Development and Validation of an HPLC-based Screening Method to Acquire Pha

Journal of Bioscience and Bioengineering VOL. 113 No. 3, 286 – 292, 2012 www.elsevier.com/locate/jbiosc

Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity Yoriko Watanabe, 1 Yousuke Ichinomiya, 1 Daisuke Shimada, 1 Azusa Saika, 1 Hideki Abe, 2 Seiichi Taguchi, 3 and Takeharu Tsuge 1,⁎ Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226–8502, Japan, 1 Bioplastic Research Team, RIKEN Biomass Engineering Program, 2–1 Hirosawa, Wako-shi, Saitama 351–0198, Japan, 2 and Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8, Kita-ku, Sapporo 060–8628, Japan 3 Received 19 June 2011; accepted 16 October 2011 Available online 15 November 2011

A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using highperformance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaCAc), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB−4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Polyhydroxyalkanoates; High-throughput screening; High-performance liquid chromatography (HPLC); Alkaline pretreatment; PHA synthase; Substrate specificity]

Polyhydroxyalkanoates (PHAs) are biological polyesters produced by a wide variety of microorganisms as an intracellular storage material for carbon and energy. PHAs have attracted industrial attention for use as biodegradable and biocompatible thermoplastics (1,2). PHAs are synthesized by PHA synthases (PhaCs) which catalyze the polymerization reaction of 3-hydroxyalkanoates (3HAs) as monomer substrates. Therefore, the substrate specificity of PhaC significantly influences the monomer composition of synthesized PHA. Poly(3-hydroxybutyrate) [P(3HB)] is the most common PHA synthesized by bacteria in nature. P(3HB) has high rigidity but is brittle with low elasticity. Therefore, flexible 3HB-based copolymers such as P(3HB-co-3-hydroxyvalerate) [P(3HB-co-3HV)], P(3HB-co-3hydroxyhexanoate) [P(3HB-co-3HHx)], P(3HB-co-3-hydroxy-4methylvalerate) [P(3HB-co-3H4MV)], and P(3HB-co-medium-chainlength-3-hydroxyalkanoate) [P(3HB-co-mcl-3HA)] are recognized as more suitable polymers for practical use (3–5). These monomer structures are shown in Fig. 1. Aeromonas caviae is capable of synthesizing P(3HB-co-3HHx) random copolymer from vegetable oils as the carbon source (6), because this bacterium possesses the PHA synthase (PhaCAc) that has

⁎ Corresponding author. Tel.: + 81 45 924 5420; fax: + 81 45 924 5426. E-mail address: [email protected] (T. Tsuge).

the unique ability to polymerize 3HB and 3HHx units. P(3HB-co3HHx) is highly desired by industry as a bio-based plastic, but this bacterium has poor ability to produce and accumulate it (less than about 30 wt.% of the cells). On the other hand, Ralstonia eutropha is a PHA over-producer (greater than 80 wt.% of the cells), while the type of PHA synthesized by this strain is limited to P(3HB) with vegetable oils as the carbon source. Thus, the higher production of P(3HB-co3HHx) was achieved from vegetable oils in recombinant R. eutropha PHB−4 transformed with a vector-borne PhaCAc gene (7,8). The resultant host-vector system, however, suffered from weak incorporation of the 3HHx unit into P(3HB-co-3HHx), limiting the 3HHx fraction to 3–4 mol% in cultivation on soybean oil. This phenomenon was mainly ascribed to the substrate specificity of PhaCAc (7). We applied directed evolution to create mutant enzymes that gain higher reactivity toward 3HHx. In the initial stage, two beneficial single mutants, N149S (asparagine 149 → serine) and D171G (aspartic acid 171 → glycine), were obtained (9). Subsequent mutation studies with PhaCAc allowed us to regulate the 3HHx fraction from 0 to 5.2 mol% (10,11). The highest 3HHx fraction (5.2 mol%) was obtained using the PhaCAc double mutant with N149S and D171G mutations (NSDG mutant) (10). To extend the regulation range of the 3HHx fraction, PhaCAc mutants with further increased reactivity toward 3HHx were required. For this, a much more efficient high-throughput screening system was necessary. Kichise et al. developed an initial assay

1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.10.015

VOL. 113, 2012

HPLC ASSAY FOR PHA SYNTHASE MUTANTS

O

*

O

O

*

*

O

3HB

O

*

*

3HV

O

3HHx

*

3H4MV

O

O

*

O

*

*

O

3HO

*

FIG. 1. Structure of monomer units in PHA synthesized in this study. 3HB, 3hydroxybutyrate; 3HV, 3-hydroxyvalerate; 3H4MV, 3-hydroxy-4-metylvalerate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate.

method to measure cellular P(3HB) content using high-performance liquid chromatography (HPLC) (12). To prepare samples for this method, P(3HB)-accumulating cells are treated with sulfuric acid at 100°C to convert P(3HB) to crotonic acid (trans-2-butenoic acid). Subsequently, the treated samples are subjected to HPLC with an ultraviolet (UV) detector to measure absorption at 210 nm due to unsaturated crotonic acid bonds (13). This method is very convenient to measure P(3HB) concentration; however, it is not applicable to other PHAs because PHA monomers longer than 3HB cannot be converted to the corresponding unsaturated fatty acids. In a previous study (12), PhaCAc mutants showing higher reactivity toward 3HHx were obtained as a result of screening for highpolymerization activity mutants based on P(3HB) accumulation levels in host Escherichia coli. Thus, establishing a sample preparation method applicable to longer PHA monomers including 3HHx would allow direct screening for substrate-specificity-altered synthases based on PHA copolymer composition using HPLC, improving screening efficiency. On the other hand, gas chromatography (GC) analysis is widely used to determine PHA composition regardless of 3HA carbon chain length. It requires derivatization process of polymer samples, which is difficult to simplify for a highthroughput assay, prior to GC analysis. In this study, a new HPLC-based screening method capable of analyzing PHA copolymer composition was developed by applying alkaline (sodium hydroxide) pretreatment instead of acid pretreatment to sample preparation for HPLC analysis. In addition, a high-throughput protocol was established by introducing a 96-well plate format for cultivation of the PHA producing host and for sample preparation. The new method was used to isolate PhaCAc mutants with high reactivity toward 3HHx from a D171 random point mutation (D171X) library to determine whether the D171G mutation is the most effective for increasing PhaCAc reactivity toward 3HHx. MATERIALS AND METHODS Bacterial strains and plasmids E. coli JM109 was used as the host strain for screening PhaCAc mutants and for P(3HB-co-3HHx) accumulation from dodecanoate, while R. eutropha PHB−4 (PHA-negative mutant, DSM541) was used for PHA copolymer production from soybean oil, octanoate, or 4-methylvalerate. Plasmid pBBR1phaPCJAcABRe was constructed by introducing a 6.4-kb XbaI–HindIII fragment of pBSEE32phbAB (12) into the same sites of a broad-host-range vector pBBR1MCS-2 (14). The resultant plasmid carries PHA polycistronic genes (accession no. D88825) for PhaPAc (granule-associated protein), PhaCAc, and PhaJAc (R-specific enoyl-CoA hydratase) with a promoter derived from A. caviae FA440 and the genes for the (R)-3HB-CoA monomer supplying enzymes PhaARe (3-ketothiolase) and PhaBRe (NADPH-dependent acetoacetyl-CoA reductase) from R. eutropha H16 (accession no. J04987). Random point mutagenesis at position 171 of PhaCAc Random point mutagenesis at position 171 of PhaCAc (Fig. 2A) was performed using an inverse polymerase chain reaction (PCR) method described by Imai et al. (15). The PCR primers used in this study were designed in inverted tail-to-tail directions to amplify pBBR1phaPCJAcABRe with the target sequence for amino acid substitution as follows: for D171X, 5′-CCT GGA GTC CNN NGG CCA GAA CCT G-3′ (the underlined sequence

287

indicates a mutation site, and N represents a random nucleotide) as the sense primer and 5′-GTC AGC TTG AGC AGC TCG GGG TTG G-3′ as the antisense primer. After PCR amplification with the primer set, the amplified linear DNA was phosphorylated and self-ligated using a BKL kit (Takara Bio Inc., Otsu). Subsequently, the self-ligated PCR products were transformed into E. coli JM109 to prepare a PhaCAc mutant library. HPLC-based screening of PhaCAc mutants Fig. 2B shows a schematic diagram of the HPLC-based screening system developed in this study. Briefly, it is consisted of site-specific random mutagenesis, preparation of a mutant library, primary assay of P(3HB) accumulation in E. coli JM109 using Nile red (9-(diethylamino)-5H-benzo[α] phenoxazin-5-one) dye on an agar plate, liquid cultivation in M9 medium plus dodecanoate (C12) using a 96-well plate for P(3HB-co-3HHx) accumulation, alkaline sample pretreatment, HPLC assay, and nucleotide sequence determination. A PhaCAc D171X mutant library constructed with E. coli JM109 was spread on Luria–Bertani (LB) agar plates (Bacto-Tryptone 10 g, Bacto-yeast extract 5 g, NaCl 10 g, and agar 15 g per liter of distilled water) supplemented with 20 gL− 1 glucose, 0.5 mg/L− 1 Nile red, and 50 mgL− 1 kanamycin and cultured at 37°C overnight. The polymerization ability of PhaCAc mutants was judged based on the intensity of the pinkish pigmentation of the cells caused by Nile red staining (16). Next, single pinkish pigmented colonies were inoculated in M9 medium (0.6 mL) containing 1.0 gL− 1 sodium dodecanoate, 1.0 gL− 1 Bacto-yeast extract, 0.4 vol% Brij35, and 50 mgL− 1 kanamycin in each 1.2-mL well of a 96-deep well culture plate (BM Equipment Co., Ltd., Tokyo). After sealing the plate with an air-permeable film (4titude, Ltd., Surrey, UK), the cells were cultured at 37°C for 72 h in a reciprocal shaker (1035 rpm, Bio Shaker, Taitec Co., Ltd., Saitama). After the grown cells were replicated on LB agar plates, the cultured 96-well plates were centrifuged using a Hitachi R6S swing rotor at 3000 rpm (1500 × g) for 10 min, and then the culture supernatants were discarded. The 96-well plates with cell pellets remaining at the bottom of each well were dried at 55°C for 3 days. For alkaline hydrolysis of the dried cells, 200 μL of 1 N NaOH was added to each well of the 96-well plates using a handheld multichannel pipettor. After heat-sealing with a polypropylene/aluminum film (4titude) using a microplate heat sealer (ABgene Ltd., Surrey, UK), the 96-well plates were heated at 100°C for 3 h on a hot plate. The cell hydrolysates were neutralized with 200 μL of 1 N HCl and then filtrated with 96-well filter plates (0.45 μm pore size polytetrafluoroethylene (PTFE) membrane, Pall Co., NY, USA) by centrifuging at 3000 rpm (1500× g) for 30 min. The filtrates were collected with a new 96-well assay plate, sealed with a cover film to prevent evaporation, and subjected to HPLC analysis. HPLC analysis was performed using a Shimadzu LC-10Avp system with an autosample injector applicable to 96-well plates. The samples were separated on two types of ion-exclusion columns, Aminex HPX-87H (300 mm × 7.8 mm I.D., Bio-Rad, CA, USA) and Fast Acid Analysis (100 mm × 7.8 mm I.D., Bio-Rad), at 60°C using 0.014 N H2SO4 with or without 20% CH3CN as a mobile phase at a flow rate of 0.7 mL/min. The chromatograms were recorded at 210 nm using a UV detector. Site-specific mutation of PhaCAc Aspartic acid (D) at position 171 of the PhaCAc N149S mutant was replaced by alanine (A), leucine (L), or histidine (H) as a second mutation to yield a doubly mutated gene (phaCAc NSDA, NSDL, or NSDH) using a QuickChange Multi Site-directed Mutagenesis Kit (Stratagene Co., CA, USA) or a similar method. The primer was designed as 5′-CTG GAG TCC NNN GGC CAG AAC CTG G-3′. The underlined NNN sequences in the primer were GCC, CTG, and CAC for alanine (A), leucine (L), and histidine (H) replacement, respectively, and were designed based on the codon usage of R. eutropha. Recombination of R. eutropha and PHA analysis To express the phaCAc gene in R. eutropha PHB−4 under our basal conditions, a 0.6-kb PstI–ScaI fragment of pBBR1phaPCJAcABRe was introduced into the same pBBREE32d13dPB sites (10,11) to yield pBBREE32d13dPB D171X carrying only the mutated phaCAc gene downstream of the pha promoter from A. caviae. These plasmids were introduced by transconjugation from E. coli S17-1 into R. eutropha PHB−4 (17). The recombinant R. eutropha strain PHB−4 was cultivated at 30°C for 72 h on a reciprocal shaker (130 strokes/min) in 500-mL flasks containing 100 mL of nitrogen-limited mineral salt (MS) medium supplemented with a carbon source (20 gL− 1 soybean oil, 5 gL− 1 sodium octanoate, or 2.5 gL− 1 4-methylvalerate). The composition of the MS medium was as follows (per liter of distilled water): 9 g of Na2HPO4·12H2O, 1.5 g of KH2PO4, 0.5 g of NH4Cl, 0.2 g of MgSO4·7H2O, and 1 mL of trace element solution (18). The pH of the medium was adjusted to 7.0. Kanamycin (50 mgL− 1) was added to the medium to maintain the expression plasmid. The PHA content in dry cells was determined by GC after methanolysis of the lyophilized cells in the presence of 15% sulfuric acid (18). GC analysis was carried out by using Shimadzu GC-14B system with a non-polar capillary column (InertCap 1, 30 m×0.25 mm, GL Sciences Inc., Tokyo) and a flame ionization detector. The polymers accumulated in the cells were extracted with chloroform for 72 h at room temperature and purified via precipitation with methanol. Molecular weight data were obtained by gel permeation chromatography (GPC) at 40°C using a Shimadzu 10A GPC system and a 10 A refractive index detector with Shodex K806M and K802 columns. Chloroform was used as the eluent at a flow rate of 0.8 mL/min, and sample concentrations of 1.0 mg/mL were applied. Polystyrene standards with a low polydispersity were used to make a calibration curve.

RESULTS Pretreatment by acid or alkaline for HPLC analysis Karr et al. established an HPLC technique for rapid analysis of P(3HB) with

288

WATANABE ET AL.

J. BIOSCI. BIOENG.,

A

N149 D171

C319 (Active site)

PhaCAc 1

Non93 conserved region

α/β Hydrolase fold region

270

B

516

594 aa

Mutation Mutagenic primer

Nile red selection E. coli JM109

PCR phaCAc

Mutant library

Mutagenesis

Inoculate in M9 + C12 medium on 96-well plate Cultivation 37 C, 72 h

Plasmid extraction from duplicated cells, DNA sequencing

Neutralization, Filtration

Selection C4 C6

(min)

(min)

Alkaline treatment on 96-well plate

HPLC assay FIG. 2. (A) The amino-acid-substituted position (D171) in PHA synthase of A. caviae (PhaCAc). (B) Schematic flow diagram of the HPLC-based assay. Site-specific random mutagenesis, preparation of a mutant library, primary assay of P(3HB) accumulation in E. coli JM109 using Nile red dye on an agar plate, liquid cultivation in M9 medium plus dodecanoate (C12) using a 96-well plate for P(3HB-co-3HHx) accumulation, alkaline sample pretreatment, HPLC assay, and nucleotide sequence determination were carried out.

Relative intensity (A210)

A C4

0

10

20

30

40 (min)

20

30

40 (min)

B Relative intensity (A210)

sulfuric acid pretreatment, which is now widely used (19). Thus, we treated the cells accumulating P(3HB-co-19 mol% 3HHx) with sulfuric acid and performed HPLC analysis. The 3HB-derived peak (crotonic acid) at 12.5 min was detected (Fig. 3A), while the 3HHx-derived peak (hexenoic acid) did not appear. Acid pretreatment was not applicable to the compositional analysis of P(3HB-co-3HHx). According to Del Don et al., the composition of P(3HB-co-3HV) was successfully analyzed by HPLC with alkaline (sodium hydroxide) pretreatment (20). To verify alkaline pretreatment in this study, the cells were treated with a sodium hydroxide solution and subjected to HPLC analysis. Fig. 3B shows a chromatogram of the cells accumulating P(3HB-co-19 mol% 3HHx). Unlike acid pretreatment, alkaline pretreatment allowed the detection of both 3HB- and 3HHx-derived peaks. Thus, alkaline pretreatment is useful in copolymer compositional analysis. Furthermore, to verify the efficacy of alkaline pretreatment, cells accumulating a different type of PHA copolymer (21), P(3HB-co-21 mol% 3HA) containing 7 mol% 3HHx, 8 mol% 3HO, 5 mol% 3HD, and 1 mol% 3HDD as 3HA units, were treated in the same manner. The HPLC chromatogram is shown in Fig. 4. All components of the copolymer other than 3HHD were detected by this method. The 3HDD fraction was too low to be detected clearly. The retention time of each 3HA-derived peak was consistent with that of the corresponding 2-alkenoic acid used as a standard reference material. Alkaline pretreatment is thus applicable to PHA consisting of a 3HB unit as well as medium-chain-length 3HA units. However, detection sensitivity of 3HA units decreased with increasing 3HA alkyl side-chain length. HPLC conditions for rapid analysis To apply HPLC analysis to a high-throughput assay for PHA composition, we tried to shorten the HPLC analysis time by modifying the HPLC conditions. The basal HPLC condition, which used a Bio-Rad Aminex HPX-87H ion-exchange column (column length 300 mm) and 0.014 N H2SO4 as a mobile phase, required 40 min for analysis (Figs. 3 and 4). Using a shorter column (Bio-Rad Fast Acid Analysis, column length 100 mm) and a

C4

C6

0

10

FIG. 3. HPLC analysis of P(3HB-co-19 mol% 3HHx)-accumulating cells [R. eutropha PHB−4/phaCAc (10)] treated with (A) 1 N H2SO4 and (B) 1 N NaOH. C4, 3HB-derived peak (crotonic acid); C6, 3HHx-derived peak (hexenoic acid). The column was a BioRad Aminex HPX-87H (column length 300 mm).

Relative intensity (A210)

VOL. 113, 2012

HPLC ASSAY FOR PHA SYNTHASE MUTANTS

C4 C6 C8 C10 C12 0

10

20

30

40 (min)

FIG. 4. HPLC analysis of P(3HB-co-21 mol% 3HA)-accumulating cells [R. eutropha PHB−4 harboring Pseudomonas sp. 61–3 PHA synthase gene (21)] treated with 1 N NaOH (solid line) and the corresponding 2-alkenoic acid used as a standard reference material (dotted line). P(3HB-co-21 mol% 3HA) consisted of 79 mol% 3HB (C4), 7 mol% 3HHx (C6), 8 mol% 3HO (C8), 5 mol% 3HD (C10), and 1 mol% 3HDD (C12). The column was a Bio-Rad Aminex HPX-87H (column length 300 mm).

Relative intensity (A210)

20% CH3CN-containig mobile phase, the analysis time was shortened to 10 min. The chromatogram of P(3HB-co-19 mol% 3HHx) analyzed using the modified conditions is shown in Fig. 5. The 3HB- and 3HHxderived peaks were well separated, even within the shortened analysis time. The area ratio of 3HB- to 3HHx-derived peak in the HPLC analysis was 87:13, whereas the compositional molar ratio of 3HB to 3HHx in the polymer was 81:19. Therefore, the correction coefficients for the 3HB and 3HHx units to calculate copolymer composition from HPLC peak areas are determined to be 1.00 and 1.58, respectively. Screening of PhaCAc mutants with an HPLC assay Previously, PhaCAc mutants with a D171G mutation were isolated by evolutionary engineering, which aimed to acquire a high-polymerization activity enzyme (12). Because substitution by glycine (G) at position 171 is known to increase reactivity toward 3HHx, the PhaCAc D171X mutant library was suitable for validation of the HPLC-based assay using the D171G mutant as a beneficial control. Until now, the mutational effect at position 171 had not been examined by replacement with amino acids other than glycine (G). Thus, another objective of this study was to verify whether the D171G mutation is the most effective to increase PhaCAc reactivity toward 3HHx. The D171X mutant library was constructed by introducing sitespecifically mutagenized pBBR1phaPCJAcABRe into E. coli JM109. To perform a high-throughput assay, a 96-well plate format was introduced for cell cultivation and sample pretreatment, as described in Materials and methods. The 343 clones able to accumulate PHA, identified by Nile red staining, were subjected to cultivation for accumulation of P(3HB-co3HHx) followed by alkaline pretreatment and an HPLC assay. Of these, 111 clones accumulated P(3HB-co-3HHx) copolymers, whereas other

clones accumulated P(3HB) homopolymers. E. coli harboring nonmutated phaCAc genes (wild type) synthesized P(3HB-co-3HHx) with a 3HHx fraction in the range of 7–13 mol%. From the HPLC assay, 22 mutants showed significantly or slightly higher 3HHx fractions than the wild type. By repeated cultivation, we confirmed that particular mutants listed in Table 1 showed good reproducibility in terms of high 3HHx fractions. Nucleotide sequencing revealed these mutants were replaced by glycine (G), alanine (A), valine (V), leucine (L), methionine (M), glutamine (Q), and histidine (H), with the diversity of the codon at position 171. P(3HB-co-3HHx) synthesis by PhaCAc mutants R. eutropha is the best prospective workhorse for industrial production of PHA. To examine the ability of PhaCAc mutants to produce PHA under more practical conditions, R. eutropha PHB−4 were used as a production host using soybean oil, one of the most abundant vegetable oils in the world. The mutated region of these phaCAc genes was transferred to the pBBREE32d13dPB and expressed in R. eutropha. Cultivation results are listed in Table 2. All PhaCAc mutants synthesized P(3HB-co-3HHx) with higher 3HHx fractions than the wild-type enzyme, which was in good agreement with the results of the HPLC-based assay. Of the examined mutants, the highest 3HHx fraction was found in an N149S mutant. Newly found mutants D171L/H/A showed equal or higher 3HHx fractions than the previously isolated mutant D171G. We previously reported the synergistic effect of the combination of N149S and D171G mutations in PhaCAc, which increased reactivity toward 3HHx (10). Thus, to investigate the combinational effect of N149S and D171A/L/H mutations, doubly mutated phaCAc genes were constructed and expressed in R. eutropha. The newly generated double mutants (NSDH, NSDA, and NADL) synthesized PHA with a composition similar to the NSDG mutant, suggesting a synergistic effect of double mutation on substrate specificity. A very small amount of a 3hydroxyoctanoate (3HO) unit was detected in the polymers as well as in the NSDG mutant. Furthermore, P(3HB-co-3HHx) production was performed from octanoate, which allows 3HHx production from sources other than soybean oil (10). As a result, all strains accumulated PHA in the range of 70–80 wt.%. The 3HHx fractions of PHA are shown in Fig. 6A. 3HHx fractions higher than the wild type were observed for all mutants. The new double mutants exhibited slightly higher 3HHx fractions than the NSDG mutant. Also, 3HO units were detected up to 0.4 mol% in PHA synthesized by N149S and the double mutants. P(3HB-co-3HV-co-3H4MV) synthesis by PhaCAc mutants Recently, our group discovered a new PHA component, 3H4MV, which is able to be polymerized by PhaCAc (4,5). The isolated mutants and their double mutants were subjected to polymerization by P(3HB-co-3HVco-3H4MV) in R. eutropha PHB−4 by feeding 4-methylvalerate to compare 3H4MV incorporation. These cells accumulated 15–40 wt.% PHA. The 3H4MV and 3HV fractions are shown in Fig. 6B. The double

TABLE 1. Codons at position 171 of PhaCAc mutants showing higher 3HHx fractions than the wild-type enzyme using an HPLC assay.

C4

Clone name

At position 171 of PhaCAc Amino acid Gly(G)

C6

0

2.5

289

5

Leu(L)

7.5

10 (min)

FIG. 5. HPLC analysis of P(3HB-co-19 mol% 3HHx)-accumulating cells [R. eutropha PHB−4/phaCAc (10)] treated with 1 N NaOH. The column was a Bio-Rad Fast Acid Analysis (column length 100 mm).

Ala(A) Val(V) Met(M) Gln(Q) His(H)

Codon GGG GGT GGC TTA CTT TTG GCC GCT GTT ATG CAA CAC

11-H5, 11-H7 11-F11, 15-D12 15-C4 11-B2, 15-D8 11-B7 15-B1 11-G4 11-C8 11-H3 11-E4 11-A10 11-G8

WATANABE ET AL.

J. BIOSCI. BIOENG.,

Wild-type N149Sb D171V D171M D171Q D171G b D171H D171A D171L N149S/D171G (NSDG) b N149S/D171H (NSDH) N149S/D171A (NSDA) N149S/D171L (NSDL)

PHA composition a PHA Dry content cell weight (wt.%) 3HB 3HHx 3HO (g/L) (mol%) (mol%) (mol%) 1.9 ± 0.1 2.0 ± 0.3 2.3 ± 0.1 2.2 ± 0.1 2.4 ± 0.1 2.3 ± 0.2 2.2 ± 0.1 2.1 ± 0.1 2.3 ± 0.1 2.3 ± 0.2 2.8 ± 0.1 2.6 ± 0.1 2.6 ± 0.2

Molecular weight Mn (× 104)

Mw/ Mn

75 ± 3

96.7

3.3

0

34

2.5

79 ± 2

94.9

5.1

0

21

2.4

80 ± 1

96.1

3.9

0

34

2.5

77 ± 1

96.0

4.0

0

28

2.5

84 ± 1

95.8

4.2

0

25

2.9

81 ± 1

95.6

4.4

0

24

2.5

82 ± 1

95.4

4.6

0

33

2.5

83 ± 1

95.2

4.8

0

29

3.1

84 ± 3

95.2

4.8

0

40

2.6

71 ± 1

94.6

5.2

0.2

33

2.9

76 ± 1

94.9

5.0

0.1

28

3.6

77 ± 2

94.7

5.2

0.1

35

3.5

81 ± 1

94.6

5.3

0.1

35

3.5

25

3HO 3HHx

20 15 10 5 0

B

60

3HV 3H4MV

40

20

0

a

PHA composition was determined by GC. bPhaCAc mutants that were generated in previous studies (10–12). Mn: number-average molecular weight. Mw/Mn: polydispersity index. Results are the averages ± standard deviations from three separate experiments (standard deviations of PHA composition are less than 5% of the mean).

mutants exhibited higher 3H4MV incorporation than the wild-type and the single mutants. The trend of substrate specificities of the mutants was similar regardless of the carbon source (Fig. 6C).

C

55

3H4MV + 3HV (mol%)

PhaCAc

A 3HHx + 3HO (mol%)

TABLE 2. PHA production from soybean oil by recombinant R. eutropha PHB−4 expressing PhaCAc mutants.

3H4MV + 3HV (mol%)

290

NSDA

50

NSDG

NSDH

40

L

35

Q

30

G V

WT

25

This study demonstrated that alkaline pretreatment of PHAaccumulating cells enabled rapid and convenient analysis of PHA composition by HPLC. Due to the ease of sample preparation, a highthroughput HPLC assay based on PHA copolymer composition was possible. In contrast, conventional sample pretreatment for an HPLC assay, which uses acid instead of alkaline, was unable to analyze 3HA units other than 3HB. The reason why 3HA was detected using alkaline pretreatment is related to the reaction mechanism during sample pretreatment. For sensitive detection of constituent monomers of PHA, it is necessary to generate 2-alkenoic acid, which has strong UV absorption due to unsaturated bonds, by eliminating the αproton of 3HA unit (22–24). The nucleophiles in this reaction are water molecules and hydroxide ions in acidic and basic conditions, respectively. The electron density at the α-carbon of 3HA increases with an increasing number of alkyl side chains. Therefore, proton elimination from longer 3HA units by nucleophilic attack becomes less efficient. Thus, water molecules, having weak nucleophilicity for α-protons, may not act as a nucleophile, but hydroxide ions maintain nucleophilicity toward 3HA with longer alkyl side chains. This may result in detection of mcl-PHA monomers with alkaline pretreatment. GC and nuclear magnetic resonance (NMR) analyses are generalized methods for determination of PHA composition. As a pretreatment for GC analysis, PHA samples are subjected to acidic

S H

A

M

20 10

DISCUSSION

NSDL

45

12

14

16

18

20

22

3HHx + 3HO (mol%) FIG. 6. (A) 3HHx fraction of P(3HB-co-3HHx) copolymers produced from octanoate, and (B) 3H4MV and 3HV fractions of P(3HB-co-3HV-co-3H4MV) copolymers produced from 4-methylvaleric acid by R. eutropha PHB−4 expressing PhaCAc mutants. PHA composition was determined by GC. A 3HO unit was detected as a minor component of P(3HB-co-3HHx). Results are averages ± standard deviations from three separate experiments. (C) Correlation between 3HHx + 3HO fractions (shown in panel A) and 3H4MV + 3HV fractions (shown in panel B) incorporated into PHA.

methanolysis and then extracted using organic solvents (25). Solvent extraction is a time-consuming step of sample pretreatment for GC analysis. For NMR analysis, PHA extraction from cells and purification are required. Therefore, these methods are preferable for precise quantitative analysis of PHA composition but are not well suited for a high-throughput assay. The sample pretreatment for the HPLC assay established in this study consisted of simple procedures: NaOH addition, heating at 100°C, neutralization with HCl, and sample filtration. Thus, all procedures can be completed on a 96-well plate. In addition, by optimizing HPLC conditions, analysis of P(3HB-co3HHx) was shortened to 10 min. The analysis time will be further shortened using the latest equipment, such as ultra high-performance liquid chromatography (UHPLC). Although the HPLC assay is rapid and convenient, the overlap of PHA- and cell-derived peaks should be carefully considered. Therefore, the HPLC-based method is

VOL. 113, 2012 preferable for a high-throughput assay but not well suited for precise quantitative analysis. To validate the HPLC assay, a D171X random point mutation library of PhaCAc was screened. Isolation of D171G mutants validated the accuracy of the HPLC assay because the mutants showed higher reactivity toward 3HHx than the wild-type enzyme, as previously demonstrated (12). Additionally, this study aimed to verify whether the D171G mutant is the most effective to increase PhaCAc reactivity toward 3HHx in the D171X library. The screening proved that the HPLC assay functioned well because of successful isolation of the D171G mutant and other new mutants showing equal or slightly higher reactivity toward 3HHx than the D171G mutant. These isolated mutants had a variety of codons at position 171 (Table 1), supporting the diversity of the D171X library. In particular, D171G/L/A mutants were repeatedly isolated with different codons. This implies that the replacement by glycine (G), leucine (L), and alanine (A) leads to increase in the reactivity toward 3HHx. On the other hand, the PHA composition might be differentiated by difference in the expression levels of PhaCAc mutants. Previous study demonstrated that D171G mutant was expressed in E. coli at the same level of wild-type enzyme, as revealed by western blot analysis (12). Thus, it might be assumed that expression levels among D171G/L/A mutants are not significantly different. The amino acids at position 171 of the isolated mutants were more hydrophobic than aspartic acid (D) of the wild-type enzyme. Histidine (H) can be either positively charged or uncharged at neutral pH; it can be uncharged in the D171H mutant based on the screening results. It is presumed that hydrophobicity at position 171 is related to the substrate specificity of PhaCAc. However, hydrophobicity was not the only factor determining specificity because some hydrophobic amino acids, such as isoleucine (I) and phenylalanine (F), were not isolated. The structure of the amino acids seems to be a factor affecting substrate specificity. To our knowledge, the N149S mutation in PhaCAc is the most effective single mutation increasing reactivity toward 3HHx, as seen from Table 2 and Fig. 6 (10–12). We examined the amino acids at position 149, other than serine (S), in terms of substrate specificity by site-specific mutagenesis and preliminary HPLC-based screening of the N149X mutant library. However, no mutant showed higher reactivity toward 3HHx than the N149S mutant (data not shown). From this result, we concluded that the most effective 149th amino acid for increasing reactivity toward 3HHx is serine (S). Consequently, the combined effect of the N149S and D171H/A/L mutations was investigated in this study. These double mutations also exhibited synergistic effects on substrate specificity as well as the NSDG mutation. In P(3HB-co-3H4MV) synthesis, an increase in the 3H4MV fraction is also desired (26). Because 3H4MV is a structural isomer of 3HHx, there is interest in studying these PhaCAc mutants to assess their ability to polymerize 3H4MV. As a result of this study, the mutants were found to have an increased ability to polymerize 3H4MV in proportion to 3HHx (Fig. 6C). This observation provides important insights into the mutational effect on the substrate binding pocket of PhaCAc. These mutations make the substrate binding pocket deeper and wider due to the structural difference between 3H4MV and 3HHx. Because there is a correlation between the polymerization of both units, the screening approach used in this study is applicable to mutants with an increased ability to polymerize 3H4MV. In conclusion, a new HPLC screening method based on PHA copolymer composition was developed by applying alkaline sample pretreatment and introducing a 96-well plate format. The new method was used to isolate PhaCAc mutants with increased reactivity toward 3HHx from a D171X mutant library. For the 171st amino acid, histidine (H), alanine (A), and leucine (L) were effective as well as

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glycine (G). The double mutants, NSDH, NSDA, and NSDL, also exhibited a synergistic effect on the alteration of substrate specificity, as previously demonstrated by the NSDG mutant. The acquired mutants have an increased ability to polymerize not only 3HHx but also 3H4MV. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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