Endogenous Ethanol Affects Biopolyester Molecular Weight in

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Endogenous Ethanol Affects Biopolyester Molecular Weight in Recombinant Escherichia coli Ayaka Hiroe,*,† Manami Hyakutake,† Nicholas M. Thomson,‡ Easan Sivaniah,‡,§ and Takeharu Tsuge†,* †

Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan ‡ Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, 19 JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom S Supporting Information *

ABSTRACT: In biopolyester synthesis, polyhydroxyalkanoate (PHA) synthase (PhaC) catalyzes the polymerization of PHA in bacterial cells, followed by a chain transfer (CT) reaction in which the PHA polymer chain is transferred from PhaC to a CT agent. Accordingly, the frequency of CT reaction determines PHA molecular weight. Previous studies have shown that exogenous alcohols are effective CT agents. This study aimed to clarify the effect of endogenous ethanol as a CT agent for poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis in recombinant Escherichia coli, by comparing with that of exogenous ethanol. Ethanol supplementation to the culture medium reduced P(3HB) molecular weights by up to 56% due to ethanol-induced CT reaction. NMR analysis of P(3HB) polymers purified from the culture supplemented with 13C-labeled ethanol showed the formation of a covalent bond between ethanol and P(3HB) chain at the carboxyl end. Cultivation without ethanol supplementation resulted in the reduction of P(3HB) molecular weight with increasing host-produced ethanol depending on culture aeration. On the other hand, production in recombinant BW25113(ΔadhE), an alcohol dehydrogenase deletion strain, resulted in a 77% increase in molecular weight. Analysis of five E. coli strains revealed that the estimated number of CT reactions was correlated with ethanol production. These results demonstrate that host-produced ethanol acts as an equally effective CT agent as exogenous ethanol, and the control of ethanol production is important to regulate the PHA molecular weight.

increase the molecular weight. This is because long chain polymers are easily entangled with each other. The weightaverage molecular weight (Mw) of P(3HB) produced by native PHA-producing bacteria is usually in the range from 0.2 × 106 to 2 × 106, whereas ultrahigh-molecular-weight P(3HB) [UHMW-P(3HB)] produced by certain recombinant E. coli is ≥3.0 × 106. Previous reports showed that UHMW-P(3HB) can be processed into strong films or fibers by hot- or colddrawing.5 As molecular weight regulation is important for the design of material property, an establishment of its regulation method is desirable. The P(3HB) polymerization reaction can be divided into three steps: initiation, propagation, and termination.6−8 First, 3HB-CoA monomer reacts with the PhaC active site (initiation step). The PhaC active site consists of two thiol groups (-SH). One thiol group binds to the incoming 3HB-CoA monomer, while the other binds to the growing polymer chain. Polymer propagation proceeds by transfer of the chain between the two thiol groups (propagation step). Chain transfer (CT) is thought to occur where the polymer chain is transferred to a CT agent.

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized and accumulated as intracellular carbon and energy storage materials by more than 200 bacteria.1,2 PHAs have attracted much attention as eco-friendly plastic materials because they are synthesized from renewable biomass-derived feedstocks such as sugars and plant oils.3 In addition, PHAs possess superior properties such as thermoplasticity, biocompatibility, and biodegradability. A homopolymer of (R)-3-hydroxybutyrate [P(3HB)] is the most common type of PHA accumulated by bacteria in nature, and its production has been extensively studied. Ralstonia eutropha is the most widely studied native producer of P(3HB). In this bacterium, the polymer is synthesized from acetylcoenzyme A (acetyl-CoA) via a reaction involving three enzymes: 3-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaA and PhaB provide (R)-3-hydroxybutyryl-CoA (3HB-CoA), and then, PhaC polymerizes the 3HB moiety of 3HB-CoA to generate P(3HB). Therefore, expression of these three enzymes allows non-PHA-producers like Escherichia coli to synthesize P(3HB).4 P(3HB) has high rigidity but is brittle with low elasticity. Therefore, the practical uses of P(3HB) are limited. One method to improve the physical property of P(3HB) is to © 2013 American Chemical Society

Received: June 25, 2013 Accepted: September 16, 2013 Published: September 16, 2013 2568

dx.doi.org/10.1021/cb400465p | ACS Chem. Biol. 2013, 8, 2568−2576

ACS Chemical Biology

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Table 1. Effect of Supplemental Ethanol on P(3HB) Biosynthesis in E. coli JM109 Harboring pGEM-3aSD-phaCReAB Plasmida molecular weight ethanol (g L−1) 0 0.5 1 2 4 8 10

dry cell wt (g L−1) 8.7 8.7 8.8 8.8 8.8 8.4 8.3

± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.2 0.1 0.1

P(3HB) content (wt %) 74 72 71 73 71 74 73

± ± ± ± ± ± ±

3 2 2 3 2 1 2

P(3HB) production (g L−1) 6.4 6.2 6.3 6.5 6.5 6.3 6.0

± ± ± ± ± ± ±

0.3 0.2 0.2 0.2 0.4 0.2 0.2

Mn ( × 106) 1.3 1.1 1.0 0.84 0.75 0.58 0.56

± ± ± ± ± ± ±

0.1 0.2 0.1 0.04 0.11 0.10 0.01

Mw/Mn

Mn reduction (%)

relative chain no.b

± ± ± ± ± ± ±

0 12 20 33 40 55 56

1.0 1.1 1.2 1.5 1.7 2.2 2.1

2.1 2.4 2.5 2.6 2.7 2.7 2.7

0.1 0.4 0.1 0.1 0.1 0.2 0.1

Cells were cultivated in 100 mL LB medium containing glucose (20 g L−1) at 37°C for 72 h with a range of ethanol concentrations. The results are the averages from three independent experiments ± standard errors. bNumber of P(3HB) chains relative to the control experiment (nonsupplementation).35 a

and P(3HB) production of E. coli JM109 harboring R. eutropha phaCAB genes (pGEM-3aSD-phaCReAB) was investigated (Table 1). E. coli cells grew well regardless of ethanol concentration (0−10 g L−1) and reached stationary phase within 24 h of cultivation. Dry cell weights after 72 h were 8.3− 8.8 g L−1, and P(3HB) content and production were respectively 71−74 wt % and 6.0−6.5 g L−1, which are high accumulation levels. Number-average molecular weight (Mn) of P(3HB) was decreased by up to 56%, probably due to ethanolinduced CT reactions. On the other hand, relative P(3HB) chain number was increased by increasing the supplemental amount of ethanol. These results suggest that supplementary ethanol enhanced the CT reaction of P(3HB). End Structure Analysis of P(3HB) Synthesized with 13 C-Labeled Ethanol. The 13C NMR structural analysis of each P(3HB) obtained from the cultivation supplemented with 10 g L−1 of ethanol or [1-13C] ethanol are shown in Figure 1A and B. From the ethanol supplementation culture (Figure 1A), four signals at 19.7 ppm (signal 1), 67.6 ppm (signal 2), 40.7 ppm (signal 3), and 169.0 ppm (signal 4) were detected and the P(3HB) structure was confirmed to agree with previous studies.18 On the other hand, the labeled ethanol culture (Figure 1B) gave a new signal at 60.6 ppm (signal 5) along with the previous four signals (1, 2, 3 and 4). This signifies the incorporation of [1-13C] ethanol into the carboxyl end of P(3HB). Additionally, signals 2 (67.6 ppm) and 4 (169.0 ppm) of the 3HB unit were enhanced 3-fold. This result suggests that [1-13C] ethanol was partly converted into 3HB units through reverse reactions of alcohol dehydrogenase E (AdhE) and alcohol dehydrogenase P (AdhP), and forward reactions of PhaA, PhaB, and PhaC (see Figure 2A). The 1H NMR spectra of P(3HB) are shown in Figure 1C and D. Strong signals at 1.3 ppm (signal a), 2.6 ppm (signal c), and 5.3 ppm (signal b) derived from 3HB units were observed in both ethanol and labeled ethanol cultures. On the other hand, signal d was different between two cultures. Methylene resonances derived from the ethanol end were observed at 4.15 ppm for the ethanol culture but at 4.0 ppm and 4.3 ppm arising for the labeled ethanol culture. This peak splitting (from 4.15 ppm to 4.0 ppm and 4.3 ppm) resulted from the spin−spin coupling of 1H atoms to an adjoining magnetically active 13C atom in labeled ethanol. In the case of labeled ethanol, ends were capped by not only supplemented [1-13C] ethanol (4.0 and 4.3 ppm) but also host-produced ethanol (4.15 ppm) derived from glucose metabolism. In addition, terminal modification ratios were roughly estimated to be 83% and 90% for ethanol culture and labeled ethanol culture (Table 2),

After binding to the CT agent, the chain is removed from PhaC, which is then free to initiate the polymerization of a new chain (termination step).9 Alternatively, PhaC loses its polymerization activity, and the polymerization reaction stops. Consequently, the frequency of CT reactions regulates PHA’s molecular weight. Poly(ethylene glycol) (PEG) has been used and investigated as the CT agent in PHA synthesis.10 In 1996, Shi et al.11 discovered that cultivation of R. eutropha under PHA accumulating condition in the presence of relatively low molecular weight PEG resulted in a large decrease in PHA’s molecular weight. A similar effect had been found not only in R. eutropha11,12 but also in Alcaligenes latus,13 Pseudomonas oleovorans,14,15 Pseudomonas putida,14 Azotobacter chroococcum,16 Azotobacter vinelandii,17 and recombinant E. coli.18 Moreover, it was demonstrated by 1H NMR analysis that PEG was incorporated at the polymer carboxyl terminus through an esterification between the growing PHA chain and the PEG hydroxy group.11−18 However, since PEG is not a biological macromolecule, it cannot be a natural CT agent for PHA production in bacterial cells. As potential naturally occurring CT agents, water, 3HB, and some hydroxy compounds have been proposed.7,19−22 In Madden’s work, alcohol compounds showed a large effect of molecular weight decrease using R. eutropha as host strain for PHA production.7 The same effect of alcohols was reported also in P(3HB) production by Methylobacterium extorquens.21 However, the incorporation of monohydric alcohol at the end of a P(3HB) chain has not been confirmed as of yet by structural analysis. Moreover, in all previous reports, the phenomenon of molecular weight reduction was confirmed by supplementing hydroxy compounds into the growth medium, and CT reaction under nonsupplemental culture has not been studied. In this study, we investigated the function of ethanol as a CT agent for PHA production in recombinant E. coli. E. coli is a suitable host to observe molecular weight changes and endgroup structure of P(3HB) because of the absence of inherent PHA depolymerases 23 and PHA granule-associated proteins (phasins).24 Ethanol is a hydroxy compound, which is produced in relatively large amounts under microaerobic and anaerobic conditions to maintain cellular redox balance in E. coli, and is considered as a potential main CT agent in bacterial cells.

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RESULTS Effect of Ethanol Supplementation on P(3HB) Synthesis. The effect of ethanol supplementation on cell growth 2569



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Figure 1. End structure analysis of synthesized P(3HB) by E. coli JM109 harboring pGEM-3aSD-phaCReAB. (A,B) 125 MHz 13C NMR spectra and (C,D) 500 MHz 1H NMR spectra of P(3HB). P(3HB) polymers were purified from cells grown in LB medium containing 20 g L−1 glucose at 37 °C for 72 h in the presence of (A,C) 10 g L−1 of ethanol or (B,D) 10 g L−1 [1-13C] ethanol.

varied culture parameters were temperature (30 and 37 °C), shaking speed (90 and 130 rpm), and medium volume (50, 100, and 250 mL). The relationship between extracellular ethanol concentration after 72 h of cultivation and Mn of synthesized P(3HB) is shown in Figure 3A. The extracellular concentrations of ethanol and Mn of synthesized P(3HB) fell in the range 0.1−1.9 g L−1 and 0.3−1.8 × 106, respectively, and

respectively, indicating that almost all of the P(3HB) chains were capped by ethanol, probably via PhaC-catalyzed CT reactions. Ethanol Production is Correlated with P(3HB) Molecular Weight. To evaluate the ability of the host strain to produce ethanol, recombinant E. coli JM109 harboring pGEM3aSD-phaCReAB was cultured under various conditions. The 2570



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200 ± 10, and 117 ± 3 h−1, respectively. The growth curves under each aerobic condition are shown in Figure 3C. All cultures entered the stationary phase by 24 h. Cells grew well regardless of medium volume (aeration), and P(3HB) production after 72 h of cultivation was 5.9−6.1 g L−1 (Table 3). Time courses of extracellular ethanol concentration are shown in Figure 3D. The ethanol concentration was maximized at 24 h of cultivation and then gradually decreased due to assimilation by E. coli and evaporation from the medium. At 72 h of cultivation, ethanol concentrations were in the range 0.95− 2.08 g L−1, whereas the Mn of P(3HB) varied from 0.86 × 106 to 0.37 × 106. The molecular weight of P(3HB) was negatively correlated with the ethanol concentration. Effect of adhE Deletion on P(3HB) Molecular Weight. AdhE is a key enzyme, which converts acetyl-CoA to ethanol in a fermentation pathway of E. coli (Figure 2A). As PHA production hosts, BW25113(ΔadhE) and its parent strain BW25113 (the Keio Collection) were used to investigate the suppression effect of ethanol production on molecular weight of P(3HB). The maximum ethanol production of BW25113 and BW25113(ΔadhE) strains were 0.86 g L−1 and 0.03 g L−1, respectively (Table 4 and Figure 3D). In the adhE deletion strain, ethanol concentration remained at almost zero throughout 72 h of cultivation, and P(3HB) with a higher molecular weight (Mn = 0.76 × 106) than in the parent strain (Mn = 0.43 × 106) was produced (Table 3 and Figure 2B). Therefore, deletion of adhE led to a 77% increase in P(3HB) molecular weight, suggesting that the suppression of ethanol production is beneficial to the production of higher molecular weight PHAs. Ethanol Production Levels of Various E. coli Host Strains. To compare ethanol production of five E. coli strains (JM109, BW25113, BW25113(ΔadhE), DH5α, and XL1-Blue), the extracellular ethanol concentrations after 24 h cultivation were assayed. The results are shown in Table 4. DH5α and XL1-Blue strains, as JM109, are often used as hosts for genetic engineering. Of the strains tested, ethanol production of JM109 was the highest, whereas that of BW25113(ΔadhE) was the lowest. Surprisingly, ethanol production by XL1-Blue was also very low despite the presence of alcohol dehydrogenase genes. This explains why XL1-Blue produced P(3HB) with the highest molecular weight (Table 3). Additionally, to estimate the activation level of fermentation pathways in each E. coli strain, extracellular concentrations of organic compounds were determined by HPLC analysis (Table 4). The results showed that flux balance was different for each strain. It is of interest to note that both BW25113(ΔadhE) and XL1-Blue generated lactate, instead of ethanol, to maintain cellular redox balance. Expression Levels of PhaC in Each Strain. Ethanol production level seems to be correlated with P(3HB) molecular weight as mentioned above. However, molecular weight is not determined only by ethanol production level. As proof of this, low molecular weight P(3HB) was produced by BW25113 although ethanol production in BW25113 was lower than that of JM109. Generally, it is known that molecular weight is affected by PhaC expression level: the higher the expression of PhaC, the lower the molecular weight of P(3HB).25,26 Thus, PhaC expression level is another factor to be considered for molecular weight regulation. To quantify PhaC expression levels, Western blots were performed for five E. coli strains at 6 h of cultivation (midexponential growth phase). The detected bands of PhaC are shown in Figure 4A. Additionally, the enzymatic activity of PhaC for these samples was also measured

Figure 2. Repression effect of ethanol production on P(3HB) molecular weight in E. coli BW25113 harboring pGEM-3aSDphaCReAB. (A) E. coli fermentation pathways. The deleted adhE gene codes for the key enzyme in ethanol production. (B) BW25113 and BW25113(ΔadhE) strains harboring pGEM-3aSD-phaCReAB were grown in 100 mL LB medium supplemented with 20 g L−1 glucose at 30 °C for 72 h. P(3HB) molecular weight after 12 h (gray bar) and 72 h (white bar) cultivation for each strain were shown.

Table 2. Ethanol Capping Yield of P(3HB) Synthesized in E. coli with Supplementation of 10 g L−1 Ethanol supplementation

Pn(NMR)a

Mw(MALLS)b ( × 106)

Pn(MALLS)c

EtOH cappingd (%)

ethanol [1-13C] ethanol

4457 4029

1.00 0.98

3698 3642

83 90

a

Degree of polymerization (Pn) calculated from 1H NMR spectra by assuming that all P(3HB) terminus ends were ethoxy groups (area ratio of signal c to signal d). bAbsolute weight-average molecular weight determined by multiangle laser light scatting (MALLS). c Degree of polymerization (Pn) estimated from Mw(MALLS) and polydispersity index by GPC analysis. dEtOH capping (%) = 100 × Pn(MALLS)/Pn(NMR).

Mn of P(3HB) decreased with increasing ethanol concentration. On the other hand, polydispersities (Mw/Mn) of P(3HB) were in the range 1.9−3.6 and increased with increasing ethanol concentration (Figure 3B). This increase of polydispersity is thought to be due to a relative increase in lower molecular weight P(3HB). These results strongly suggest that hostproduced ethanol induced CT reactions during P(3HB) biosynthesis. From the culture data shown in Figure 3A and B, cultivation at 30 °C and 130 rpm with 50−250 mL of medium volume were found to be better conditions to vary the ethanol production level of recombinant JM109. The behavior of JM109 under these culture conditions was investigated in more detail. The volumetric oxygen transfer coefficients (kLa) with 50, 100, and 250 mL-cultures were measured to be 288 ± 27, 2571



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Figure 3. Host-produced ethanol and P(3HB) production. (A,B) Relationship between extracellular ethanol concentration and P(3HB) production in E. coli JM109 harboring pGEM-3aSD-phaCReAB. Cells were grown in LB medium containing 20 g L−1 glucose at 30 °C(⧫) or 37 °C (◊) with shaking at 90 or 130 rpm. Medium volumes were 50 mL (kLa = 288 ± 27 h−1), 100 mL (kLa = 200 ± 10 h−1), or 250 mL (kLa = 117 ± 3 h−1) in 500 mL flask. (A) Relationship between extracellular ethanol concentration and P(3HB) molecular weight (Mn) after 72 h cultivation. (B) Relationship between extracellular ethanol concentration and molecular weight distribution (polydispersity, Mw/Mn) of synthesized P(3HB) after 72 h cultivation. (C) Cell growth and (D) ethanol production of E. coli JM109, BW25113, and BW25113(ΔadhE) harboring pGEM-3aSD-phaCReAB. Cells were grown in LB medium containing 20 g L−1 glucose at 30 °C for 72 h. Strain/medium volume was JM109/50 mL (□), JM109/100 mL (gray square symbol), JM109/250 mL (■), BW25113/100 mL (○), and BW25113(ΔadhE)/100 mL( × ).

Table 3. P(3HB) Production of Various E. coli Strains Harboring pGEM-3aSD-phaCReAB Plasmida molecular weight strain/medium vol. JM109/50 mL JM109/100 mL JM109/250 mL BW25113/100 mL BW25113(ΔadhE)/100 mL DH5α/100 mL XL1-Blue/100 mL

−1

dry cell wt (g L ) 9.5 9.5 8.5 10.7 11.3 10.9 10.0

± ± ± ± ± ± ±

0.2 0.1 0.1 0.1 0.2 0.1 0.1

P(3HB) content (wt %) 63 62 72 73 75 63 78

± ± ± ± ± ± ±

3 1 2 1 2 1 1

−1

P(3HB) production (g L ) 6.0 5.9 6.1 7.8 8.5 6.9 7.8

± ± ± ± ± ± ±

0.2 0.2 0.3 0.1 0.3 0.2 0.1

Mn ( × 106) 0.86 0.54 0.37 0.43 0.76 0.66 0.93

± ± ± ± ± ± ±

0.12 0.09 0.03 0.08 0.09 0.05 0.03

Mw/Mn 2.2 3.3 3.1 3.2 2.3 2.6 2.1

± ± ± ± ± ± ±

0.3 0.3 0.1 0.5 0.1 0.2 0.1

Cells were cultivated in LB medium containing glucose (20 g L−1) at 30 °C for 72 h. The results are the averages from three independent experiments ± standard errors.

a

agent and regulates the molecular weight of P(3HB). This is the first report showing regulation of P(3HB) molecular weight by CT reaction with an endogenous CT agent. E. coli is widely used as a host strain for PHA production, not only UHMW-P(3HB)26 but also medium-chain-length P(3HA) (consisting of monomer units with 6−12 carbon atoms)27,28 and PHA copolymers such as P(LA-co-3HB). 29 These copolymers’ Mw values have been limited to around 104−105. The suppression of ethanol production of E. coli is expected to contribute to increasing the molecular weight of various PHAs and expand applications of the biopolymer material. In particular, the suppression of CT reactions mediated by ethanol is considered to be possible by setting the medium volume and shaking speed to be more aerobic. The aerobic

(Figure 4A). The ratio of PhaC specific activity was JM109/ BW25113/BW25113(ΔadhE)/DH5α/XL1-Blue = 1.0/4.2/ 6.7/3.0/4.8, which were in accordance with band strength from Western blotting. Even though the same plasmid (pGEM3aSD-phaCReAB) was transformed, each strain exhibited a different expression level of PhaC.

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DISCUSSION In this study, we investigated the effect of ethanol as a CT agent of P(3HB) synthesis by using recombinant E. coli strains. Our structure analyses demonstrated that supplemental ethanol indeed capped the carboxyl end of P(3HB). In addition, focusing on cultivations of E. coli strain JM109, we obtained results suggesting that host-produced ethanol acts as a CT 2572



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Table 4. Extracellular Organic Compound Profile after 24 h Cultivation of Various E. coli Strains Harboring pGEM-3aSDphaCReAB Plasmida concentration of product (g L−1) strain/medium vol. JM109/50 mL JM109/100 mL JM109/250 mL BW25113/100 mL BW25113(ΔadhE)/100 mL DH5α/100 mL XL1-Blue/100 mL

ethanol 0.95 1.35 2.08 0.86 0.03 0.44 0.05

± ± ± ± ± ± ±

0.06 0.02 0.04 0.03 0.00 0.11 0.00

acetate 0.17 0.20 0.40 0.14 0.14 0.15 0.18

± ± ± ± ± ± ±

0.00 0.04 0.05 0.00 0.00 0.00 0.02

formate 0.31 0.29 0.09 0.25 0.22 0.14 0.07

± ± ± ± ± ± ±

lactate

0.01 0.09 0.00 0.01 0.01 0.02 0.00

0.49 ± 0.00 0.52 ± 0.02

succinate 0.07 0.08 0.08 0.07 0.08 0.08 0.10

± ± ± ± ± ± ±

0.00 0.01 0.01 0.00 0.00 0.00 0.01

Cells were cultivated in LB medium containing glucose (20 g L−1) at 30 °C. Concentration of each metabolite in culture supernatant was determined by enzyme assay (for ethanol) or HPLC analysis. The results are the averages from three independent experiments ± standard errors.

a

Figure 4. Relationship between ethanol production and estimated number of CT reactions. (A) PhaC expression level after 6 h of cultivation (30 °C, 130 rpm, 100 mL of medium volume) in various E. coli harboring pGEM-3aSD-phaCReAB (J: JM109. W: BW25113. A: BW25113(ΔadhE). D: DH5α. X: XL1-Blue.) (B) Relationship between ethanol concentration and estimated number of CT reactions (NCT) normalized by number of PhaC active sites (NE). The detailed data are shown in Table 5.

In general, under microaerobic and anaerobic conditions, E. coli produces acetate as a major compound of glucose metabolism.31 Indeed, plasmid-free JM109 and XL1-Blue produced acetate in relatively large amounts (1.4−3.2 g L−1) under the culture conditions in this study (Supporting Information (SI) Table 1). However, P(3HB)-producing E. coli tended to produce ethanol or lactate as the main fermentation products (Table 4 and SI Table 1). This suggests that ethanol or lactate production were activated as a result of P(3HB) production. Consistent with an earlier report,32 our results showed that P(3HB)-producing XL1-Blue produced lactate much more than ethanol. Because of the low ethanol production, XL1-Blue is considered to be the most preferred host for P(3HB) production with higher molecular weight.33 In order to verify the ethanol-induced CT reaction, we here discuss the relationship between P(3HB) chain number and PhaC concentration. According to the prevailing model proposed by Kawaguchi and Doi,6 P(3HB) chain number NP is given by

level of cultivations is represented by an index of volumetric oxygen transfer coefficient (kLa), the solution rate of oxygen from gas phase to liquid phase. A larger value of kLa means the culture is more aerobic. The apple-shaped flasks (Sakaguchi flask) used in this study with a shaking speed of 130 rpm (reciprocal shaking) enable high kLa (up to 288 h−1) comparable to a baffled Erlenmeyer shake-flask at 250 rpm (orbital shaking).30 As shown in cultivations of JM109 (Table 3), aerobic conditions repressed fermentation metabolites such as ethanol and acetate (Table 4), thereby increasing the molecular weight of P(3HB). As for culture temperature, cultivation at 37 °C is preferable to repress ethanol production, rather than 30 °C. Additionally, higher temperatures facilitate ethanol evaporation from the culture medium. In our preliminary study, when the medium volume was 100 mL, 23% and 13% of ethanol were evaporated during 72 h of abiotic incubation at 37 and 30 °C, respectively. The temperature setting at 37 °C, which is the optimum for E. coli, leads to ethanol evaporation more than that at 30 °C and is considered to contribute toward increasing P(3HB) molecular weight. The suppression of CT reaction is considered to be possible by the deletion of ethanol biosynthesis genes such as adhE, adhC, and adhP. As shown in this study, the deletion of adhE gene can repress ethanol production and ethanol-induced CT reactions. The disturbance to intracellular redox balance associated with the repression of alcohol production can be redressed by increasing production of lactate, a non-CT agent, as shown in the culture of BW25113(ΔadhE) (Table 4).

NP = NE + NCT

(1)

where NE and NCT are number of PhaC active sites involved in P(3HB) polymerization and number of CT reactions, respectively. On the other hand, P(3HB) chain number NP is also given by34 NP = Y /M n × NA

(2)

where Y, Mn, and NA are the yield of synthesized P(3HB) (g L−1), the molecular weight of synthesized P(3HB) (g mol−1), 2573



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Table 5. Estimation of the Numbers of PhaC Involved in P(3HB) Synthesis (NE) and CT Reaction (NCT)a strain-medium vol. JM109-100 mL BW25113-100 mL BW25113(ΔadhE)-100 mL DH5α-100 mL XL1-Blue-100 mL

PhaC activity ratio 1.0 4.2 6.7 3.0 4.8

± ± ± ± ±

0.0 0.3 0.7 0.2 0.9

NP ( × 1018 L−1) 6.7 11.3 6.7 6.3 5.0

± ± ± ± ±

1.1 2.5 0.8 0.3 0.1

NCT ( × 1018 L−1) 5.7 7.1 0 3.3 0.2

± 0.6 ± 3.5 ± 0.2 ± 0.9

NE ( × 1018 L−1)

NCT/NE

± ± ± ± ±

5.7 ± 1.3 1.7 ± 0.9 0 1.1 ± 0.2 0.04 ± 0.19

1.0 4.2 6.7 3.0 4.8

0.2 0.9 0.8 0.6 1.3

a

In this calculation, NE was estimated from PhaC activity ratio of 6 h-cultured cells by assuming that NCT for strain BW25113(ΔadhE) is zero. Avogadro constant of 6.02 ×1023 was used.

and an Avogadro constant (6.23 × 1023 mol−1). Thus, NP (L−1) can be determined experimentally. From eq 1 and 2, NCT can be calculated if NE is available. Although the tentative concentration of PhaC can be measured by means of PhaC activity assay and Western blotting, it is still difficult to determine the total number of PhaC active sites involved in P(3HB) polymerization (NE) through the cultivation. Here, we estimated NE from the PhaC activity ratio of 6 h-cultured cells by assuming that NCT for strain BW25113(ΔadhE) is zero. By this assumption, NE for BW25113(ΔadhE) was determined as 6.7 × 1018 (L−1) from eq 1, and NE values for other strains were proportionately calculated as listed in Table 5. Accordingly, NCT and number of CT reactions normalized by PhaC active site (NCT/NE) were calculable. This estimation should be reliable because XL1-Blue, the low-ethanol producer, showed NCT to be almost zero. As shown in Figure 4B, NCT/NE increased with an increase in ethanol concentration, confirming that the CT reaction is associated with ethanol production. Because the ethanol-dependent CT reaction became obvious by normalizing PhaC concentration, the molecular weight of P(3HB) in E. coli is regulated by mainly two factors; namely, ethanol-induced CT reaction and PHA synthase concentration. The detailed reaction mechanism between ethanol and PHA synthase in the CT reaction requires further validation.35 In conclusion, this study demonstrated for the first time that the molecular weight of PHA is affected by CT reactions with endogenous ethanol in E. coli, together with the amount of PHA synthase (PhaC). The present study provides important information for the design of PHA production and the regulation of PHA molecular weight.

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ethanol or ethanol (control) was added to the medium to a final concentration of 10 g L−1. Ethanol nonsupplemental cultivation: JM109, BW25113, BW25113(ΔadhE), DH5α, and XL1-Blue were transformed with pGEM-3aSD-phaCReAB. The transformants were grown for 72 h at 37 or 30 °C using a reciprocal shaker (90 or 130 rpm) in 500-mL flasks containing LB medium (50 or 100 or 250 mL) supplemented with 20 g L−1 glucose and 100 μg mL−1 ampicillin. After cultivation, the collected cells were washed with water once, frozen, and lyophilized for 3 days. kLa Measurement. To elucidate oxygen supply levels at various medium volumes (50, 100, and 250 mL) in 500 mL flasks, the volumetric oxygen transfer coefficient (kLa) was measured by a sulfite oxidation method39 with suitable modifications. Shake-flasks were filled with Na2SO3 solution (0.2 N Na2SO3 and 0.1 mM CuSO4) and were shaken at 30 °C and 130 rpm. A portion of Na2SO3 solution was taken at appropriate time intervals for iodometry. In this method, by means of the oxidation reaction of Na2SO3 (Na2SO3 + 1/2 O2 → Na2SO4), residual Na2SO3 after shaking for a definite period of time is measured by titration. The amount of oxygen absorbed by the Na2SO3 solution is calculated from the concentration of residual Na2SO3, and the oxygen-supplying ability of each cultivation condition can thus be evaluated. GC, GPC, and NMR Analyses. The P(3HB) 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 described previously.40 Molecular weight of purified P(3HB) was determined by gel permeation chromatography− multiangle laser light scattering photometer (GPC-MALLS).41 The end-group structure of isolated P(3HB) was analyzed by NMR spectroscopy. Each polymer (40 mg) was dissolved in CDCl3 (1.0 mL) and subjected to 125 MHz 13C NMR and 500 MHz 1H NMR analyses. NMR spectra were recorded using a JEOL JNM-LA500 spectrometer as described previously.42 Quantification of Extracellular Metabolites. The concentration of extracellular ethanol was measured using an enzymatic method by F-kit (Roche Diagnostics, Basel, Switzerland). Culture supernatant was recovered by centrifugation (20400g, 5 min, RT). Supernatant samples were suitably diluted by ddH2O, and added to the reaction solution of F-kit according to the manufacturer’s protocol. Ethanol concentration in samples was spectrophotometrically quantified at Abs = 340 nm. Culture supernatant was filtered through a 0.45 μm syringe filter, and analyzed for ethanol, acetate, formate, lactate, and succinate by using a high-performance liquid chromatography (HPLC) system (Shimadzu, Columbia, MD) with a differential refractive index detector (RID). The analytical conditions used were as follows: 300 mm × 7.8 mm HPX-87H column (BioRad, Hercules, CA); column temperature, 60 °C; mobile phase, 0.013 N H2SO4; flow rate 0.5 mL min−1; injection volume, 100 μL; detector temperature 40 °C. Enzyme Assay. Cells at 6 h of cultivation (midexponential growth phase) were disrupted by sonication (46 W, 10 s × 10 times) and centrifuged at low speed (1500g, 5 min, 4 °C) to obtain a soluble extract containing P(3HB) granules. Using this soluble extract, the activities of the PhaC were measured with the DTNB assay, as described previously.43 The durations of enzyme activity assay were 0, 2, 4, and 6 min. For Western blotting analysis of PhaC, 5 μg each of the soluble extract containing P(3HB) granules was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

METHODS

Bacterial Strains and Plasmid. E. coli JM109 (Takara Bio Inc., Otsu, Japan), BW25113 (parent strain of Keio collection), BW25113(ΔadhE) (Keio collection),36 DH5α(Takara Bio), and XL1-Blue (Stratagene Corp., La Jolla, CA) were used as the host strains for PHA production. pGEM-3aSD-phaCReAB (pGEM-T derivative; phaRe promoter, Shine-Dalgarno (SD) sequence from pET3a-vector, phaCphaA-phaB operon from R. eutropha) was used as an expression plasmid for PHA production. To construct pGEM-3aSD-phaCReAB, first, pET15b-phaCRe37 was digested with NdeI and BamHI and the DNA fragment carrying phaCRe was ligated with NdeI-BamHI digested pET-3a to yield pET-3a-phaCRe. Next, pET-3a-phaCRe was digested with XbaI and BamHI and the DNA fragment carrying SD sequence and phaCRe was ligated with XbaI-BglII digested pGEM”ABex 38 by DNA Ligation Kit Ver2.1 (Takara Bio). Transformation of E. coli strains was performed by the general calcium chloride method. Cultivation Conditions. Ethanol supplemental cultivation: JM109 harboring pGEM-3aSD-phaCReAB plasmid was grown at 37 °C for 72 h, using a reciprocal shaker (130 strokes/min) in 500-mL Sakaguchi flasks containing 100 mL of LB medium (10 g L−1 NaCl, 10 g L−1 tryptone, and 5 g L−1 yeast extract) supplemented with 20 g L−1 glucose and 100 μg mL−1 ampicillin. Supplemental ethanol concentrations were up to 10 g L−1. For NMR analysis, [1-13C] 2574



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PAGE). PhaC bands were detected using the specific rabbit antiserum raised against the C-terminus of PhaC.

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composition, repeat unit sequence, and end group structure. Macromolecules 29, 10−17. (12) Shi, F., Ashby, R. D., and Gross, R. A. (1996) Use of poly(ethylene glycol)s to regulate poly(3-hydroxybutyrate) molecular weight during Alcaligenes eutrophus cultivations. Macromolecules 29, 7753−7758. (13) Ashby, R., Shi, F. Y., and Gross, R. A. (1997) Use of poly(ethylene glycol) to control the end group structure and molecular weight of poly(3-hydroxybutyrate) formed by Alcaligenes latus DSM 1122. Tetrahedron 53, 15209−15223. (14) Kim, O. (2000) Biological effects of poly(ethylene glycol) on the microbial poly(β-hydroxyalkanoates) produced by Pseudomonas microorganisms. J. Polym. Res. 7, 91−96. (15) Sanguanchaipaiwong, V., Gabelish, C. L., Hook, J., Scholz, C., and Foster, L. J. (2004) Biosynthesis of natural-synthetic hybrid copolymers: Polyhydroxyoctanoate diethylene glycol. Biomacromolecules 5, 643−649. (16) Saha, S. P., Patra, A., and Paul, A. K. (2006) Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201. J. Ind. Microbiol. Biotechnol. 33, 377−383. (17) Zanzig, J., and Scholz, C. (2003) Effects of poly(ethylene glycol) on the production of poly(β-hydroxybutyrate) by Azotobacter vinelandii UWD. J. Polym. Environ. 11, 145−154. (18) Tomizawa, S., Saito, Y., Hyakutake, M., Nakamura, Y., Abe, H., and Tsuge, T. (2010) Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent. Appl. Microbiol. Biotechnol. 87, 1427− 1435. (19) Mothes, G., Schnorpfeil, C., and Ackermann, J. U. (2007) Production of PHB from Crude Glycerol. Eng. Life Sci. 7, 475−479. (20) Cavalheiro, JaM., de Almeida, M. C. M., Grandfls, C., and da Fonseca, M. (2009) Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Proc. Biochem. 44, 509−515. (21) Taidi, B., Anderson, A. J., Dawes, E. A., and Byrom, D. (1994) Effect of carbon source and concentration on the molecular mass of poly(3-hydroxybutyrate) produced by Methylobacterium extorquens and Alcaligenes eutrophus. Appl. Microbiol. Biotechnol. 40, 786−790. (22) Thomson, N. M., Hiroe, A., Tsuge, T., Summers, D. K., and Sivaniah, E. (2013) Efficient molecular weight control of bacterially synthesized polyesters by alcohol supplementation. J. Chem. Technol. Biotechnol., DOI: 10.1003/jctb.4198. (23) Kusaka, S., Abe, H., Lee, S. Y., and Doi, Y. (1997) Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 47, 140−143. (24) Tian, S. J., Lai, W. J., Zheng, Z., Wang, H. X., and Chen, G. Q. (2005) Effect of over-expression of phasin gene from Aeromonas hydrophila on biosynthesis of copolyesters of 3-hydroxybutyrate and 3hydroxyhexanoate. FEMS Microbiol Lett. 244, 19−25. (25) Sim, S. J., Sneel, K. D., Hogan, S. A., Stubbe, J., Rha, C., and Sinskey, A. J. (1997) PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat. Biotechnol. 15, 63−67. (26) Hiroe, A., Tsuge, K., Nomura, C. T., Itaya, M., and Tsuge, T. (2012) Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl. Environ. Microbiol. 78, 3177−3184. (27) Sato, S., Ishii, N., Hamada, Y., Abe, H., and Tsuge, T. (2012) Utilization of 2-alkenoic acids for biosynthesis of medium-chain-length polyhydroxyalkanoates in metabolically engineered Escherichia coli to construct a novel chemical recycling system. Polym. Degrad. Stab. 97, 329−336. (28) Tappel, R., Wang, Q., and Nomura, C. T. (2012) Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanote) polymers synthesized by Escherichia coli. J. Biosci. Bioeng. 113, 480−486. (29) Taguchi, S., Yamada, M., Matsumoto, K., Tajima, K., Satoh, Y., Munekata, M., Ohno, K., Kohda, K., Shimamura, T., Kambe, H., and

ASSOCIATED CONTENT

S Supporting Information *

Extracellular organic compound profile of wild type E. coli and P(3HB)-producing E. coli. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81 45 924 5420. Fax: +81 45 924 5426. E-mail: hiroe.a. [email protected]. *Tel.: +81 45 924 5420. Fax: +81 45 924 5426. E-mail: tsuge.t. [email protected]. Present Address §

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Y. Nakamura (Tokyo Institute of Technology, Yokohama, Japan) for NMR analysis. E. coli strains BW25113 and BW25113(ΔadhE) were obtained from National BioResource Project (NIG, Japan): E. coli. This work was supported by funding from the New Energy and Industrial Technology Development Organization (NEDO), Grant-inAid for Scientific Research (KAKENHI 23310060), and JST, CREST.

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REFERENCES

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Endogenous Ethanol Affects Biopolyester Molecular Weight in

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