enzymatic polymerization offers many advantages, including easier control of polymer structure and Tomohiro Hiraishi1\* and Seiichi Taguchi2,3

\*Address all correspondence to: thiraish@riken.jp

1 Bioengineering Laboratory, RIKEN Advanced Science Institute, Hirosawa, Wako-shi, Saitama, Japan

2 Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku, Sapporo, Japan

3 JST, CREST, Sanbancho, Chiyoda-ku, Tokyo, Japan

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