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Production of blue-colored polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo and PHB from recombinant Escherichia coli
Dyes and Pigments 173 (2020) 107889
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Production of blue-colored polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo and PHB from recombinant Escherichia coli
T
Hye-Rim Junga, Tae-Rim Choia, Yeong Hoon Hana, Ye-Lim Parka, Jun Young Parka, Hun-Suk Songa, Soo-Yeon Yanga, Shashi Kant Bhatiaa, Ranjit Gurava, HyunA. Parkb, Seyun Namgungb, Choi Kwon-Youngb,∗∗, Yung-Hun Yanga,c,∗ a b c
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South Korea Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, 16499, South Korea Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul, 05029, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Colored polyhydroxyalkanoate Indigo Polyhydroxybutyrate Escherichia coli One-pot process
Polyhydroxyalkanoate (PHA) is a promising substitute for petroleum-based plastics. For economically feasible and sustainable production of PHA, various strains and feedstocks have been reported till date, using which, polyhydroxybutyrate (PHB) could be produced on a large scale. However, the sensory characteristics of PHA, such as color, limit its further application in industries related to packaging, textiles, and medical materials. In this study, we investigated the feasibility of simultaneous production of indigo and PHB in recombinant Escherichia coli to generate colored bioplastics. With that objective, one-pot production and extraction of both indigo and PHB were successfully performed, and biotransformation of indole to indigo was reinforced by the introduction of PHA synthetic genes. Upon further investigation of the factors responsible for the increased indigo production, groEL and grpE, related to heat-shock proteins, and CYP102G4, were found to be up-regulated when analyzed by semi-quantitative reverse transcription-PCR. In the thermograms analyzed by thermal gravimetric analysis and differential scanning calorimetry, there was no notable difference between PHB and PHB-indigo film, implying that indigo did not affect the thermal properties of PHB itself. Overall, this study demonstrates the production of blue-colored PHB and enhanced production of indigo by the introduction of PHA synthetic genes.
1. Introduction Polyhydroxybutyrate (PHB), a member of the polyhydroxyalkanoate (PHA) family, is a bio-based and biodegradable polymer with the potential to replace petroleum-based plastics [1–3]. PHB is one of the easily produced bioplastics that can be made from inexpensive carbon-rich substrates, such as industrial waste streams and lignocellulose hydrolysates, thereby enabling economic production of PHA, besides the raw material cost, procurement of fermentation equipment, and optimization of downstream processes [1,3,4]. Amongst the several strains that are capable of producing various PHAs, recombinant Escherichia coli harboring PHA synthetic genes are regarded as the ideal hosts for PHA production, considering their utilization of inexpensive carbon sources, absence of depolymerases,
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relative independence from C/N ratio, and ease of modification of their pathways by using genetic tools [5–8]. Although different strains and strategies have been employed till date, several limitations regarding the conferral of chemical and physical properties, suitable for widespread use, still remain. Many studies have attempted to alter the composition of PHAs [2,9–11]. Additionally, the sensory properties of PHA, such as color of the fermentation broth, often need to be improved. However, the sensory qualities of the produced PHAs have not been investigated in detail. Indigo is a widely used pigment in the textile industry, and can be naturally extracted from the leaves of certain plants as a plant-derived secondary metabolite [12]. However, the plants require a long cultivation period and their productivity may be affected by climate changes. On the other hand, chemical synthesis of indigo involves toxic
Corresponding author. Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South Korea. Corresponding author. Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, 16499, South Korea. E-mail addresses: [email protected] (K.-Y. Choi), [email protected] (Y.-H. Yang).
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https://doi.org/10.1016/j.dyepig.2019.107889 Received 26 June 2019; Received in revised form 19 August 2019 Available online 18 September 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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materials from petroleum-based resources such as aniline [13]. With increase in interest in the microbial production of various materials using eco-friendly processes, many studies have now reported on biological synthesis of indigo [14]. Recombinant E. coli expressing cytochrome P450 monooxygenase (CYPs) from Streptomyces cattleya has been used to successfully produce indigo from indole [13,14]. The yellowish color of PHB may be eliminated by one-pot production and co-extraction of indigo and PHB, thereby producing a colored bioplastic. Because one-pot synthesis is a simple method of producing and recovering PHB and the dye separately, we evaluated the feasibility of simultaneous production of indigo and PHB in recombinant E. coli, and co-extraction of both, to produce colored PHB. Unexpectedly, we found the biotransformation of indole to indigo to be reinforced by the introduction of PHA synthetic genes, thus suggesting possible synergistic effects. To evaluate whether PHB may be altered by co-extraction with indigo, a PHB-indigo film was characterized by scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). These experiments moved a step ahead from previous efforts to improve the physical and chemical properties of PHB, by altering its composition, which will enable greater use of PHB and avoid issues associated with sensory characteristics, such as color.
acid [17] (as a heme precursor) were added with different concentrations of indole to evaluate biotransformation of the substrate [13,14]. For flask-culture, the cells were inoculated into 100 mL of production medium in 250-mL screw cap flasks at 1:100 (v/v) dilutions. The cultures were continuously shaken in an incubator at 200 rpm and 30 °C. 2.3. Analytical methods Indigo production was analyzed using high-performance liquid chromatography (YL-9100, Korea) based on absorbance at 620 nm. The products were separated on a C8 column (Eclipse Plus C8, 250 × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA) and eluted with acetonitrile/water (50:50, v/v) at 1.0 mL/min. The column temperature was maintained at 25 °C. To analyze indigo yield, the product was extracted using dimethyl sulfoxide and filtered through a 0.22-μm polyvinylidene fluoride syringe filter (Chromdisc, Korea). PHB production and quantity were determined using gas chromatography, as previously described, with slight modifications [1,2,5,19]. For analysis, culture samples were centrifuged at 10,000×g for 10 min, washed twice with deionized water, and suspended in 1 mL of water. The suspended samples were lyophilized, weighed, and placed in Teflon-stoppered glass vials. For methanolysis of PHB samples, 1 mL of chloroform and 1 mL of a methanol/sulfuric acid (85:15 v/v) mixture were added to the vials and incubated at 100 °C for 2 h. The samples were then cooled to room temperature and incubated on ice for approximately 10 min. After adding 1 mL of ice-cold water, samples were thoroughly mixed, by vortex mixing, for 1 min and centrifuged at 2000×g. The organic phase (bottom) was extracted using a pipette and moved to clean borosilicate glass tubes containing anhydrous sodium sulfate. The samples were then injected into a gas chromatography instrument (Young Lin Tech, Korea) using a HP-FFAP column (30 m × 0.32 mm × 0.25 μm) (Agilent Technologies). The split ratio was 1:10. Helium was used as the carrier gas and its flow rate was maintained at 3.0 mL/min. A 2-μL portion of the organic phase was injected using an autosampler. The inlet was maintained at 210 °C. The column oven was held at 80 °C for 5 min, heated to 220 °C at 20 °C/min, and then held at 220 °C for another 5 min. Peak detection was performed using a flame ionization detector, which was maintained at 230 °C. PHB content was calculated PHB (g / L ) as DCW (g / L) (%), by weight.
2. Material and methods 2.1. Chemicals All chemicals used in this study were of analytical grade. Indole was purchased from Alfa Aesar (Haverhill, MA, USA) [15]. Synthetic indigo was purchased from Sigma-Aldrich (St. Louis, MO, USA) [15]. Other medium components were purchased from Difco (Detroit, MI, USA) [15]. Solvents for extraction of indigo and PHB were purchased from Thermo Fisher Scientific (Waltham, MA, USA) [15]. 2.2. Bacterial strains, media, and culture conditions All strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5 and KSYH(DE3) were used as host strains for gene cloning and PHB production, respectively. For cell preparation and transformant selection, strains were cultured in lysogeny broth [16,16] agar and/or in liquid broth. LB agar was prepared by dissolving 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of distilled water. For pre-culture, a single colony from an LB agar plate was inoculated into 3 mL of LB medium. The culture was incubated overnight in a shaking incubator at 37 °C and 200 rpm. To evaluate indigo and PHB production, transformants were cultured in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and different concentrations of indole. Appropriate antibiotics (100 μg/ mL of spectinomycin and 50 μg/mL of kanamycin for E. coli transformants) were added as required. Upon reaching an OD600 of 0.8, 0.3 mM isopropyl-β-D-thiogalactoside and 0.25 mM δ-aminolevulinic
2.4. Transcriptional analysis Total RNA was extracted from E. coli using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The purity and quantity of extracted RNA were evaluated by 1% agarose gel electrophoresis and a NanoDrop system (Thermo Fisher Scientific). First-strand cDNA was synthesized from total RNA using Invitrogen SuperScript III (Carlsbad, CA, USA) and random hexamer primers
Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid E. coli strains DH5 KSYH(DE3) KSYH(DE3)::CYP102G4 YH090 Plasmids pLW487 pET28a::CYP102G4
Description
Reference
General cloning strain BW25113 derivative containing DE3, ΔaraBAD, ΔrhaBAD KSYH(DE3) containing pET28a::CYP102G4 KSYH(DE3) containing pLW487
Invitrogen [6] [13] [6]
Spectinomycin-resistant pEP2-based plasmid carrying PCR products of bktB, phaB, and phaC, under trc promoter from Ralstonia eutropha H16 pET28a (+) carrying PCR product of CYP102G4 from Streptomyces cattleya
[18]
2
[13]
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Fig. 1. Engineered metabolic pathways for the production of indigo and polyhydroxybutyrate (PHB) in recombinant E. coli. The following genes are involved in indigo and PHB production and are depicted in Fig. 1. BktB; β-ketothiolase, PhaB; Acetoacetyl-CoA reductase, PhaC; polyhydroxyalkanoate synthase, and CYP102G4; Cytochrome P450 monooxygenase.
Fig. 2. Time-dependent monitoring of indigo and polyhydroxybutyrate (PHB) production in recombinant E. coli cells. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole. (A): DCW (dry cell weight, g/L), (B): PHB content (w/w %), (C): Residual biomass (g/L), and (D): indigo concentration (μM). Residual biomass is defined as dry cell weight, excluding that of indigo and PHB. Cells were cultivated for 24 h. Error bars represent the standard deviation of two replicates.
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Fig. 3. One-pot production of polyhydroxybutyrate (PHB) and indigo using E. coli cells expressing CYP102G4 and PHA synthetic genes. Indole concentration ranged from 1 to 7 μM. (A): DCW (dry cell weight, g/L), (B): PHB content (w/w %), (C): Residual biomass (g/L), and (D): indigo concentration (μM). Residual biomass was defined as the dry cell weight, excluding that of indigo and PHB. Cells were cultivated for 24 h. Error bars represented the standard deviation of two replicates.
according to the manufacturer's protocol. Changes in the expression level of different genes were compared by semi-quantitative reverse transcription PCR using the primers listed in Table S1. The expression of rrsA, encoding 16S ribosomal RNA, was evaluated as an internal control. PCR products using gene-specific primers were obtained from different cycles (20–35), and the amounts of PCR products were compared by electrophoresis on 1% agarose gels.
Whatman No. 1 filter paper to remove any residual cell debris. The refined solvent was dried at room temperature, whereby the solvent evaporated until a plastic film was formed. To form the PHB film, YH090 cells were grown as described previously, and extracted as mentioned above [1,5,6]. The extracted PHB-indigo and PHB films were subsequently used for further analysis. 2.6. Characterization of the extracted polymer
2.5. Polymer extraction
Extracted PHB-indigo and PHB film were analyzed separately by SEM, TGA, DSC, and decolorization test. In each analysis, 10 mg of sample was used. For SEM, TM3030Plus tabletop microscope (Hitachi, Tokyo, Japan) was used without surface coating of samples [21]. SEM images were obtained at an accelerating voltage of 5–15 kV. Thermal gravimetric analysis (TGA) was performed as described previously, using a PerkinElmer TGA 4000 instrument (PerkinElmer, Waltham, MA, USA). The samples were heated from 0 °C to 900 °C at a rate of 10 °C/ min, under nitrogen at a flow rate of 20 mL/min [20]. For differential scanning calorimetry (DSC), DSC Q1000 (V9.9 Build 303; TA Instruments, New Castle, DE, USA) was used [20]. The samples were heated from −60 °C to 170 °C at a rate of 10 °C/min. The melting temperature
Conventional solvent-cast method was used for preparing PHB film and PHB-indigo film [2,10,18,20]. To form the PHB-indigo film, YH090::CYP102G4 cells were grown in 50 mL of M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole, in a 250mL baffled flask, for 24 h. The blue-colored culture broth (50 mL) was centrifuged, and a blue-colored cell pellet was collected; the produced indigo aggregated in the cell pellet rather than in the supernatant. The blue-colored pellet was suspended, washed twice with deionized water, and lyophilized. The dried cells were submerged in chloroform (20 mL) and extracted by agitation at 60 °C for 16 h. The polymer-containing chloroform was collected by centrifugation, followed by filtration using
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for further experiments. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM of indole as a precursor for indigo for 24 h (Fig. 2). After 24 h, 52.7% PHB production and 0.6 mM indigo production were observed. Thus, simultaneous PHB and indigo production was achieved by combining two biotransformation systems. 3.2. Reinforced biotransformation of indole to indigo by the introduction of PHA synthetic genes High concentrations of indole (1–6 mM) used as an indigo precursor inhibited cell growth due to toxicity, resulting in lower productivity [23]. Previous reports showed that PHA production and mobilization could enhance tolerance to stresses caused by toxic inhibitors, osmotic pressure, and carbon starvation [1,24–26]. A previous study indicated that PHA synthetic genes improved tolerance to toxic inhibitors, such as furfural, vanillin, and 4-hydroxybenzaldehyde. Additionally, high PHB accumulation in recombinant E. coli could cause stress and induce the expression of heat shock proteins related to stress resistance [24]. Thus, we tested indole concentrations ranging from 1 to 7 mM (Fig. 3). YH090::CYP102G4 cells showed higher residual biomass, which suggested that the introduction of PHA synthetic genes affected indole tolerance (Fig. 2C). In addition to cell growth, indigo production was increased by 1.81-fold from 457.15 to 829.58 μM in the presence of 5 mM indole (Fig. 2D). The most substantial difference, a 4.96-fold increase in indigo production, was observed when 5 mM indole was used (Fig. 3D). Based on these results, PHA synthetic genes affected indole tolerance and indigo production, and increased PHB production, suggesting synergistic effects rather than inhibition by cofactor depletion. In order to evaluate whether stress responses were responsible for increased indigo production at higher concentrations of indole, transcriptional analysis was performed using semi-quantitative RT-PCR (Fig. 4). This analysis was independently performed three times under identical conditions for reliable results. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole for 24 h. Seven genes were selected: trnA; encoding tryptophanase, which converts tryptophan to indole, CYP102G4; encoding monooxygenase, which converts indole to indigo, groES, groEL, dnaK, dnaJ, and grpE, which were related to heat-shock proteins. Based on the amounts of PCR products obtained after different cycle numbers (20–35), PCR products from 30 cycles were analyzed to compare expression levels (Fig. S1). The semi-quantitative RT-PCR data showed increases in CYP102G4, groEL, and grpE genes, which suggested that the introduction of PHB synthetic genes increased tolerance and activity by upregulating some chaperones and CYP102G4 (Fig. 4). In contrast to these results, there was no notable expression of trnA, which might be related to feedback inhibition caused by supplemental indole.
Fig. 4. Transcriptional analysis by semi-quantitative RT-PCR. Changes in expression levels following the introduction of PHA synthetic genes were investigated in KSYH(DE3)::CYP102G4 and YH090::CYP102G4 cells supplemented with 5 mM indole. Total RNA was extracted at 24 h.
(Tm) was determined from the DSC curve. To evaluate the degree of decolorization of PHB-indigo film after solvent treatment, seven different solvents, namely deionized water, 10% SDS solution, ethanol, methanol, chloroform, ethyl acetate, and methyl ethyl ketone, were used. The PHB-indigo film (10 mg) was immersed in 1 mL of each solvent and incubated for 6 h at 25 °C. The solvent-treated PHB-indigo film was completely dried at room temperature and degree of decolorization was evaluated. 3. Results and discussion 3.1. One-pot production of indigo and PHB in recombinant E. coli Biotransformation of indole to indigo was previously investigated using a self-sufficient CYP102G4 [13,14,22]. Escherichia coli expressing CYP102G4 from S. cattleya produced indigo successfully in LB medium supplemented with indole and tryptophan, and these cells were applied to produce indigoid [13]. A one-pot system for the biosynthesis of indigo and PHB in recombinant E. coli was generated by combining PHB production and indigo production (Fig. 1). Genes involved in indigo and PHB production were as follows: BktB; β-ketothiolase, PhaB; acetoacetyl-CoA reductase, PhaC; polyhydroxyalkanoate synthase; CYP102G4; cytochrome P450 monooxygenase. PHB was produced with YH090 cells containing a pLW487 vector and bktB-phaB-phaC under the trc promoter [6] by adding a pET28a vector harboring CYP102G4 [14]. To evaluate the feasibility of simultaneous indigo and PHB production in recombinant E. coli expressing CYP102G4 and PHA synthetic genes, various media were tested in an effort to balance both PHB and indigo production (data not shown). We compared complex media such as lysogeny broth [16], terrific broth (TB), and super optimal broth (SOB) with M9 minimal media. However, no distinct PHB production or differences in indigo production were found within the complex media. Thus, M9 minimal medium was selected
3.3. One-pot extraction of indigo and PHB for colored bioplastic To evaluate one-pot extraction of indigo and PHB, the blue-colored pellet obtained following culturing of YH090::CYP102G4 cells was evaluated by SEM, TGA, and DSC analysis. Changes in the morphology of PHB-indigo film were compared to those of the PHB film. The SEM micrographs of PHB films and PHB-
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Fig. 5. SEM micrographs of PHB and PHB-indigo films. Changes in surface morphology were investigated via film. A: PHB film, (B-C): PHB-indigo film.
indigo film are depicted in Fig. 5. PHB-indigo film exhibited lower porosity with smaller pore sizes compared to PHB film (Fig. 5A–C). Additionally, the surface of PHB-indigo film was rougher than that of PHB film, showing aggregated small particles. The pores of PHB may have been filled with indigo during the extraction process. Additionally, the thermal properties of the PHB film and PHB-indigo film were investigated. TGA revealed no difference in thermal stability, showing a Td of 307.55 °C for PHB film and 303.19 °C for PHB-indigo film (Fig. 6A and B). Similarly, there was no notable difference in the thermograms obtained by DSC analysis (Fig. 6C and D). Thus, one-pot extraction of
indigo and PHB did not affect the thermal properties of the PHB-indigo film. 3.4. Examination of decolorization with different solvents and PHB-indigo film Because colored plastic and colored textiles exhibit decolorization upon repetitive washing and drying [13], we also examined decolorization of the PHB-indigo film. To investigate the effect of solvents on extracted PHB-indigo film, seven different solvents, deionized water, 10% SDS solution, ethanol, methanol, chloroform, ethyl acetate, and
Fig. 6. Thermal properties of the extracted polymer. Thermal degradation spectra of PHB and PHB-indigo films were analyzed by TGA. (A): PHB film, (B): PHB-indigo film. DSC thermograms of PHB film (C) and PHB-indigo film (D).
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Fig. 7. Effect of solvents on colored PHB and resultant degree of decolorization. (A): Degree of decolorization of the PHB-indigo film following washing with solvents (distilled water, 10% SDS solution, absolute ethanol, absolute methanol, chloroform, ethyl acetate, methyl ethyl ketone). PHB-indigo film (10 mg) was incubated in 1 mL of each solvent at 25 °C for 6 h. (B): Color of extracted polymer was compared. Decolorized plastic was prepared after washing with ethyl acetate.
methyl ethyl ketone, were screened. The changes in solvent color are depicted in Fig. 7. The detergent solution containing SDS, ethanol, and methanol resulted in no difference in color following incubation, which suggested that detergent and alcohols do not affect the PHB-indigo film (Fig. 7A). However, chloroform completely dissolved the PHB-indigo film. This was expected considering that chloroform was used to extract the PHBindigo film. Interestingly, ethyl acetate and MEK dissolved only indigo products from the PHB-indigo film without altering the morphology of the film (Fig. 7B). Based upon the degree of color in the solvents, ethyl acetate dissolved indigo products more specifically than MEK. Thus, water, detergent, and alcohols did not affect the degree of color of the PHB-indigo film. Ethyl acetate and MEK redissolved the indigo products in the PHB-indigo film, resulting in decolorization.
of the National Research Foundation of Korea (NRF) funded by the Ministry of Science ICT and Future Planning (2017M3A9E4077234) and National Research Foundation of Korea (NRF) (NRF2015M1A5A1037196 and NRF-2019R1F1A1058805). In addition, this work was also supported by polar academic program (PAP, PE18900). This work was also supported by Next-Generation BioGreen21 Program (SSAC, PJ01312801), Rural Development Administration. The consulting service of the Microbial Carbohydrate Resource Bank (MCRB, Seoul, South Korea) is greatly appreciated.
4. Conclusions
Conflicts of interest
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107889.
The authors declare no competing interests.
Many studies have shown that PHB could be easily produced with various strains and from various feedstocks [1,24,27–29]. However, PHB exhibited relatively poor chemical and physical properties, such as odor and color. We altered the color of PHB and investigated the feasibility of simultaneous production of indigo and PHB in recombinant E. coli by combining two biotransformation systems, which resulted in the first colored PHB-indigo film. Although we formed different colors by changing the initial indole molecules as described previously [13], we developed a simple method for one-pot production of blue-colored PHB. Additionally, biotransformation of indole to indigo was found to be reinforced by the introduction of PHA synthetic genes, which suggested synergistic effects. One-pot production and co-extraction of indigo and PHB were successfully performed, and colored PHB was obtained. The yield was increased by 4.6-fold, and higher tolerance to indole was observed after introduction of PHA synthetic genes. As described previously in several reports [1,25,28], PHA synthetic genes enhanced the yield and productivity of the fermentation products. We observed increased bioconversion yield in a relatively short period of time with increased resistance to the initial toxic substrates such as indole. The use of higher initial substrate level resulted in higher productivity. Although the mechanisms involved in this process require further analysis, we demonstrated the feasibility of one-pot production of PHBindigo. Moreover, use of PHA synthetic genes could enhance the robustness of the cell system.
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Acknowledgments This work was supported by Research Program to solve social issues 7
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[21] Nishimura M, Ichikawa K, Ajima M. Features and applications of Hitachi tabletop microscope TM3030Plus. 2016. [22] Kim J, Lee P-g, Jung E-o, Kim B-G. In vitro characterization of CYP102G4 from Streptomyces cattleya: a self-sufficient P450 naturally producing indigo. Biochim Biophys Acta Protein Proteonomics 2018;1866(1):60–7. [23] Chant EL, Summers DK. Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids. Mol Microbiol 2007;63(1):35–43. [24] Han M-J, Yoon SS, Lee SY. Proteome analysis of metabolically engineeredescherichia coli producing poly (3-hydroxybutyrate). J Bacteriol 2001;183(1):301–8. [25] Wang Q, Yu H, Xia Y, Kang Z, Qi Q. Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application. Microb Cell Factories 2009;8(1):47. [26] James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl Environ Microbiol 1999;65(2):822–7. [27] Dietrich D, Illman B, Crooks C. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava. BMC Res Notes 2013;6(1):219. [28] Kadouri D, Jurkevitch E, Okon Y. Involvement of the reserve material poly-β-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 2003;69(6):3244–50. [29] Wei X-X, Zheng W-T, Hou X, Liang J, Li Z-J. Metabolic engineering of Escherichia coli for poly (3-hydroxybutyrate) production under microaerobic condition. BioMed Res Int 2015;2015.
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Production of blue-colored polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo and PHB from recombinant Escherichia coli
T
Hye-Rim Junga, Tae-Rim Choia, Yeong Hoon Hana, Ye-Lim Parka, Jun Young Parka, Hun-Suk Songa, Soo-Yeon Yanga, Shashi Kant Bhatiaa, Ranjit Gurava, HyunA. Parkb, Seyun Namgungb, Choi Kwon-Youngb,∗∗, Yung-Hun Yanga,c,∗ a b c
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South Korea Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, 16499, South Korea Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul, 05029, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Colored polyhydroxyalkanoate Indigo Polyhydroxybutyrate Escherichia coli One-pot process
Polyhydroxyalkanoate (PHA) is a promising substitute for petroleum-based plastics. For economically feasible and sustainable production of PHA, various strains and feedstocks have been reported till date, using which, polyhydroxybutyrate (PHB) could be produced on a large scale. However, the sensory characteristics of PHA, such as color, limit its further application in industries related to packaging, textiles, and medical materials. In this study, we investigated the feasibility of simultaneous production of indigo and PHB in recombinant Escherichia coli to generate colored bioplastics. With that objective, one-pot production and extraction of both indigo and PHB were successfully performed, and biotransformation of indole to indigo was reinforced by the introduction of PHA synthetic genes. Upon further investigation of the factors responsible for the increased indigo production, groEL and grpE, related to heat-shock proteins, and CYP102G4, were found to be up-regulated when analyzed by semi-quantitative reverse transcription-PCR. In the thermograms analyzed by thermal gravimetric analysis and differential scanning calorimetry, there was no notable difference between PHB and PHB-indigo film, implying that indigo did not affect the thermal properties of PHB itself. Overall, this study demonstrates the production of blue-colored PHB and enhanced production of indigo by the introduction of PHA synthetic genes.
1. Introduction Polyhydroxybutyrate (PHB), a member of the polyhydroxyalkanoate (PHA) family, is a bio-based and biodegradable polymer with the potential to replace petroleum-based plastics [1–3]. PHB is one of the easily produced bioplastics that can be made from inexpensive carbon-rich substrates, such as industrial waste streams and lignocellulose hydrolysates, thereby enabling economic production of PHA, besides the raw material cost, procurement of fermentation equipment, and optimization of downstream processes [1,3,4]. Amongst the several strains that are capable of producing various PHAs, recombinant Escherichia coli harboring PHA synthetic genes are regarded as the ideal hosts for PHA production, considering their utilization of inexpensive carbon sources, absence of depolymerases,
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relative independence from C/N ratio, and ease of modification of their pathways by using genetic tools [5–8]. Although different strains and strategies have been employed till date, several limitations regarding the conferral of chemical and physical properties, suitable for widespread use, still remain. Many studies have attempted to alter the composition of PHAs [2,9–11]. Additionally, the sensory properties of PHA, such as color of the fermentation broth, often need to be improved. However, the sensory qualities of the produced PHAs have not been investigated in detail. Indigo is a widely used pigment in the textile industry, and can be naturally extracted from the leaves of certain plants as a plant-derived secondary metabolite [12]. However, the plants require a long cultivation period and their productivity may be affected by climate changes. On the other hand, chemical synthesis of indigo involves toxic
Corresponding author. Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South Korea. Corresponding author. Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, 16499, South Korea. E-mail addresses: [email protected] (K.-Y. Choi), [email protected] (Y.-H. Yang).
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https://doi.org/10.1016/j.dyepig.2019.107889 Received 26 June 2019; Received in revised form 19 August 2019 Available online 18 September 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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materials from petroleum-based resources such as aniline [13]. With increase in interest in the microbial production of various materials using eco-friendly processes, many studies have now reported on biological synthesis of indigo [14]. Recombinant E. coli expressing cytochrome P450 monooxygenase (CYPs) from Streptomyces cattleya has been used to successfully produce indigo from indole [13,14]. The yellowish color of PHB may be eliminated by one-pot production and co-extraction of indigo and PHB, thereby producing a colored bioplastic. Because one-pot synthesis is a simple method of producing and recovering PHB and the dye separately, we evaluated the feasibility of simultaneous production of indigo and PHB in recombinant E. coli, and co-extraction of both, to produce colored PHB. Unexpectedly, we found the biotransformation of indole to indigo to be reinforced by the introduction of PHA synthetic genes, thus suggesting possible synergistic effects. To evaluate whether PHB may be altered by co-extraction with indigo, a PHB-indigo film was characterized by scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). These experiments moved a step ahead from previous efforts to improve the physical and chemical properties of PHB, by altering its composition, which will enable greater use of PHB and avoid issues associated with sensory characteristics, such as color.
acid [17] (as a heme precursor) were added with different concentrations of indole to evaluate biotransformation of the substrate [13,14]. For flask-culture, the cells were inoculated into 100 mL of production medium in 250-mL screw cap flasks at 1:100 (v/v) dilutions. The cultures were continuously shaken in an incubator at 200 rpm and 30 °C. 2.3. Analytical methods Indigo production was analyzed using high-performance liquid chromatography (YL-9100, Korea) based on absorbance at 620 nm. The products were separated on a C8 column (Eclipse Plus C8, 250 × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA) and eluted with acetonitrile/water (50:50, v/v) at 1.0 mL/min. The column temperature was maintained at 25 °C. To analyze indigo yield, the product was extracted using dimethyl sulfoxide and filtered through a 0.22-μm polyvinylidene fluoride syringe filter (Chromdisc, Korea). PHB production and quantity were determined using gas chromatography, as previously described, with slight modifications [1,2,5,19]. For analysis, culture samples were centrifuged at 10,000×g for 10 min, washed twice with deionized water, and suspended in 1 mL of water. The suspended samples were lyophilized, weighed, and placed in Teflon-stoppered glass vials. For methanolysis of PHB samples, 1 mL of chloroform and 1 mL of a methanol/sulfuric acid (85:15 v/v) mixture were added to the vials and incubated at 100 °C for 2 h. The samples were then cooled to room temperature and incubated on ice for approximately 10 min. After adding 1 mL of ice-cold water, samples were thoroughly mixed, by vortex mixing, for 1 min and centrifuged at 2000×g. The organic phase (bottom) was extracted using a pipette and moved to clean borosilicate glass tubes containing anhydrous sodium sulfate. The samples were then injected into a gas chromatography instrument (Young Lin Tech, Korea) using a HP-FFAP column (30 m × 0.32 mm × 0.25 μm) (Agilent Technologies). The split ratio was 1:10. Helium was used as the carrier gas and its flow rate was maintained at 3.0 mL/min. A 2-μL portion of the organic phase was injected using an autosampler. The inlet was maintained at 210 °C. The column oven was held at 80 °C for 5 min, heated to 220 °C at 20 °C/min, and then held at 220 °C for another 5 min. Peak detection was performed using a flame ionization detector, which was maintained at 230 °C. PHB content was calculated PHB (g / L ) as DCW (g / L) (%), by weight.
2. Material and methods 2.1. Chemicals All chemicals used in this study were of analytical grade. Indole was purchased from Alfa Aesar (Haverhill, MA, USA) [15]. Synthetic indigo was purchased from Sigma-Aldrich (St. Louis, MO, USA) [15]. Other medium components were purchased from Difco (Detroit, MI, USA) [15]. Solvents for extraction of indigo and PHB were purchased from Thermo Fisher Scientific (Waltham, MA, USA) [15]. 2.2. Bacterial strains, media, and culture conditions All strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5 and KSYH(DE3) were used as host strains for gene cloning and PHB production, respectively. For cell preparation and transformant selection, strains were cultured in lysogeny broth [16,16] agar and/or in liquid broth. LB agar was prepared by dissolving 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of distilled water. For pre-culture, a single colony from an LB agar plate was inoculated into 3 mL of LB medium. The culture was incubated overnight in a shaking incubator at 37 °C and 200 rpm. To evaluate indigo and PHB production, transformants were cultured in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and different concentrations of indole. Appropriate antibiotics (100 μg/ mL of spectinomycin and 50 μg/mL of kanamycin for E. coli transformants) were added as required. Upon reaching an OD600 of 0.8, 0.3 mM isopropyl-β-D-thiogalactoside and 0.25 mM δ-aminolevulinic
2.4. Transcriptional analysis Total RNA was extracted from E. coli using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The purity and quantity of extracted RNA were evaluated by 1% agarose gel electrophoresis and a NanoDrop system (Thermo Fisher Scientific). First-strand cDNA was synthesized from total RNA using Invitrogen SuperScript III (Carlsbad, CA, USA) and random hexamer primers
Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid E. coli strains DH5 KSYH(DE3) KSYH(DE3)::CYP102G4 YH090 Plasmids pLW487 pET28a::CYP102G4
Description
Reference
General cloning strain BW25113 derivative containing DE3, ΔaraBAD, ΔrhaBAD KSYH(DE3) containing pET28a::CYP102G4 KSYH(DE3) containing pLW487
Invitrogen [6] [13] [6]
Spectinomycin-resistant pEP2-based plasmid carrying PCR products of bktB, phaB, and phaC, under trc promoter from Ralstonia eutropha H16 pET28a (+) carrying PCR product of CYP102G4 from Streptomyces cattleya
[18]
2
[13]
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Fig. 1. Engineered metabolic pathways for the production of indigo and polyhydroxybutyrate (PHB) in recombinant E. coli. The following genes are involved in indigo and PHB production and are depicted in Fig. 1. BktB; β-ketothiolase, PhaB; Acetoacetyl-CoA reductase, PhaC; polyhydroxyalkanoate synthase, and CYP102G4; Cytochrome P450 monooxygenase.
Fig. 2. Time-dependent monitoring of indigo and polyhydroxybutyrate (PHB) production in recombinant E. coli cells. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole. (A): DCW (dry cell weight, g/L), (B): PHB content (w/w %), (C): Residual biomass (g/L), and (D): indigo concentration (μM). Residual biomass is defined as dry cell weight, excluding that of indigo and PHB. Cells were cultivated for 24 h. Error bars represent the standard deviation of two replicates.
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Fig. 3. One-pot production of polyhydroxybutyrate (PHB) and indigo using E. coli cells expressing CYP102G4 and PHA synthetic genes. Indole concentration ranged from 1 to 7 μM. (A): DCW (dry cell weight, g/L), (B): PHB content (w/w %), (C): Residual biomass (g/L), and (D): indigo concentration (μM). Residual biomass was defined as the dry cell weight, excluding that of indigo and PHB. Cells were cultivated for 24 h. Error bars represented the standard deviation of two replicates.
according to the manufacturer's protocol. Changes in the expression level of different genes were compared by semi-quantitative reverse transcription PCR using the primers listed in Table S1. The expression of rrsA, encoding 16S ribosomal RNA, was evaluated as an internal control. PCR products using gene-specific primers were obtained from different cycles (20–35), and the amounts of PCR products were compared by electrophoresis on 1% agarose gels.
Whatman No. 1 filter paper to remove any residual cell debris. The refined solvent was dried at room temperature, whereby the solvent evaporated until a plastic film was formed. To form the PHB film, YH090 cells were grown as described previously, and extracted as mentioned above [1,5,6]. The extracted PHB-indigo and PHB films were subsequently used for further analysis. 2.6. Characterization of the extracted polymer
2.5. Polymer extraction
Extracted PHB-indigo and PHB film were analyzed separately by SEM, TGA, DSC, and decolorization test. In each analysis, 10 mg of sample was used. For SEM, TM3030Plus tabletop microscope (Hitachi, Tokyo, Japan) was used without surface coating of samples [21]. SEM images were obtained at an accelerating voltage of 5–15 kV. Thermal gravimetric analysis (TGA) was performed as described previously, using a PerkinElmer TGA 4000 instrument (PerkinElmer, Waltham, MA, USA). The samples were heated from 0 °C to 900 °C at a rate of 10 °C/ min, under nitrogen at a flow rate of 20 mL/min [20]. For differential scanning calorimetry (DSC), DSC Q1000 (V9.9 Build 303; TA Instruments, New Castle, DE, USA) was used [20]. The samples were heated from −60 °C to 170 °C at a rate of 10 °C/min. The melting temperature
Conventional solvent-cast method was used for preparing PHB film and PHB-indigo film [2,10,18,20]. To form the PHB-indigo film, YH090::CYP102G4 cells were grown in 50 mL of M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole, in a 250mL baffled flask, for 24 h. The blue-colored culture broth (50 mL) was centrifuged, and a blue-colored cell pellet was collected; the produced indigo aggregated in the cell pellet rather than in the supernatant. The blue-colored pellet was suspended, washed twice with deionized water, and lyophilized. The dried cells were submerged in chloroform (20 mL) and extracted by agitation at 60 °C for 16 h. The polymer-containing chloroform was collected by centrifugation, followed by filtration using
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for further experiments. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM of indole as a precursor for indigo for 24 h (Fig. 2). After 24 h, 52.7% PHB production and 0.6 mM indigo production were observed. Thus, simultaneous PHB and indigo production was achieved by combining two biotransformation systems. 3.2. Reinforced biotransformation of indole to indigo by the introduction of PHA synthetic genes High concentrations of indole (1–6 mM) used as an indigo precursor inhibited cell growth due to toxicity, resulting in lower productivity [23]. Previous reports showed that PHA production and mobilization could enhance tolerance to stresses caused by toxic inhibitors, osmotic pressure, and carbon starvation [1,24–26]. A previous study indicated that PHA synthetic genes improved tolerance to toxic inhibitors, such as furfural, vanillin, and 4-hydroxybenzaldehyde. Additionally, high PHB accumulation in recombinant E. coli could cause stress and induce the expression of heat shock proteins related to stress resistance [24]. Thus, we tested indole concentrations ranging from 1 to 7 mM (Fig. 3). YH090::CYP102G4 cells showed higher residual biomass, which suggested that the introduction of PHA synthetic genes affected indole tolerance (Fig. 2C). In addition to cell growth, indigo production was increased by 1.81-fold from 457.15 to 829.58 μM in the presence of 5 mM indole (Fig. 2D). The most substantial difference, a 4.96-fold increase in indigo production, was observed when 5 mM indole was used (Fig. 3D). Based on these results, PHA synthetic genes affected indole tolerance and indigo production, and increased PHB production, suggesting synergistic effects rather than inhibition by cofactor depletion. In order to evaluate whether stress responses were responsible for increased indigo production at higher concentrations of indole, transcriptional analysis was performed using semi-quantitative RT-PCR (Fig. 4). This analysis was independently performed three times under identical conditions for reliable results. Cells were cultivated in M9 minimal medium containing 2% glucose, 0.5% yeast extract, and 5 mM indole for 24 h. Seven genes were selected: trnA; encoding tryptophanase, which converts tryptophan to indole, CYP102G4; encoding monooxygenase, which converts indole to indigo, groES, groEL, dnaK, dnaJ, and grpE, which were related to heat-shock proteins. Based on the amounts of PCR products obtained after different cycle numbers (20–35), PCR products from 30 cycles were analyzed to compare expression levels (Fig. S1). The semi-quantitative RT-PCR data showed increases in CYP102G4, groEL, and grpE genes, which suggested that the introduction of PHB synthetic genes increased tolerance and activity by upregulating some chaperones and CYP102G4 (Fig. 4). In contrast to these results, there was no notable expression of trnA, which might be related to feedback inhibition caused by supplemental indole.
Fig. 4. Transcriptional analysis by semi-quantitative RT-PCR. Changes in expression levels following the introduction of PHA synthetic genes were investigated in KSYH(DE3)::CYP102G4 and YH090::CYP102G4 cells supplemented with 5 mM indole. Total RNA was extracted at 24 h.
(Tm) was determined from the DSC curve. To evaluate the degree of decolorization of PHB-indigo film after solvent treatment, seven different solvents, namely deionized water, 10% SDS solution, ethanol, methanol, chloroform, ethyl acetate, and methyl ethyl ketone, were used. The PHB-indigo film (10 mg) was immersed in 1 mL of each solvent and incubated for 6 h at 25 °C. The solvent-treated PHB-indigo film was completely dried at room temperature and degree of decolorization was evaluated. 3. Results and discussion 3.1. One-pot production of indigo and PHB in recombinant E. coli Biotransformation of indole to indigo was previously investigated using a self-sufficient CYP102G4 [13,14,22]. Escherichia coli expressing CYP102G4 from S. cattleya produced indigo successfully in LB medium supplemented with indole and tryptophan, and these cells were applied to produce indigoid [13]. A one-pot system for the biosynthesis of indigo and PHB in recombinant E. coli was generated by combining PHB production and indigo production (Fig. 1). Genes involved in indigo and PHB production were as follows: BktB; β-ketothiolase, PhaB; acetoacetyl-CoA reductase, PhaC; polyhydroxyalkanoate synthase; CYP102G4; cytochrome P450 monooxygenase. PHB was produced with YH090 cells containing a pLW487 vector and bktB-phaB-phaC under the trc promoter [6] by adding a pET28a vector harboring CYP102G4 [14]. To evaluate the feasibility of simultaneous indigo and PHB production in recombinant E. coli expressing CYP102G4 and PHA synthetic genes, various media were tested in an effort to balance both PHB and indigo production (data not shown). We compared complex media such as lysogeny broth [16], terrific broth (TB), and super optimal broth (SOB) with M9 minimal media. However, no distinct PHB production or differences in indigo production were found within the complex media. Thus, M9 minimal medium was selected
3.3. One-pot extraction of indigo and PHB for colored bioplastic To evaluate one-pot extraction of indigo and PHB, the blue-colored pellet obtained following culturing of YH090::CYP102G4 cells was evaluated by SEM, TGA, and DSC analysis. Changes in the morphology of PHB-indigo film were compared to those of the PHB film. The SEM micrographs of PHB films and PHB-
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Fig. 5. SEM micrographs of PHB and PHB-indigo films. Changes in surface morphology were investigated via film. A: PHB film, (B-C): PHB-indigo film.
indigo film are depicted in Fig. 5. PHB-indigo film exhibited lower porosity with smaller pore sizes compared to PHB film (Fig. 5A–C). Additionally, the surface of PHB-indigo film was rougher than that of PHB film, showing aggregated small particles. The pores of PHB may have been filled with indigo during the extraction process. Additionally, the thermal properties of the PHB film and PHB-indigo film were investigated. TGA revealed no difference in thermal stability, showing a Td of 307.55 °C for PHB film and 303.19 °C for PHB-indigo film (Fig. 6A and B). Similarly, there was no notable difference in the thermograms obtained by DSC analysis (Fig. 6C and D). Thus, one-pot extraction of
indigo and PHB did not affect the thermal properties of the PHB-indigo film. 3.4. Examination of decolorization with different solvents and PHB-indigo film Because colored plastic and colored textiles exhibit decolorization upon repetitive washing and drying [13], we also examined decolorization of the PHB-indigo film. To investigate the effect of solvents on extracted PHB-indigo film, seven different solvents, deionized water, 10% SDS solution, ethanol, methanol, chloroform, ethyl acetate, and
Fig. 6. Thermal properties of the extracted polymer. Thermal degradation spectra of PHB and PHB-indigo films were analyzed by TGA. (A): PHB film, (B): PHB-indigo film. DSC thermograms of PHB film (C) and PHB-indigo film (D).
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Fig. 7. Effect of solvents on colored PHB and resultant degree of decolorization. (A): Degree of decolorization of the PHB-indigo film following washing with solvents (distilled water, 10% SDS solution, absolute ethanol, absolute methanol, chloroform, ethyl acetate, methyl ethyl ketone). PHB-indigo film (10 mg) was incubated in 1 mL of each solvent at 25 °C for 6 h. (B): Color of extracted polymer was compared. Decolorized plastic was prepared after washing with ethyl acetate.
methyl ethyl ketone, were screened. The changes in solvent color are depicted in Fig. 7. The detergent solution containing SDS, ethanol, and methanol resulted in no difference in color following incubation, which suggested that detergent and alcohols do not affect the PHB-indigo film (Fig. 7A). However, chloroform completely dissolved the PHB-indigo film. This was expected considering that chloroform was used to extract the PHBindigo film. Interestingly, ethyl acetate and MEK dissolved only indigo products from the PHB-indigo film without altering the morphology of the film (Fig. 7B). Based upon the degree of color in the solvents, ethyl acetate dissolved indigo products more specifically than MEK. Thus, water, detergent, and alcohols did not affect the degree of color of the PHB-indigo film. Ethyl acetate and MEK redissolved the indigo products in the PHB-indigo film, resulting in decolorization.
of the National Research Foundation of Korea (NRF) funded by the Ministry of Science ICT and Future Planning (2017M3A9E4077234) and National Research Foundation of Korea (NRF) (NRF2015M1A5A1037196 and NRF-2019R1F1A1058805). In addition, this work was also supported by polar academic program (PAP, PE18900). This work was also supported by Next-Generation BioGreen21 Program (SSAC, PJ01312801), Rural Development Administration. The consulting service of the Microbial Carbohydrate Resource Bank (MCRB, Seoul, South Korea) is greatly appreciated.
4. Conclusions
Conflicts of interest
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107889.
The authors declare no competing interests.
Many studies have shown that PHB could be easily produced with various strains and from various feedstocks [1,24,27–29]. However, PHB exhibited relatively poor chemical and physical properties, such as odor and color. We altered the color of PHB and investigated the feasibility of simultaneous production of indigo and PHB in recombinant E. coli by combining two biotransformation systems, which resulted in the first colored PHB-indigo film. Although we formed different colors by changing the initial indole molecules as described previously [13], we developed a simple method for one-pot production of blue-colored PHB. Additionally, biotransformation of indole to indigo was found to be reinforced by the introduction of PHA synthetic genes, which suggested synergistic effects. One-pot production and co-extraction of indigo and PHB were successfully performed, and colored PHB was obtained. The yield was increased by 4.6-fold, and higher tolerance to indole was observed after introduction of PHA synthetic genes. As described previously in several reports [1,25,28], PHA synthetic genes enhanced the yield and productivity of the fermentation products. We observed increased bioconversion yield in a relatively short period of time with increased resistance to the initial toxic substrates such as indole. The use of higher initial substrate level resulted in higher productivity. Although the mechanisms involved in this process require further analysis, we demonstrated the feasibility of one-pot production of PHBindigo. Moreover, use of PHA synthetic genes could enhance the robustness of the cell system.
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Acknowledgments This work was supported by Research Program to solve social issues 7
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