Citrate enhanced cAMP fermentation performance in Arthrobacter sp. CCTCC 2013431
Citrate can be utilized as auxiliary energy substrate to improve intracellular ATP level and direct more carbon flux into pentose phosphate pathway leading to the increase of nucleotides production (Chen et al. 2010a; ). In our previous study, 3 g/L-broth sodium citrate added at 24 h was determined as optimal manipulating condition for cAMP fermentatiom conducted in shake flasks (Li et al. 2018). To investigate the metabolic mechanism, batch fermentations with the optimal condition were conducted in a 7 L stirred tank bioreactor. As presented in Fig. 1, due to the addition of citrate, final cAMP concentration and conversion yield from glucose reached 4.34 g/L and 0.076 g/g which were improved by 30.7% and 29.8%, respectively, when compared with those of control (without citrate addition). It was 24 h after which cAMP contents as well as synthesis rates were enhanced obviously and maintained at higher levels than those of control suggesting that citrate addition did accelerate cAMP production. The final OD600 and glucose consumption amounts in fermentation with citrate added were almost equal to those of control, however, OD600 in control fermentation achieved its peak value at 43 h and then decreased gradually suggesting that cell viability and metabolic intensity had started to decline during the later period, while in citrate added fermentation, cell concentrations kept increasing during the whole period (Fig. 1a). The data of cell viability and CO2 assay were in support of this deduction directly. The fluorescence intensity of cell suspension after suitable dilution and CTC treatment was detected as an indicator of cell viability (respiration activity), and CO2 content in discharge gas could represent cell aerobic respiration level. As shown in Fig. 1cd, after 24 h, cell viability and CO2 contents were improved significantly by citrate and maintained at higher levels than those of control, however, cell viability and CO2 contents were almost equal under the two conditions during 0–24 h, suggesting that aerobic respiration and energy metabolism level was enhanced obviously after 24 h in citrate added fermentation.
In addition, after 24 h, hypoxanthine concentrations were maintained at lower levels about 0.2 g/L in both fermentations suggesting that cAMP was not synthesized by salvage pathway but via purine nucleotide de novo pathway. In this case, sufficient supplies of phosphoribosyl pyrophosphate (PRPP) and precursor amino acids were required for high-level cAMP biosynthesis. PRPP formation related to carbon flux distribution between glycolysis and pentose phosphate pathway whereas precursor amino acids mainly included aspartate as well as glutamate whose carbon skeletons derived from tricarboxylate cycle. Moreover, cAMP is synthesized from ATP directly catalyzed by adenylate cyclase and abundant ATP supply is beneficial for cATP synthesis. Hence we attempt to reveal the regulation mechanism of citrate on cAMP biosynthesis via investigating the change of key enzymes activities, energy metabolism levels and amino acids synthesis.
Citrate enhanced intracellular NADH and ATP level during fermentation using Arthrobacter sp. CCTCC 2013431
In living cell, cAMP is synthesized from ATP directly catalyzed by adenylate cyclase and abundant ATP supply is beneficial for cAMP synthesis. In aerobic fermentation process, ATP is mainly formed via NADH oxidation reaction coupled with electron transport chain, namely oxidative phosphorylation reaction. As an auxiliary energy substance, citrate could strengthen energy metabolism and enhance ATP regeneration for lactic acid (Kang et al. 2016; Comasio et al. 2019), pyruvate (Zhou et al.2010) and nucleotide production (Chen et al.2010a) efficiently. Therefore, we hypothesized that citrate might increase intracellular ATP level, further promoting cAMP synthesis. To confirm the assumption, NADH and ATP levels in fermentations with/without citrate addition were determined and the results were shown in Fig. 2.
As shown in Fig. 2a, owing to citrate added, intracellular NADH/NAD+ ratios were improved obviously and maintained at higher levels when compared with those of control. The production of cAMP by Arthrobacter sp. CCTCC 2013431 was an aerobic fermentation process and NADH regeneration was mainly achieved by strengthened NAD+-linked dehydrogenase reactions presence in tricarboxylate cycle. In the case, citrate enhanced tricarboxylate cycle metabolic intensity and NADH regeneration, further improved NADH/NAD+ ratios. Moreover, NADH can be reoxidized to NAD+ by electron transport chain which accompanied by plenty of ATP synthesis. High NADH/NAD+ ratios suggested that energy metabolism was strengthened by citrate and ATP might be generated more efficiently.
ATP assay results also verified our deduction. Intracellular ATP/AMP ratios were enhanced obviously as result of the addition of citrate when compared with those of control (Fig. 2b), which in agreement with NADH/NAD+ ratios assay results. What deserve to be mentioned is that ATP and AMP contents in citrate added fermentation were both higher than those of control indicating that citrate was not only strengthened the phosphorylation reactions rendering energy for ATP formation but also enhanced purine nucleotide (AMP) synthesis. Continuous supplement of AMP and energy for ATP phosphorylation provided sufficient substance and energy for cAMP fermentation production.
Citrate Improved Intracellular Amino Acids Levels For Camp Synthesis
Precursor amino acids were necessary for cAMP synthesis via de novo pathway and the change of intracellular amino acids amounts could influence cell growth, physiological status and product synthesis. So intracellular amino acids contents were assayed to investigate the causes of higher cAMP formation capacity in citrate added fermentation. 18 kind of intracellular amino acids were detected by HPLC according to AccQ-Tag method and 6 of them were increased obviously in concentrations as result of citrate addition when compared with those of control, as displayed in Fig. 3. Due to citrate addition, intracellular lysine, aspartate, glutamate, glutamine, threonine and arginine contents were improved by 59.4%, 167,7%, 96.9%, 673.9%, 365.2% and 76.7% (at 50 h), respectively, which were beneficial for cell growth and cAMP production. Among the 6 kind of amino asids, aspartate and glutamine were necessary for cAMP synthesis via de novo pathway which were deemed as precursor amino acids. Moreover, lysine, aspartate and threonine were synthesized on basis of oxaloacetate as the carbon skeleton donator, and α-ketoglutarate was the carbon skeleton donator for glutamate, glutamine and arginine biosynthesis. Oxaloacetate and α-ketoglutarate were derived from tricarboxylate cycle. It can be deduced that exogenous citrate activated the metabolic intensity of tricarboxylate cycle, enhanced some metabolites levels (such as oxaloacetate and α-ketoglutarate) resulting in the significant improvement of precursor amino acids levels together with NADH/NAD+ ratios, which further accelerated cAMP production.
The effects of citrate on activities of key enzymes in cAMP synthesis related metabolic pathway
In certain metabolic pathway, the change of key enzymes activities reflected metabolic flux distribution state and the metabolic pathway for cAMP formation had been elucidated in previous study (Niu et al. 2013). To investigate the metabolic response of cAMP biosynthesis towards citrate, the activities of key enzymes in tricarboxylate cycle, glycolysis, pentose phosphate pathway and some amino acids synthesis pathway were assayed and the detected results were exhibited in Table 1 and Fig. 4.
Table 1
Comparison of key enzymes activities in cAMP fermentations with/without citrate addition.
Time (h)
|
G6PDH
|
PK
|
PEPC
|
CS
|
ICDH
|
AAT
|
NADPH-GDH
|
A
|
B
|
A
|
B
|
A
|
B
|
A
|
B
|
A
|
B
|
A
|
B
|
A
|
B
|
26
|
3.42 ± 0.12
|
3.72 ± 0.14
|
7.33 ± 0.13
|
7.14 ± 0.12
|
4.85 ± 0.13
|
5.02 ± 0.16
|
0.43 ± 0.05
|
0.40 ± 0.02
|
1.89 ± 0.15
|
1.95 ± 0.12
|
5.23 ± 0.11
|
5.62 ± 0.13
|
0.76 ± 0.02
|
0.83 ± 0.03
|
36
|
3.33 ± 0.13
|
4.02 ± 0.12
|
9.46 ± 0.14
|
6.86 ± 0.18
|
4.68 ± 0.14
|
5.16 ± 0.14
|
0.45 ± 0.02
|
0.32 ± 0.03
|
1.78 ± 0.14
|
2.03 ± 0.11
|
5.45 ± 0.13
|
7.43 ± 0.13
|
0.64 ± 0.03
|
0.86 ± 0.02
|
50
|
3.05 ± 0.10
|
3.63 ± 0.14
|
8.12 ± 0.12
|
6.44 ± 0.14
|
4.35 ± 0.10
|
4.82 ± 0.13
|
0.42 ± 0.03
|
0.30 ± 0.02
|
1.68 ± 0.12
|
1.85 ± 0.13
|
4.92 ± 0.12
|
6.32 ± 0.13
|
0.60 ± 0.02
|
0.75 ± 0.02
|
60
|
2.54 ± 0.11
|
3.15 ± 0.13
|
5.45 ± 0.12
|
4.75 ± 0.15
|
3.81 ± 0.16
|
4.14 ± 0.12
|
0.27 ± 0.02
|
0.20 ± 0.03
|
1.72 ± 0.11
|
1.82 ± 0.13
|
4.25 ± 0.13
|
5.46 ± 0.13
|
0.43 ± 0.02
|
0.58 ± 0.03
|
Note: A: control, B: with 3 g/L-broth sodium citrate added at 24 h, the unite of enzyme activities was 10− 2 U/mg-protein. |
6-phosphoglucose dehydrogenase (G6PDH) was the key enzyme connecting glycolysis and pentose phosphate pathway, whose activity reflected the distribution of carbon flux between two routes. Pyruvate kinase (PK) was the rate-limiting enzyme whose activity related to the metabolic intensity of glycolysis pathway directly. As shown in Table 1, due to the addition of citrate, the activities of G6PDH were promoted obviously with a increment of 21.6% (at 36 h), meanwhile, PK activities were decreased evidently with a reduction of 27.5% (at 36 h), when compared with those of control. It indicated that more carbon flux was distributed to pentose phosphate pathway for cAMP synthesis and glycolysis pathway was weakened correspondingly.
The activities of phosphoenolpyruvate carboxylase (PEPC), citrate synthases (CS) and isocitrate dehydrogenase (ICDH) were also assayed to illustrate the influence of citrate on tricarboxylate cycle. As presented in Table 1, citrate inhibited the activities of CS evidently with a decrease of 28.9% (at 36 h), while ICDH and PEPC were stimulated to some extent with increasments of 14.1% and 10.3% (at 36 h), respectively, as compared to control. CS is the rate-limiting enzyme in tricarboxylate cycle which catalyzed the reaction of oxaloacetic acid and acetyl-coA for citric acid synthesis. Here, CS and PK were inhibited greatly by citrate simultaneously suggesting that less carbon flux was injected into tricarboxylate cycle via citric acid synthesis reaction. However, enhanced ICDH activities and 2 g/L exogenous sodium citrate consumption (Fig. 1b) indicated that exogenous citrate was utilized and injected into tricarboxylate cycle as cosubstrate via the transformation reaction from citrate to isocitric acid catalyzed by ICDH. In addition, improved PEPC activities indicated that more phosphoenolpyruvate was transformed to oxaloacetate by which insufficient metabolites in tricarboxylate cycle would be replenished. Therefore, it suggested that the metabolic intensity of tricarboxylate cycle would be enhanced to some extent owing to exogenous citrate utilization and more oxaloacetate synthesis, which had been proved by improved NADH/NAD+ ratios. Furthermore, some derivatives such as amino acids derived from tricarboxylate cycle might be produced in excess and accumulated.
In order to find the cause for enhanced 6 amino acids mentioned above, the activities of aspartate aminotransferase (AAT) and NADPH-dependent glutamate dehydrogenase (NADPH-GDH) were detected. As shown in Table 1, AAT and NADPH-GDH activities were improved by citrate significantly with respective increments of 36.3% and 34.4% higher than those of control at 36 h, which in favour of related amino acids synthesis. It was in good agreement with the amino acids assay result.
The effects of citrate on intracellular redox state during cAMP fermentation period
Intracellular NADPH/NADP+ ratio was another important influence factor for cAMP synthesis, owing to the feedback inhibition of high ratios on G6PDH. As shown in Fig. 5a, NADPH/NADP+ ratios were decreased obviously by citrate with 24.5% reduction than control at 36 h. In general, citrate directed more carbon flux into pentose phosphate pathway supplying substrate basis for cAMP synthesis, meanwhile, more NADPH was produced and accumulated, furthermore, NADPH/NADP+ ratio would be improved to some extent, in turn, high NADPH/NADP+ ratio would inhibit the activity of G6PDH and restrain carbon flux injection into pentose phosphate pathway for cAMP synthesis in high proportion. Here, NADPH/NADP+ ratios were not improved with higher G6PDH activities correspondingly in citrate added fermentation, on the contrary, the ratios were decreased significantly suggesting that there was other NADPH consuming route existed.
As described in previous study, high level energy metabolism was accompanied by more serious electron leakage from electron transport chain resulting in excessive ROS synthesis and severe oxidative stress (Li et al. 2020). Then, cell components would suffer from serious damage from oxidative stress which could be reflected by MDA level. So, change of intracellular ROS and MDA contents were assayed which reflected the cell oxidative stress state and damage degree, respectivyly. As shown in Fig. 5bc, intracellular ROS contents in fermentation with citrate added were higher than those of control obviously with average 30.1% increments after 36 h, meanwhile, MDA levels in fermentations with/without citrate addition were almost equal at different fermentation time and increased gradually over time during whole fermentation period. It indicated that excessive NADPH derived from pentose phosphate pathway was partly consumed for the reduction of ROS aroused by citrate addition for intracellular redox balance maintenance, avoiding more serious cell damage than control. In addition, excessive NADPH was also utilized for abundant glutamate synthesis in citrate added fermentation due to its higher NADPH-GDH activities. Consequently, excessive NADPH was consumed by above two routes efficiently resulting in lower NADPH/NADP+ ratios and higher glutamate contents in citrate added fermentation, by which feedback suppression for pentose phosphate pathway was relieved efficiently. Finally, enhanced carbon flux, amino acids and energy metabolism levels promoted cAMP production synergistically.