Incorporation of Glycolate Units Promotes Hydrolytic Degradation in

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Incorporation of glycolate units promotes hydrolytic degradation in flexible poly(glycolate-co-3hydroxybutyrate) synthesized by engineered Escherichia coli Ken'ichiro Matsumoto, Tetsufumi Shiba, Yukikazu Hiraide, and Seiichi Taguchi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00194 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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ACS Biomaterials Science & Engineering

Incorporation of glycolate units promotes hydrolytic degradation in flexible poly(glycolate-co-3hydroxybutyrate) synthesized by engineered Escherichia coli Ken'ichiro Matsumoto,1,2,* Tetsufumi Shiba, 1 Yukikazu Hiraide, 1 and Seiichi Taguchi1,3 1

Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering,

Hokkaido University, N13W8, Kitaku, Sapporo 060-8628, Japan, 2PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan, and 3CREST, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan KEYWORDS. poly(glycolide), poly(glycolic acid), polyhydroxybutyrate, biocompatible material

ABSTRACT. Glycolate (GL)-based polyhydroxyalkanoate (PHA), P[GL-co-3-hydroxybutyrate (3HB)], was characterized with respect to its physical properties and hydrolytic degradability. The copolymers were produced from GL and xylose in recombinant Escherichia coli JW1375 (∆ldhA) expressing an engineered PHA synthase and monomer supplying enzymes. The GL molar ratio in the copolymer was regulated in the range of 0 to 16 mol% dependent on the

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concentration of GL supplemented in the medium. Unlike P(3HB) homopolymers which are rigid and opaque, the transparency and elasticity of P(GL-co-3HB) films could be tuned dependent on the GL molar ratio. For example, Young's modulus of the films varied in the range of 1620 to 54 MPa. The hydrothermal treatment of P(GL-co-3HB)s resulted in the generation of water-soluble oligomers, and their concentration was positively correlated with the GL molar ratio in the polymer, indicating that the GL units in the polymer chain promoted the hydrolytic degradation of the polymer. The results of this study demonstrate that the GL molar ratio is a potent determinant for regulating the elasticity and hydrolytic degradability of P(GL-co-3HB).

Introduction Glycolate (GL)-based polymers are aliphatic polyesters that exhibit superior biocompatibility and bioabsorbability. Polyglycolic acid (PGA) degrades in the body within 60–90 days, and as such, GL-based polymers are used for biomedical applications such as sutures, drug delivery,1 vaccine adjuvant,2 and tissue engineering.3-4 Polyhydroxyalkanoates (PHAs) are biocompatible and biodegradable materials that have gained importance also due to their structural diversity.5-6 Physical properties of PHAs such as stretchiness and pliancy can be regulated by genetically modifying PHA-producing bacteria and relevant enzymes.7 The hydrolysis of PHAs in mammalian tissues is very slow and degradation typically takes more than a year.8 Their relatively low acidity confers them with minimal risk compared to other biopolymers such as PGAs. In 2011, our group succeeded in incorporating GL units into PHA using an engineered PHA synthase

and

propionoyl-CoA

transferase.9

The

engineered

PHA

synthase,

a


2

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Ser325Thr/Gln481Lys mutant of PhaC1Ps from Pseudomonas sp. 61-3,10 had substrate specificity towards 2-hydroxyacyl-CoAs such as lactate (LA), 2-hydroxybutyrate (2HB), and GL as well as towards short-chain-length (SCL) and medium-chain-length (MCL) 3-hydroxyacylCoAs.9, 11 The broad substrate specificity greatly expanded the structural variety of PHAs, in that PHAs containing natural and unusual monomer units and their numerous copolymers could be synthesized using the engineered enzyme. For example, copolymers with LA and 3hydroxybutyrate (3HB),11-12 LA and SCL/MCL units,9 LA and 2HB,13 GL, SCL/MCL units and LA,9,

14

have been reported. In order to design the polymers with beneficial properties, it is

particularly important to understand their structure-property relationships. For instance, the primary structure and physical properties of P(LA-co-3HB)15 and the co-crystallization behavior of P(2HB-co-LA) have been reported,13,

16

but the properties of GL-based PHA remain

incompletely characterized. Thus, this study characterized the physical properties and hydrolytic degradability of a GL-based PHA [P(GL-co-3HB)] as the initial step for evaluating the potential of the material as a new bioabsorbable material.

Experimental Bacterial strains, plasmids, and culture condition The

expression

vector

pTV118NpctphaC1Ps(ST/QK)AB,11

which

harbors

the

pct,

phaC1Ps(ST/QK), phaA and phaB genes was previously constructed.17 Escherichia coli JW1375, which harbors the lactate dehydrogenase gene (ldhA) knockout mutation (Keio collection strains)18, was purchased from the National BioResource Project, Japan. The use of this strain reduced the incorporation of lactate units to trace amounts, which was beneficial for avoiding the complex structure-property relationships of the terpolymers. Polymer production was performed


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as previously described.12 In brief, the recombinant cells were grown in 100 mL LB medium containing various concentrations of glycolate, 20 g l-1 xylose, and 100 mg l-1 ampicillin in a shake flask at 30ºC for 48 h. The concentrations of glucose, xylose, and glycolate were determined using HPLC equipped with a refractive index detector as previously described.12 Polymer extraction and analyses The polymer was extracted with chloroform as previously described.15 The monomer composition of the polymer was determined by 1H NMR. The monomer composition of the copolymer was also confirmed by gas chromatography analysis as previously described (data not shown).10 The molecular weights of the extracted P(GL-co-3HB)s were estimated by gel permeation chromatography (GPC; JUSCO, Japan) equipped with a Shodex GPC KF-805 column (Showa Denko K. K., Tokyo, Japan) with polystyrene standards. Differential scanning calorimetry (DSC) data were recorded on a Perkin-Elmer DSC8500 using solvent-cast films (10 mg), which was prepared with chloroform as previously described.11 Stress-strain tests of solvent-cast films (10 mm × 5 mm × 30-70 µm) were performed using an EZ test (Shimadzu, Japan) at room temperature with a strain rate of 100 mm/min, according to a method described previously.19 NMR analysis NMR spectra were acquired with a JEOL ECS400 spectrometer. 1H and

13

C NMR data

recorded in CDCl3 used tetramethyl silane (TMS) (δ 0) and residual internal CHCl3 (δ 77.0), respectively, as standards. Approximately 30 mg extracted polymer was dissolved in CDCl3. Poly(glycolate-co-3-hydroxybutyrate): 1H NMR (CDCl3, 400 MHz): δ 1.27 (d, 3H, J = 6.4), 2.47 (dd, H, J = 16.2 Hz and 6.0 Hz), 2.61 (dd, H, J = 15.6 Hz and 7.2 Hz), 4.60 (s, 2H), 5.26 (m, H). 13

C NMR (CDCl3, 100 MHz): δ 19.7, 40.1, 40.7, 60.8, 67.6, 68.7, 166.7, 169.1.


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Hydrolytic degradation assay Polymer emulsions were prepared as previously described.20 In brief, 40 mg polymers were dissolved in 1 mL dichloromethane, which was combined with 20 mL ultrapure water. The mixture was sonicated three times for 30 sec that generating milky emulsion that was incubated overnight at 50ºC to evaporate the dichloromethane. Then the emulsion was placed in a test tube with a screw cap, and incubated at 100ºC for 5 days to accelerate hydrolytic degradation. The gas-tight tubes kept the water volume constant during the incubation. The emulsion remaining in the solution was removed by centrifugation and filtration (hydrophilic PTFE membrane filter, 0.2 µm pore). The insoluble fraction was lyophilized and subjected to GPC. The soluble fraction was subjected to capillary electrophoresis (CE) analysis, which was performed on an Agilent 7100 using a 72 cm long capillary with a diameter of 50 µm (G 1600-62211). Electrophoresis was performed at 25 °C and -15 kV, and α-AFQ108 was used as the running buffer (Otsuka Electronics Co., Ltd, Osaka, Japan). The target was detected with indirect UV detection at 400 nm. The soluble fraction was diluted to approximately 100 µg/mL and subjected to electrospray ionization mass spectrometry (ESI-MS) analysis using a MicroTOF (Bruker Daltonics). The sample was directly injected using a syringe pump at room temperature at a flow rate of 180 µl/h. The ESI voltage was 4.5 kV in the negative mode and the drying temperature was 200 °C. Nitrogen was used as the nebulizer (1.6 bar) and drying gas (9.0 ml/min).

Results Efficient production of P(GL-co-3HB)s with different GL molar ratios An obstacle that was encountered in this study was the low polymer productivity of the GLbased polymer9 compared to that of other typical microbial polyesters.21 To improve polymer


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production, the use of xylose, an abundant bio-based resource obtained from lignocelluosic biomass, was effective because GL and xylose were simultaneously utilized by E. coli (Fig. S1). The recombinant cells harboring polymer biosynthetic genes were grown on xylose with different concentrations of GL. The cells accumulated P(GL-co-3HB) with various GL molar ratios dependent upon the GL concentration supplemented in the medium (Table 1). Polymer production was remarkably improved up to 4.3–4.6 g/L compared to the first report (31 mg/L).9 The synthesized polymers were subjected to NMR analysis (Fig. 1), and the 1H and

13

C NMR

resonances of the GL units were consistent with a previous report.9 The 1H NMR resonances of the 3HB unit exhibited a low-field shift as indicated by the asterisk in Figure 1A. In addition, the 13

C resonance of the carbonyl carbon of 3HB (δ 169.3) also exhibited a shifted peak (δ 169.0 and

169.5). The shifted resonances indicate the presence of GL-3HB and/or 3HB-GL dyads in the polymer chain, similar to the NMR results of the P(LA-co-3HB) copolymer.17 The proton signal of the GL units was predominantly observed at δ 4.60 as a single peak, suggesting that the majority of GL units in P(GL-co-3HB) were located in the 3HB-GL*-3HB triad rather than in the 3HB-GL*-GL, GL-GL*-3HB or GL-GL*-GL triads. In addition, the polymer degradation experiment indicates that the distribution of GL units in the polymer chain was not biased (see below). Based on these results, the obtained P(GL-co-3HB) was determined to be a random copolymer.

The effects of GL units on the physical properties of the polymer The obtained P(GL-co-3HB)s were processed into solvent-cast films (Fig. 2). The films of P(GL-co-3HB)s are flat and semitransparent compared to P(3HB), which became more opaque


6

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and distorted during the processing. The transparency of the films was positively correlated with the GL molar ratio of the polymers. The thermal properties of P(GL-co-3HB) were measured using DSC. There was a clear trend for copolymers with higher GL molar ratios to exhibit lower melting temperatures compared to the P(3HB) homopolymer (Fig. 3, Table 2). In addition, the enthalpy of fusion (∆H) of the polymers significantly decreased with an increase in GL molar ratio. The crystallinity of the polymers was roughly estimated based on the ∆H of the fully crystallized P(3HB) (146 J/g).22 For example, the crystallinity of P(15 mol% GL-co-3HB) was 4%, whereas that of P(3HB) was 54%. These results indicate that the crystallinity of the polymer was reduced by introduction of GL units. This fact was consistent with the semitransparency of the P(GL-co-3HB) films (Fig. 2). The polymers were subjected to stress-strain tests, and as shown in Table 2, P(GL-co-3HB)s possessed a lower Young's modulus and a higher elongation at break compared to P(3HB). The flexible property of the copolymers was most likely due to the lowered crystallinity of the polymers, which was consistent with their thermal property and transparency.

Hydrolytic degradation of P(GL-co-3HB) The hydrolytic degradability of P(GL-co-3HB)s was evaluated in order to justify the availability of the target polymers for biomedical applications. To accelerate the hydrolysis of the polymer, the polymer emulsion was incubated at 100°C. The copolymers containing a 4, 9, 12, and 16 mol% GL molar ratio, which were obtained using the conditions noted in Table 1, were used. After five days, the turbidity of the P(16 mol% GL-co-3HB) emulsion was significantly decreased, suggesting that degradation of the copolymer took place (data not


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shown). Therefore, the hydrothermal treatment was terminated, and the sample was analyzed according to the procedure in Scheme 1. The relative amount of water-soluble fraction increased with increasing GL molar ratio (Fig. 4A), namely, the water-insoluble fraction decreased with the incorporation of higher concentrations of GL units in the polymer chains. The molecular weight of the water-insoluble fraction also decreased with treatment, and the reduction in molecular weight was positively correlated with the GL molar ratio (Fig. 4B). The water-soluble fraction was subjected to CE analysis to measure the amounts of GL and 3HB monomers. From the copolymers with 12 and 16 mol% GL units, less than 5 % GL and only trace amounts of 3HB monomers were detected. The amounts of GL and 3HB monomers were smaller than the total amount of the water-soluble fraction (Fig. 4A), and as such, the major soluble degradation products were expectedly oligomers. Indeed, ESI-MS analysis of the water-soluble fractions derived from the copolymers with 9, 12 and 16 mol% GL (Fig. 5) revealed that the oligomers were 3 to 18 mer in length and were mainly composed of 3HB units with or without one or two GL units. These results indicate that hydrolytic degradation of the polymer was promoted by the presence of GL units in the polymer chain.

Discussion To consider the biomedical applications of polymer materials, it is important to study their hydrolytic degradation.23-24 This study performed an introductory analysis of the degradation behavior of P(GL-co-3HB), which was synthesized for the first time, compared to the P(3HB) homopolymer. The factors affecting the non-enzymatic degradation behavior of polymers are their primary structure, crystallinity, and form that is closely related to the surface area. 25-26 The


8

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use of emulsion, which has a large surface to volume ratio, enabled relatively homogeneous and rapid degradation. In terms of the primary structure, the GL units in P(GL-co-3HB) were mostly located in the 3HB-GL*-3HB triad (Fig. 1); therefore, the degradation behavior of the copolymers should be mainly determined by the relative hydrolysis rates of GL-3HB and/or 3HB-GL linkages over 3HB-3HB linkage. Taken together with the generation of oligomers and GL monomer from the copolymers, as well as the low frequency of 3HB monomer generation (Figs. 4A, 5), the hydrolysis should occur at GL-3HB and/or 3HB-GL linkages more rapidly than at the 3HB-3HB linkage. It has been discussed that the high hydrolysis rate of GL units is due to its -OCH2- group in the main chain,26 which may allow water molecules to attack the ester bond. In addition, if the copolymer is a random copolymer, the GL unit in P(16 mol% GL-co-3HB) will appear at every six units on average, which was in approximate accordance with the observed size of the oligomers (Fig. 5). Taken together, it was concluded that GL-containing sequences were more readily hydrolyzed than 3HB-homopolymer sequences. Regarding crystallinity, P(GL-co-3HB) exhibited lower crystallinity than that of P(3HB) (Table 2), which positively affected the hydrolytic degradability of the copolymer. Therefore, the introduction of GL units should accelerate the hydrolytic degradation of the polymer by both mechanisms; primary structure and crystallinity. Additional studies are needed to cast P(GL-co-3HB) into films and fibers and evaluate their degradation behavior at ambient conditions in vivo.27 The physical properties of polymer are critical factors for the practical applications and processing of polymer materials. As a typical example, it is known that the brittleness of P(3HB) limits its range of applications. Here it was shown that P(GL-co-3HB) exhibited remarkably improved stretchiness compared to the rigid homopolymers of 3HB and glycolic acid (Fig. 3). The beneficial effects of GL units on the mechanical properties were due to the reduction of


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crystallinity of the P(3HB) backbone as supported by the lowered ∆H values of the copolymer (Table 2). In addition, the transparency of the films was also consistent with the lowered crystallinity (Fig. 2). Therefore, the introduction of GL units led to several benefits such as increased degradability, flexibility, and transparency of the polymer. In addition, the GL molar ratio and ∆H values were linearly correlated (Table 2), suggesting that the crystallinity of the polymer could be further reduced with increasing GL molar ratio.

Conclusions This study reported the efficient production of GL-based PHA, P(GL-co-3HB), and for the first time, described the physical properties and hydrolytic degradability of the copolymer. The production of the polymer was approximately 140-fold greater than the first report.9 GL units in the polymer chain significantly accelerated the hydrolytic degradation of the polymer. GL-based PHA with elevated degradability can be a candidate for biomedical applications and also for use as a biodegradable material. Furthermore, this effect of GL units in promoting hydrolytic degradation may be applicable to a wide range of PHAs, and as such, GL units could be a useful determinant for regulating the degradability of PHAs.


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A

e

a

c

b

e f

d

d c

a

* *

* (ppm)

B e c

d

* *

f

*

b

*

.

a

*

(ppm)

Figure 1. NMR analysis of P(11 mol% GL-co-3HB). A: 1H NMR, B: 13C NMR. The resonances with an asterisk observed in P(GL-co-3HB) were not detected in P(3HB) (data not shown).


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a

b

c

d

Page 12 of 21

e

Figure 2. Solvent-cast films of P(GL-co-3HB) and P(3HB). (a) P(5 mol% GL-co-3HB), (b) P(9 mol% GL-co-3HB), (c) P(11 mol% GL-co-3HB), (d) P(15 mol% GL-co-3HB) and (e) P(3HB).


12

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40 Stress (MPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

e a

20

b

10

c

d

0 0%

50% Strain

100%

Figure 3. Stress-strain tests of P(GL-co-3HB) and P(3HB). (a) P(5 mol% GL-co-3HB), (b) P(9 mol% GL-co-3HB), (c) P(11 mol% GL-co-3HB), (d) P(15 mol% GL-co-3HB) and (e) P(3HB).


13


A Relative water-soluble fraction and monomers

0.5 0.4 0.3 0.2 0.1 0 0

5

10

15

20

GL molar ratio (mol%)

B 6

weight-averaged molecular weight of water-insoluble fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

5

10

4

10

3

10

0

5

10

15

20

GL molar ratio (mol%)

Figure 4. Analysis of the degradation products of P(GL-co-3HB) with various GL molar ratios. (A) The relative amount of water-soluble fraction; total (diamond), GL monomer (square) and 3HB monomer (triangle). (B) The weight-averaged molecular weight of P(GL-co-3HB)s before hydrothermal treatment (diamond) and water-insoluble fraction after treatment (square). Data are the average of two trials.


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(c) (b) (a)

*

(f) (e) (d)

m/z

Figure 5. ESI-MS analysis of the water-soluble fraction of P(16 mol% GL-co-3HB). The bracket corresponds to the molecular mass of the 3HB unit (m/z = 86). The peaks are assigned as [M-H]ions of H-(3HB)n-OH (a), H-(GL)(3HB)n-OH (b), H-(GL)2(3HB)n-OH (c), H-(3HB)n-OH + H2O (d), H-(GL)2(3HB)n-OH + H2O (e), and H-(GL)(3HB)n-OH + H2O (f). The peak with an asterisk corresponds to H-(3HB)6-OH. The copolymer with 9 and 12 mol% GL exhibited similar results (data not shown).


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water-insoluble fraction

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GPC

chloroform extraction PTFE membrane treated emulsion

water-soluble fraction

CE and ESI-MS

Scheme 1. Analytical procedures of P(GL-co-3HB) emulsion. The emulsion after hydrothermal treatment was separated into water-soluble and insoluble fractions. The lyophilized fractions were weighed to determine the weight loss. The water-insoluble fraction was subjected to GPC to determine the reduction in molecular weight of the polymer. The water-soluble fraction was subjected to CE to evaluate the GL and 3HB monomers, and to ESI-MS to detect the oligomers.


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Table 1. Production of P(GL-co-3HB) polymers in recombinant E. colia Glycolate conc. (g/L)

Cell dry weight (g/L)

Polymer production (g/L)

Polymer content (wt%)

Monomer (mol%)

composition

GL

3HB

0

7.7 ± 0.3

4.3 ± 0.2

56 ± 3

0±0

100 ± 0

2.5

7.5 ± 0.1

4.6 ± 0.1

61 ± 3

5±2

95 ± 2

5.0

7.4 ± 0.2

4.4 ± 0.1

60 ± 1

9±2

91 ± 2

7.5

7.6 ± 0.3

4.6 ± 0.2

61 ± 3

11 ± 2

89 ± 2

10.0

7.6 ± 0.5

4.3 ± 0.3

57 ± 3

15 ± 2

85 ± 2

a

Recombinant E. coli JW1375 (∆ldhA) were grown on LB medium containing 2% xylose at 30 °C for 48 h. The polymers contained a small amount of lactate, which was less than 1 mol%. Data are the average and SD of three trials. Table 2. Thermal and mechanical properties of P(GL-co-3HB) Monomer composition (mol%)

Molecular (× 10-4)

GL

Mn

3HB

Mw

weight

PDI

Thermal properties

Mechanical properties

Tg

Tm

∆H

(°C)

(°C)

(J/g)

Tensile strength (MPa)

Young's modulus (MPa)

Elongati on at break (%)

0

100

11

40

3.6

1.1

155, 173

79.3

29 ± 2

1620 ± 97

15 ± 1

5

95

7.6

33

4.3

3.0

139, 154

48.3

25 ± 3

806 ± 90

9±4

9

91

7.0

29

4.2

3.9

132, 149

29.7

17 ± 1

400 ± 21

19 ± 4

11

89

4.3

11

2.5

2.7

126, 142

11.3

11 ± 2

132 ± 29

37 ± 7

15

85

3.8

15

4.0

4.1

122, 137

5.3

7±1

54 ± 9

99 ± 7

The mechanical properties were the average of three trials after discarding the two samples that exhibited the largest and smallest elongation at break from five samples. PDI: polydispersity index.


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ASSOCIATED CONTENT Supporting Information. Utilization of glycolate and sugars by E. coli (Fig. S1). This material is available free of charge at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Ken'ichiro Matsumoto: [email protected] Author Contributions KM designed the research and wrote paper. TS performed the experiments. YH performed the degradation experiment. ST took part in the writing of the paper. All of the authors contributed to the writing of the manuscript, and approved the final version of the manuscript. Funding Sources This research was supported by PRESTO (the Precursory Research for Embryonic Science and Technology) program from the Japan Science and Technology Agency (JST), and was partly supported by ALCA (Advanced Lowe Carbon Technology Research and Development) program from JST and JSPS KAKENHI (Grant Number 23681015 to KM). ACKNOWLEDGMENT We thank Dr. Hideki Abe (RIKEN Institute) for the valuable discussion on physical property analysis, and Dr. Tomoki Erata (Hokkaido University) for the valuable discussion on the interpretation of NMR results. We thank NBRP for providing the Keio collection strain, and Ms. Kasumi Kobayashi for her technical assistance.


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REFERENCES 1. Liu, Y. R.; Xiao, L.; Joo, K. I.; Hu, B. L.; Fang, J. X.; Wang, P., In situ modulation of dendritic cells by injectable thermosensitive hydrogels for cancer vaccines in mice. Biomacromolecules 2014, 15 (10), 3836-3845. DOI: 10.1021/Bm501166j. 2. Hanson, M. C.; Bershteyn, A.; Crespo, M. P.; Irvine, D. J., Antigen delivery by lipidenveloped PLGA microparticle vaccines mediated by in situ vesicle shedding. Biomacromolecules 2014, 15 (7), 2475-81. DOI: 10.1021/bm500337r. 3. Zhang, K. R.; Tang, X.; Zhang, J.; Lu, W.; Lin, X.; Zhang, Y.; Tian, B.; Yang, H.; He, H. B., PEG-PLGA copolymers: Their structure and structure-influenced drug delivery applications. J. Control. Release 2014, 183, 77-86. DOI: 10.1016/j.jconrel.2014.03.026. 4. Romagnoli, C.; D'Asta, F.; Brandi, M. L., Drug delivery using composite scaffolds in the context of bone tissue engineering. Clin Cases Miner Bone Metab 2013, 10 (3), 155-61. 5. Keshavarz, T.; Roy, I., Polyhydroxyalkanoates: bioplastics with a green agenda. Curr. Opin. Microbiol. 2010, 13 (3), 321-326. DOI: 10.1016/j.mib.2010.02.006. 6. Lizarraga-Valderrama, L. R.; Nigmatullin, R.; Taylor, C.; Haycock, J. W.; Claeyssens, F.; Knowles, J. C.; Roy, I., Nerve tissue engineering using blends of poly(3-hydroxyalkanoates) for peripheral nerve regeneration. Eng Life Sci 2015, 15 (6), 612-621. DOI: 10.1002/elsc.201400151. 7. Matsumoto, K.; Taguchi, S., Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes. Current Opinion in Biotechnology 2013, 24 (6), 1054-1060. DOI: 10.1016/j.copbio.2013.02.021. 8. Boccaccini, A. R.; Ma, P. X., Tissue engineering using ceramics and polymers. Second edition. ed.; p xxix, 698 pages. 9. Matsumoto, K.; Ishiyama, A.; Sakai, K.; Shiba, T.; Taguchi, S., Biosynthesis of glycolate-based polyesters containing medium-chain-length 3-hydroxyalkanoates in recombinant Escherichia coli expressing engineered polyhydroxyalkanoate synthase. J. Biotechnol. 2011, 156 (3), 214-217. DOI: 10.1016/j.jbiotec.2011.07.040. 10. Takase, K.; Matsumoto, K.; Taguchi, S.; Doi, Y., Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays. Biomacromolecules 2004, 5 (2), 480-485. 11. Taguchi, S.; Yamada, M.; Matsumoto, K.; Tajima, K.; Satoh, Y.; Munekata, M.; Ohno, K.; Kohda, K.; Shimamura, T.; Kambe, H.; Obata, S., A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme. Proc. Natl. Acad. Sci. USA 2008, 105 (45), 17323-17327. 12. Nduko, J. M.; Matsumoto, K.; Ooi, T.; Taguchi, S., Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactateoverproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme. Metab Eng 2013, 15, 159-66. DOI: 10.1016/j.ymben.2012.11.007. 13. Matsumoto, K.; Terai, S.; Ishiyama, A.; Sun, J.; Kabe, T.; Song, Y.; Nduko, J. M.; Iwata, T.; Taguchi, S., One-pot microbial production, mechanical properties and enzymatic degradation of isotactic P[(R)-2-hydroxybutyrate] and its copolymer with (R)-lactate. Biomacromolecules 2013, 14 (6), 1913-1918. DOI: 10.1021/bm400278j.


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Page 20 of 21

14. Li, Z. J.; Qiao, K.; Shi, W.; Pereira, B.; Zhang, H.; Olsen, B. D.; Stephanopoulos, G., Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli. Metab Eng 2016, 35, 1-8. DOI: 10.1016/j.ymben.2016.01.004. 15. Yamada, M.; Matsumoto, K.; Uramoto, S.; Motohashi, R.; Abe, H.; Taguchi, S., Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli. J. Biotechnol. 2011, 154 (4), 255-260. DOI: 10.1016/j.jbiotec.2011.05.011. 16. Kabe, T.; Matsumoto, K.; Terai, S.; Hikima, T.; Takata, M.; Miyake, M.; Taguchi, S.; Iwata, T., Co-crystallization phenomena in biosynthesized isotactic poly[(R)-lactate-co-(R)-2hydroxybutyrate]s with various lactate unit ratios. Polym. Degrad. Stab. 2016, In press. DOI: 10.1016/j.polymdegradstab.2016.03.005. 17. Yamada, M.; Matsumoto, K.; Nakai, T.; Taguchi, S., Microbial production of lactateenriched poly[(R)-lactate-co-(R)-3-hydroxybutyrate] with novel thermal properties. Biomacromolecules 2009, 10 (4), 677-681. DOI: 10.1021/bm8013846. 18. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H., Construction of Escherichia coli K-12 in-frame, singlegene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2:2006.0008. 19. Doi, Y.; Kitamura, S.; Abe, H., Microbial synthesis and characterization of poly(3hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 1995, 28, 4822-4828. 20. Sun, J.; Matsumoto, K.; Nduko, J. M.; Ooi, T.; Taguchi, S., Enzymatic characterization of a depolymerase from the isolated bacterium Variovorax sp. C34 that degrades poly(enriched lactate-co-3-hydroxybutyrate). Polym. Degrad. Stab. 2014, 110, 44-49. DOI: 10.1016/j.polymdegradstab.2014.08.013. 21. Sudesh, K.; Bhubalan, K.; Chuah, J. A.; Kek, Y. K.; Kamilah, H.; Sridewi, N.; Lee, Y. F., Synthesis of polyhydroxyalkanoate from palm oil and some new applications. Appl Microbiol Biotechnol 2011, 89 (5), 1373-1386. DOI: 10.1007/s00253-011-3098-5. 22. Barham, P. J.; Keller, A.; Otun, E. L.; Holmes, P. A., Crystallization and morphology of a bacterial thermoplastic poly-3-hydroxybutyrate. J Mater Sci 1984, 19 (9), 2781-2794. DOI: Doi 10.1007/Bf01026954. 23. Ulery, B. D.; Nair, L. S.; Laurencin, C. T., Biomedical applications of biodegradable polymers. J Polym Sci B Polym Phys 2011, 49 (12), 832-864. DOI: 10.1002/polb.22259. 24. Makadia, H. K.; Siegel, S. J., Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers-Basel 2011, 3 (3), 1377-1397. DOI: 10.3390/Polym3031377. 25. Mochizuki, M.; Mukai, K.; Yamada, K.; Ichise, N.; Murase, S.; Iwaya, Y., Structural effects upon enzymatic hydrolysis of poly(butylene succinate-co-ethylene succinate)s. Macromolecules 1997, 30 (24), 7403-7407. DOI: 10.1021/Ma970036k. 26. Singh, V.; Tiwari, M., Structure-processing-property relationship of poly(glycolic acid) for drug delivery systems 1: Synthesis and catalysis. Int J Polym Sci 2010, Article ID 652719. DOI: 10.1155/2010/652719. 27. Ying, T. H.; Ishii, D.; Mahara, A.; Murakami, S.; Yamaoka, T.; Sudesh, K.; Samian, R.; Fujita, M.; Maeda, M.; Iwata, T., Scaffolds from electrospun polyhydroxyalkanoate copolymers: Fabrication, characterization, bioabsorption and tissue response. Biomaterials 2008, 29 (10), 1307-1317. DOI: 10.1016/j.biomaterials.2007.11.031.


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Insert Table of Contents Graphic and Synopsis Here

Incorporation of glycolate units promotes hydrolytic degradation in flexible poly(glycolateco-3-hydroxybutyrate) synthesized by engineered Escherichia coli

Ken'ichiro Matsumoto, Tetsufumi Shiba, Yukikazu Hiraide, and Seiichi Taguchi


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Incorporation of Glycolate Units Promotes Hydrolytic Degradation in

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