N-acetylneuraminic acid fermentation method capable of reducing fermentation byproducts

Abstract

The invention relates to an N-acetylneuraminic acid fermentation method capable of reducing fermentation byproducts, and belongs to the technical field of fermentation. Escherichia coli is used as a chassis strain for fermentation synthesis of N-acetylneuraminic acid, sodium hexametaphosphate is added in the fermentation process to reduce generation of byproducts in the synthesis process of N-acetylneuraminic acid, and fermentation conditions are further optimized to improve the synthesis yield of N-acetylneuraminic acid. According to the fermentation method, the N-acetylneuraminic acid can be efficiently synthesized, the yield of the N-acetylneuraminic acid is increased, meanwhile, by-products difficult to separate are reduced, and a technical basis is provided for subsequent fermentation amplification and industrial production of the N-acetylneuraminic acid.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a fermentation method for reducing fermentation byproducts of N-acetylneuraminic acid. Background Technology

[0002] N-acetylneuraminic acid (NeuAc) is an amino sugar widely found in breast milk and red meat. Due to its various physiological activities, including promoting infant brain development, improving memory and learning abilities, and enhancing immune function, it has been widely used in the food, health product, and pharmaceutical industries. With the continuous deepening of research in related fields and the growth of industrial demand, the demand for industrial production of NeuAc is increasing daily.

[0003] Currently, the synthesis of NeuAc mainly includes five methods: natural product extraction, chemical synthesis, enzymatic catalysis, whole-cell catalysis, and microbial fermentation. However, the first four methods generally suffer from low yield, high cost, cumbersome operation, and environmental pollution, making it difficult to meet the needs of large-scale, sustainable industrial production. In contrast, microbial fermentation has advantages such as inexpensive raw materials, simple operation, green and environmentally friendly production process, and relatively low cost, and has become a research hotspot and development direction for the large-scale production of NeuAc.

[0004] Against this backdrop, *Escherichia coli*, as a typical representative of model microorganisms, is considered an ideal chassis cell for NeuAc synthesis due to its clear genetic background, well-defined metabolic pathways, strong gene editability (such as the widespread application of the CRISPR-Cas9 system), ease of integration of exogenous genes, short fermentation cycle, and good metabolic plasticity. However, the metabolic pathways of *E. coli* are very complex, involving multiple enzymes and intermediates. Many enzymes have broad substrate scope and can catalyze multiple reactions, which may lead to the generation of non-target products. Therefore, it is necessary to further optimize the fermentation conditions of *E. coli* to increase the yield of N-acetylneuraminic acid while reducing fermentation byproducts. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem of high by-products and low yield in the production of N-acetylneuraminic acid by fermentation of Escherichia coli in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a fermentation method for N-acetylneuraminic acid (NNA) with reduced fermentation byproducts. This invention uses *Escherichia coli* as the substrate strain for the fermentation synthesis of NNA. Sodium hexametaphosphate is added during fermentation to reduce the generation of byproducts during NNA synthesis, and fermentation conditions are further optimized to increase the yield of NNA. The method of this invention enables efficient synthesis of NNA, increasing yield while reducing the occurrence of difficult-to-separate byproducts, providing a technical foundation for subsequent fermentation scale-up and industrial production of NNA.

[0007] The first objective of this invention is to provide a fermentation method for N-acetylneuraminic acid that reduces fermentation byproducts. The fermentation method involves inoculating Escherichia coli into a fermentation medium containing sodium hexametaphosphate for fermentation culture. During the fermentation process, isopropyl-β-D-thiogalactoside, glucose, and ammonium sulfate are added. After purification, the N-acetylneuraminic acid is obtained. The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and metal ions.

[0008] Furthermore, the amount of sodium hexametaphosphate added is 0.01-0.5 g / L.

[0009] Furthermore, the fermentation medium contains ammonium phosphate, yeast powder, tryptone, glucose, trisodium citrate dihydrate, malic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium chloride, vitamin B1, and trace element stock solution.

[0010] Furthermore, the trace element mother liquor includes EDTA, zinc sulfate heptahydrate, manganese chloride dihydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, sodium permanganate dihydrate, calcium chloride dihydrate, ferric sulfate heptahydrate, boric acid, and potassium iodide.

[0011] Furthermore, the concentration of glucose in the fermentation medium is 10-20 g / L, preferably 15 g / L.

[0012] Furthermore, the fermentation temperature is 25-37℃.

[0013] Furthermore, the isopropyl-β-D-thiogalactoside (IPTG) is added 3-14 hours after Escherichia coli is inoculated into the fermentation medium.

[0014] Furthermore, during the fermentation process, 10 g / L of glucose was added simultaneously with the addition of isopropyl-β-D-thiogalactoside.

[0015] Furthermore, the concentration of the isopropyl-β-D-thiogalactoside is 3-100 mM.

[0016] Furthermore, the *E. coli* includes *E. coli* Nissle 1917.

[0017] Furthermore, the fermentation time is 31-47 hours.

[0018] The beneficial effects of this invention are:

[0019] This invention optimizes fermentation using *E. coli* as the substrate strain. Sodium hexametaphosphate is added during fermentation to reduce the generation of byproducts in N-acetylneuraminic acid synthesis, and fermentation conditions are further optimized to increase the yield of N-acetylneuraminic acid. The method of this invention enables efficient synthesis of N-acetylneuraminic acid, increasing yield while reducing the occurrence of difficult-to-separate byproducts, providing a technical foundation for subsequent fermentation scale-up and industrial production of N-acetylneuraminic acid. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Figure 1 This is a graph showing the effect of different culture media on the yield of N-acetylneuraminic acid;

[0022] Figure 2 This is a graph showing the effect of different carbon sources on the production of N-acetylneuraminic acid.

[0023] Figure 3 The graph shows the effect of different fermentation times on the yield of N-acetylneuraminic acid.

[0024] Figure 4 This is a graph showing the effects of IPTG concentration and addition time on N-acetylneuraminic acid.

[0025] Figure 5 The figure shows the experimental results for the optimal induction concentration and induction time of IPTG.

[0026] Figure 6 The graph shows the effect of different fermentation temperatures on the yield of N-acetylneuraminic acid.

[0027] Figure 7 The graph shows the effect of sodium hexametaphosphate addition on the yield of N-acetylneuraminic acid.

[0028] Figure 8 This is the liquid chromatogram before fermentation optimization;

[0029] Figure 9 This is a liquid chromatogram of optimized fermentation conditions without the addition of sodium hexaphosphate;

[0030] Figure 10This is a liquid chromatogram after adding sodium hexametaphosphate under optimal fermentation conditions. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0032] (a) Culture medium

[0033] LB medium for Escherichia coli: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0034] IS medium: magnesium sulfate heptahydrate 2.5 g / L, vitamin B1 0.45 g / L, trace element stock solution 2 mL / L, ammonium sulfate 10 g / L, glucose 10 g / L, trisodium citrate dihydrate 2 g / L, malic acid 1 g / L, disodium hydrogen phosphate 6.8 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L.

[0035] RM medium: glucose 40g / L, ammonium sulfate 5.5g / L, yeast extract 5g / L, magnesium sulfate heptahydrate 2.5g / L, vitamin B1 0.45g / L, trace element stock solution 2mL / L, sodium dihydrogen phosphate 4.2g / L, potassium dihydrogen phosphate 8.7g / L.

[0036] MM medium: ammonium phosphate 10g / L, yeast extract 2.5g / L, tryptone 5g / L, glucose 15g / L, trisodium citrate dihydrate 2g / L, malic acid 1g / L, disodium hydrogen phosphate 6.8g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.5g / L, ammonium chloride 1g / L, vitamin B1 0.45g / L, trace element stock solution 2mL / L.

[0037] Trace element stock solution: EDTA 15g / L, zinc sulfate heptahydrate 4.5g / L, manganese chloride dihydrate 0.84g / L, cobalt chloride hexahydrate 0.3g / L, copper sulfate pentahydrate 0.3g / L, sodium permanganate dihydrate 0.4g / L, calcium chloride dihydrate 4.5g / L, ferric sulfate heptahydrate 3g / L, boric acid 1g / L, potassium iodide 0.1g / L.

[0038] (II) Strain Culture Methods

[0039] One loopful of E. coli Nissle 1917 glycerol culture was taken from the inoculation loop and streaked onto an LB agar plate, incubated at 37°C for 12 hours. Then, the colonies were transferred from the plate to a shaker tube containing 2 mL of LB liquid medium and incubated at 37°C and 220 rpm for 12 hours to obtain a seed culture. This seed culture was used as the inoculation source for subsequent experiments.

[0040] (III) Fermentation Cultivation Method

[0041] The seed culture was transferred at a 2% inoculum to 24-well plates containing 1.5 mL of fermentation medium, labeled, and incubated at 37°C and 220 r·min. -1 The mixture was cultured in a shaker under shaking conditions. After the induction time, IPTG, 10 g / L glucose, and 8 g / L ammonium sulfate were added. Fermentation ended after 48 h, and the yield of the target product was determined by collecting the fermentation supernatant.

[0042] (iv) N-acetylneuraminic acid detection

[0043] This invention employs high-performance liquid chromatography (HPLC) to determine the synthesis yield of NeuAc in fermentation broth. An HPLC detection system (Waters, USA) equipped with a Bio-Rad Aminex® HPX-87H column (7.8 × 300 mm) and a UV detector (210 nm) was used to detect the content of each target metabolite in the sample. The column temperature was set at 40°C, and the mobile phase was 10 mmol·L⁻¹. -1 H2SO4, flow rate 0.5 mL·min -1 Under these detection conditions, the retention time of NeuAc was 9.7 min.

[0044] (v) Sample processing methods

[0045] Take 1 mL of fermentation broth into a 1.5 mL centrifuge tube and incubate at 4℃ and 12000 r·min. -1 Cells were obtained by centrifugation for 10 min under the specified conditions. 200 μL of fermentation supernatant was then added to a solution containing 600 μL of 35 mmol / L... -1 In a centrifuge tube containing H2SO4, mix thoroughly and centrifuge again for 10 min under the same conditions. Remove air bubbles and impurities by passing through a 0.22 m aqueous filter membrane, then add to a sample bottle to obtain the sample to be tested.

[0046] Example 1: Optimization of basal culture medium composition during fermentation

[0047] like Figure 1As shown, fermentation was carried out at 37℃ with 0.2 mM IPTG induction for 5 h using IS, RM, and MM media, respectively, to compare the effects of different nitrogen source components on NeuAc synthesis. The results showed that the MM medium group had the highest NeuAc yield, reaching 1.88 g / L, which was 4.06 times that of the IS medium group and 6.16 times that of the RM medium group. This indicates that an appropriate amount of nitrogen source in MM medium significantly promoted the synthesis of NeuAc by *E. coli* Nissle 1917. Therefore, MM medium was used for fermentation culture in subsequent experiments.

[0048] Example 2: Fermentation optimization with different carbon sources

[0049] like Figure 2 As shown, to investigate the differences in carbon source utilization and NeuAc synthesis under different carbon source conditions, this invention compared the carbon source utilization rate, consumption rate, and NeuAc synthesis of *E. coli* Nissle 1917 with different carbon sources on MM medium, using 15 g glycerol, 15 g glucose, and 7.5 g glucose + 7.5 g glycerol (hereinafter referred to as hemiglucose hemiglucose) as carbon sources. The NeuAc yield was 2.99 g / L in the glucose group, 0.995 g / L in the glycerol group, and 0.99 g / L in the hemiglucose hemiglucose group. The yield in the glucose group was 201% and 202% higher than that in the glycerol and hemiglucose hemiglucose groups, respectively. In conclusion, *E. coli* Nissle 1917 exhibited the highest NeuAc yield under glucose as the carbon source condition. Therefore, glucose was selected as the sole carbon source in subsequent experiments to further optimize NeuAc production.

[0050] Example 3: Fermentation Time Optimization

[0051] like Figure 3 As shown, with the carbon and nitrogen source components of the culture medium determined, the NeuAc-producing strain was continuously fermented at 37℃ with 0.2 mM IPTG for 5 h of induction. Samples were taken at intervals during fermentation to determine the optimal fermentation time. The results showed that the NeuAc yield continuously increased during the fermentation process from 0 to 31 h, and fluctuated slightly from 31 to 47 h, but the yield tended to stabilize.

[0052] Example 4: Optimization of IPTG Concentration and Induction Time

[0053] like Figure 4As shown, adjusting the IPTG induction concentration and induction time can balance gene expression intensity and cellular metabolic state, thereby increasing NeuAc yield. In deep-well plates at 37°C on MM medium, this example added IPTG at gradient concentrations of 0.1-3 mM at 5 h, 8 h, 11 h, and 14 h of fermentation to screen for optimal induction fermentation conditions. NeuAc yield was measured at 47 h of fermentation. The results showed that the highest NeuAc yield (3.95 g / L) was achieved at 5 h with the addition of 3 mM IPTG. NeuAc yield decreased overall with increasing induction time, and excessively high IPTG concentrations also inhibited NeuAc yield at the same time point. A second round of optimization experiments was conducted to further determine the optimal induction conditions.

[0054] like Figure 5 As shown, a second round of induction experiments was conducted to further determine the optimal induction concentration and induction time of IPTG. Under MM medium conditions at 37℃, IPTG concentrations ranging from 3 to 100 mM were added at 0 h, 3 h, and 5 h of fermentation for optimization. NeuAc yield was measured at 47 h of fermentation. The results showed that NeuAc yield generally increased with time in the 0-5 h range; NeuAc yield generally increased in the 0-75 mM IPTG concentration range, while the yield remained relatively stable in the 75-100 mM range. In all three groups, NeuAc yield reached its peak at 3 mM IPTG, at 2.11 g / L (0 h), 1.49 g / L (3 h), and 3.98 g / L (5 h), respectively. In conclusion, the optimal concentration of the inducing agent for NeuAc production from Nissle 1917 fermentation is 3 mM, and the optimal addition time is 5 h of fermentation.

[0055] Example 5: Fermentation Temperature Optimization

[0056] like Figure 6 As shown, to determine the optimal temperature for NeuAc production by fermentation of E. coli Nissle 1917, this experiment compared NeuAc yield at 25℃, 30℃, and 37℃. The experiment was conducted using MM medium, 3 mM IPTG, and a 5-hour induction period. The results showed that the highest NeuAc yield was observed at 37℃, reaching 5.13 g / L, which was 1.23 times that of the 25℃ group and 1.12 times that of the 30℃ group. Therefore, within the range of 25–37℃, the optimal temperature for NeuAc production by fermentation of Nissle 1917 is 37℃.

[0057] Example 6: Addition of sodium hexametaphosphate

[0058] Sodium hexametaphosphate (SHP) is an inexpensive inorganic chemical that can serve as a phosphate donor in the synthesis of high-energy phosphate compounds such as ATP. Appropriate addition can provide a recombinant energy supply for complex metabolic processes. In this example, under optimal fermentation conditions, different amounts of SHP (0, 0.01, 0.05, 0.1, and 0.5 g / L) were added to test the optimal addition amount. Ultimately, with the addition of 0.05 g / L SHP, the NeuAc yield reached 5.97 g / L, a 16% increase compared to the control without SHP. This result demonstrates that by adding a very small amount of inexpensive substrate, NeuAc yield can be optimized and the generation of byproducts (such as...) can be reduced. Figure 7 (As shown).

[0059] Test case

[0060] like Figures 8-10 As shown, the fermentation broth after optimization was analyzed by HPLC. The main products after fermentation were detected at a wavelength of 210 nm. Before optimization, in addition to the target product NeuAc, there were also a large amount of pyruvic acid and acetic acid residues. In addition, there were also residues of ManNAc, the direct precursor of NeuAc. When only the fermentation conditions were optimized but no sodium hexaphosphate was added, the by-products still remained at a high level. However, after adding sodium hexaphosphate, the three main by-products were significantly reduced. The organic acids in the fermentation broth were mainly the target product NeuAc.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fermentation method for reducing N-acetylneuraminic acid fermentation byproducts, characterized in that, The fermentation method involves inoculating Escherichia coli into a fermentation medium containing sodium hexametaphosphate for fermentation culture. During the fermentation process, isopropyl-β-D-thiogalactoside, glucose, and ammonium sulfate are added. After purification, the N-acetylneuraminic acid is obtained. The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and metal ions.

2. The fermentation method according to claim 1, characterized in that, The concentration of the sodium hexametaphosphate is 0.01-0.5 g / L.

3. The fermentation method according to claim 1, characterized in that, The fermentation medium includes ammonium phosphate, yeast extract, tryptone, glucose, trisodium citrate dihydrate, malic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium chloride, vitamin B1, and trace elements.

4. The fermentation method according to claim 3, characterized in that, The trace elements include ethylenediaminetetraacetic acid, zinc sulfate heptahydrate, manganese chloride dihydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, sodium permanganate dihydrate, calcium chloride dihydrate, ferric sulfate heptahydrate, boric acid, and potassium iodide.

5. The fermentation method according to claim 1, characterized in that, The inoculation amount of Escherichia coli is 1-10%.

6. The fermentation method according to claim 1, characterized in that, The fermentation culture temperature is 25-37℃.

7. The fermentation method according to claim 1, characterized in that, The isopropyl-β-D-thiogalactoside was added 3-14 hours after Escherichia coli was inoculated into the fermentation medium.

8. The fermentation method according to claim 1, characterized in that, The concentration of the isopropyl-β-D-thiogalactoside is 3-100 mM.

9. The fermentation method according to claim 1, characterized in that, The Escherichia coli includes Escherichia coli Nissle1917.

10. The fermentation method according to claim 1, characterized in that, The fermentation time is 31-47 hours.

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