Although DAAO has been successfully applied to produce of 7-ACA [27, 12], the technology of using RgDAAO to transform d-PPT is still immature, and production by fermentation is always at a disadvantage in industrial applications. Therefore, in this study, the feed fermentation mode was optimized for the recombinant strain expressing DAAO.
Many factors affected fed-batch fermentation, such as dissolved oxygen (DO), temperature, pH, and culture methods. Exploring the effects of various feed fermentation processes on the production of DAAO is necessary to improve both biomass and enzyme activity.
In the optimization process, the final concentration of lactose was 20 g/L. Initially, the feeding mode was kept constant at a rate of 15 mL/h (Fig. 2a); the resulting biomass was 14.6 g DCW/L, and the specific activity was approximately 210 U/g DCW. As shown in Fig. 2b and Fig. 2c, changing the feeding medium flow rate has little effect on the fermentation yield. The comparison of biomass and enzyme activity is shown in Fig. 2d.
When the feeding rate is too slow, the nutrients in the fermentation broth are insufficient, resulting in low bacterial biomass. A fast feeding rate will result in excessive growth of the bacteria, leading to the accumulation of fermentation by-products such as acetic acid, which will affect the growth of the bacteria and the expression of enzymes. Therefore, choosing an appropriate feeding rate will have a positive effect on fermentation. In this work, the optimal feeding rate was determined to be 20 mL/h. Because the constant-speed feeding caused the unfavorable accumulation of glycerin during the late fermentation stage, the feeding mode was changed to variable-speed feeding, which was determined by the concentration of residual glycerin. The concentration of the residual glycerol was controlled at 1 g/L, 3 g/L, and 5 g/L respectively. The results indicated that there was an advantage of maintaining the concentration of the remaining glycerol at 1 g/L through variable-speed feeding in the fermentation broth (Fig. 3) When the concentration of residual glycerol in the fermentation broth was maintained at a low level (1 g/L), the biomass was further increased based on the best constant-speed feeding mode (20 mL/h). This feeding mode is more adapted to the growth rate of the bacteria, which improves the biomass and enzyme activity.
Because dissolved oxygen (DO) in aerobic fermentation will significantly affect cell growth, product synthesis, and normal cell metabolism, fermentation using the DO-STAT feedback feeding mode was used. DO-STAT is a fermentation regulation method that uses the increase in dissolved oxygen caused by the lack of nutrients as a feed signal. Because of the delay of dissolved oxygen monitoring process, the floating range for the DO in the fermenter was set at 10% ± 5%, 30% ± 5%, and 40% ± 5%. Biomass (18.3 g DCW/L) and specific activity (251 U/g DCW) are highest when DO is approximately 30% ± 5% (Table 1). The biomass of the recombinant stain expressing RgDAAO increased 1.25 times as a result of the optimization of the feeding mode. The results demonstrate that the DO-STAT feeding mode is the best for the recombinant strain expressing RgDAAO when DO is controlled at 30% ± 5%.
Table 1
The effect of different DO on the fermentation of the recombinant strain
Dissolved oxygen
|
Biomass (g DCW/L)
|
Specific activity (U/g DCW)
|
10% ± 5%
|
15.2
|
225.9
|
30% ± 5%
|
18.3
|
251.1
|
40% ± 5%
|
13.5
|
233.6
|
The by-product H2O2 in the fermentation process of DAAO
The recombinant strain expressing DAAO usually cannot grow to high biomass even during fed-batch fermentation. In order to determine the mechanism for the inhibitory effect of DAAO on host cell growth, the by-products in the fermentation broth were analyzed. During the fermentation process, H2O2 was observed to accumulate rapidly after induction. Studies have shown that H2O2 is harmful to cell metabolism even at concentrations of a few micromoles [20, 28].
During the process described above for optimizing the feeding mode of the recombinant strain expressing DAAO, the concentration of H2O2 in the fermentation broth was analyzed. In DO-STAT feeding mode, when DO was controlled at 30% ± 5%, 20 g/L lactose was used to induce the expression of DAAO. The concentration of H2O2 produced during the fermentation process is shown in Fig. 4a. H2O2 accumulated rapidly after induction, continued to increase, and accumulated slowly in the late fermentation period. After induction, the growth of the strain gradually entered the stable phase, and DAAO began to be expressed, which lead to the accumulation of H2O2.
Studies have shown that DAAO catalyzes d-Ala, which is an amino acid necessary for the synthesis of E. coli cell walls, and also produces H2O2. In order to prove that d-Ala can act as a substrate of RgDAAO catalysis, 5 g/L of the recombinant strain expressing DAAO was mixed with an initial concentration of 20 mM/L d-Ala and incubated at 30 °C and pH 8.0 in a total volume was 30 mL. After reacting for 5 h, the residual concentration of d-Ala was 50% of the initial concentration, and the accumulation of H2O2 was detected during the reaction as shown in Fig. 4b. This indirectly shows that the rapid accumulation of H2O2 during the fermentation process after induction is a result of the conversion of d-Ala by DAAO. Therefore, removal of H2O2 from the fermentation broth is important for optimal cell growth and enzyme expression.
Fed-batch fermentation expressing DAAO-CAT to relieve H2O2 toxicity
In order to reduce the effects of H2O2 toxicity on cell metabolism, Ju et al. proposed replacing the critical methionine residues in DAAO with leucine [29]. In this work, CAT was added to the fermentation system in order to remove H2O2. A recombinant strain that co-expressed DAAO and CAT was constructed by our laboratory. The purpose of DAAO and CAT co-expression is to remove H2O2 from the fermentation broth during the fermentation process. In this study, fed-batch fermentation for the expression of DAAO-CAT was first studied and optimized. According to the fermentation characteristics of the recombinant strain expressing DAAO, lower glycerol concentrations and proper DO which is at 30% ± 5% are optimal for cell growth and enzyme expression, which has also been confirmed for the fermentation of the recombinant strain expressing DAAO-CAT (Fig. 5a). The biomass of the recombinant strain expressing DAAO-CAT was increased by 1.5 times (21.6 g DCW/L) and the specific activity was increased by 1.4 times (292 U/g DCW) during the fed-batch fermentation during DO-STAT feeding where DO was controlled at 30% ± 5%.
The growth curves of the two recombinant strains are shown in Fig. 5b, and the concentration of H2O2 in the fermentation broth of DAAO and DAAO-CAT are shown in Fig. 5c. The biomass is significantly different after induction. Compared with the fermentation time for DAAO to reach the maximum biomass, the fermentation time for DAAO-CAT to reach the same biomass only takes 17 h, which was shortened by approximately 4 h. When DAAO and CAT were co-expressed, cell growth was significantly accelerated, and after induction, the biomass of the DAAO-CAT strain was higher than that of the DAAO recombinant strain. Although CAT was co-expressed, part of the H2O2 in the fermentation broth was removed, reducing the accumulation and allowing the strain to be more resistant to H2O2, which is reflected in an accelerated cell growth. However, the reduction of H2O2 also promotes the forward reaction rate of DAAO, which produces additional H2O2. Studies indicated that catalase forms an intermediate compound I (Cpd I) when decomposing H2O2. When the H2O2 concentration is low, Cpd I may react with external one-electron/hydrogen donors resulting in the formation of Compound II (Cpd II, an oxoferryl derivative without radical site [porFeIV = O]) [30]. In contrast to Cpd II of heme peroxidases, Cpd II of catalase is not effectively reduced to the native enzyme; therefore, Cpd II accumulation leads to catalase inactivation [31]. Co-expression of CAT can only remove part of the growth restriction during the fermentation process and cannot fundamentally prevent the inhibitory effect of DAAO on the growth of host cells during the fermentation process.
Based on research on H2O2 in the fermentation process, the expression of DAAO will lead to the production of H2O2, so it is necessary to balance the specific activity and the biomass. Because the high concentration of lactose will make the fermentation broth sticky and not conducive to cell growth, the lactose was added in two doses if the total concentration was greater than 20 g/L. As shown in Fig. 5d, the specific activity decreased with lactose concentration. In contrast, the biomass increased with decreasing lactose concentrations, especially for the 12.5 g/L and 15 g/L doses, which indirectly indicates that the expression of DAAO inhibited the growth of host cells. Based on the results from this study, the optimal lactose concentration is 27.5 g/L, resulting in a biomass of 20.2 g DCW/L and a specific activity of 395 U/g DCW, which was 1.85 times higher than the specific activity before optimization.
d-Ala relieves the inhibitory effect of DAAO
Based on the fed-batch fermentation of the recombinant strain expressing DAAO-CAT, the growth curve (Fig. 5b) indicates that cell growth is still inhibited during the late fermentation stage (biomass only reaching 21.6 g DCW/L), although reaching the same fermentation level compared with that before shortening the fermentation time. During the decomposition of d-Ala by DAAO, H2O2 was produced as a by-product while the synthesis of the E. coli cell wall was weakened. In this work, based on the previously optimized fermentation conditions, the influence of d-Ala on fermentation was explored.
Study showed that d-Ala can be transported into cells [32]. Because the expression of DAAO inhibits the growth of host cells, and is generated after induction with lactose, d-Ala was added under the following culture conditions: fed-batch fermentation with DO-STAT feeding mode (DO was controlled at 30% ± 5%), cells were cultured at 37°C until the OD600 reached approximately 20, and a total of 27.5 g/L lactose mixed with d-Ala was added in two additions to induce the cells at 28°C. Because the addition of d-Ala promotes the forward conversion reaction of DAAO, excess d-Ala is detrimental to biomass production and enzyme activity. The optimal concentration of d-Ala resulted in a large increase in biomass, reaching 26.03 g DCW/L, and the specific activity was minimally negatively affected, maintaining 390 U/g DCW (Fig. 6a). d-Ala relieves the inhibition of DAAO on the growth of host cells, and results in a similar biomass to that resulting from induction with a low concentration of lactose.
In this work, the co-expression of CAT was used to remove H2O2, the concentration of lactose was optimized to adjust the relationship between biomass and specific activity, and lactose mixed with d-Ala was added for induction, which relieved the inhibitory effect of DAAO on host cell growth during fed-batch fermentation (Fig. 6b). Compared with previous studies, the biomass of the recombinant strain co-expressing DAAO and CAT in fed-batch fermentation under the optimal fermentation conditions in this study reached a high level. Table 2 summarizes the results of some of the higher biomass production obtained in the previous literature in comparation with the results obtained in this study. Although the biomass obtained in some studies is similar, the fermentation time in this study is the shortest, which is a great advantage in industrial applications.
Table 2
Summary of biomass of the fermentation of DAAO from previous literature and this study
DAAO
|
Fermentation
Volume (L)
|
OD600
|
Biomass
|
Fermentation
Time (h)
|
References
|
Rg
|
2.0
|
80.3
|
26.03 g DCW/L
|
24.0
|
This study
|
Rt
|
2.0
|
/
|
48.50 g /L
|
24.0
|
[33]
|
Tv
|
2.0
|
55.0
|
/
|
28.5
|
[34]
|
Tv
|
3.5
|
/
|
24.00 g DCW/L
|
26.0
|
[18]
|
Tv
|
15
|
80.0
|
/
|
48.0
|
[17]
|
Tv
|
15
|
79.0
|
/
|
38.0
|
[35]
|
Scale-up cultivation
To adapt the production of DAAO by fermentation to industrial applications, scale-up cultivation was performed in a 50 L fermenter containing 30 L medium and a 5000 L fermenter containing 3000 L medium based on the optimal condition in 5 L fermenter. Fed-batch fermentation in the 50 L fermenter containing 30 L fermenter medium resulted in a DAAO specific activity of 353.1 U/g DCW and a biomass production of 19.68 g DCW/L. Furthermore, a DAAO specific activity of 341.7 U/g DCW and a biomass production of 18.43 g DCW/L was obtained in the fed-batch 5000 L fermentation, (fermentation process is shown in Fig. 7). These fermentation results suggested that the process of scale-up did not have a major impact on the specific activity of DAAO, but the biomass has been reduced by the difference in parameter control and the change in the composition of the feeding medium, the inorganic salt and nitrogen source in the compound feeding medium contribute to the increase of cell density.