Metabolic Flux Analysis of Simultaneous Production of Vitamin B12 and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii

The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanism 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.


Introduction
To set up a cost-effective semi-continuous fermentation process to realize the simultaneous production of vitamin B 12 and propionic acid, a new ISPR process characterized by expanded bed adsorption was built [22]. Membrane separation was introduced into the fermentation system to realize fixed-bed removal of propionic acid, based on vitamin B 12 ex situ conversion [23].
In this study, to further investigate the regulatory mechanisms of vitamin B 12 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 B 12 and propionic acid.

Materials and Methods
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.
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 NH 3 ·H 2 O or H 3 PO 4 . In the batch fermentation process, 0.90 mg/L DMB was added as a precursor for vitamin B 12 biosynthesis at 84 h [24].
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 B 12 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. [23]. 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 H 2 SO 4 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 B 12 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 B 12 and heme [24]. After removing cell debris by centrifugation at 10,000×g for 10 min, the supernatant was used to analyze the concentration of vitamin B 12 and heme.
HPLC analysis of vitamin B 12 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 [22]. 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 B 12 was used as an external standard.
The concentration of heme was measured by colorimetry [25]. 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 B 12 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 B 12 synthesis. Details are shown in Fig. 2 [26][27][28].
To facilitate analysis and calculation, a simplified ideal metabolic network was established based on the following principles [29]: (1) metabolic flux analysis was based on the pseudosteady-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 δaminolevulinate (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 B 12 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:  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 (r 1 ), and the production rates of acetic acid (r 11 ), propionic acid (r 13 ), lactic acid (r 17 ), succinic acid (r 21 ), heme (r 24 ), and vitamin B 12 (r 27 ). 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 B 12 and propionic acid, including pH, temperature, fermentation time, inoculum size, the carbon and nitrogen sources and their concentrations, as well as fermentation strategies [27]. On the basis of previous studies, the effects of glucose feeding and membrane separation-coupled operation on the simultaneous production of vitamin B 12 and propionic acid were analyzed. Generally, cell growth was accompanied by the synthesis of Table 1 Metabolic reactions of simultaneous production of vitamin B 12 and propionic acid by Propionibacterium freudenreichii
To overcome the feedback inhibition of propionic acid synthesis, a membrane separationcoupled 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 B 12 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 B 12 and propionic acid by Propionibacterium is shown in Table 3. Compared with the expanded bed reactor (EBAreactor) [22,33], 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 B 12 and propionic acid. However, the coupled system still needs to be improved, 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 B 12 and propionic acid synthesis was established based on the current literature [29,35].  During the fermentation process of P freudenreichii, the consumption rate of glucose (r 1 ), and the production rates of acetic acid (r 11 ), propionic acid (r 13 ), lactic acid (r 17 ), succinic acid (r 21 ), heme (r 24 ), and vitamin B 12 (r 27 ), 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 B 12 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 B 12 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 B 12 synthesis, by condensation with glycine [36]. 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 B 12 . 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 B 12 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 B 12
can be regulated at both the transcriptional and metabolic levels [37,38]. A cobalamin riboswitch is used to quickly respond to the environment [39]. It was reported that a complex interdependent and interactional relationship exists among the tetrapyrrole compounds [26].
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 B 12 . 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 B 12 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 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 B 12 [40,41]. However, because of its low concentration, α-pyrroleribose was a key rate-limiting factor in vitamin B 12 synthesis, which led to the accumulation of intracellular Ado-cbi-GDP [42]. During the fermentation process, additional DMB was required to promote the conversion of Ado-cbi-GDP to vitamin B 12 .
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 B 12 synthesis. Details are shown in Fig. 7. The specific effects observed were as follows. (1) Increased synthesis of pyruvate by increased flux of 3phosphoglycerate 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 B 12 synthesis) was lower than that to heme. In fact, the concentration of heme was slightly higher than that of vitamin B 12 . 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.

Conclusions
The simultaneous production of vitamin B 12 and propionic acid by P. freudenreichii has attracted wide attention. In the membrane separation-coupled fermentation process described here, the concentration of vitamin B 12 and propionic acid reached 21.6 mg/L and 50.12 g/L Fig. 6 Metabolic flux distribution at uroporphyrinogen III node. (a) Batch fermentation; (b) fed-batch fermentation; (c) coupled fermentation 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 B 12 and propionic acid.