Engineering of Aeromonas caviae Polyhydroxyalkanoate Synthase Through Site-Directed Mutagenesis for Enhanced Polymerization of the 3-Hydroxyhexanoate Unit

Polyhydroxyalkanoate (PHA) synthase is an enzyme that polymerizes the acyl group of hydroxyacyl-coenzyme A (CoA) substrates. Aeromonas caviae PHA synthase (PhaCAc) is an important biocatalyst for the synthesis of a useful PHA copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. Previously, a PhaCAc mutant with double mutations in asparagine 149 (replaced by serine [N149S]) and aspartate 171 (replaced by glycine [D171G]) was generated to synthesize a 3HHx-rich P(3HB-co-3HHx) and was named PhaCAc NSDG. In this study, to further increase the 3HHx fraction in biosynthesized PHA, PhaCAc was engineered based on the three-dimensional structural information of PHA synthases. First, a homology model of PhaCAc was built to target the residues for site-directed mutagenesis. Three residues, namely tyrosine 318 (Y318), serine 389 (S389), and leucine 436 (L436), were predicted to be involved in substrate recognition by PhaCAc. These PhaCAc NSDG residues were replaced with other amino acids, and the resulting triple mutants were expressed in the engineered strain of Ralstonia eutropha for application in PHA biosynthesis from palm kernel oil. The S389T mutation allowed the synthesis of P(3HB-co-3HHx) with an increased 3HHx fraction without a significant reduction in PHA yield. Thus, a new workhorse enzyme was successfully engineered for the biosynthesis of a higher 3HHx-fraction polymer.


INTRODUCTION
Polyhydroxyalkanoates (PHAs) are bio-based polyesters produced by a wide range of microorganisms as carbon and energy storage materials. The wild-type strain H16 of Ralstonia eutropha (or Cupriavidus necator) is one of the best-known PHA-producing bacteria (Sudesh et al., 2000;Steinbüchel and Hein, 2001). There has been long-standing interest in using PHAs as biodegradable bioplastics that could serve as alternatives to petrochemical plastics. Recently, PHAs have attracted attention as biodegradable and biocompatible thermoplastics for use in a wide range of agricultural, marine, and medical applications because of their excellent biodegradability (Akiyama et al., 2003).
Polyhydroxyalkanoates mainly consist of short-chain length (SCL; C3 to C5) and/or medium-chain-length (MCL; C6 and longer) monomers (Rehm, 2003). Among the SCL-PHAs, poly[(R)-3-hydroxybutyrate] [P(3HB)] is the most common bacterial PHA in nature. Although P(3HB) is a highly crystalline, hard, and brittle polymer, it begins to decompose at a temperature close to its melting point, making it difficult to process this polymer (Lehrle and Williams, 1994). Copolymerization of MCL monomers with a 3HB unit leads to notable changes in the physical properties of PHA, depending on the molecular structure and copolymer composition (Noda et al., 2005). The best-studied SCL/MCL-PHA copolymer is poly(3HB-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)]. In this polymer, an important aspect is to control the level of the 3HHx monomer fraction for practical application in many fields. For example, the elongation at break increases from 5 to 760% by increasing the 3HHx fraction from 0 to 15 mol% (Doi et al., 1995;Chen et al., 2000;Andreeßen et al., 2014). P(3HB-co-3HHx) with 10-15 mol% 3HHx fraction can be used as an alternative to conventional plastics such as polypropylene and polyethylene (Shimamura et al., 1994;Chen et al., 2000;Andreeßen et al., 2014). However, it is difficult to efficiently produce P(3HBco-3HHx) with such a high 3HHx fraction. Thus, significant efforts have been made to increase the 3HHx fraction in P(3HBco-3HHx) biosynthesis (Jian et al., 2010;Budde et al., 2011;Arikawa and Matsumoto, 2016a).
The bacterium Aeromonas caviae is an original strain that can produce P(3HB-co-3HHx) from plant oils and fatty acids (Shimamura et al., 1994). Aeromonas caviae PHA synthase (PhaC Ac ) shows substrate specificity toward 3HB and 3hydroxyvalerate monomers, as well as the 3HHx monomer . From this point of view, PhaC Ac is a valuable biocatalyst for production of P(3HB-co-3HHx). However, the polymer production capacity of A. caviae is not superior to that of other PHA producers. With the help of genetic engineering, recombinant R. eutropha expressing PhaC Ac was generated, which demonstrated remarkable enhancement of P(3HB-co-3HHx) production from plant oils Doi, 1997, 1998;Kahar et al., 2004).
Additionally, to increase the 3HHx fraction in P(3HB-co-3HHx), various strategies have been developed. One effective approach is to increase the expression of (R)-specific enoylcoenzyme A (CoA) hydratase (PhaJ4b Re ), which provides R-3-hydroxyacyl-CoA precursors for PHA synthesis from the β-oxidation cycle, to reinforce the supply of the 3HHx monomer (Arikawa and Matsumoto, 2016a). In contrast, the 3HHx fraction in the polymer was increased by deleting the gene for the 3HB supplier acetoacetyl-CoA reductase (PhaB Re ) to suppress the 3HB monomer supply; however, the PHA yield decreased (Budde et al., 2011).
Another approach to increase the 3HHx fraction in PHA is the engineering of PHA synthase (Kichise et al., 2002;Tsuge et al., 2004Tsuge et al., , 2007aWatanabe et al., 2012). In previous studies, PhaC Ac was modified via evolutionary engineering approaches, and several mutation sites (e.g., asparagine 149, aspartate 171, valine 214, and phenylalanine 518) enhanced the 3HHx polymerization capacity (Amara et al., 2002;Kichise et al., 2002;Tsuge et al., 2004). Furthermore, a double mutant of PhaC Ac , termed the NSDG mutant, which has two amino acid substitutions of asparagine 149 by serine (N149S) and aspartate 171 by glycine (D171G), was generated as a superior enzyme capable of synthesizing P(3HB-co-3HHx) with a higher 3HHx fraction than the wild-type enzyme (Tsuge et al., 2007b). However, since then, no PhaC Ac mutant with further high 3HHx polymerization ability has been created.
The three-dimensional structure of a protein provides important information for understanding its biochemical function and catalytic mechanism. Homology modeling aims to build three-dimensional protein structure models using experimentally determined structures of related family members as templates. Thus, homology modeling is a powerful tool for understanding and predicting the three-dimensional structure of unknown proteins to determine beneficial mutation sites and improve protein properties (Stoilov et al., 1998;Lee et al., 2011). Recently, some research groups have determined the partial crystal structure of R. eutropha PHA synthase (PhaC Re ), which is classified into the same group (class I) as PhaC Ac based on its substrate specificity and subunit structure (Wittenborn et al., 2016;Kim et al., 2017). According to their crystal structures, three active residues, Cys319, Asp480, and His508, in PhaC Re are in close proximity. Additionally, amino acid residues that make up the substrate pocket have been identified (Wittenborn et al., 2016;Kim et al., 2017). Moreover, structural information on the available PHA synthases has been increasing (Chek et al., 2017(Chek et al., , 2019(Chek et al., , 2020. In this study, using a newly constructed homology model of PhaC Ac , three amino acid residues were predicted to be constituents of the substrate pocket and involved in substrate recognition. Based on this prediction, site-specific mutagenesis was conducted on PhaC Ac NSDG to introduce an additional third mutation. The resulting triple mutants were expressed in the strain 005dC1Z126TRCB, an engineered R. eutropha strain, grown on palm kernel oil as a carbon source for PHA biosynthesis. It was found that the triple mutant PhaC Ac NSDG/S389T is capable of synthesizing P(3HB-co-3HHx) with a higher 3HHx fraction than the parental PhaC Ac NSDG. Furthermore, the selected PhaC Ac triple mutants were isolated as PHA granule-associated enzymes from R. eutropha and characterized through enzyme kinetic analysis to understand how the catalytic function changed.

Bacterial Strains and Plasmids
Bacterial strains and gene expression plasmids used are listed in Table 1. All Escherichia coli strains were grown in Luria-Bertani (LB) medium. The E. coli strains JM109 and S17-1 were used for plasmid construction and as donors in the intergeneric conjugation experiments, respectively. All R. eutropha strains were grown in nutrient broth (Difco Laboratories, Detroit, MI, United States).
To delete the phaC Ac NSDG gene in the R. eutropha CnTRCB strain (Arikawa and Matsumoto, 2016a), the gene This study deletion plasmid pNS2X-sacB-phaC1AdS (Sato et al., 2013) was introduced into the CnTRCB strain by conjugation from the donor strain E. coli S17-1. The deletion of phaC was confirmed through PCR. The resulting strain was named 005dC1Z126TRCB, which retained phaA and phaB involved in the 3HB monomer supply and provided greater proportions of 3HHx than the H16 strain, by enhancing the expression of phaJ4b Re .

Homology Modeling of A. caviae PHA Synthase
A template-based modeling method using HyperChem (HYPERCUBE, INC., Gainsville, FL, United States) (Froimowitz, 1993) was used to predict the structure of PhaC Ac using PDB:5T6O (residues 201-589) from PhaC Re as a template.

Plasmid Construction and Site-Directed Mutagenesis
Plasmids expressing wild-type PhaC Ac , the double mutant NSDG, and the triple mutants NSDG-Y318/S389/L436X were constructed based on the pCUP3 vector, which is stably maintained in R. eutropha (Sato et al., 2013). The wild-type phaC Ac (WT-phaC Ac ) and phaC Ac NSDG genes were obtained through PCR with MunI_PhaCAc_F and SpeI_PhaCAc_R as primers, using the plasmid pColdI::phaC Ac and the genomic DNA of the R. eutropha strain KNK005 as a template, respectively (Sato et al., 2013;Ushimaru et al., 2014). These fragments were digested by MunI and SpeI, and then cloned into the same sites of the pCUP3 vector. The P trp fragment, which was amplified by PCR using pKK388-1 (Clontech, Palo Alto, CA, United States) as a template (Arikawa and Matsumoto, 2016a), was digested with MunI and ligated with MunI-digested pCUP3 vectors carrying WT-phaC Ac and phaC Ac NSDG genes to yield pCUP3-P trp -WT-phaC Ac and pCUP3-P trp -phaC Ac NSDG, respectively. Site-directed mutagenesis of phaC Ac NSDG gene was performed by overlap extension PCR (Ho et al., 1989). Reverse primers containing a point mutation were designed as listed in Supplementary Table S1, and primers containing a restriction enzyme site were designed as (pCUP3_IF_MunI_trp_F) 5 -ACA TTGCGCTGAAAGAAGGGCCAATTGTGCTTCTGGCGTC-3 and (pCUP3_SpeI_IF_R) 5 -GCTCGGATCCACTAGTCGGCT GCCGACTGGT-3 (the underlined sequences indicate the MunI and SpeI sites, and the bold sequences indicate in-fusion alignment). Using the corresponding primers in phaC Ac _Y318/S389/L436X_R and phaC Ac _Y318/S389/L436X_F (Supplementary Table S1), the DNA fragments were amplified.
The resulting fragments after one round of PCR were used as templates, and PCR was performed again using the outside primers with MunI and SpeI sites. The resulting phaC Ac NSDG fragments with point mutations were digested using MunI and SpeI, and then inserted into the corresponding restriction sites in the pCUP3 vector. The resulting pCUP3-P trp -NSDG-Y318/S389/L436X plasmids were introduced into an engineered strain of R. eutropha 005dC1Z126TRCB strain, in which phaC gene was disrupted. Transformation was performed through electroporation, as described previously (Sato et al., 2013;Arikawa et al., 2016b).

PHA Accumulation From Palm Kernel Oil
Polyhydroxyalkanoate production was performed in 50 mL of mineral salt (MS) medium (Kato et al., 1996) with 1.29 g/L (NH 4 ) 2 SO 4 and 1.5 w/v% palm kernel oil as a sole carbon source for 72 h. Kanamycin was added to the medium at a concentration of 50 mg/L to maintain the plasmid in the cells. After cultivation, the collected cells were washed with water and ethanol to remove the remaining carbon sources and then lyophilized (Arikawa et al., 2016b). The PHA content in the cells was determined by gas chromatography (GC) after methanolysis of approximately 15 mg of lyophilized cells in the presence of 15% (v/v) sulfuric acid, as previously described (Lakshman and Shamala, 2006).

Kinetic Analysis of the Granule-Associated PHA Synthase
The PHA synthase activity assay was performed, wherein the amount of CoA released was measured using 5,5-dithiobis(2nitrobenzoic acid) (DTNB) with the following modifications: PHA synthase assay was initiated by adding the granuleassociated PhaC Ac , which was obtained from 24 h of R. eutropha culture broth by ultracentrifugation as previously described (Valentin and Steinbüchel, 1994;Harada et al., 2019). After

Analysis of the PHA Synthase Concentration Through Western Blotting
The concentration of the granule-associated PhaC Ac was determined as previously described (Harada et al., 2019), after incubation with rabbit antiserum against a peptide from the C-terminus of PhaC Ac , followed by incubation with a goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP; Santa Cruz Biotechnology, CA, United States). Proteins were visualized using the ECL Plus Western Blotting Detection Reagent (Bio-Rad, Hercules, CA, United States). Data were recorded using a CCD camera FAS-1000 (Toyobo, Osaka, Japan). Quantitative analysis of PhaC Ac concentration on PHA granules was performed using calibration curves prepared using purified PhaC Ac (130-520 ng). Band intensities were quantified using the ImageJ software 1 .

Amino Acid Residues That Determine the Substrate Pocket Size of PhaC Ac
To identify the beneficial mutation site for increasing the 3HHx fraction, a homology model of PhaC Ac was first built by targeting  (Figures 2A,B). This is in good agreement with the experimental observation that PhaC Ac has a broader substrate specificity than PhaC Re (Fukui and Doi, 1997). From the comparison of these structural models, two amino acid residues adjacent to the active center (PhaC Ac vs. PhaC Re : Y318 vs. F318, S389 vs. T393) were found to be different. It was presumed that Y318 and S389 determine the depth and width of the substrate pocket of PhaC Ac , respectively. The substrate entrance tunnel of these models was further compared (Figures 2C,D), and additional differences were found (PhaC Ac vs. PhaC Re : L436 vs. Y440). In PhaC Ac , L436 mainly contributes to expanding the substrate entrance tunnel, because there is a significant difference in the amino acid size at the homologous positions in these structural models.

PHA Synthesis by PhaC Ac NSDG With an Additional Y318 Mutation
As the Y318 of PhaC Ac was predicted to determine the depth of the substrate pocket based on the homology model, we investigated the effect of the amino acid size at this position on 3HHx polymerization ability. To replace Y318, we selected Leu, Ile, and Met, which are smaller than Tyr, with the aim of expanding the substrate pocket space. The three PhaC Ac mutants with NSDG mutations and either Y318L/I/M mutations were generated by sitedirected mutagenesis and expressed in the engineered R. eutropha strain 005dC1Z126TRCB to induce P(3HBco-3HHx) biosynthesis from palm kernel oil. The results are presented in Table 2. The strain expressing the wildtype enzyme accumulated 80.3 wt% P(3HB-co-3HHx) of dried cells, with 7.4 mol% of 3HHx fraction. Meanwhile,  the strain expressing PhaC Ac NSDG accumulated 85.7 wt% P(3HB-co-3HHx) of dried cells with 13.1 mol% of 3HHx fraction. A very small amount (less than 0.1 mol%) of 3hydroxyoctanoate (3HO) was also detected, which is consistent with previous study (Tsuge et al., 2007b). PhaC Ac NSDG was confirmed to have the ability to synthesize P(3HB-co-3HHx) with a higher 3HHx fraction than the wild-type enzyme. Compared to NSDG and NSDG/Y318X, a slight increase in the 3HHx fraction was observed in the strain expressing the NSDG/Y318I mutant, whereas the other two strains showed a considerable decrease in the 3HHx fraction. As for the NSDG/Y318L mutant, it showed a slight increase (0.8 mol%) in the 3HO fraction. On the contrary, expression of the NSDG/Y318I mutant notably decreased polymer accumulation (11.7 wt%) in the cells compared to the parental NSDG strain (85.7 wt%). Thus, additional mutagenesis of Y318 was not beneficial.

PHA Synthesis by PhaC Ac NSDG With an Additional S389 Mutation
S389 in PhaC Ac contributes to cavity formation near the active center. It is homologous to T393 in PhaC Re , and the cavity space in PhaC Ac is larger due to the volume of one methyl group. To further expand the cavity space, the amino acid residue at position 389 was replaced with Ala (S389A), which is a smaller amino acid.
To examine the opposite effect on the amino acid size, this residue was also replaced with the larger amino acid Thr (S389T) with the aim of narrowing the space. The two PhaC Ac mutants with NSDG mutations and either S389A/T mutations were generated by site-directed mutagenesis and evaluated for P(3HB-co-3HHx) biosynthesis. The results are presented in Table 3. The additional S389A mutation did not alter the 3HHx fraction. However, the S389T mutation in PhaC Ac NSDG increased the 3HHx fraction to 14.9 mol% without a significant decrease in PHA yield. Since the 3HHx fraction increased due to replacement with the bulkier amino acid in the mutant, further replacements were conducted using Val, Leu, Ile, and Cys, which have bulkier side chains than Ser based on their van der Waals volumes (Darby and Creighton, 1995;Tsuge et al., 2009). As a result, a slight increase in the 3HHx fraction up to 13.8 mol% was observed by introducing S389V/L/I/C mutations in PhaC Ac NSDG. Of the mutations tested, the S389T mutation was the most effective in increasing the 3HHx fraction, followed by S389V. It was found that mutagenesis at position 318 in PhaC Ac may enhance the 3HHx polymerization ability, although replacement with relatively bulky amino acids was effective.

PHA Synthesis by PhaC Ac NSDG With Additional Mutation for L436
L436 is an amino acid located slightly outside the active center, which corresponds to Y440 in PhaC Re . As predicted by homology modeling, the cavity of PhaC Ac is larger than that of PhaC Re because of the difference in the amino acid side size at this position. To examine the effect of mutagenesis for L436 on the 3HHx polymerization ability of PhaC Ac NSDG, sitedirected mutagenesis was performed. To examine the expanding effect of the pocket space, L436A/V mutations were introduced into PhaC Ac NSDG. In addition, L436Y/I mutations were introduced to examine the opposite narrowing effect (Darby and Creighton, 1995;Tsuge et al., 2009). The results are listed in Table 4. PHA accumulation was observed for all strains with polymer contents greater than 80 wt%. However, these mutations showed a decrease in the 3HHx fraction; The L436A and L436Y mutations showed 21% and 66% reductions in the 3HHx fraction, respectively, when compared to the parental NSDG strain. Based on this observation, the residue at position 436 may be involved in substrate recognition, but mutagenesis at this position did not result in an increase in the 3HHx fraction of the polymer.

Kinetic Analysis of PhaC Ac NSDG With S389V/T/C Mutations
To obtain a better understanding of the polymerization ability of the 3HHx monomer of PhaC Ac , granule-associated PHA synthases were prepared and used for enzyme kinetic analysis. The granule-associated PHA synthase does not exhibit a lag phase (Gerngross et al., 1994;Taguchi et al., 2002) because the enzyme is already activated and thus is suitable for use in accurate kinetic analysis. To determine the PhaC Ac concentrations on the surface of the isolated PHA granules, western blotting was performed using an antibody against PhaC Ac . The kinetic parameters determined for wild-type PhaC Ac , NSDG mutant, and NSDG/S389X mutants are listed in Table 5. The NSDG   mutant and NSDG/S389X showed a lower Michaelis constant (K m ) for the R-3HHx-CoA substrate than the wild-type PhaC Ac but was not significant for the R-3HB-CoA substrate. In addition, the NSDG mutant and NSDG/S389X mutants showed a higher turnover number (k cat ) for both substrates than the wild-type PhaC Ac , except for NSDG/S389V toward R-3HHx-CoA. Kinetic analysis revealed that the substrate affinity and turnover number, especially for R-3HHx-CoA, increased in the NSDG mutant. Among the mutants tested, the K m values of S389V/C mutants for R-3HHx-CoA, which were 0.46 mM and 0.53 mM, respectively, showed smaller values than that of the parental NSDG strain (0.73 mM). The decrease in K m value indicates the increased affinity between enzyme and substrate, thus providing evidence of the reinforced ability of 3HHx polymerization by these mutations. In contrast, by introducing the S389T mutation into PhaC Ac NSDG, the K m value slightly increased for both R-3HB-CoA and R-3HHx-CoA. Furthermore, the k cat value significantly increased for both substrates by up to 3.4-fold compared to the parental NSDG enzyme. Thus, the increase in the 3HHx fraction caused by the S389T mutation could be attributed to the increased catalytic turnover of the enzyme, rather than the increased affinity between the substrate and the enzyme.

DISCUSSION
This study aimed to increase the 3HHx fraction in P(3HBco-3HHx) by engineering PhaC Ac . Based on evolutionary engineering, we had already generated a PhaC Ac NSDG mutant as a workhorse to synthesize a high 3HHx-fraction polymer. The mutation positions of NSDG are at the N-terminal region of PhaC Ac , and these amino acid residues are predicted to not be involved in the formation of the substrate pocket. Thus, to further modify the PhaC Ac NSDG for higher 3HHx-fraction polymer synthesis, we attempted to change the cavity space of the substrate pocket by replacing certain amino acids. Recently, two research groups have published the partial crystal structure of PhaC Re (Wittenborn et al., 2016;Kim et al., 2017). PhaC Re can polymerize up to C5 monomers, whereas PhaC Ac is capable of polymerizing up to C6 monomers. The difference in substrate specificity may be caused by the size of the substrate pocket near the active center . From this viewpoint, the three-dimensional structures around the cavity pocket space of PhaC Re and the homology model of PhaC Ac were compared, mainly focusing on the difference in the spread of amino acid side chains. As possible determining residues for the pocket size of PhaC Ac , three amino acids, namely Y318, S389, and L436, were identified in this study.
Our homology model suggests that Y318 may be an important residue that determines the pocket size ( Figure 2B). Interestingly, this position is Ala in PHA synthases from Pseudomonas spp. (class II) that can polymerize MCL monomers up to C14. Therefore, it is reasonable to hypothesize that a mutation at this position has a significant influence on the pocket depth. The amino acid at this position in PhaC Re (F318) has been suggested to stabilize the structure of the substrate pocket . Indeed, mutagenesis at this position of PhaC Re led to a decrease in 75% of the synthase activity . In our study, mutation of Y318 of PhaC Ac also resulted in a significant reduction in polymer synthesis ( Table 2). Y318 maintains the structure of the substrate pocket and is strongly related to the polymerization ability in the same manner as PhaC Re .
The docking simulation using the crystal structure of PhaC Re suggested that Y440 is located in the substrate entrance tunnel and contributes to the structural stabilization of the β-mercaptoethylamine/pantothenate (β-MP) moiety of R-3HB-CoA . Y440 stabilizes the substrate orientation by interacting with neighboring amino acids to efficiently catalyze the polymerization reaction. In PhaC Ac , the corresponding L436 was considered to regulate the space of the substrate entrance tunnel based on the homology model ( Figure 2D). In fact, mutagenesis of L436 limited the substrate specificity of PhaC Ac and reduced the 3HHx fraction in the biosynthesized polymer (Table 4). Among the NSDG/L436X mutants examined, the most remarkable reduction in the 3HHx fraction was observed for the NSDG/L436Y mutant, probably due to the narrowest pocket space by replacement with the largest amino acid Tyr.
However, the effect of 3HHx polymerization ability cannot always be explained by the reduction and expansion of pocket space due to amino acid replacement. In this study, we found that the 3HHx fraction in PHA increased after narrowing the substrate pocket by mutagenesis of S389 (Table 3). However, this observation was opposite to our hypothesis.
To better understand the effect of S389 mutagenesis, the kinetics of the enzymes with the S389X mutation were investigated. Kinetic analysis provided new information on the changes in catalytic function due to S389X mutations. It was revealed that substrate affinity for R-3HHx-CoA was increased by S389V/C mutations, whereas the catalytic turnover of the enzyme was increased by the S389T mutation. Thus, the increase in the 3HHx fraction caused by the S389T mutation may be due to the increased catalytic turnover of the enzyme, rather than the change in binding affinity between the enzyme and substrate. The relationship between pocket size narrowing and 3HHx polymerization ability may be explained by stabilization of the substrate orientation when the substrate accesses the active site. The proper orientation of the substrate may increase the efficiency of the catalytic reaction. However, further studies are required to elucidate the underlying mechanisms of mutagenesis.

CONCLUSION
In conclusion, by comparing the substrate pocket structures of PhaC Re and PhaC Ac , a new beneficial mutation position at S389 was found to enhance the 3HHx polymerization ability of PhaC Ac NSDG. Since the discovery of the NSDG mutation, additional mutations conferring a superior ability of 3HHx polymerization have not been found by an evolutionary engineering approach. Thus, this is a successful example of PHA synthase engineering by effectively exploiting the findings from the three-dimensional structure of proteins.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.