Appl Biochem Biotechnol DOI 10.1007/s12010-015-1644-9
Indigoids Biosynthesis from Indole by Two Phenol-Degrading Strains, Pseudomonas sp. PI1 and Acinetobacter sp. PI2 Jing Wang 1 & Xuwang Zhang 2 & Jiangli Fan 1 & Zhaojing Zhang 2 & Qiao Ma 2 & Xiaojun Peng 1
Received: 7 February 2015 / Accepted: 21 April 2015 # Springer Science+Business Media New York 2015
Abstract In this study, two phenol-degrading bacterial strains, designated as PI1 and PI2, were isolated from activated sludge for the production of indigoids from indole. According to the 16S ribosomal RNA (rRNA) gene sequence analysis, strains PI1 and PI2 were identified as Pseudomonas sp. and Acinetobacter sp., respectively. Liquid chromatography/time-of-flight/ mass spectrometry (LC/TOF/MS) was applied to analyze the metabolites during the biotransformation of indole by the phenol-degrading strains. The results indicated that both strains could catalyze the formation of four indigoids with the same prominent molecular ion (M-H)− peak at m/z 261.067 and molecular formula of C16H10N2O2, including indigo and a purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. Isatin and 7-hydroxyindole were detected as the intermediates. Thus, the possible pathways for the production of indigoids from indole were proposed. Subsequently, the optimal conditions for the production of indigo from indole were determined using response surface methodology, and 11.82±0.30 and 17.19± 0.49 mg/L indigo were produced by strains PI1 and PI2, respectively. The present study should provide potential candidates for microbial production of indigoids. Keywords Indigoids . Indole . Phenol-degrading strains . Biotransformation . Response surface methodology
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1644-9) contains supplementary material, which is available to authorized users.
* Jing Wang [email protected] 1
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
2
Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
Appl Biochem Biotechnol
Introduction Indole is proven to be an important biological signal molecule. Researches have shown it can be produced by at least 85 gram-positive and gram-negative bacterial species due to the existence of tryptophanase which catalyzes the transformation of tryptophan into indole, pyruvate, and ammonium [1]. The produced indole plays multiple roles in controlling virulence, drug resistance, biofilm formation, plasmid stability, and transition to stationary phase to coordinate microbial behaviors [2, 3]. In multispecies communities, many non-indole-producing bacteria can transform indole into downstream metabolites, whereas the indole transformation pathways are poorly concerned. Furthermore, indole also belongs to the typical N-heterocyclic pollutants [4]. It is abundant in coking wastewater influents, which may harm microbes, plants, and animals. Thus, the removal of indole is a necessity before discharging the wastewater. Therefore, the investigation on indole biotransformation should be paid more attention. The biotransformation researches of indole could be divided into two parts, i.e., indole biodegradation into ring-opening products and indigoid bioproduction through indole oxidation process. Indigoids are among the oldest pigments extensively used in dyestuff, food, cosmetic, and pharmaceutical industries [5, 6]. The production of the valuable products inspires scientists to invest efforts on indole-indigoid studies. During the last 30 years, many bacteria and enzymes were identified to be able to convert indole into indigoid pigments. For example, styrene monooxygenase from Pseudomonas putida S12 and CA-3 could catalyze indole into high-purity indigo [7]. Escherichia coli expressing naphthalene dioxygenase from P. putida PpG7 and flavin-containing monooxygenase from Methylophaga aminisulfidivorans MPT could synthesize indigo and indirubin [8, 9]. Cytochrome P450 2A6 could mediate formation of two blue and two pink/purple products as well as a novel end product 6Hoxazolo[3,2-a:4,5-b′]diindole [10]. As an important and ubiquitous multicomponent oxygenase, phenol hydroxylase (PHO) could also oxidize indole leading to the formation of indigoids. Kim et al. found PHO from Pseudomonas sp. KL28 to be a versatile catalyst for indigoids production from indole derivatives [11]. We have previously isolated and sequenced a Pseudomonas sp. QM and proved that it can mediate indigo synthesis. During the transformation process, two novel indigo isomers (red and purple) were produced [12, 13]. Similarly, we cloned and expressed a novel PHO from Arthrobacter sp. W1 and the purple product was also detected. According to the high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analyses, the structure was identified as 2-(7-oxo1H-indol-6(7H)-ylidene) indolin-3-one [14]. The produced novel indigoids might have broad applications especially in pharmaceutical field [6, 11]. All the above researches indicated that the indole transformation pathways in phenol-degrading bacteria were diverse, which is worthy of further investigation. Herein, two phenol-degrading strains, designated as PI1 and PI2, were isolated from activated sludge of lab bioreactors designed for the treatment of coking wastewater. The indole oxidation possibility of the strains was verified, and transformation products were analyzed. Furthermore, the transformation conditions of the two strains were optimized to improve indigo yields. The present study flourished our understanding of indole biotransformation by phenol-degrading strains.
Appl Biochem Biotechnol
Materials and Methods Chemicals and Media Indole was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), indigo was purchased from Acros Organics (New Jersey, USA), and phenol was purchased from J&K Scientific Ltd. (Beijing, China). All other chemicals were of analytical grade or above. The medium used in this study was mineral salt medium (MSM, pH 6.7), which consisted of (NH4)2SO4 2 g/L, KH2PO4 2 g/L, Na2HPO4·12H2O 3.28 g/L, and FeCl3 0.25 mg/L. Phenol, indole, and yeast extract were added to the media with certain concentrations according to experimental requirements. Solid medium contained 2.0 % (w/v) agar in MSM. The media were autoclaved at 121 °C for 20 min before use.
Isolation and Identification of Phenol-Degrading Strains for Indigo Production The phenol-degrading strains were isolated from activated sludge of lab bioreactors designed for the treatment of coking wastewater by enrichment method. Briefly, the activated sludge samples were firstly inoculated into a flask containing 100 mL MSM supplemented with 100 mg/L phenol and cultivated at 30 °C under continuous shaking (150 r/min) for 48 h. Then, 10 % (v/v) of the broth was inoculated into fresh MSM supplemented with 100 mg/L phenol. After about 2-week domestication, 10 % (v/v) of the broth was inoculated into MSM containing 100 mg/L phenol and 100 mg/L indole. During the cultivation, blue bubbles could be observed on the surface of culture broth. Subsequently, the culture was further plated and transferred several times using solid phenol-indole-MSM. After about 2 months of screening and enriching, two of the blue colonies, designated as PI1 and PI2, were selected for further characterization and identified by a 16S ribosomal RNA (rRNA) gene sequence analysis. The 16S rRNA genes of strains PI1 and PI2 were amplified by PCR using the primer set 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). The PCR amplicons were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The partial sequences were compared with those in GenBank using BLAST program, and the related sequences were aligned using ClustalX (1.8). Then, the aligned sequences were used to construct phylogenetic tree by MEGA 5.1 using neighbor-joining method with 1000 bootstrap replicates.
Optimization of Indigo Production by Response Surface Methodology To produce indigo from indole, the biotransformation assays were performed in 100-mL flasks with 30 mL MSM containing 100 mg/L indole, 100 mg/L phenol, and 0.1 % yeast extract (w/v) at 30 °C under continuous shaking (150 r/min) for 48 h. The inoculation volume was set as 5 % (v/v). Response surface methodology (RSM) was applied to evaluate the optimal conditions for indigo production by the phenol-degrading strains based on a 23 central composite design, including the concentrations of indole (factor X1), phenol (X2) and yeast extract (X3) (Table 1). Design Expert 8.0.6 (Stat Ease, Minneapolis, USA) was used to generate the RSM design matrix and analyze the experimental data. The regression equations were fitted using the coded values of variables, and the analysis of variance (ANOVA) was applied to analyze the significance of the model and each coefficient (linear or quadratic) [15–17]. The model terms were considered to be significant when the p value (probability>F) was less than 0.05. The
Appl Biochem Biotechnol Table 1 RSM design and results for indigo production by two phenol-degrading strains No.
Factor X1 indole (mg/L)
Factor X2 phenol (mg/L)
Factor X3 yeast extract (%)
PI1 (indigo, mg/L)
PI2 (indigo, mg/L)
Experimental
Experimental
Predicted
Predicted
1
50 (−1)
100 (−1)
0.05 (−1)
2.70
3.24
3.01
1.30
2
150 (1)
100 (−1)
0.05 (−1)
0.24
0.03
0.53
0.77
3
50 (−1)
300 (1)
0.05 (−1)
3.78
2.89
4.74
5.76
4
150 (1)
300 (1)
0.05 (−1)
0.03
0.58
0.01
0.03 6.05
5
50 (−1)
100 (−1)
0.15 (1)
2.04
1.69
5.42
6
150 (1)
100 (−1)
0.15 (1)
0.75
2.56
7.05
6.97
7 8
50 (−1) 150 (1)
300 (1) 300 (1)
0.15 (1) 0.15 (1)
0.04 2.74
1.00 3.92
6.86 0.01
7.18 0.81
9
16 (−1.7)
200 (0)
0.1 (0)
0.87
0.65
1.21
1.74
10
184 (1.7)
200 (0)
0.1 (0)
0.67
0.07
0.01
0.16
11
100 (0)
32 (−1.7)
0.1 (0)
3.22
3.16
4.43
5.82
12
100 (0)
368 (1.7)
0.1 (0)
6.93
4.03
6.54
4.04
13
100 (0)
200 (0)
0.02 (−1.7)
1.87
2.27
2.75
3.47
14
100 (0)
200 (0)
0.18 (1.7)
8.54
4.45
16.59
12.79
15 16
100 (0) 100 (0)
200 (0) 200 (0)
0.1 (0) 0.1 (0)
12.75 10.95
11.29 11.29
16.00 15.60
15.92 15.92
17
100 (0)
200 (0)
0.1 (0)
11.90
11.29
15.42
15.92
18
100 (0)
200 (0)
0.1 (0)
11.06
11.29
15.90
15.92
19
100 (0)
200 (0)
0.1 (0)
10.53
11.29
15.74
15.92
20
100 (0)
200 (0)
0.1 (0)
9.85
11.29
16.26
15.92
The coded values of the independent variables are shown in the parenthesis
optimal conditions were obtained according to the analyses of the regression equations. All experiments were carried out in triplicate, and the average values were used for calculation.
Analytical Methods To quantify the yields of indigo and the residual concentrations of indole, the biotransformation mixtures were centrifuged at 10,000×g for 10 min. The supernatant was extracted using an equal volume of chloroform, and the samples of chloroform layer were analyzed by HPLC to determine the concentrations of indole. The pellets were resuspended in dimethylsulfoxide (DMSO) with 10 min of ultrasonic treatment, followed by centrifugation at 10,000×g for 10 min to obtain blue supernatant, which was analyzed by HPLC to determine the amount of indigo. HPLC was performed on a Shimadzu LC-20A (Japan) system equipped with a Hypersil ODS2 column (5 μm, 250×4.6 mm). The compounds were eluted using a linear gradient from 60 to 70 % methanol/water (v/v) over 20 min at 1 mL/min and monitored at 280 and 265 nm for indigo and indole, respectively. To detect the metabolites during the biotransformation of indole, the blue DMSO supernatant was also analyzed by liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS) (Agilent 6244, USA) equipped with a standard electrospray ionization (ESI) source in negative ionization mode.
Appl Biochem Biotechnol
Results and Discussion Isolation and Identification of Phenol-Degrading Strains Two phenol-degrading strains, PI1 and PI2, were isolated from the activated sludge of lab bioreactors, which were able to co-metabolize indole to produce indigo in the presence of phenol, but could not use indole as the sole carbon source for growth. To identify the isolates taxonomically, 1444 and 1363 bp fragments of 16S rRNA gene were amplified from strains PI1 and PI2, respectively. Based on a sequence analysis, strain PI1 exhibited 99 % homology to bacterial species Pseudomonas citronellolis BG6903 (GenBank accession number AM088480), while strain PI2 had high identity (99 %) with Acinetobacter sp. N12 (GenBank accession number AB208676). Therefore, strain PI1 was identified as Pseudomonas sp., and strain PI2 belonged to genus Acinetobacter. The 16S rRNA gene sequences of strains PI1 and PI2 were deposited in GenBank database under the accession number KP269079 and KP330203, respectively. Neighbor-joining phylogenetic analysis was performed using the 16S rRNA gene sequences of strains PI1 and PI2 and the close relatives, together with the sequences of many different indigo-producing bacteria (Fig. 1). As reported previously, various indigo-producing bacterial strains of genera Pseudomonas and Acinetobacter have been isolated with different abilities of aromatic hydrocarbon degradation, including naphthalene-degrading strains P. putida PpG7, Pseudomonas sp. J26, and Pseudomonas aeruginosa HOB1; styrenedegrading strains P. putida S12 and P. putida CA-3; p-cumate-degrading strain P. putida F1; m- and p-toluate-degrading strain P. putida mt-2; phenol-degrading strains Pseudomonas monteilii QM, Acinetobacter sp. ST-500, and Acinetobacter sp. PP-2; etc. [7, 15, 18–22]. Among the reported Pseudomonas spp., most of them belonged to P. putida group, such as P. putida and P. monteilii [23], which was famous for its high capability to degrade recalcitrant xenobiotics and to produce a brand range of chemicals [24]. Here, strain PI1 (P. citronellolis) was more likely to belong to P. aeruginosa group, just like P. aeruginosa HOB1 [23]. As for strain PI2, it was another representative of Acinetobacter spp. used for indigo bioproduction.
Mechanisms of Indigoids Production by the Two Phenol-Degrading Strains To identify the products produced from indole by the two phenol-degrading strains, LC/TOF/ MS analysis was performed, and similar results were obtained from both strains (Fig. 2). Four products with the prominent molecular ion (M-H)− peak at m/z 261.067 were detected at the retention times of 17.6 min (I), 19.5 min (II), 20.9 min (III), and 22.5 min (IV), respectively, which had the same molecular formula with indigo, i.e., C16H10N2O2 (Fig. 2a–d). The UV-Vis spectrum of product III was observed with absorption maxima at wavelengths of 241, 286, 340, and 615 nm (Fig. S1c), which was identical with the commercial indigo standard [7, 25]. Therefore, the product III was identified as indigo. For the product IV, the UV-Vis absorption maxima were observed at wavelengths of 266, 315, 390, and 577 nm (Fig. S1d). In our previous study, the recombinant E. coli expressing the phenol hydroxylase from Arthrobacter sp. W1 was used for the biotransformation of indole, and a new purple product, 2-(7-oxo-1Hindol-6(7H)-ylidene) indolin-3-one, was obtained [14], which had the same UV-Vis spectrum and the MS fragmentation patterns with product IV. Thus, the product IV was suggested to be the purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. The UV-Vis spectra of products I and II were apparently different from those of products III and IV (Fig. S1a and b),
Appl Biochem Biotechnol
Fig. 1 Phylogenetic tree of the newly isolated strains PI1 and PI2. The tree was constructed using neighborjoining method, and the numbers indicated the bootstrap values derived from 1000 replicates. The corresponding GenBank accession numbers of the partial 16S rRNA gene sequences were shown in parenthesis
but we were unable to further identify their chemical structures. However, indirubin, the red isomer of indigo, was not detected, which should have the same prominent molecular ion (M-H)− peak at m/z 261.067 (C16H10N2O2) with the absorption maxima at wavelengths of 289, 363, and 544 nm (data not shown). Besides, product V with a retention time of 10.1 min and a prominent molecular ion (M-H)− peak at m/z 146.0246 (C8H5NO2) was identified as isatin by comparing its mass spectral data with standard chemical and previous studies (Fig. 2e) [26, 27]. Product VI was detected at retention time of 12.7 min with a prominent molecular ion (M-H)− peak at m/z 132.0454 (C8H7NO) (Fig. 2f), which was likely to be 7-hydroxyindole. Previous study showed that the phenol-degrading strain Pseudomonas sp. KL33 could produce 7-hydroxyindole from indole [11]. According to the LC/TOF/MS analysis and previous studies, the possible pathways of indigoid production from indole by the two phenol-degrading strains were proposed as shown in Fig. 3. Phenol hydroxylase might be the functional enzyme in both strains, PI1 and PI2, responsible for the oxidation of indole [11, 14]. Indole could be oxidized by phenol hydroxylase through C-3 oxidation pathway to form 3-hydroxyindole (indoxyl), which was extremely unstable and could be spontaneously dimerized by air oxidation to form indigo [28, 29]. In addition, indoxyl could be further hydroxylated to produce isatin [14, 28]. Besides, isatin could also be obtained through the decomposition of indigo [26, 28]. Previous studies indicated that the C-3 oxidation pathway of indole biotransformation usually occurred in accompany with C2 and C-7 oxidation pathways, leading to the formation of 2-hydroxyindole (2-oxindole) and 7-hydroxyindole, respectively [14, 29, 30]. Condensation of indoxyl and 2-hydroxyindole/ isatin could yield indirubin [14, 26, 28, 29], and 2-hydroxyindole was more preferable for indirubin production [17]. Furthermore, condensation of isatin and 7-hydroxyindole would lead to the formation of the purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one [14]. Therefore, the absence of indirubin and the presence of the purple product in this study indirectly suggested that product VI was 7-hydroxyindole. The products I and II might be generated through some new pathways catalyzed by other oxygenases. In our recent efforts, the genome sequence of strain PI1 was obtained using an Illumina HiSeq 2000 sequencer. The results
Appl Biochem Biotechnol
Fig. 2 Mass spectra of products produced from indole by the phenol-degrading strains. a–d Four indigoids with the same prominent molecular ion (M-H) peak at m/z 261.067, including indigo (c) and 2-(7-oxo-1H-indol6(7H)-ylidene) indolin-3-one (d). e Isatin. f 7-Hydroxyindole
Appl Biochem Biotechnol
Fig. 2 (continued)
indicated that a rich set of candidate protein-coding sequences was annotated for metabolism of aromatic compounds, including biphenyl, benzoate, salicylate, chloroaromatics, etc., among which there should be some functional genes responsible for the production of products I and II.
Appl Biochem Biotechnol
Fig. 3 Proposed pathways for the production of indigoids and other metabolites from indole by the phenoldegrading strains
Optimization of Indigo Production by Response Surface Methodology The time courses of indigo production from indole by strains PI1 and PI2 were examined with 100 mg/L indole, 100 mg/L phenol, and 0.1 % yeast extract (Fig. S2). The results indicated that both indole transformation and indigo production could become stable in 48 h, and 7.28± 0.27 and 10.93±0.29 mg/L indigo were produced by strains PI1 and PI2, respectively. To further optimize the process of indigo production, RSM was applied to investigate the effects of indole (X1), phenol (X2), and yeast extract (X3) on indigo yields. A 23 central composite design with 20 groups of experiments was carried out for each strain, and the results are shown in Table 1. The second-order polynomial equations were fitted Table 2 ANOVA for response surface model of indigo production Source
PI1
PI2
F value
p valuea
F value
p value
Model
7.43
0.0021
19.93
Indigoids Biosynthesis from Indole by Two Phenol-Degrading Strains, Pseudomonas sp. PI1 and Acinetobacter sp. PI2 Jing Wang 1 & Xuwang Zhang 2 & Jiangli Fan 1 & Zhaojing Zhang 2 & Qiao Ma 2 & Xiaojun Peng 1
Received: 7 February 2015 / Accepted: 21 April 2015 # Springer Science+Business Media New York 2015
Abstract In this study, two phenol-degrading bacterial strains, designated as PI1 and PI2, were isolated from activated sludge for the production of indigoids from indole. According to the 16S ribosomal RNA (rRNA) gene sequence analysis, strains PI1 and PI2 were identified as Pseudomonas sp. and Acinetobacter sp., respectively. Liquid chromatography/time-of-flight/ mass spectrometry (LC/TOF/MS) was applied to analyze the metabolites during the biotransformation of indole by the phenol-degrading strains. The results indicated that both strains could catalyze the formation of four indigoids with the same prominent molecular ion (M-H)− peak at m/z 261.067 and molecular formula of C16H10N2O2, including indigo and a purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. Isatin and 7-hydroxyindole were detected as the intermediates. Thus, the possible pathways for the production of indigoids from indole were proposed. Subsequently, the optimal conditions for the production of indigo from indole were determined using response surface methodology, and 11.82±0.30 and 17.19± 0.49 mg/L indigo were produced by strains PI1 and PI2, respectively. The present study should provide potential candidates for microbial production of indigoids. Keywords Indigoids . Indole . Phenol-degrading strains . Biotransformation . Response surface methodology
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1644-9) contains supplementary material, which is available to authorized users.
* Jing Wang [email protected] 1
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
2
Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, China), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
Appl Biochem Biotechnol
Introduction Indole is proven to be an important biological signal molecule. Researches have shown it can be produced by at least 85 gram-positive and gram-negative bacterial species due to the existence of tryptophanase which catalyzes the transformation of tryptophan into indole, pyruvate, and ammonium [1]. The produced indole plays multiple roles in controlling virulence, drug resistance, biofilm formation, plasmid stability, and transition to stationary phase to coordinate microbial behaviors [2, 3]. In multispecies communities, many non-indole-producing bacteria can transform indole into downstream metabolites, whereas the indole transformation pathways are poorly concerned. Furthermore, indole also belongs to the typical N-heterocyclic pollutants [4]. It is abundant in coking wastewater influents, which may harm microbes, plants, and animals. Thus, the removal of indole is a necessity before discharging the wastewater. Therefore, the investigation on indole biotransformation should be paid more attention. The biotransformation researches of indole could be divided into two parts, i.e., indole biodegradation into ring-opening products and indigoid bioproduction through indole oxidation process. Indigoids are among the oldest pigments extensively used in dyestuff, food, cosmetic, and pharmaceutical industries [5, 6]. The production of the valuable products inspires scientists to invest efforts on indole-indigoid studies. During the last 30 years, many bacteria and enzymes were identified to be able to convert indole into indigoid pigments. For example, styrene monooxygenase from Pseudomonas putida S12 and CA-3 could catalyze indole into high-purity indigo [7]. Escherichia coli expressing naphthalene dioxygenase from P. putida PpG7 and flavin-containing monooxygenase from Methylophaga aminisulfidivorans MPT could synthesize indigo and indirubin [8, 9]. Cytochrome P450 2A6 could mediate formation of two blue and two pink/purple products as well as a novel end product 6Hoxazolo[3,2-a:4,5-b′]diindole [10]. As an important and ubiquitous multicomponent oxygenase, phenol hydroxylase (PHO) could also oxidize indole leading to the formation of indigoids. Kim et al. found PHO from Pseudomonas sp. KL28 to be a versatile catalyst for indigoids production from indole derivatives [11]. We have previously isolated and sequenced a Pseudomonas sp. QM and proved that it can mediate indigo synthesis. During the transformation process, two novel indigo isomers (red and purple) were produced [12, 13]. Similarly, we cloned and expressed a novel PHO from Arthrobacter sp. W1 and the purple product was also detected. According to the high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analyses, the structure was identified as 2-(7-oxo1H-indol-6(7H)-ylidene) indolin-3-one [14]. The produced novel indigoids might have broad applications especially in pharmaceutical field [6, 11]. All the above researches indicated that the indole transformation pathways in phenol-degrading bacteria were diverse, which is worthy of further investigation. Herein, two phenol-degrading strains, designated as PI1 and PI2, were isolated from activated sludge of lab bioreactors designed for the treatment of coking wastewater. The indole oxidation possibility of the strains was verified, and transformation products were analyzed. Furthermore, the transformation conditions of the two strains were optimized to improve indigo yields. The present study flourished our understanding of indole biotransformation by phenol-degrading strains.
Appl Biochem Biotechnol
Materials and Methods Chemicals and Media Indole was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), indigo was purchased from Acros Organics (New Jersey, USA), and phenol was purchased from J&K Scientific Ltd. (Beijing, China). All other chemicals were of analytical grade or above. The medium used in this study was mineral salt medium (MSM, pH 6.7), which consisted of (NH4)2SO4 2 g/L, KH2PO4 2 g/L, Na2HPO4·12H2O 3.28 g/L, and FeCl3 0.25 mg/L. Phenol, indole, and yeast extract were added to the media with certain concentrations according to experimental requirements. Solid medium contained 2.0 % (w/v) agar in MSM. The media were autoclaved at 121 °C for 20 min before use.
Isolation and Identification of Phenol-Degrading Strains for Indigo Production The phenol-degrading strains were isolated from activated sludge of lab bioreactors designed for the treatment of coking wastewater by enrichment method. Briefly, the activated sludge samples were firstly inoculated into a flask containing 100 mL MSM supplemented with 100 mg/L phenol and cultivated at 30 °C under continuous shaking (150 r/min) for 48 h. Then, 10 % (v/v) of the broth was inoculated into fresh MSM supplemented with 100 mg/L phenol. After about 2-week domestication, 10 % (v/v) of the broth was inoculated into MSM containing 100 mg/L phenol and 100 mg/L indole. During the cultivation, blue bubbles could be observed on the surface of culture broth. Subsequently, the culture was further plated and transferred several times using solid phenol-indole-MSM. After about 2 months of screening and enriching, two of the blue colonies, designated as PI1 and PI2, were selected for further characterization and identified by a 16S ribosomal RNA (rRNA) gene sequence analysis. The 16S rRNA genes of strains PI1 and PI2 were amplified by PCR using the primer set 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). The PCR amplicons were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The partial sequences were compared with those in GenBank using BLAST program, and the related sequences were aligned using ClustalX (1.8). Then, the aligned sequences were used to construct phylogenetic tree by MEGA 5.1 using neighbor-joining method with 1000 bootstrap replicates.
Optimization of Indigo Production by Response Surface Methodology To produce indigo from indole, the biotransformation assays were performed in 100-mL flasks with 30 mL MSM containing 100 mg/L indole, 100 mg/L phenol, and 0.1 % yeast extract (w/v) at 30 °C under continuous shaking (150 r/min) for 48 h. The inoculation volume was set as 5 % (v/v). Response surface methodology (RSM) was applied to evaluate the optimal conditions for indigo production by the phenol-degrading strains based on a 23 central composite design, including the concentrations of indole (factor X1), phenol (X2) and yeast extract (X3) (Table 1). Design Expert 8.0.6 (Stat Ease, Minneapolis, USA) was used to generate the RSM design matrix and analyze the experimental data. The regression equations were fitted using the coded values of variables, and the analysis of variance (ANOVA) was applied to analyze the significance of the model and each coefficient (linear or quadratic) [15–17]. The model terms were considered to be significant when the p value (probability>F) was less than 0.05. The
Appl Biochem Biotechnol Table 1 RSM design and results for indigo production by two phenol-degrading strains No.
Factor X1 indole (mg/L)
Factor X2 phenol (mg/L)
Factor X3 yeast extract (%)
PI1 (indigo, mg/L)
PI2 (indigo, mg/L)
Experimental
Experimental
Predicted
Predicted
1
50 (−1)
100 (−1)
0.05 (−1)
2.70
3.24
3.01
1.30
2
150 (1)
100 (−1)
0.05 (−1)
0.24
0.03
0.53
0.77
3
50 (−1)
300 (1)
0.05 (−1)
3.78
2.89
4.74
5.76
4
150 (1)
300 (1)
0.05 (−1)
0.03
0.58
0.01
0.03 6.05
5
50 (−1)
100 (−1)
0.15 (1)
2.04
1.69
5.42
6
150 (1)
100 (−1)
0.15 (1)
0.75
2.56
7.05
6.97
7 8
50 (−1) 150 (1)
300 (1) 300 (1)
0.15 (1) 0.15 (1)
0.04 2.74
1.00 3.92
6.86 0.01
7.18 0.81
9
16 (−1.7)
200 (0)
0.1 (0)
0.87
0.65
1.21
1.74
10
184 (1.7)
200 (0)
0.1 (0)
0.67
0.07
0.01
0.16
11
100 (0)
32 (−1.7)
0.1 (0)
3.22
3.16
4.43
5.82
12
100 (0)
368 (1.7)
0.1 (0)
6.93
4.03
6.54
4.04
13
100 (0)
200 (0)
0.02 (−1.7)
1.87
2.27
2.75
3.47
14
100 (0)
200 (0)
0.18 (1.7)
8.54
4.45
16.59
12.79
15 16
100 (0) 100 (0)
200 (0) 200 (0)
0.1 (0) 0.1 (0)
12.75 10.95
11.29 11.29
16.00 15.60
15.92 15.92
17
100 (0)
200 (0)
0.1 (0)
11.90
11.29
15.42
15.92
18
100 (0)
200 (0)
0.1 (0)
11.06
11.29
15.90
15.92
19
100 (0)
200 (0)
0.1 (0)
10.53
11.29
15.74
15.92
20
100 (0)
200 (0)
0.1 (0)
9.85
11.29
16.26
15.92
The coded values of the independent variables are shown in the parenthesis
optimal conditions were obtained according to the analyses of the regression equations. All experiments were carried out in triplicate, and the average values were used for calculation.
Analytical Methods To quantify the yields of indigo and the residual concentrations of indole, the biotransformation mixtures were centrifuged at 10,000×g for 10 min. The supernatant was extracted using an equal volume of chloroform, and the samples of chloroform layer were analyzed by HPLC to determine the concentrations of indole. The pellets were resuspended in dimethylsulfoxide (DMSO) with 10 min of ultrasonic treatment, followed by centrifugation at 10,000×g for 10 min to obtain blue supernatant, which was analyzed by HPLC to determine the amount of indigo. HPLC was performed on a Shimadzu LC-20A (Japan) system equipped with a Hypersil ODS2 column (5 μm, 250×4.6 mm). The compounds were eluted using a linear gradient from 60 to 70 % methanol/water (v/v) over 20 min at 1 mL/min and monitored at 280 and 265 nm for indigo and indole, respectively. To detect the metabolites during the biotransformation of indole, the blue DMSO supernatant was also analyzed by liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS) (Agilent 6244, USA) equipped with a standard electrospray ionization (ESI) source in negative ionization mode.
Appl Biochem Biotechnol
Results and Discussion Isolation and Identification of Phenol-Degrading Strains Two phenol-degrading strains, PI1 and PI2, were isolated from the activated sludge of lab bioreactors, which were able to co-metabolize indole to produce indigo in the presence of phenol, but could not use indole as the sole carbon source for growth. To identify the isolates taxonomically, 1444 and 1363 bp fragments of 16S rRNA gene were amplified from strains PI1 and PI2, respectively. Based on a sequence analysis, strain PI1 exhibited 99 % homology to bacterial species Pseudomonas citronellolis BG6903 (GenBank accession number AM088480), while strain PI2 had high identity (99 %) with Acinetobacter sp. N12 (GenBank accession number AB208676). Therefore, strain PI1 was identified as Pseudomonas sp., and strain PI2 belonged to genus Acinetobacter. The 16S rRNA gene sequences of strains PI1 and PI2 were deposited in GenBank database under the accession number KP269079 and KP330203, respectively. Neighbor-joining phylogenetic analysis was performed using the 16S rRNA gene sequences of strains PI1 and PI2 and the close relatives, together with the sequences of many different indigo-producing bacteria (Fig. 1). As reported previously, various indigo-producing bacterial strains of genera Pseudomonas and Acinetobacter have been isolated with different abilities of aromatic hydrocarbon degradation, including naphthalene-degrading strains P. putida PpG7, Pseudomonas sp. J26, and Pseudomonas aeruginosa HOB1; styrenedegrading strains P. putida S12 and P. putida CA-3; p-cumate-degrading strain P. putida F1; m- and p-toluate-degrading strain P. putida mt-2; phenol-degrading strains Pseudomonas monteilii QM, Acinetobacter sp. ST-500, and Acinetobacter sp. PP-2; etc. [7, 15, 18–22]. Among the reported Pseudomonas spp., most of them belonged to P. putida group, such as P. putida and P. monteilii [23], which was famous for its high capability to degrade recalcitrant xenobiotics and to produce a brand range of chemicals [24]. Here, strain PI1 (P. citronellolis) was more likely to belong to P. aeruginosa group, just like P. aeruginosa HOB1 [23]. As for strain PI2, it was another representative of Acinetobacter spp. used for indigo bioproduction.
Mechanisms of Indigoids Production by the Two Phenol-Degrading Strains To identify the products produced from indole by the two phenol-degrading strains, LC/TOF/ MS analysis was performed, and similar results were obtained from both strains (Fig. 2). Four products with the prominent molecular ion (M-H)− peak at m/z 261.067 were detected at the retention times of 17.6 min (I), 19.5 min (II), 20.9 min (III), and 22.5 min (IV), respectively, which had the same molecular formula with indigo, i.e., C16H10N2O2 (Fig. 2a–d). The UV-Vis spectrum of product III was observed with absorption maxima at wavelengths of 241, 286, 340, and 615 nm (Fig. S1c), which was identical with the commercial indigo standard [7, 25]. Therefore, the product III was identified as indigo. For the product IV, the UV-Vis absorption maxima were observed at wavelengths of 266, 315, 390, and 577 nm (Fig. S1d). In our previous study, the recombinant E. coli expressing the phenol hydroxylase from Arthrobacter sp. W1 was used for the biotransformation of indole, and a new purple product, 2-(7-oxo-1Hindol-6(7H)-ylidene) indolin-3-one, was obtained [14], which had the same UV-Vis spectrum and the MS fragmentation patterns with product IV. Thus, the product IV was suggested to be the purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one. The UV-Vis spectra of products I and II were apparently different from those of products III and IV (Fig. S1a and b),
Appl Biochem Biotechnol
Fig. 1 Phylogenetic tree of the newly isolated strains PI1 and PI2. The tree was constructed using neighborjoining method, and the numbers indicated the bootstrap values derived from 1000 replicates. The corresponding GenBank accession numbers of the partial 16S rRNA gene sequences were shown in parenthesis
but we were unable to further identify their chemical structures. However, indirubin, the red isomer of indigo, was not detected, which should have the same prominent molecular ion (M-H)− peak at m/z 261.067 (C16H10N2O2) with the absorption maxima at wavelengths of 289, 363, and 544 nm (data not shown). Besides, product V with a retention time of 10.1 min and a prominent molecular ion (M-H)− peak at m/z 146.0246 (C8H5NO2) was identified as isatin by comparing its mass spectral data with standard chemical and previous studies (Fig. 2e) [26, 27]. Product VI was detected at retention time of 12.7 min with a prominent molecular ion (M-H)− peak at m/z 132.0454 (C8H7NO) (Fig. 2f), which was likely to be 7-hydroxyindole. Previous study showed that the phenol-degrading strain Pseudomonas sp. KL33 could produce 7-hydroxyindole from indole [11]. According to the LC/TOF/MS analysis and previous studies, the possible pathways of indigoid production from indole by the two phenol-degrading strains were proposed as shown in Fig. 3. Phenol hydroxylase might be the functional enzyme in both strains, PI1 and PI2, responsible for the oxidation of indole [11, 14]. Indole could be oxidized by phenol hydroxylase through C-3 oxidation pathway to form 3-hydroxyindole (indoxyl), which was extremely unstable and could be spontaneously dimerized by air oxidation to form indigo [28, 29]. In addition, indoxyl could be further hydroxylated to produce isatin [14, 28]. Besides, isatin could also be obtained through the decomposition of indigo [26, 28]. Previous studies indicated that the C-3 oxidation pathway of indole biotransformation usually occurred in accompany with C2 and C-7 oxidation pathways, leading to the formation of 2-hydroxyindole (2-oxindole) and 7-hydroxyindole, respectively [14, 29, 30]. Condensation of indoxyl and 2-hydroxyindole/ isatin could yield indirubin [14, 26, 28, 29], and 2-hydroxyindole was more preferable for indirubin production [17]. Furthermore, condensation of isatin and 7-hydroxyindole would lead to the formation of the purple product, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one [14]. Therefore, the absence of indirubin and the presence of the purple product in this study indirectly suggested that product VI was 7-hydroxyindole. The products I and II might be generated through some new pathways catalyzed by other oxygenases. In our recent efforts, the genome sequence of strain PI1 was obtained using an Illumina HiSeq 2000 sequencer. The results
Appl Biochem Biotechnol
Fig. 2 Mass spectra of products produced from indole by the phenol-degrading strains. a–d Four indigoids with the same prominent molecular ion (M-H) peak at m/z 261.067, including indigo (c) and 2-(7-oxo-1H-indol6(7H)-ylidene) indolin-3-one (d). e Isatin. f 7-Hydroxyindole
Appl Biochem Biotechnol
Fig. 2 (continued)
indicated that a rich set of candidate protein-coding sequences was annotated for metabolism of aromatic compounds, including biphenyl, benzoate, salicylate, chloroaromatics, etc., among which there should be some functional genes responsible for the production of products I and II.
Appl Biochem Biotechnol
Fig. 3 Proposed pathways for the production of indigoids and other metabolites from indole by the phenoldegrading strains
Optimization of Indigo Production by Response Surface Methodology The time courses of indigo production from indole by strains PI1 and PI2 were examined with 100 mg/L indole, 100 mg/L phenol, and 0.1 % yeast extract (Fig. S2). The results indicated that both indole transformation and indigo production could become stable in 48 h, and 7.28± 0.27 and 10.93±0.29 mg/L indigo were produced by strains PI1 and PI2, respectively. To further optimize the process of indigo production, RSM was applied to investigate the effects of indole (X1), phenol (X2), and yeast extract (X3) on indigo yields. A 23 central composite design with 20 groups of experiments was carried out for each strain, and the results are shown in Table 1. The second-order polynomial equations were fitted Table 2 ANOVA for response surface model of indigo production Source
PI1
PI2
F value
p valuea
F value
p value
Model
7.43
0.0021
19.93
