The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanisms of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.
Research Article
Metabolic flux analysis of simultaneous production of vitamin B12 and propionic acid in a coupled fermentation process by Propionibacterium freudenreichii
https://doi.org/10.21203/rs.3.rs-296552/v1
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Propionibacterium freudenreichii
Vitamin B12
Propionic acid
Membrane separation coupled fermentation
Metabolic flux analysis
Vitamin B12 (cobalamin) is a complex cobalt-containing cyclic tetrapyrrole coenzyme that is widely used in the pharmaceutical and food industries. The industrial production of vitamin B12 by chemical methods is very difficult, complicated and expensive, with > 70 steps. Therefore, the production of vitamin B12 is exclusively preformed using aerobic and anaerobic fermentation by microorganisms; vitamin B12 synthesis involves > 30 genes [1]. The main strains used for industrial production of vitamin B12 are Pseudomonas denitrificans, Propionibacterium freudenreichii, and Propionibacterium shermanii [2].
Propionic acid is an important chemical intermediate, and among the top 30 candidate platform chemicals according to the US Department of Energy [3]. Propionic acid and its salts can be used as antibacterial agents, anti-inflammatory drugs, herbicides, food preservatives, and artificial foods [4]. Industrially, propionic acid is produced from petroleum-based chemicals via the hydrocarboxylation of ethylene, which causes substantial damage to the environment. As an alternative, renewable route, the preparation of propionic acid or its salts by microbial fermentation has been investigated extensively and is considered a safe and pollution-free process [5].
P. freudenreichii is a “generally recognized as safe” bacterial strain. However, the production of vitamin B12 by anaerobic fermentation suffers from formation of byproducts such as propionic acid, acetic acid, and succinic acid. In the traditional batch fermentation process, the concentration of propionic acid can reach 20–30 g/L, which causes feedback inhibition of cell growth [6]. Considerable effort has been made to improve the fermentation efficiency. A highly-efficient vitamin B12-producing strain was constructed by Rhone-Poulenc Rorer, Inc. (France) using random mutagenesis [7]. Some necessary genes were overexpressed, such as the cobF–cobM cluster, cobA, and cobE, while some genes, such as hemZ, were silenced with an antisense RNA strategy [8, 9]. Finally, an engineered strain of P. freudenreichii for vitamin B12 production was successfully constructed [10–12]. However, the engineered strain was unbalanced at the systems level because of the limited available genetic tools [13].
Traditional strategies are still crucial for industrial production, for example, medium optimization (glucose, glycerol, corn steep liquor, and Co2+); cell immobilization [Ca alginate beads, polyethylenimine-treated Poraver (PEI-Poraver)]; and process regulation [5,6-dimethylbenzimidazole (DMB) addition, in-situ product removal (ISPR) processes] [14–15]. ISPR can remove propionic acid in time to effectively overcome its feedback inhibition effect, and help to realize a semi-continuous fermentation process. Various ISPR processes have been developed using in situ cell retention reactors [16], plant fibrous-bed bioreactors [17–19], and PEI-Poraver immobilized bioreactors [4]. However, these studies focused on production of only a single product, and few works have reported on simultaneous production of vitamin B12 and propionic acid.
To set up a cost-effective semi-continuous fermentation process to realize the simultaneous production of vitamin B12 and propionic acid, a new ISPR process characterized by expanded bed adsorption was built [20]. Membrane separation was introduced into the fermentation system to realize fixed-bed removal of propionic acid, based on vitamin B12 ex situ conversion [21].
In this study, to further investigate the regulatory mechanisms of vitamin B12 and propionic acid biosynthesis, the metabolic flux in the coupled fermentation process was analyzed based on the metabolic network of P. freudenreichii. In view of the complexity of P. freudenreichii metabolism, a simplified metabolic network was established. The metabolic flux distribution at key nodes in different fermentation conditions was analyzed, and the influencing factors were assessed. Our findings provide theoretical guidance for the optimization of simultaneous production of vitamin B12 and propionic acid.
Microorganism and medium
P. freudenreichii CICC 10019 was obtained from the Chinese Industrial Microorganism Conservation Center. The stock culture was incubated in agar slants at 30 °C, stored at 4 °C, and transferred to fresh agar monthly. For long-term preservation, the stock was stored at −80 °C.
Inoculum medium contained glucose (35 g/L), corn steep liquor (21 g/L), (NH4)2SO4 (5 g/L), KH2PO4 (4 g/L), CoCl2 (5 mg/L), and distilled water (pH 6.8–7.0). Fermentation medium contained glucose (60 g/L), corn steep liquor (41 g/L), KH2PO4 (4.6 g/L), CoCl2 (12.7 mg/L), and distilled water (pH 6.8–7.0). For medium preparation, glucose was autoclaved separately.
Corn steep liquor was purchased from Lingshanhe Plant Protein Manufacturing Co., Ltd. (Xingtai, Hebei, China). Glucose and inorganic salts were of analytical grade and produced by Shanghai Macklin Biochemical Co., Ltd. (China).
Batch/fed-batch fermentation of P. freudenreichii
P. freudenreichii CICC 10019, stored on a agar slant, was activated at 30 °C for 24 h. One loopful of culture from the activated deep agar slant was inoculated aseptically into 50 mL of inoculum medium and cultivated statically at 30 °C for 48 h. This culture was used to inoculate 500 mL of fresh inoculum medium. After 48 h, the culture was used to inoculate fermentation medium in a 7-L fermenter filled to 70% capacity, and cultivated at 30 °C, 50 rpm. During the fermentation, no sterile air was required and the pH was controlled at 7.0 by the automatic addition of NH3·H2O or H3PO4. In the batch fermentation process, 0.90 mg/L DMB was added as a precursor for vitamin B12 biosynthesis at 84 h [22].
In fed-batch fermentation of P. freudenreichii, 500 g/L glucose were added when the glucose concentration fell below 10 g/L, to maintain cell growth and product synthesis.
During the fermentation process, the concentrations of glucose, propionic acid, acetic acid, succinic acid, and lactic acid were determined every 12 h. After DMB addition, the concentrations of vitamin B12 and heme were also analyzed.
Membrane separation-coupled fermentation of P. freudenreichii
The membrane separation-coupled fermentation process of P. freudenreichii was established by Wang et al. [21]. During the fermentation process, a multifunctional membrane separation device equipped with a G13 plunger diaphragm pump (RNF 0460-011) and a 0.22-μm spiral membrane module (Type 1812) was used to separate P. freudenreichii cells online, while the penetrant flowed through a chromatography column packed with ZGA302 anion exchange resin and propionic acid in the filtrate was adsorbed. Details are shown in Fig. 1. After optimizing the adsorption conditions, the concentration of propionic acid was reduced to <10 g/L. Then, the deacidified filtrate was returned to the fermenter.
Cell concentration assay
Cell growth was analyzed photometrically at 600 nm. Before measuring the cell concentration, P. freudenreichii cells were centrifuged at 10,000 × g for 10 min, and then resuspended in isovolumetric phosphate buffer (20 mM, pH 7.0) to eliminate the effect of corn steep liquor and other fermentation components.
Assay of organic acids and glucose
After centrifugation, fermentation supernatant was used to analyze the concentration of organic acids and glucose.
High-performance liquid chromatography (HPLC) analysis of propionic acid, lactic acid, acetic acid, and succinic acid was performed using a Shimadzu LC-20AT HPLC system fitted with a Bio-Rad HPX-87H column (5 μm, 380 mm × 4.6 mm) and an SPD-20A UV detector operated at 215 nm. The mobile phase was 5 mM H2SO4 with a flow-rate of 0.6 mL/min at 55 °C. The injection volume was 10 μL. Commercially available organic acids were used as external standards.
Residual glucose was determined enzymatically using an SBA-40E biological sensor (Shandong, China). The tested supernatant was diluted to a final glucose concentration of 0–1 g/L, and then loaded onto the biological sensor with an injection volume of 25 μL. The glucose concentration was calculated according to a glucose standard solution.
Assay of vitamin B12 and heme
After centrifugation, P. freudenreichii cells were washed and resuspended in 20 mM phosphate buffer (pH 7.0), then disrupted in a boiling water for 30 min to release intracellular vitamin B12 and heme [22]. After removing cell debris by centrifugation at 10,000 × g for 10 min, the supernatant was used to analyze the concentration of vitamin B12 and heme.
HPLC analysis of vitamin B12 was performed with a Hypersil GOLD C18 column (5 μm × 4.6 mm × 250 mm; Dikma) at 254 nm with the same HPLC system as described above [20]. The mobile phase consisted of 0.02 M sodium acetate buffer solution (pH 3.5, adjusted with acetic acid), and acetonitrile (gradient of 5%–24%, 0–30 min). The elution was carried out at 25 °C with a flow-rate of 1.0 mL/min. Commercially available vitamin B12 was used as an external standard.
The concentration of heme was measured by colorimetry [23]. Commercially available hemin was used as external standard. First, hemin standard solutions (1–10 µg/mL) were prepared. Pyridine-NaOH (2 mL, 33% pyridine and 0.1 mol/L NaOH) solution was added to 1 mL of hemin standard solution. After mixing, 3 mg sodium sulfite were added and reacted for 30 min to reduce the heme iron. A linear standard calibration curve was obtained using absorbance at 557 nm. When measuring a fermentation sample, there was no need to add sodium sulfite.
Establishment of metabolic flux balance model of vitamin B12 and propionic acid
The metabolic network of P. freudenreichii is very complicated. According to the literature and preliminary experimental data, the most important metabolic pathways of propionic acid and other related metabolites with glucose as the carbon source were determined, including glycolysis, the Wood-Werkman pathway, succinate synthesis, lactate synthesis, acetate synthesis, and vitamin B12 synthesis. Details are shown in Fig. 2 [24-26].
To facilitate analysis and calculation, a simplified ideal metabolic network was established based on the following principles: (1) Metabolic flux analysis was based on the pseudo-steady-state assumption. In the late stage of fermentation, P. freudenreichii cells were in the non-growth period and growth rate was ignored to simplify the model; (2) The pentose phosphate pathway and glyoxylate pathway were ignored; (3) A complex interdependent and interactional relationship exists among the tetrapyrrole compounds, which are derived from δ-aminolevulinate (ALA). However, the synthesis of chlorophyll and coenzyme F430 was ignored; (4) Reactions with fixed stoichiometry and with intermediates without branch points were simplified into a single reaction equation. (5) In the cell growth arrest stage, the total amount of cell maintenance energy and the consumption of ATP were not equal, due to the existence of many invalid cycles. Thus, the balance of total ATP was not considered.
The established metabolic network of simultaneous production of vitamin B12 and propionic acid in P. freudenreichii included 27 reaction equations, as shown in Table 1. According to the metabolic network and the stoichiometric balance equation of each reaction, the relationship among the reaction rates was obtained. It was assumed that the intermediate metabolites were in a pseudo-steady-state, that is, their concentration change rate was zero. On the basis of the material balance law, the accumulation rate of metabolites can be calculated from the formula:
where ri(t) is the accumulation rate of intermediate metabolite i [mmol/(L·h)]; rj(t) and rk(t) are the reaction rates of the jth and kth reactions of substance i, [mmol/(L·h)]; and aij and aik are the stoichiometric coefficients corresponding to each reaction. An equation based on this principle can be established for each metabolite in the metabolic network.
According to the pseudo-steady-state assumption, ri(t) = 0. The m intermediate metabolites and n reactions in the metabolic network constitute m metabolic flux balance equations with degree of freedom of F = n − m. The overall metabolic flux distribution can be determined by measuring F reaction rates. The metabolic flux balance model of vitamin B12 and propionic acid established in this study included 27 reaction rates (J) and 23 equilibrium equations (K), with degree of freedom (F) = 4. Details are shown in Table 2. Therefore, the metabolic flux distribution can be determined only by measuring just 4-step reaction rates.
During the fermentation of P freudenreichii, the concentration of seven substances can be measured, and the corresponding reaction rates were obtained by differentiation: the consumption rate of glucose (r1), and the production rates of acetic acid (r11), propionic acid (r13), lactic acid (r17), succinic acid (r21), heme (r24), and vitamin B12 (r27). Taking these parameters as known variables, substituting them into the metabolic balance equations, listing the matrix, and using the Linprog function in Matlab software, the ideal metabolic flux distribution was obtained. During the calculation process, the glucose consumption rate was assumed to be 100 mmol/(L·h).
Fermentation of P. freudenreichii in different conditions
In the fermentation process of P. freudenreichii, many process variables can affect the production of vitamin B12 and propionic acid, including pH, temperature, fermentation time, inoculum size, the carbon and nitrogen sources and their concentrations, as well as fermentation strategies [25]. On the basis of previous studies, the effects of glucose feeding and membrane separation-coupled operation on the simultaneous production of vitamin B12 and propionic acid were analyzed. Generally, cell growth was accompanied by the synthesis of propionic acid, acetic acid, and succinic acid in the initial stage of P. freudenreichii fermentation, while the synthesis of vitamin B12 occurred later. The consumption of glucose and the production of organic acids such as propionic acid, acetic acid, and succinic acid, as well as vitamin B12 were analyzed. As shown in Fig. 3, in batch fermentation, the concentrations of vitamin B12 and propionic acid were 10.5 mg/L and 28.82 g/L at 132 h, with productivity of 0.08 mg/(L·h) and 0.22 g/(L·h), respectively.
In fed-batch fermentation, when the glucose concentration fell to <10 g/L, a high concentration of glucose was fed into the fermentor to maintain cell growth and product synthesis. After 132 h, the concentration of vitamin B12 and propionic acid reached 16.0 mg/L and 32.70 g/L, increased by 52.38% and 13.46% respectively, compared with the batch fermentation process. Therefore, feeding glucose during the fermentation process can improve the fermentation efficiency of P. freudenreichii. However, propionic acid fermentation suffers from end-product feedback inhibition, which leads to a decrease in productivity of propionic acid [27]. Our results showed that in the initial stage (0–72 h) and the later stage (72–132 h) of the fermentation process, the productivity of propionic acid was 0.34 g/(L·h) and 0.13 g/(L·h), respectively, with an overall productivity of 0.25 g/(L·h).
To overcome the feedback inhibition of propionic acid synthesis, a membrane separation-coupled fermentation process of P. freudenreichii was established. When the concentration of propionic acid reached about 20–25 g/L, P. freudenreichii cells were separated online using a multifunctional membrane device, and the penetrant flowed through a ZGA302 anion chromatography column to adsorb propionic acid. When the propionic acid concentration was <10 g/L, the flow-through liquid was returned to the fermenter. After this deacidification, the feedback inhibition effect of propionic acid was relieved, which further promoted cell growth and product synthesis. In this set-up, the concentrations of vitamin B12 and propionic acid reached 21.6 mg/L and 50.12 g/L at 132 h, with productivity of 0.16 mg/(L·h) and 0.38 g/(L·h) respectively, increased by 35.00% and 49.12% respectively, compared with the fed-batch fermentation process. The concentration of succinic acid in the coupled fermentation process was 22.09 g/L, increased by 52.03%, implying that the metabolism of propionic acid and succinic acid was inherently related. However, the concentration of acetic acid decreased by 31.86% compared with the fed-batch fermentation process, indicating that more metabolic flux flowed to propionic acid synthesis in the coupled fermentation process. Details are shown in Fig. 3.
The current state of simultaneous production of vitamin B12 and propionic acid by Propionibacterium is shown in Table 3. Compared with the expanded bed reactor (EBA-reactor)[20,28], the membrane separation-coupled process does not require complicated ISPR operation and is easy for industrial application. Therefore, this process can improve the fermentation efficiency of P. freudenreichii and realize the simultaneous production of vitamin B12 and propionic acid. However, the coupled system still needs to be integrated, for example, the removal efficiency of propionic acid around neutral pH (6.5–7.0) was still low, and the adsorption conditions should be further optimized, which is ongoing in our laboratory.
Metabolic flux analysis of key nodes
The cellular metabolic network of P. freudenreichii is a complex reaction system with steps catalyzed simultaneously by multiple enzymes, and is affected by interaction of the internal and external environments. To analyze the intracellular metabolic flux distribution in different conditions, a simplified ideal metabolic network of vitamin B12 and propionic acid synthesis was established based on the current literature.
During the fermentation process of P freudenreichii, the consumption rate of glucose (r1), and the production rates of acetic acid (r11), propionic acid (r13), lactic acid (r17), succinic acid (r21), heme (r24), and vitamin B12 (r27), can be calculated by differentiation, and then the ideal metabolic flux distribution was obtained using the Linprog function in Matlab software. The influence of culture conditions on product synthesis was assessed by analyzing the metabolic flux at key nodes, which provided theoretical guidance for the optimization of simultaneous production of vitamin B12 and propionic acid.
Metabolic flux analysis of pyruvate node
In the simplified metabolic network of P. freudenreichii, pyruvate was the first key metabolic node. Pyruvate directly or indirectly participates in the synthesis of organic acids, including: (1) direct generation of lactate via catalysis by lactate dehydrogenase; (2) generation of acetyl-CoA, an intermediate in acetate production, via catalysis by pyruvate dehydrogenase; and (3) generation of oxaloacetate via the pyruvate carboxylation pathway, and hence production of succinate. It was found that the flux at the pyruvate node to the above three branches was 84.64:3.91:30.44 in the batch fermentation process, while the flux distribution was 83.47:2.51:33.52 and 75.44:2.32:34.64 respectively in the fed-batch and coupled fermentation processes. Details are shown in Fig. 4. Therefore, the addition of glucose to the culture and the removal of propionic acid changed the metabolic flux distribution at the pyruvate node. That is, the flux to the lactate and acetate branches decreased, while the flux to the oxaloacetate branch increased accordingly, which was consistent with the experimental results. Especially in the later stage of fermentation, the flux to the lactate branch dropped to a negative value.
Metabolic flux analysis of succinyl-CoA node
Succinyl-CoA is another key node in the metabolism of vitamin B12 and propionic acid. In the metabolic process, pyruvate can directly generate oxaloacetate via catalysis by pyruvate carboxylase. Then, oxaloacetate can be used to produce succinate via a three-step enzymatic reaction. It was reported that the last step of propionate synthesis was the transfer of CoA from propionyl-CoA to succinic acid, also generating succinyl-CoA. Succinyl-CoA can be used to synthesize ALA, a vital intermediate of vitamin B12 synthesis, by condensation with glycine [29]. In addition, succinyl-CoA can be converted to methylmalonyl-CoA, an important intermediate of propionic acid synthesis.
According to our results (Fig. 5), the flux distribution at the succinyl-CoA node to ALA and methylmalonyl-CoA was 0.02:95.11 in the batch fermentation process. However, the flux to the ALA branch increased significantly (from 0.02 to 4.40) in the fed-batch process, which directly led to a 52.38% increase in the concentration of vitamin B12. The propionic acid concentration only increased by 13.42% because of feedback inhibition. However, when propionic acid was partly removed in the coupled fermentation process and its feedback inhibition effect was relieved, the flux to the ALA and methylmalonyl-CoA branches both increased, with proportion 4.52:100. As a result, the concentrations of vitamin B12 and propionic acid increased by 105.71% and 73.91%, respectively, compared with those in the batch fermentation process.
Metabolic flux analysis of uroporphyrinogen III node
Microbial synthesis of vitamin B12 can be regulated at both the transcriptional and metabolic levels [29-31]. A cobalamin riboswitch is used to quickly respond to the environment [32]. It was reported that a complex interdependent and interactional relationship exists among the tetrapyrrole compounds [24].
According to the metabolic network, there were two metabolic branches at the uroporphyrinogen III node: one is the synthesis of heme, and the other is the synthesis of sirohydrochlorin, a precursor of vitamin B12. It was found that the metabolic flux to the heme branch significantly increased when glucose was fed or the coupled process was adopted. However, the increase in the metabolic flux to the heme branch did not reduce the metabolic flux to the sirohydrochlorin branch, which implied that the total flux through the uroporphyrinogen III node increased. Details are shown in Fig. 6. In the late stage of vitamin B12 synthesis, the metabolic flux was divided into two branches in equal proportion at the sirohydrochlorin node, one of which was the synthesis of siroheme. Relevant enzymes (e.g., HemE, HemZ, HemY, HemH, SirB) can be modified (silenced or knocked-out) to reduce the metabolic flux to the heme and siroheme branches, to increase the concentration of vitamin B12.
Metabolic flux distribution in the coupled fermentation process
According to the metabolic network of P. freudenreichii, α-pyrroleribose is activated to form the lower ligand base DMB that is linked to adenosylcobinamide-GDP (Ado-cbi-GDP) to synthesize vitamin B12 [33,34]. However, because of its low concentration, α-pyrroleribose was a key rate-limiting factor in vitamin B12 synthesis, which led to the accumulation of intracellular Ado-cbi-GDP [35]. During the fermentation process, additional DMB was required to promote the conversion of Ado-cbi-GDP to vitamin B12.
In our experiments, DMB was added to the fermentor at 84 h. To investigate the effect of DMB addition on the metabolic flux distribution, we analyzed the data from the coupled fermentation process for 0–84 and 84–132 h. After DMB addition, the metabolic flux significantly flowed toward vitamin B12 synthesis. Details are shown in Fig. 7. The specific effects observed were: (1) Increased synthesis of pyruvate by increased flux of 3-phosphoglycerate and phosphoenolpyruvate. (2) Decreased synthesis of organic acids. The synthesis of organic acids mainly occurred in the early and middle stages of the fermentation process. After DMB addition, the flux to lactic acid, acetic acid, succinic acid, and propionic acid decreased sharply, which was consistent with the experimental results. (3) Increased synthesis of succinyl-CoA and its flux proportion to the ALA branch, which provided the possibility of efficient synthesis of uroporphyrinogen III. (4) The flux increase at the uroporphyrinogen III node further increased the flux to heme and sirohydrochlorin. However, the flux proportion to the sirohydrochlorin branch (the direction of vitamin B12 synthesis) was lower than that to heme. In fact, the concentration of heme was slightly higher than that of vitamin B12. Detailed studies should be carried out with the aid of systems biology and synthetic biology to investigate the complex relationship among the tetrapyrrole compounds (e.g., cobalamin, chlorophyll, heme, and coenzyme F430) and its regulatory mechanisms.
The simultaneous production of vitamin B12 and propionic acid by P. freudenreichii has attracted wide attention. In the membrane separation-coupled fermentation process described here, the concentration of vitamin B12 and propionic acid reached 21.6 mg/L and 50.12 g/L after 132 h, increased by 105.71% and 73.91% respectively compared with a batch fermentation process. To investigate the distribution of intracellular materials and energy, a simplified ideal metabolic network of P. freudenreichii was established. By analyzing the flux distribution at key metabolic nodes in different conditions, the effects of glucose feeding, propionic acid removal, and DMB addition on the fermentation efficiency were assessed. The results showed that all these methods can improve the metabolic flux through key nodes and increase the product yield, which provides theoretical guidance for the optimization of P. freudenreichii fermentation processes for production of vitamin B12 and propionic acid.
CRediT authorship contribution statement
Yuhan Zhang: Methodology, Validation, Writing-Original Draft; Xiaolian Li: Writing-Review & Editing; Ziqiang Wang: Conceptualization, Writing-Review & Editing, Funding acquisition, Project administration; Yunshan Wang: Resources, Supervision, Project administration; Yuanyuan Ma: Resources, Supervision; Zhiguo Su: Resources, Supervision.
Acknowledgment
This work was financially supported by the National Natural Science Foundation of China (Grant no. 21506227).
Conflict of Interest
The authors declare that they have no conflict of interest.
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Table 1 Metabolic reactions of simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii
|
Flux |
Metabolic reactions |
|
r1 |
Glucose + ATP → Glucose-6-phosphate + ADP + H+ |
|
r2 |
Glucose-6-phosphate → Fructose-6-phosphate |
|
r3 |
fructose-6-phosphate + ATP → Fructose-1,6-diphosphate + ADP + H+ |
|
r4 |
Fructose-1,6-diphosphate → Dihydroxyacetone phosphate + Glyceraldehyde-3-phosphate |
|
r5 |
Dihydroxyacetone phosphate → Glyceraldehyde-3-phosphate |
|
r6 |
Glyceraldehyde-3-phosphate + NAD+ + Pi → 1,3-diphosphoglycerate + NADH + H+ |
|
r7 |
1,3-Diphosphoglycerate + ADP → 3-Phosphoglycerate + ATP |
|
r8 |
3-Phosphoglycerate → 2-Phosphoglycerate |
|
r9 |
2-Phosphoglycerate → Phosphoenolpyruvate + H2O |
|
r10 |
Phosphoenolpyruvate + ADP +H+ → Pyruvate + ATP |
|
r11 |
Pyruvate + NADH + H+ → Lactate + NAD+ |
|
r12 |
Pyruvate + CoA + NAD+ → Acetyl-CoA + CO2 + NADH |
|
r13 |
Acetyl-CoA + ADP → Acetic acid + CoA + ATP |
|
r14 |
Pyruvate + CO2 + ATP + H2O → Oxaloacetate + ADP + 2Pi + 2H+ |
|
r15 |
Oxaloacetate + NADH + H+ → L-malate + NAD+ |
|
r16 |
L-malic acid → Fumaric acid + H2O |
|
r17 |
Fumaric acid + FADH2 → Succinic acid + FAD |
|
r18 |
Succinic acid + ATP + CoA → Succinyl-CoA + Pi + ADP |
|
r19 |
Succinyl-CoA → Methylmalonyl-CoA |
|
r20 |
Methylmalonyl-CoA → propionyl-CoA+CO2 |
|
r21 |
Propionyl-CoA + ADP → Propionic acid + CoA + ATP |
|
r22 |
Succinyl-CoA + Glycine → δ-aminolevulinic acid + ADP |
|
r23 |
δ-aminolevulinic acid → …… → Uroporphyrinogen III |
|
r24 |
Uroporphyrinogen III → …… → heme |
|
r25 |
Uroporphyrinogen III → …… → Sirohydrochlorin |
|
r26 |
Sirohydrochlorin → Siroheme |
|
r27 |
Sirohydrochlorin →……→ Cob(II)yrinic acid a,c diamide→ ……→ Adenosylcobalamin |
Table 2 Equations of metabolic flux of simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii
|
Intermediate |
Equations |
|
Glucose-6-phosphate |
r1-r2=0 |
|
Fructose-6-phosphate |
r2-r3=0 |
|
Fructose-1,6-diphosphate |
r3-r4=0 |
|
Glyceraldehyde-3-phosphate |
r4+r5-r6=0 |
|
Dihydroxyacetone phosphate |
r4-r5=0 |
|
1,3-diphosphoglycerate |
r6-r7=0 |
|
3-Phosphoglycerate |
r7-r8=0 |
|
2-Phosphoglycerate |
r8-r9=0 |
|
Phosphoenolpyruvate |
r9-r10=0 |
|
Acetyl-CoA |
r12-r13=0 |
|
Oxaloacetate |
r14-r15=0 |
|
L-malic acid |
r15-r16=0 |
|
Fumaric acid |
r16-r17=0 |
|
Succinyl-CoA |
r18-r19-r22=0 |
|
Methylmalonyl-CoA |
r19-r20=0 |
|
Propionyl-CoA |
r20-r21=0 |
|
δ-aminolevulinic acid |
r22-r23=0 |
|
Uroporphyrinogen III |
r23-r24-r25=0 |
|
Sirohydrochlorin |
r25-r26-r27=0 |
|
NADH |
r6+r12-r11-r15=0 |
|
Pi |
2r14+r18-r6=0 |
|
CoA |
r13+r21-r12-r18-r22=0 |
|
H+ |
r1+r3+r6+2r14-r10-r11-r15=0 |
Table 3 The current state-of vitamin B12 and propionic acid production by Propionibacterium
|
Microorganism |
Carbon source |
Culture system |
Vitamin B12 (mg/L) |
Propionic acid (g/L) |
References |
|
P. acidipropionici DSM 8250 |
beet molasses |
Two-stage |
34.8 |
17.0 |
[36] |
|
P. acidipropionici DSM 8250 |
reed molasses |
Two-stage |
28.8 |
17.7 |
[36] |
|
P. acidipropionici DSM 8250 |
sugar (household refined product) |
Two-stage |
33 |
30 |
[36] |
|
P. shermanii PZ-3 |
glucose |
Hollow-fiber module |
52 |
~12 |
[37] |
|
P. freudenreichii CICC 10019 |
glucose |
EBA bioreactor |
42.6 |
52.5 |
[20] |
|
P. freudenreichii CICC 10019 |
corn stalk hydrolysates |
EBA bioreactor |
47.6 |
91.4 |
[28] |
|
P. freudenreichii T82 |
glucose/fructose |
Batch |
4-14 ug L-1 |
23-40 |
[38] |
|
P. freudenreichii CICC 10019 |
glucose |
Membrane coupled |
21.6 |
50.12 |
This study |