Appl Biochem Biotechnol DOI 10.1007/s12010-014-0743-3
Production of Indirubin from Tryptophan by Recombinant Escherichia coli Containing Naphthalene Dioxygenase Genes from Comamonas sp. MQ Xuwang Zhang & Yuanyuan Qu & Qiao Ma & Chunlei Kong & Hao Zhou & Xiangyu Cao & Wenli Shen & E Shen & Jiti Zhou
Received: 25 September 2013 / Accepted: 16 January 2014 # Springer Science+Business Media New York 2014
Abstract Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 30 °C when cells were grown to OD600 ≈1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37±1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98±2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production. Keywords Indirubin . Tryptophan . 2-oxindole . Naphthalene dioxygenase . Response surface methodology
Introduction Indirubin, a red isomer of indigo, is deemed to be an undesirable by-product during the biological and chemical production of indigo in a dyeing industry, which is found to be the active ingredient of Danggui Longhui Wan, a traditional Chinese medicine for the treatment of
X. Zhang : Y. Qu (*) : Q. Ma : C. Kong : H. Zhou : X. Cao : W. Shen : E. Shen : J. Zhou 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 e-mail: [email protected]
Appl Biochem Biotechnol
chronic myelocytic leukemia [1, 2]. Moreover, indirubin possesses the ability to inhibit cyclindependent kinases and glycogen synthase kinase-3β, thereby exhibiting a definite efficiency against various diseases including Alzheimer’s disease, diabetes, etc. [2–5]. Therefore, the production of indirubin will be of wide interest owing to the significant implications for pharmaceutical industries [6–8]. Since the 1980s, many recombinant Escherichia coli strains expressing mono- or dioxygenases have been constructed to produce indigo and indirubin from tryptophan or indole [8–13]. Generally, tryptophan is assumed to be oxidized to indole by tryptophanase, a natural enzyme in E. coli, and then indole can be converted to 3-hydroxyindole (indoxyl), isatin, and/or 2-oxindole by heterologous oxygenases, e.g., naphthalene dioxygenase (NDO) (Fig. 1) [9, 13, 14]. Two molecules of indoxyl are spontaneously dimerized in the presence of oxygen to form indigo, whereas indoxyl and 2-oxindole/isatin are condensed to generate indirubin (Fig. 1) [6, 9, 15, 16]. However, most of the previous studies were mainly focused on indigo production because of the low yield and inefficiency of indirubin production. To improve indirubin production, many attempts have been carried out. The wild-type toluene ortho-monooxygenase of Burkholderia cepacia G4 produced isoindigo as the major product from indole, but almost no indirubin was detected [13]. By random and saturation mutagenesis, the mutants A113S, A113F, and A113I obtained the capability to produce indirubin as the major product [13]. The nonrecombinant E. coli is found to be capable of producing indirubin from indican, but the indirubin yield is unsatisfactory [7]. During the process of indirubin production, isatin and 2-oxindole are found to be the essential precursors (Fig. 1) [6, 7, 16]. While it has been reported that the addition of isatin could promote the production of indirubin [17, 18], less has been done to examine the effects of 2-oxindole on indirubin production. As previously reported, various environmental factors, including induction timing, induction temperature, IPTG concentration, medium composition, etc., may play the important roles
Fig. 1 Proposed pathways for the production of indigo and indirubin from tryptophan by recombinant E. coli [6, 9, 13, 16]
Appl Biochem Biotechnol
in the biotechnological production processes of recombinant E. coli [19–22]. Therefore, it is necessary to investigate the optimal parameters in order to improve the production of indirubin. In our previous work, the naphthalene-degrading strain Comamonas sp. MQ was isolated from activated sludge, and the recombinant E. coli harboring the NDO genes from strain MQ was also constructed [18, 23]. Both the wild strain MQ and the recombinant E. coli possessed a good ability to producing indigo from indole, but almost no indirubin was produced. Herein, the recombinant E. coli was utilized for the production of indirubin from tryptophan, and the culture conditions were investigated, including induction conditions and medium composition. The effects of 2-oxindole and isatin on indirubin production were also determined in detail.
Materials and Methods Chemicals The synthetic indirubin used as standard was purchased from the National Institutes for Food and Drug Control (China). L-Tryptophan, isatin, 2-oxindole, kanamycin, and isopropyl-β-Dthiogalactopyranoside (IPTG) were purchased from J&K Scientific Ltd. (China). All the other chemicals and solvents used in this study were of analytical grade or above. Bacterial Strain and Culture Media The indigo-producing strain Comamonas sp. MQ was previously isolated in our laboratory [18]. A 5,145-bp fragment of nag gene (GenBank accession number JN655512) coding for NDO was amplified from strain MQ and successfully expressed in E. coli BL21 (DE3) (named ND_IND). The recombinant strain ND_IND and the plasmids used in this study have previously been described [23]. The seed medium for the recombinant strain ND_IND was Luria–Bertani (LB) broth (pH 7.0) containing peptone (10 g/l), yeast extract (5 g/l), and NaCl (10 g/l). Tryptophan medium (pH 7.0) was used for the production of indirubin, which consisted of tryptophan (2 g/l), yeast extract (5 g/l), and NaCl (10 g/l) [24]. To maintain selection pressure, kanamycin (30 μg/ml) was added to the media at the beginning of cultivation. Indirubin Production Assays For inoculum, a single colony of strain ND_IND from a freshly prepared LB agar plate was inoculated into a 25-ml LB broth and cultivated at 30 °C overnight. The seed cultures were inoculated (2 %, v/v) into a series of 100-ml flasks containing 30-ml tryptophan media, which were then incubated with continuous shaking at 37 °C. For NDO expression, IPTG was added to the culture broth when the cells were grown to the desired optical density at 600 nm (OD600) and then the cells were induced at an appropriate temperature for 20 h. To improve the production of indirubin, the induction conditions for NDO expression were optimized over a range of induction timing (OD600 0.2–1.4), induction temperature (15–37 °C), and IPTG concentration (0–1.25 mM). Further enhancement of indirubin production was investigated by adding two putative intermediates, 2-oxindole and isatin, to the cultures after induction using acetone as cosolvent. The effects of addition time and concentrations of 2-oxindole and isatin on indirubin production were determined
Appl Biochem Biotechnol
individually. All experiments were performed in triplicate, and the average values were used for calculation. Effects of Medium Composition on Indirubin Production Response surface methodology (RSM) was applied to analyze the effects of medium composition on indirubin production, and a three-factor, five-level central composite design was adopted. The concentrations of tryptophan (X1), yeast extract (X2), and NaCl (X3) were selected as the independent variables. Twenty groups of independent experiments were designed, including six replicates of central points for error estimation (Table 1). In developing the regression equation, the test variables were coded using the following equation: xi ¼
X i −X 0 ΔX
ð1Þ
where xi was the coded value of the independent variable, Xi was the real value of the independent variable, X0 was the real value of the independent variable at the central point, and ΔX was the step change of Xi [25–28]. The software, Design Expert 8.0.6 (Stat Ease, Minneapolis, USA), was used to set up and analyze the RSM design matrix.
Table 1 Experimental design and the results of RSM for indirubin production No.
Tryptophan (X1) (g/l)
Yeast extract (X2) (g/l)
NaCl (X3) (g/l)
Indirubin (mg/l) Experimental
Predicted
1
2.00 (−1.0)
3.00 (−1.0)
5.00 (−1.0)
5.26
3.60
2
4.00 (1.0)
3.00 (−1.0)
5.00 (−1.0)
1.49
1.96
3
2.00 (−1.0)
7.00 (1.0)
5.00 (−1.0)
1.53
0.98
4
4.00 (1.0)
7.00 (1.0)
5.00 (−1.0)
5.85
4.78
5
2.00 (−1.0)
3.00 (−1.0)
15.00 (1.0)
0.82
1.58
6
4.00 (1.0)
3.00 (−1.0)
15.00 (1.0)
2.77
2.98
7 8
2.00 (−1.0) 4.00 (1.0)
7.00 (1.0) 7.00 (1.0)
15.00 (1.0) 15.00 (1.0)
3.92 8.64
3.12 9.96
9
1.32 (−1.68)
5.00 (0)
10.00 (0)
0.65
1.87
10
4.68 (1.68)
5.00 (0)
10.00 (0)
6.95
6.24
11
3.00 (0)
1.64 (−1.68)
10.00 (0)
0.00
–
12
3.00 (0)
8.36 (1.68)
10.00 (0)
13
3.00 (0)
5.00 (0)
14
3.00 (0)
5.00 (0)
15 16
3.00 (0) 3.00 (0)
5.00 (0) 5.00 (0)
17
3.00 (0)
18
3.00 (0)
19 20
3.12
3.63
3.11
4.62
18.41 (1.68)
8.29
7.27
10.00 (0) 10.00 (0)
8.76 9.08
8.85 8.85
5.00 (0)
10.00 (0)
8.73
8.85
5.00 (0)
10.00 (0)
8.62
8.85
3.00 (0)
5.00 (0)
10.00 (0)
9.17
8.85
3.00 (0)
5.00 (0)
10.00 (0)
8.82
8.85
1.59 (−1.68)
The coded values of the independent variables were shown in the parenthesis
Appl Biochem Biotechnol
Analytical Methods To quantify the yields of indirubin, the culture broth was centrifuged at 11,000×g for 10 min, and the pellets were resuspended in an equal volume of dimethylsulfoxide (DMSO) followed by ultrasonic treatment for 10 min to ensure dissolution of cell-associated indirubin. The mixture was centrifuged, and the supernatant was analyzed by high performance liquid chromatography (HPLC) equipped with a Hypersil ODS2 column (5 μm, 250×4.6 mm). Samples were eluted using a linear gradient from 60/40 to 70/30 (v/v) (methanol/water) over 20 min at 1 ml/min and monitored at 289 nm for indirubin.
Results and Discussion Optimization of Induction Conditions for NDO Expression Various conditions could greatly affect the production of indirubin by recombinant E. coli, such as the induction conditions for NDO expression and medium composition. Generally, indole was supposed to be produced accompanying with the expression of tryptophanase during the metabolism of the host cells. But no indirubin was produced until IPTG was added to the media, when the recombinant strain started expressing NDO. Therefore, the optimal induction conditions for NDO expression, including induction timing, IPTG concentration, and induction temperature, were investigated. As shown in Fig. 2a, the indirubin yields increased when OD600 for induction was increased from 0.20 to 1.27. However, the indirubin yields decreased when IPTG was added at higher OD600 levels. No detectable indirubin was produced in the absence of IPTG (Fig. 2b), indicating that the host E. coli cells were unable to produce indirubin. Preliminary experiments also demonstrated that the host E. coli without the nag gene could not produce indirubin from tryptophan (data not shown). The highest yields of indirubin were obtained when induced with 0.5 mM IPTG. High concentrations (above 0.75 mM) of IPTG were unfavorable for indirubin production. In previous studies, it was found that a high concentration of IPTG would inhibit the production of recombinant protein, and low IPTG concentrations (0.2–0.5 mM) would be more efficient for protein expression [19, 22]. Temperature could affect the expression of the heterologous enzyme in recombinant E. coli, thus posing a great impact on indirubin production. The effects of induction temperature on indirubin production were investigated at 15, 20, 25, 30, and 37 °C (Fig. 2c). The yields of indirubin increased versus the induction temperature and reached a maximum at 30 °C. However, when induced at a higher temperature (37 °C), the indirubin yield decreased dramatically. The results indicated that the production of indirubin was improved when the recombinant strain ND_IND was induced at the proper phase of growth (OD600 ≈1.20) with an appropriate concentration of IPTG (0.5 mM) at a suitable temperature (30 °C). Effects of Medium Composition on Indirubin Production As the tryptophan medium consisted of tryptophan, yeast extract, and NaCl, each medium composition could perform particular functions in indirubin production. To improve the production of indirubin, medium composition was further adjusted by RSM using a 23 central composite design. RSM is an effective statistical tool for optimization design, and it has been widely used to optimize the process parameters in biotechnological areas [21, 25–28]. In this
Appl Biochem Biotechnol Fig. 2 Effects of induction timing (a), IPTG concentration (b), and induction temperature (c) on indirubin production. The concentrations of indirubin were measured by HPLC
Appl Biochem Biotechnol
study, 20 groups of experiments were carried out according to the RSM design matrix, in which the indirubin yields were measured as the responses (Table 1). The quadratic polynomial equation was fitted using the coded units to correlate the independent variables with indirubin production, which was as follows: Y Indirubin ¼ 8:85 þ 1:30X 1 þ 1:09X 2 þ 0:79X 3 þ 1:36X 1 X 2 þ 0:76X 1 X 3 þ 1:04X 2 X 3 −1:70X 21 −2:50X 22 −1:03X 32 ð2Þ where Y is indirubin yield and X1, X2, and X3 are the coded values of the test variables, i.e., concentrations of tryptophan, yeast extract, and NaCl, respectively. The predicted values of the indirubin yields were also listed in Table 1, which were calculated based on the regression equation. The coefficient of determination (R2) was found to be 0.9338, indicating that 93.38 % of the total variation for indirubin yield was attributed to the test variables. Meanwhile, the adjusted R2 was 0.8838, suggesting an acceptable agreement between the experimental and predicted values of indirubin yields. Additionally, according to the ANOVA, the F statistic of the model was 17.05, corresponding to a p value less than 0.0001 (Table 2), indicating that the model was significant. X1, X2, X3, X1X2, X2X3, X12, X22, and X32 were significant model terms due to their p values that were all less than 0.05 (Table 2). The response surface plots were shown in Fig. 3, indicating the interactive effects between two independent variables on indirubin production. Tryptophan (factor X1) was a critical factor for indirubin production (p value
Production of Indirubin from Tryptophan by Recombinant Escherichia coli Containing Naphthalene Dioxygenase Genes from Comamonas sp. MQ Xuwang Zhang & Yuanyuan Qu & Qiao Ma & Chunlei Kong & Hao Zhou & Xiangyu Cao & Wenli Shen & E Shen & Jiti Zhou
Received: 25 September 2013 / Accepted: 16 January 2014 # Springer Science+Business Media New York 2014
Abstract Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 30 °C when cells were grown to OD600 ≈1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37±1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98±2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production. Keywords Indirubin . Tryptophan . 2-oxindole . Naphthalene dioxygenase . Response surface methodology
Introduction Indirubin, a red isomer of indigo, is deemed to be an undesirable by-product during the biological and chemical production of indigo in a dyeing industry, which is found to be the active ingredient of Danggui Longhui Wan, a traditional Chinese medicine for the treatment of
X. Zhang : Y. Qu (*) : Q. Ma : C. Kong : H. Zhou : X. Cao : W. Shen : E. Shen : J. Zhou 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 e-mail: [email protected]
Appl Biochem Biotechnol
chronic myelocytic leukemia [1, 2]. Moreover, indirubin possesses the ability to inhibit cyclindependent kinases and glycogen synthase kinase-3β, thereby exhibiting a definite efficiency against various diseases including Alzheimer’s disease, diabetes, etc. [2–5]. Therefore, the production of indirubin will be of wide interest owing to the significant implications for pharmaceutical industries [6–8]. Since the 1980s, many recombinant Escherichia coli strains expressing mono- or dioxygenases have been constructed to produce indigo and indirubin from tryptophan or indole [8–13]. Generally, tryptophan is assumed to be oxidized to indole by tryptophanase, a natural enzyme in E. coli, and then indole can be converted to 3-hydroxyindole (indoxyl), isatin, and/or 2-oxindole by heterologous oxygenases, e.g., naphthalene dioxygenase (NDO) (Fig. 1) [9, 13, 14]. Two molecules of indoxyl are spontaneously dimerized in the presence of oxygen to form indigo, whereas indoxyl and 2-oxindole/isatin are condensed to generate indirubin (Fig. 1) [6, 9, 15, 16]. However, most of the previous studies were mainly focused on indigo production because of the low yield and inefficiency of indirubin production. To improve indirubin production, many attempts have been carried out. The wild-type toluene ortho-monooxygenase of Burkholderia cepacia G4 produced isoindigo as the major product from indole, but almost no indirubin was detected [13]. By random and saturation mutagenesis, the mutants A113S, A113F, and A113I obtained the capability to produce indirubin as the major product [13]. The nonrecombinant E. coli is found to be capable of producing indirubin from indican, but the indirubin yield is unsatisfactory [7]. During the process of indirubin production, isatin and 2-oxindole are found to be the essential precursors (Fig. 1) [6, 7, 16]. While it has been reported that the addition of isatin could promote the production of indirubin [17, 18], less has been done to examine the effects of 2-oxindole on indirubin production. As previously reported, various environmental factors, including induction timing, induction temperature, IPTG concentration, medium composition, etc., may play the important roles
Fig. 1 Proposed pathways for the production of indigo and indirubin from tryptophan by recombinant E. coli [6, 9, 13, 16]
Appl Biochem Biotechnol
in the biotechnological production processes of recombinant E. coli [19–22]. Therefore, it is necessary to investigate the optimal parameters in order to improve the production of indirubin. In our previous work, the naphthalene-degrading strain Comamonas sp. MQ was isolated from activated sludge, and the recombinant E. coli harboring the NDO genes from strain MQ was also constructed [18, 23]. Both the wild strain MQ and the recombinant E. coli possessed a good ability to producing indigo from indole, but almost no indirubin was produced. Herein, the recombinant E. coli was utilized for the production of indirubin from tryptophan, and the culture conditions were investigated, including induction conditions and medium composition. The effects of 2-oxindole and isatin on indirubin production were also determined in detail.
Materials and Methods Chemicals The synthetic indirubin used as standard was purchased from the National Institutes for Food and Drug Control (China). L-Tryptophan, isatin, 2-oxindole, kanamycin, and isopropyl-β-Dthiogalactopyranoside (IPTG) were purchased from J&K Scientific Ltd. (China). All the other chemicals and solvents used in this study were of analytical grade or above. Bacterial Strain and Culture Media The indigo-producing strain Comamonas sp. MQ was previously isolated in our laboratory [18]. A 5,145-bp fragment of nag gene (GenBank accession number JN655512) coding for NDO was amplified from strain MQ and successfully expressed in E. coli BL21 (DE3) (named ND_IND). The recombinant strain ND_IND and the plasmids used in this study have previously been described [23]. The seed medium for the recombinant strain ND_IND was Luria–Bertani (LB) broth (pH 7.0) containing peptone (10 g/l), yeast extract (5 g/l), and NaCl (10 g/l). Tryptophan medium (pH 7.0) was used for the production of indirubin, which consisted of tryptophan (2 g/l), yeast extract (5 g/l), and NaCl (10 g/l) [24]. To maintain selection pressure, kanamycin (30 μg/ml) was added to the media at the beginning of cultivation. Indirubin Production Assays For inoculum, a single colony of strain ND_IND from a freshly prepared LB agar plate was inoculated into a 25-ml LB broth and cultivated at 30 °C overnight. The seed cultures were inoculated (2 %, v/v) into a series of 100-ml flasks containing 30-ml tryptophan media, which were then incubated with continuous shaking at 37 °C. For NDO expression, IPTG was added to the culture broth when the cells were grown to the desired optical density at 600 nm (OD600) and then the cells were induced at an appropriate temperature for 20 h. To improve the production of indirubin, the induction conditions for NDO expression were optimized over a range of induction timing (OD600 0.2–1.4), induction temperature (15–37 °C), and IPTG concentration (0–1.25 mM). Further enhancement of indirubin production was investigated by adding two putative intermediates, 2-oxindole and isatin, to the cultures after induction using acetone as cosolvent. The effects of addition time and concentrations of 2-oxindole and isatin on indirubin production were determined
Appl Biochem Biotechnol
individually. All experiments were performed in triplicate, and the average values were used for calculation. Effects of Medium Composition on Indirubin Production Response surface methodology (RSM) was applied to analyze the effects of medium composition on indirubin production, and a three-factor, five-level central composite design was adopted. The concentrations of tryptophan (X1), yeast extract (X2), and NaCl (X3) were selected as the independent variables. Twenty groups of independent experiments were designed, including six replicates of central points for error estimation (Table 1). In developing the regression equation, the test variables were coded using the following equation: xi ¼
X i −X 0 ΔX
ð1Þ
where xi was the coded value of the independent variable, Xi was the real value of the independent variable, X0 was the real value of the independent variable at the central point, and ΔX was the step change of Xi [25–28]. The software, Design Expert 8.0.6 (Stat Ease, Minneapolis, USA), was used to set up and analyze the RSM design matrix.
Table 1 Experimental design and the results of RSM for indirubin production No.
Tryptophan (X1) (g/l)
Yeast extract (X2) (g/l)
NaCl (X3) (g/l)
Indirubin (mg/l) Experimental
Predicted
1
2.00 (−1.0)
3.00 (−1.0)
5.00 (−1.0)
5.26
3.60
2
4.00 (1.0)
3.00 (−1.0)
5.00 (−1.0)
1.49
1.96
3
2.00 (−1.0)
7.00 (1.0)
5.00 (−1.0)
1.53
0.98
4
4.00 (1.0)
7.00 (1.0)
5.00 (−1.0)
5.85
4.78
5
2.00 (−1.0)
3.00 (−1.0)
15.00 (1.0)
0.82
1.58
6
4.00 (1.0)
3.00 (−1.0)
15.00 (1.0)
2.77
2.98
7 8
2.00 (−1.0) 4.00 (1.0)
7.00 (1.0) 7.00 (1.0)
15.00 (1.0) 15.00 (1.0)
3.92 8.64
3.12 9.96
9
1.32 (−1.68)
5.00 (0)
10.00 (0)
0.65
1.87
10
4.68 (1.68)
5.00 (0)
10.00 (0)
6.95
6.24
11
3.00 (0)
1.64 (−1.68)
10.00 (0)
0.00
–
12
3.00 (0)
8.36 (1.68)
10.00 (0)
13
3.00 (0)
5.00 (0)
14
3.00 (0)
5.00 (0)
15 16
3.00 (0) 3.00 (0)
5.00 (0) 5.00 (0)
17
3.00 (0)
18
3.00 (0)
19 20
3.12
3.63
3.11
4.62
18.41 (1.68)
8.29
7.27
10.00 (0) 10.00 (0)
8.76 9.08
8.85 8.85
5.00 (0)
10.00 (0)
8.73
8.85
5.00 (0)
10.00 (0)
8.62
8.85
3.00 (0)
5.00 (0)
10.00 (0)
9.17
8.85
3.00 (0)
5.00 (0)
10.00 (0)
8.82
8.85
1.59 (−1.68)
The coded values of the independent variables were shown in the parenthesis
Appl Biochem Biotechnol
Analytical Methods To quantify the yields of indirubin, the culture broth was centrifuged at 11,000×g for 10 min, and the pellets were resuspended in an equal volume of dimethylsulfoxide (DMSO) followed by ultrasonic treatment for 10 min to ensure dissolution of cell-associated indirubin. The mixture was centrifuged, and the supernatant was analyzed by high performance liquid chromatography (HPLC) equipped with a Hypersil ODS2 column (5 μm, 250×4.6 mm). Samples were eluted using a linear gradient from 60/40 to 70/30 (v/v) (methanol/water) over 20 min at 1 ml/min and monitored at 289 nm for indirubin.
Results and Discussion Optimization of Induction Conditions for NDO Expression Various conditions could greatly affect the production of indirubin by recombinant E. coli, such as the induction conditions for NDO expression and medium composition. Generally, indole was supposed to be produced accompanying with the expression of tryptophanase during the metabolism of the host cells. But no indirubin was produced until IPTG was added to the media, when the recombinant strain started expressing NDO. Therefore, the optimal induction conditions for NDO expression, including induction timing, IPTG concentration, and induction temperature, were investigated. As shown in Fig. 2a, the indirubin yields increased when OD600 for induction was increased from 0.20 to 1.27. However, the indirubin yields decreased when IPTG was added at higher OD600 levels. No detectable indirubin was produced in the absence of IPTG (Fig. 2b), indicating that the host E. coli cells were unable to produce indirubin. Preliminary experiments also demonstrated that the host E. coli without the nag gene could not produce indirubin from tryptophan (data not shown). The highest yields of indirubin were obtained when induced with 0.5 mM IPTG. High concentrations (above 0.75 mM) of IPTG were unfavorable for indirubin production. In previous studies, it was found that a high concentration of IPTG would inhibit the production of recombinant protein, and low IPTG concentrations (0.2–0.5 mM) would be more efficient for protein expression [19, 22]. Temperature could affect the expression of the heterologous enzyme in recombinant E. coli, thus posing a great impact on indirubin production. The effects of induction temperature on indirubin production were investigated at 15, 20, 25, 30, and 37 °C (Fig. 2c). The yields of indirubin increased versus the induction temperature and reached a maximum at 30 °C. However, when induced at a higher temperature (37 °C), the indirubin yield decreased dramatically. The results indicated that the production of indirubin was improved when the recombinant strain ND_IND was induced at the proper phase of growth (OD600 ≈1.20) with an appropriate concentration of IPTG (0.5 mM) at a suitable temperature (30 °C). Effects of Medium Composition on Indirubin Production As the tryptophan medium consisted of tryptophan, yeast extract, and NaCl, each medium composition could perform particular functions in indirubin production. To improve the production of indirubin, medium composition was further adjusted by RSM using a 23 central composite design. RSM is an effective statistical tool for optimization design, and it has been widely used to optimize the process parameters in biotechnological areas [21, 25–28]. In this
Appl Biochem Biotechnol Fig. 2 Effects of induction timing (a), IPTG concentration (b), and induction temperature (c) on indirubin production. The concentrations of indirubin were measured by HPLC
Appl Biochem Biotechnol
study, 20 groups of experiments were carried out according to the RSM design matrix, in which the indirubin yields were measured as the responses (Table 1). The quadratic polynomial equation was fitted using the coded units to correlate the independent variables with indirubin production, which was as follows: Y Indirubin ¼ 8:85 þ 1:30X 1 þ 1:09X 2 þ 0:79X 3 þ 1:36X 1 X 2 þ 0:76X 1 X 3 þ 1:04X 2 X 3 −1:70X 21 −2:50X 22 −1:03X 32 ð2Þ where Y is indirubin yield and X1, X2, and X3 are the coded values of the test variables, i.e., concentrations of tryptophan, yeast extract, and NaCl, respectively. The predicted values of the indirubin yields were also listed in Table 1, which were calculated based on the regression equation. The coefficient of determination (R2) was found to be 0.9338, indicating that 93.38 % of the total variation for indirubin yield was attributed to the test variables. Meanwhile, the adjusted R2 was 0.8838, suggesting an acceptable agreement between the experimental and predicted values of indirubin yields. Additionally, according to the ANOVA, the F statistic of the model was 17.05, corresponding to a p value less than 0.0001 (Table 2), indicating that the model was significant. X1, X2, X3, X1X2, X2X3, X12, X22, and X32 were significant model terms due to their p values that were all less than 0.05 (Table 2). The response surface plots were shown in Fig. 3, indicating the interactive effects between two independent variables on indirubin production. Tryptophan (factor X1) was a critical factor for indirubin production (p value
