Molecular Weight Change of Polyhydroxyalkanoate (PHA) Caused by

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Molecular Weight Change of Polyhydroxyalkanoate (PHA) Caused by the PhaC Subunit of PHA Synthase from Bacillus cereus YB-4 in Recombinant Escherichia coli Satoshi Tomizawa,† Manami Hyakutake,† Yuta Saito,† Jumiarti Agus,† Kouhei Mizuno,‡ Hideki Abe,§ and Takeharu Tsuge*,† †

Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan ‡ Division of Biochemical Engineering, Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 802-0985, Japan § Bioplastic Research Team, RIKEN Biomass Engineering Program, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ABSTRACT: Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308T (PhaRCBm), B. cereus YB-4 (PhaRCYB4), and hybrids (PhaRBmCYB4 and PhaRYB4CBm) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRCBm synthesized P(3HB) with a relatively high molecular weight (Mn = 890  103) during 72 h of cultivation, whereas PhaRCYB4 synthesized low-molecular-weight P(3HB) (Mn = 20  103). The molecular weight of P(3HB) synthesized by PhaRCYB4 decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaRBmCYB4, but not for PhaRYB4CBm. These results suggest that the molecular weight change is caused by the PhaCYB4 subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaCYB4 was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.

’ INTRODUCTION A wide variety of bacteria synthesize polyhydroxyalkanoates (PHAs), which are biodegradable and biocompatible polyesters under nutrient-limited conditions for intracellular carbon and energy storage.1
4 Poly(3-hydroxybutyrate) [P(3HB)] is the most common bacterial PHA. The bacterium Ralstonia eutropha has been extensively studied as a P(3HB) producer. Its P(3HB) biosynthesis pathway initiates from acetyl-CoA and involves 3-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC).1
4 The expression of these enzymes allows the production of P(3HB) in nonPHA-producing bacteria such as Escherichia coli.5 E. coli harboring R. eutropha P(3HB) biosynthesis genes (phaCAB) can synthesize high-molecular-weight P(3HB).6,7 PHA synthases are grouped into four classes (classes I
IV) based on the substrate specificities and subunit composition of the enzymes.3 Classes I, III, and IV PHA synthases have substrate specificities toward short-chain-length monomers (C3
C5), whereas class II PHA synthase utilizes medium-chain-length monomers (C6
C14). Classes I and II have a single subunit (PhaC). In contrast, classes III and IV are composed of two subunits, namely, PhaE and PhaC, which form the PhaEC complex and PhaR and PhaC, which form the PhaRC complex, respectively. Either the PhaE or the PhaR subunit is necessary for PHA polymerization; r 2011 American Chemical Society

however, the exact roles of these subunits in polymerization are unclear.8
10 In previous studies, recombinant E. coli harboring classes I
IV synthase genes were generated, and the molecular weights of the synthesized P(3HB) were characterized.11,12 These studies reported that the molecular weight of P(3HB) depends on the characteristics of the PHA synthase expressed. In E. coli expressing PHA synthase from Bacillus sp. INT005 (PhaRCBsp),11,13 the P(3HB) synthesized by PhaRCBsp at a culture time of 14 h showed bimodal distribution with a maximum polydispersity index (PDI) of 9.1. In addition, the molecular weight of P(3HB) decreased from 4400  103 to 48  103 during cultivation, but the dry cell weight (DCW) and P(3HB) content remained almost unchanged. The culture temperature also affected the molecular weight of the P(3HB) synthesized by these cells. At a culture temperature of 37 °C, the molecular weight decreased with the culture time but was almost unchanged at 25 °C. Because E. coli has no PHA depolymerase, this phenomenon suggests that PhaRCBsp is involved in both the polymerization of P(3HB) and its random scission.13 Received: April 5, 2011 Revised: May 25, 2011 Published: May 27, 2011 2660

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Table 1. Bacterial Strains and Plasmids Used in the Study strain/plasmid

relative characteristics

reference or source

Strain Escherichia coli JM109

recA1, endA1, gyrA96, thi-1, hsdR17(rK
mKþ), e14
(mcrA
),

TaKaRa Bio.

supE44, relA1, Δ(lac-proAB)/F’ [traD36, proABþ, lacIq, lacZΔM15] Bacillus megaterium

wild type

NBRC15308T

Bacillus cereus YB-4

wild type

Mizuno et al.14

Plasmid pT7-Blue

T-vector for cloning of PCR products

Novagen

pT7-phaRBm

pT7-Blue derivative; phaRmg from B. megaterium

present study

pT7-phaCBm pT7-phaRCYB4

pT7-Blue derivative; phaCmg from B. megaterium pT7-Blue derivative; phaRC YB4 from B. cereus YB-4

present study present study

pGEM’’ABex

pGEM-T derivative; phaRe promoter, phaARe and phaBRe from R. eutropha

Takase et al.15

pGEM- phaRBmCBmAB

pGEM”ABex derivative; phaRBm and phaCBm from B. megaterium

present study

pGEM- phaRYB4CYB4AB

pGEM”ABex derivative; phaRYB4 and phaCYB4 from B. cereus YB-4

present study

pGEM- phaRYB4CBmAB

pGEM”ABex derivative; phaRYB4 from B. cereus YB-4 and phaCBm from B. megaterium

present study

pGEM- phaRBmCYB4AB

pGEM”ABex derivative; phaRBm from B. megaterium and phaCYB4 from B. cereus YB-4

present study

pGEM- phaRYB4AB

pGEM”ABex derivative; phaRYB4 from B. cereus YB-4

present study

pGEM- phaCYB4AB

pGEM”ABex derivative; phaCYB4 from B. cereus YB-4

present study

The aim of this study was to characterize the molecular weights of P(3HB) synthesized by class IV PHA synthases from B. cereus YB-4 (PhaRCYB4) and B. megaterium NBRC15308T (PhaRCBm) by using E. coli JM109 as the host. The molecular weights of P(3HB) synthesized by E. coli expressing PhaRCBm or PhaRCYB4 at several culture temperatures were analyzed. Furthermore, a recombinant E. coli expressing hybrid PHA synthases of B. cereus YB-4 and B. megaterium, namely, PhaRYB4CBm or PhaRBmCYB4, was cultured to identify the key subunit of PHA synthase that determines the molecular weight of P(3HB). The results revealed that the PhaCYB4 subunit has a previously unknown effect on the molecular weight of P(3HB) synthesized by E. coli.

’ EXPERIMENTAL SECTION Phylogenetic Tree. The amino acid sequences of six PHA synthases consisting of PhaRC subunits from Bacillus sp. INT005 (accession no. AB077026), B. cereus SPV (DQ486135), B. cereus YB-4 (AB525763), B. cereus E33L (CP000001), B. weihenstephanensis KBAB4 (CP000903), B. megaterium NBRC15308T (AB525783), and B. megaterium ACTT11561 (AF109909) were used to analyze phylogenetic relationships. Sequence data alignments were performed using Clustal W (ver. 1.83), and a phylogenetic tree was drawn by TreeView. Bacterial Strains, Plasmids, and Media. The bacterial strains and plasmids used in this study are listed in Table 1. The E. coli JM109 strain was used as the host for P(3HB) production. For preculturing, the strains were grown in Luria
Bertani (LB) media (yeast extract, 5 g; peptone, 10 g; and NaCl, 10 g in deionized water, 1 L). To maintain the plasmid within the cell, ampicillin (100 mg/L) was added to the media. DNA Manipulation and Plasmid Construction. To construct a plasmid for phaRCBm gene expression, polymerase chain reaction (PCR) was performed to amplify the coding region of phaRBm and phaCBm by using B. megaterium NBRC15308T genomic DNA as a template. For phaRBm amplification, the following PCR primers were used: forward 50 -TTC GAA AGA AGG AGA TAT ACA TAT GAA TCG TGA AGA ATT TTC CCA GCT C-30 and reverse 50 -CCT GCA GGT AAT GTT GTT ATA AAA TCC CCT-30 (underlined sequences show Csp45I and Sse8387I sites, respectively). For phaCBm

amplification, the following PCR primers were used: forward 50 -CCT GCA GGA GAA GGA GAT ATA CAT ATG GCA ATT CCT TAC GTG CAA GAG TGG G-30 and reverse 50 -AGA TCT TAC TTT GTC TCA GCC TCG TCT TTA-30 (underlined sequences show Sse8387I and BglII sites, respectively). The plasmid pT7-Blue was used to clone the PCR products (0.6 and 1.1 kb), and the resultant plasmids were named pT7-phaRBm and pT7-phaCBm, respectively. Next, pT7-phaRBm and pT7-phaCBm were digested at Csp45I, Sse8387I, and BglII, and two DNA fragments were ligated with the Csp45I-BglII digested pGEM”ABex to yield pGEM-phaRBmCBmAB. To construct a plasmid for phaRCYB4 gene expression, PCR was performed to amplify the coding region of phaRYB4 and phaCYB4 by using B. cereus YB-4 genomic DNA as a template. For phaRYB4 amplification, the following PCR primers were used: forward 50 -TTC GAA AGA AGG AGA TAT ACA TAT GAT TGA TCA AAA ATT CGA TCC ACT A-30 and reverse 50 -CCT GCA GGT CTT TCA TAA GTT ATA AAC AAG CGC30 (underlined sequences show Csp45I and Sse8387I sites, respectively). For phaCBm amplification, the following PCR primers were used: forward 50 -AGA AGG AGA TAT ACA TAT GAC ATA CAT TCG CAA CAG AAT GGG AAA AG-30 and reverse 50 -AGA TCT AAC ATA GGC TAG TTC CAT TTT TTA T-30 (underlined sequence shows BglII site). The PCR products (0.6 and 1.1 kb) were bound, and a 1.7-kb DNA fragment was inserted into the pT7-Blue plasmid. The resultant plasmids were named pT7-phaRCYB4. Next, pT7-phaRCYB4 was digested at Csp45I and BglII, and a 1.7-kb DNA fragment was inserted into the pGEM”ABex digested at the same sites, and the resultant plasmids were named pGEMphaRYB4CYB4AB. To yield pGEM-phaRYB4CBmAB, two plasmids, pT7-phaRCYB4 and pT7-phaCBm, were digested at Csp45I plus Sse8387I and Sse8387I plus BglII, respectively, and then, the DNA fragments were ligated. The DNA fragment (phaRYB4CBm) was inserted into pGEM”ABex digested at Csp45I and BglII, and the resultant plasmids were named pGEMphaRYB4CBmAB. pGEM-phaRBmCYB4AB was constructed in the same manner. Figure 1 shows four expression plasmids (pGEM-phaRCAB series) carrying PHA synthase genes. For solely expressing the PhaRYB4 or PhaCYB4 subunit, pGEMphaRYB4CYB4AB was digested at Sse8387I and BglII or Csp45I and Sse8387I, and self-ligated using Takara BKL kit (Takara Bio Inc., Otsu, Japan) to yield pGEM-phaCYB4AB or pGEM-phaRYB4AB. 2661

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Figure 1. Plasmids (pGEM-phaRCAB series) used in this study. PHA synthase genes (phaRC) from Bacillus megaterium NBRC15308T and B. cereus YB-4 were used for P(3HB) synthesis. PRe, phaARe, and phaBRe are pha promoter, β-ketothiolase gene, and NADPH-dependent acetoacetyl-CoA reductase gene, respectively, derived from Ralstonia eutropha.

Synthesis and Isolation of P(3HB). The recombinant E. coli JM109 was cultivated in 500 mL flasks containing 100 mL LB media plus glucose (20 g/L) on a reciprocal shaker (130 rpm) at various temperatures (25, 30, and 37 °C) for 72 h. Post incubation, the cells were collected, washed with water, and then lyophilized. The polymers accumulated in the cells were extracted with chloroform for 72 h at room temperature and purified by precipitation with methanol. Gas Chromatography and Gel Permeation Chromatography. The P(3HB) content in the cells was determined by gas chromatography (GC) after methanolysis of lyophilized cells in the presence of 15% (v/v) sulfuric acid, as described previously.16 The molecular weights and their distribution were determined by gel permeation chromatography (GPC) at 40 °C, using a Shimadzu 10A GPC system equipped with a 10A refractive index detector with Shodex K-806 M and K-802 columns. Chloroform, at a flow rate of 0.8 mL/min, was used as the eluent, and the sample was prepared at a concentration of 1.0 mg/mL.17 The molecular weight was calibrated with low polydispersity polystyrene standards. Transmission Electron Microscopy. The cells cultured for 12 or 72 h were observed under a transmission electron microscope (TEM) by ultrathin sectioning and negative staining methods. In the ultrathin sectioning method,18 the cells were washed, resuspended in 100 mM phosphate buffer (pH 6.8), and fixed overnight at 4 °C in a buffer containing 2.5% glutaraldehyde. Post fixation, samples were treated with 2% osmium tetroxide in the same phosphate buffer for 2 h on ice. After washing with the same buffer, the cells were embedded in 2% agar and dehydrated by ethanol at room temperature. The samples were treated with ether and infiltrated with a solution of mixing ratios of ether/Quetol 812 resin (Nisshin EM Corp., Tokyo, Japan). The samples were then embedded in Quetol 812 resin that was allowed to polymerize at 60 °C for 48 h. The ultrathin sections were stained with uranyl acetate and Reynold’s lead citrate stain. In the negative staining method, 6 μL of the sample solution was spotted on a hydrophilic collodion-carbon-coated copper grid (Nisshin EM Corp.) and allowed to adsorb for 1 min. Excess sample was removed by carefully touching the side of a grid with filter paper; then, 6 μL of distilled water was spotted on the grid and removed after a short time. Samples were stained using 6 μL of uranyl acetate. After 1 min, excess stain was removed, and the grid was allowed to air-dry for 12 h. Both micrographs were obtained with a HITACHI-H-7500 electron microscope operated at 60 or 80 kV.

’ RESULTS AND DISCUSSION Homology among Class IV PHA Synthases. Figure 2 shows the phylogenetic trees of the PhaR and PhaC subunits belonging to the class IV PHA synthases. From this relationship, the PhaRC

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Figure 2. Phylogenetic tree of (A) PhaR and (B) PhaC subunits of class IV PHA synthases. PhaRCs from Bacillus sp. NIT005, B. cereus YB-4, and B. megaterium NBRC15308T have been examined for P(3HB) production in E. coli JM109; the former two PhaRCs showed a decrease in the P(3HB) molecular weight.12,13.

subunits of class IV PHA synthases can be divided into two groups, that is, B. cereus and B. megaterium groups. The PhaRCYB4 subunits from B. cereus YB-4 and the corresponding subunits belonging to the B. cereus group (e.g., Bacillus sp. INT005 and B. cereus SPV) showed amino acid sequence identities as high as 99%. On the other hand, the PhaCYB4 and PhaCBm subunits of the B. megaterium group (e.g., strains NBRC15308T and ACTT11561) showed relatively low homology, with sequence identities of 71%. In the case of the PhaR subunit, the homology between PhaRYB4 and PhaRBm was 47% in amino acid sequence identity. B. cereus YB-4 was isolated as a PHA producer from the polluted soil in Kitakyushu City, Japan.14 The molecular weight (Mn) of PHA accumulated in B. cereus YB-4 was 720  103 in the mid phase of cultivation (14 h), but it decreased to 85  103 in the late phase (72 h).14 Because this strain is a natural PHA producer, generally the cells possess intracellular PHA depolymerases for the mobilization of PHA. Thus, PHA depolymerases are thought to be the most likely agent inducing the change in the molecular weight of B. cereus YB-4. Another possible explanation for the decrease in the molecular weight of PHA in B. cereus YB-4 could be that PhaRCYB4 not only possesses PHA polymerization activity but also PHA degradation activity. Our previous study suggested that PhaRCBsp from Bacillus sp. INT005, which is homologous to PhaRCYB4, may contribute to the change in the molecular weight of PHA in recombinant E. coli.13 Because E. coli has no PHA depolymerase, the molecular weight of PHA is usually unchanged in the mid and late phases of cultivation. Indeed, the molecular weight of PHA did not change when six PHA synthases belonging to classes I
III were expressed in E. coli.11,13 However, a significant decrease in the PHA molecular weight was observed, in particular, for PhaRCBsp, which belongs to class IV.13 It is not known whether other class IV synthases show a similar behavior. Molecular Weight of P(3HB) after a 72 h Cultivation. To assess whether the change in the molecular weight of PHA is a common occurrence in other class IV synthases, we attempted to express the PHA synthases from B. megaterium NBRC15308T (PhaRCBm) and B. cereus YB-4 (PhaRCYB4) in E. coli JM109, in a manner similar to that used for PhaRCBsp.13 Table 2 lists the results of cultivation for 72 h at 37 °C. The cells expressing PhaRCBm showed a low level of P(3HB) (26 wt %) and moderate molecular weight (Mn = 890  103). PhaRCYB4 expression resulted in high P(3HB) accumulation (80 wt %), but the molecular weight was low at 20  103. A large difference in the molecular weights of P(3HB) synthesized by the cells expressing PhaRCBm and PhaRCYB4 was observed. In these cells, the decrease in the molecular weight of P(3HB) was thought to occur during the 2662

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Table 2. P(3HB) Synthesis by Recombinant E. coli JM109a molecular weight plasmid (relevant markers)

dry cell weight (g/L)

P(3HB) content (wt %)

Mn (103)

Mw (103)

PDI

pGEM-phaRBmCBmAB (PhaRCBm)

2.6 ( 0.2

26 ( 1

890

1870

2.1

pGEM-phaRYB4CYB4AB (PhaRCYB4)

9.3 ( 0.1

80 ( 1

20

42

2.1

pGEM-phaRYB4CBmAB (PhaRYB4CBm)

2.9 ( 0.2

39 ( 1

3000

6000

2.0

pGEM-phaRBmCYB4AB (PhaRBmCYB4)

9.4 ( 0.2

66 ( 3

55

77

1.4

pGEM- phaCYB4AB (PhaCYB4)

1.9 ( 0.1

ND

pGEM- phaRYB4AB (PhaRYB4)

1.8 ( 0.1

ND

a

Cells were cultured in LB media containing glucose (20 g/L) for 72 h at 37°C. Mn, number-average molecular weight; Mw, weight-average molecular weight; PDI, polydispersity index (Mw/Mn); ND, not detectable. Results are the average ( standard deviations from three separate experiments.

Figure 3. P(3HB) synthesis by PhaRCBm in recombinant E. coli JM109 cultured at (A) 37 °C, (B) 30 °C, and (C) 25 °C. Cells were cultured in shake flasks containing LB media plus glucose (20 g/L). Each experiment was carried out in triplicate, and the average value is shown. Mn, number-average molecular weight; Mw, weight-average molecular weight; PDI, polydispersity index (Mw/Mn).

cultivation of E. coli expressing PhaRCYB4, as previously observed for PhaRCBsp.13 Change in the Molecular Weight of the P(3HB) Synthesized by PhaRCBm. To investigate the time-dependent change in the molecular weight of P(3HB), E. coli expressing PhaRCBm was first cultured in LB media plus glucose (20 g/L) for 72 h. In addition, the effect of culture temperature on the molecular weight of P(3HB) was investigated at 37, 30, and 25 °C, based on reports demonstrating that the changes in the molecular weight of P(3HB) are suppressed at lower temperatures.13 The results of these experiments are shown in Figure 3. After 12 h of cultivation at 37 °C, the DCW and P(3HB) content were almost constant at 3.0 g/L and 25 wt %, respectively. At 30 °C, DCW and P(3HB) content increased to 4.7 g/L and 28 wt %, respectively, until 48 h and then slightly decreased by 72 h. At 25 °C, DCW and P(3HB) content increased to 4.7 g/L and 18 wt %, respectively, until 24 h and did not change until 72 h. Thus, the optimum culture temperature to achieve high DCW and P(3HB) content by cells expressing PhaRCBm was found to be 30 °C. The Mn at each temperature was almost constant regardless of the culture time, and these values at 72 h of cultivation were 890  103, 1250  103, and 1070  103 at 37, 30, and 25 °C,

respectively. Further, the PDI (Mw/Mn) of P(3HB) remained almost unchanged at around 2.0
2.5. This trend is commonly observed in the recombinant E. coli expressing PHA synthase in the mid and late phases of cultivation.6,11,13 For example, in the case of E. coli expressing PHA synthase from R. eutropha (PhaCRe) cultured at 37 °C, the Mn of P(3HB) was constant at around 1100  103 after 14 h of cultivation.13 Change in the Molecular Weight of P(3HB) Synthesized by PhaRCYB4. The expression of PhaRCYB4 was used to investigate the time-dependent molecular weight changes of P(3HB) in E. coli. The results are shown in Figure 4. At a culture temperature of 37 °C, the DCW and P(3HB) content rapidly increased to 9.6 g/L and 67 wt %, respectively, until 24 h, and then stabilized. At 30 °C, the DCW and P(3HB) content increased to 9.4 g/L and 60 wt %, respectively, until 24 h, and then remained unchanged until 72 h. At 25 °C, the DCW and P(3HB) content increased to 9.5 g/L and 45 wt %, respectively, until 48 h, and remained stable thereafter. The cell growth rate and accumulation level of P(3HB) decreased with decreasing culture temperature. As expected, the molecular weight of P(3HB) decreased with culture time at all the temperatures tested. At a culture temperature of 37 °C, the Mn decreased from 150  103 to 20  103 from 2663

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Figure 4. P(3HB) synthesis by PhaRCYB4 in recombinant E. coli JM109 cultured at (A) 37 °C, (B) 30 °C and (C) 25 °C. Cells were cultured in shake flasks containing LB media plus glucose (20 g/L). Each experiment was carried out in triplicate, and the average value is shown. Mn, number-average molecular weight; Mw, weight-average molecular weight; PDI, polydispersity index (Mw/Mn).

12 to 72 h of cultivation. However, a rapid decrease in Mn was observed only in the early phases of cultivation. At 30 °C, the Mn changed to 280  103 to 21  103 during cultivation. At 25 °C, the Mn was as high as 970  103 at 12 h and then gradually decreased to 310  103 at 72 h of cultivation. The decrease in Mn at lower temperatures was relatively slower than that at higher culture temperatures. This decrease in the molecular weight was very similar to that observed in the case of PhaRCBsp,13 where Mn changed from 440  103 to 48  103 from 12 to 60 h of cultivation. The PDI of P(3HB) tended to be high during rapid molecular weight changes. Thus, the highest PDIs of 2.5 and 4.9 were observed at 12 h of cultivation when the cells were cultured at 37 and 30 °C, respectively. At 25 °C, the attenuated decrease in Mn resulted in a higher PDI in the late phase (PDI = 4.2) than that in the earlier phase (PDI = 3.0). Figure 5 shows the distribution of the molecular weights of P(3HB) synthesized by PhaRCBm and PhaRCYB4. PhaRCBm-synthesized polymer samples extracted at 12 and 72 h of cultivation showed almost the same GPC curves. Meanwhile, the molecular weight distribution of the PhaRCYB4synthesized polymers shifted toward a lower mass with an increase in the culture time. TEM Observation of E. coli Expressing PhaRCYB4. The P(3HB) granules in the cells were observed by TEM using ultrathin sectioning and negative staining methods. The ultrathin sectioning method is widely used for direct observation of intracellular PHA granules, while the negative staining method allows observation of the cell’s appearance under near-natural conditions. Figure 6 shows micrographs of cells expressing PhaRCYB4 at 12 and 72 h of cultivation. The negative staining method revealed that the average cell size at 12 h of cultivation was 3.14 ( 1.17 μm in length and 1.19 ( 0.17 μm in width (n = 66). At 72 h, the average cell size was 3.12 ( 0.90 μm in length and 1.27 ( 0.25 μm in width (n = 41). Interestingly, the P(3HB) granules were visible even by negative staining observation. The ultrathin sectioning method allowed the detection of several P(3HB) granules in each cell. The average sizes of P(3HB) granules were 375 ( 115 nm (n = 190) and

Figure 5. Molecular weight distributions of P(3HB) synthesized by (A) PhaRCBm and (B) PhaRCYB4 at 37 °C. Solid and dashed lines denote P(3HB) synthesized for 12 and 72 h of cultivation, respectively.

480 ( 130 nm (n = 126) at 12 and 72 h of cultivation, respectively. This suggests that, with increasing culture time, the size of the P(3HB) granules gradually increased, while the molecular weight decreased. Molecular Weight Change of P(3HB) Synthesized by Hybrid Synthases. To understand which subunits of PhaRC are involved in the change in the molecular weight, further cultivations were carried out by expressing two types of hybrid PHA synthases, namely, PhaRYB4CBm and PhaRBmCYB4. This technique was based on prior reports using class III synthases.19 The results of P(3HB) synthesis by the hybrid PHA synthases after 72 h of cultivation are listed in Table 2. PhaRYB4CBm synthesized P(3HB) of 40 wt % and Mn as high as 3000  103. However, PhaRBmCYB4 accumulated more P(3HB) (66 wt %) with a low Mn of 55  103. These results revealed an important feature of class IV PHA synthases: PhaCYB4 can be functionalized with the help of PhaRBm even though their origins are different and their amino acid identity is not high (47%). The opposite combination of PhaRYB4CBm was also acceptable. To confirm the in vivo polymerization activity of the sole subunits of PhaRYB4 and PhaCYB4, these subunits were individually expressed in E. coli along with the monomer-supplying enzymes (PhaABRe). As shown in Table 2, no P(3HB)s was accumulated, indicating that the PhaCYB4 subunit can be functionalized by the presence of the PhaRBm subunit. 2664

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Figure 6. Electron micrographs of (A, C) negatively stained and (B, D) ultrathin sections of E. coli expressing PhaRCYB4, cultured at 37 °C for (A,B) 12 h and (C,D) 72 h. The scale bars represent 1 μm.

The use of the hybrid synthase provided another important clue about the mechanisms underlying the change in the molecular weight. A comparison of the results listed in Table 2 indicates that the molecular weights of P(3HB) were always low when PhaCYB4 was employed for P(3HB) synthesis. Therefore, PhaCYB4 was speculated to be involved in lowering the molecular weight. Figure 7 shows the time-dependent change in the molecular weight in cultures employing PhaRYB4CBm and PhaRBmCYB4. In case of PhaRYB4CBm, the DCW, P(3HB) content, Mn, and PDI were kept at 2.9
3.4 g/L, 33
38 wt %, 2610
3100  103, and 1.8
2.3, respectively, from 12 to 72 h of cultivation. As for PhaRBmCYB4, the DCW and P(3HB) content increased to 9.5 g/L and 68 wt %, respectively, until 24 h of cultivation, and were stable thereafter. However, only the Mn of P(3HB) decreased from 360  103 to 55  103 from 12 to 72 h of cultivation. This behavior is similar to that of P(3HB) synthesized by PhaRCYB4, suggesting that the PhaCYB4 subunit regulates the changes in the molecular weight of P(3HB). Kinetic Analysis of Molecular Weight Change. The current results and those of our previous study13 suggest that the most probable explanation for the decrease in the molecular weight is the degradation of the P(3HB) polymer chain through random scission occurring in the cells. To test this hypothesis, a kinetic analysis was performed. As a matter of convenience, the molecular weights at 12 h of cultivation were set as the initial state (t = 0) in this kinetic analysis. Because the chain scission is completely random, the number-average degree of polymerization (Pn,t) at time t (in h) is given by the following:20 1=Pn, t
1=Pn, 0 ¼ kd t where, Pn,0 is the initial number-average degree of polymerization (at 12 h of cultivation) and kd is the rate constant (h
1) of intracellular degradation. The data pertaining to PhaRCYB4 and

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Figure 7. P(3HB) synthesis by hybrid PhaRC in E. coli JM109 cultured at 37 °C: (A) PhaRYB4CBm, (B) PhaRBmCYB4. Cells were cultured in shake flasks containing LB media plus glucose (20 g/L). Each experiment was carried out in triplicate, and the average value is shown. Mn, number-average molecular weight; Mw, weight-average molecular weight; PDI, polydispersity index (Mw/Mn).

PhaRBmCYB4 obtained at 37 °C are shown in Figure 8. The plot of inverse Pn against t revealed a linear relationship, indicating that the intracellular degradation of P(3HB) proceeds through a random scission of the polymer chain. The kd values for PhaRCYB4 and PhaRBmCYB4 at 37 °C were estimated to be 5.99  10
5 and 2.08  10
5, respectively. This suggests that the degradation ability of the hybrid synthase PhaRBmCYB4 is relatively lower than that of the original synthase PhaRCYB4. This degradation ability might be associated with in vivo synthase activity, because higher P(3HB) content is likely to cause lower molecular weights (Table 2). In fact, low culture temperature allowed P(3HB) to slow down the decrease in the molecular weights, probably due to depressed synthase activity at lower temperatures. In addition, R. eutropha strain PHB
4 (PHA negative strain) was examined as another production host by expressing PhaRCYB4. The Mn of P(3HB) synthesized from fructose at 30 and 37 °C were 1700  103 and 790  103, respectively, after 72 h of cultivation (data not shown). Although the molecular weights were significantly higher than those of E. coli, a similar temperature-dependent behavior was observed for R. eutropha. Presumable Hydrolase Activity of PHA Synthase. PhaCs are known to catalyze the chain transfer (CT) reaction after the polymerization reaction of PHA by transferring the PHA polymer chain from PhaC to a CT agent.21 Water, 3HB, and some hydroxyl compounds have been proposed as CT agents.12,21
23 Because water acts as a CT agent, hydrolysis of the thioester bond occurs between the cysteine residue in the active site of PhaC and the growing PHA polymer chain. A hydrolase activity toward thioester bonds is believed to be an inherent function of PhaCs. Regarding PhaCYB4 and its related synthases, this event might occur not only toward the thioester bond but also toward the oxoester bond of the PHA polymer chain in rare cases, resulting in a change in the molecular weight by random scission of the polymer chain. Based on an in vitro study with a class III 2665

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Biomacromolecules

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’ REFERENCES

Figure 8. Time-dependent change in inverse of degree of polymerization (1/Pn) during intracellular P(3HB) degradation at a culture temperature of 37 °C. Molecular weights of P(3HB) at 12 h of culture were set as the initial state (t = 0). Open square, PhaRCYB4; closed square, hybrid synthase PhaRBmCYB4.

synthase,8 Tian et al. proposed that PHA synthase has a putative amino acid residue that plays the role of a general-base catalyst for hydrolase activity, and is probably located on the enzyme surface. The decreasing molecular weight in E. coli, however, was not observed for class III synthase in a previous study.11 PhaCYB4 and its related synthases might be suitable models to investigate the hydrolase activity of PHA synthases and the mechanism of the termination step of PHA polymerization in vivo.

’ CONCLUSIONS In this study, we investigated the unusual changes in the molecular weight of P(3HB) that occur during the cultivation of E. coli expressing class IV PHA synthases. The bacterial strain expressing PhaRCYB4 showed decrease in the molecular weight of P(3HB), while that expressing PhaRCBm did not show a significant change in the molecular weight. Thus, unusual changes in the molecular weight could be observed only for the PhaRCs from the B. cereus group. Furthermore, hybrids formed by the PhaRCs of B. cereus YB-4 and B. megaterium were expressed in E. coli. These hybrids were functional for P(3HB) polymerization, but the decrease in the molecular weight was observed only for PhaRBmCYB4. These results strongly suggest that the decrease in the molecular weight is caused by the PhaCYB4 subunit. The results of a kinetic analysis indicated that the decrease in the molecular weight proceeded via a random scission of the polymer chain. PhaCYB4 possibly possesses hydrolase activity, which breaks the PHA polymer chain in association with a CT reaction. Further studies to confirm the hydrolase activity of PhaCYB4 are underway.

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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The authors thank Keiko Yamamichi, Yoko Yamamoto, and Mamie Suzuki (Tokyo Institute of Technology) for their technical support in TEM observations. This work was supported by a Grant-in-Aid for scientific research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and the New Energy and Industrial Technology Development Organization (NEDO). 2666

dx.doi.org/10.1021/bm2004687 |Biomacromolecules 2011, 12, 2660–2666