A method for fermentative production of 5-hydroxyvaleric acid and 6-hydroxyhexanoic acid
By utilizing substrates such as glucose, ammonium sulfate, and 6-aminohexanoic acid through fermentation and optimizing fermentation parameters, the environmental friendliness issues of chemical synthesis of 5-hydroxyvalerate and 6-hydroxyhexanoic acid have been resolved, achieving high-efficiency production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DONGHUA UNIV
- Filing Date
- 2026-01-30
- Publication Date
- 2026-06-12
AI Technical Summary
Existing chemical methods for synthesizing 5-hydroxyvalerate and 6-hydroxyhexanoic acid require expensive catalysts, high temperatures, and petroleum resources, and generate byproducts. There is a lack of environmentally friendly biological production methods.
5-Hydroxyvalerate and 6-hydroxyhexanoic acid are produced by fermentation. Glucose, ammonium sulfate and 6-aminohexanoic acid are used as substrates in batch fermentation. Dissolved oxygen and ammonia nitrogen concentrations are controlled during fermentation, respiratory quotient (RQ) is monitored, and fermentation parameters such as rotation speed and air volume are optimized to achieve high-efficiency production.
It achieves efficient and environmentally friendly production of 5-hydroxyvalerate and 6-hydroxyhexanoic acid, simplifies operations, and has industrialization potential.
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Figure CN122189113A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fermentation engineering technology, and specifically relates to a method for fermentation production of 5-hydroxyvalerate and 6-hydroxyhexanoic acid. Background Technology
[0002] 5-Hydroxyvalerate (5-HV) and 6-hydroxyhexanoate (6-HHA) possess bifunctional groups, with a hydroxyl group at one end and a carboxyl group at the other. These active functional groups at both ends give them more important chemical properties than ordinary compounds, leading to their wide applications in medical and chemical industries. 5-Hydroxyvalerate can serve as an intermediate in the synthesis of various drugs and is a precursor for a variety of widely used compounds, including δ-caprolactone, glutaric acid, 1,5-pentanediol, and polyhydroxyalkanoates. 6-Hydroxyhexanoate is an antifibrinolytic agent with excellent anti-inflammatory and insulin-sensitizing effects. Furthermore, it is a precursor for the synthesis of ε-caprolactone, polyε-caprolactone, and other medical polymer degradable materials.
[0003] Currently, 5-hydroxyvalerate and 6-hydroxyhexanoic acid are mostly synthesized chemically. 5-hydroxyvalerate can be obtained by decomposing 2-furanic acid and reducing glutaric acid, while 6-hydroxyhexanoic acid can be synthesized from adipic acid and hexanediol. These synthetic methods require petroleum resources as raw materials, and the processes often require expensive catalysts, hydrogenation, high temperatures, and the generation of byproducts. Compared to chemical methods, biological methods for synthesizing 5-hydroxyvalerate and 6-hydroxyhexanoic acid offer significant advantages, including renewable raw materials, milder reaction conditions, and environmental friendliness, effectively reducing dependence on petroleum resources. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for producing 5-hydroxyvalerate and 6-hydroxyhexanoic acid by fermentation.
[0005] This invention provides a method for fermenting to produce 5-hydroxyvalerate and 6-hydroxyhexanoic acid, comprising:
[0006] The seed culture containing the bacterial strain is inoculated into a fermentation medium, and the target product is obtained through batch fermentation; wherein one or more of glucose, ammonium sulfate, and 6-aminocaproic acid are added during the batch fermentation process.
[0007] The fermentation process produces 5-hydroxyvalerate and 6-hydroxyhexanoic acid, wherein the substrate for the fermentation of 5-hydroxyvalerate is glucose, and the substrate for the fermentation of 6-hydroxyhexanoic acid is 6-aminohexanoic acid.
[0008] Preferably, the process of adding glucose is as follows: glucose solution is added during fermentation so that the glucose concentration in the fermentation broth is 0.5-1.0% (w / v).
[0009] Preferably, the process of adding ammonium sulfate is as follows: ammonium sulfate is added during fermentation so that the ammonia nitrogen content in the fermentation broth is 0-0.15% (w / v), more preferably 0-0.05% (w / v).
[0010] Preferably, the process of adding 6-aminohexanoic acid is as follows: the amount and rate of 6-aminohexanoic acid added are implemented according to the generation rate and amount of 5-hydroxyvalerate.
[0011] The flow acceleration of the 6-aminohexanoic acid is the flow velocity. Where M refers to the relative molecular mass of 6-aminohexanoic acid, Mo refers to the relative molecular mass of 5-hydroxyvalerate, and C2V2-C1V1 refers to... The amount of 5-hydroxyvalerate produced by T, T represents the time period, and C refers to the concentration of the added 6-aminocaproic acid aqueous solution. T is T2-T1.
[0012] Preferably, the respiratory quotient (RQ) is monitored during fermentation. When the yield of 5-hydroxyvalerate is increased, the RQ decreases to 0.7-0.8 during fermentation. Through process control, dissolved oxygen is maintained at 50-80%, and the RQ value is maintained at 0.9-1.0.
[0013] Maintaining dissolved oxygen at 50-80% is achieved by increasing fermentation speed and / or increasing air volume.
[0014] When the yield of 6-hydroxyhexanoic acid is increased, the RQ during fermentation decreases to 0.7-0.8. Through process control, dissolved oxygen is maintained at 10-20%, and the RQ value is maintained at 0.6-0.7.
[0015] Maintaining dissolved oxygen at 10-20% is achieved by reducing fermentation speed and / or reducing airflow.
[0016] Preferably, the fermentation process parameters include: inoculum size 0.2-15% (v / v), aeration rate 0.4-0.6 vvm, tank pressure 0.05-0.1 MPa, pH of the fermentation broth controlled at 6.7-7.2 by adding sodium hydroxide; initial fermentation speed controlled at 300-400 rpm, the fermentation speed is related to dissolved oxygen during fermentation, initial fermentation temperature maintained at 37℃, after 9-12 h of fermentation, IPTG is added to make the final IPTG concentration in the fermentation broth 0.05 mM, and then the fermentation temperature is controlled at 30℃ until the end of fermentation.
[0017] During the early and middle stages of fermentation, dissolved oxygen is maintained at 20-40%. In the later stages of fermentation, RQ gradually decreases to 0.7-0.8, at which point the dissolved oxygen maintenance range is adjusted.
[0018] The concentration of the added sodium hydroxide solution is 10-40% (w / v).
[0019] The strains include, but are not limited to, one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast.
[0020] Furthermore, the strain is obtained by overexpressing one or more of the following on a chassis: lysine 2-monooxygenase (DavB), δ-aminopentanoamidinase (DavA), γ-aminobutyrate transaminase (GabT), aldehyde reductase (YqhD), aspartate kinase (LysC), phosphoenolpyruvate carboxylase, dihydropyridine dicarboxylic acid synthase, and diaminopimelic acid decarboxylase, to obtain a genetically engineered strain.
[0021] The fermentation medium comprises inorganic salts, nitrogen source, glucose, amino acids, and vitamins.
[0022] The inorganic salts mentioned therein include, but are not limited to, one or more of magnesium sulfate, copper sulfate, ferrous sulfate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; the nitrogen sources include, but are not limited to, one or more of ammonium sulfate, corn steep liquor, yeast extract, and urea; the amino acids include, but are not limited to, one or more of glutamic acid, threonine, and lysine; and the vitamins include, but are not limited to, one or more of vitamin B1, vitamin B2, vitamin C, and vitamin B6.
[0023] The seed culture medium containing the strain in this invention includes: strain activation and seed culture preparation.
[0024] The strain activation process is as follows: After the glycerol tube seed is naturally thawed, 300 μL of seed solution is pipetted onto a plate culture medium, then spread onto LB agar plates using a spreader, and incubated upside down in a 37℃ constant temperature incubator for 24 h. The corresponding antibiotics are added to the LB agar as needed.
[0025] Seed culture preparation: After bacterial colonies form on the agar plates, pick up the bacterial cells from the plates using an inoculation loop and transfer them to a seed shake flask. Incubate at 37°C and 200 rpm for 8-12 h on a shaker. Inoculate this seed culture into a fermenter. The components of the seed shake flask culture medium are the same as those of the agar plate culture medium.
[0026] Beneficial effects
[0027] The fermentation method provided by this invention specifically employs a feed-through strategy of one or more of glucose, ammonium sulfate, and aminocaproic acid, which can achieve efficient production of 5-hydroxyvalerate and 6-hydroxycaproic acid.
[0028] The fermentation method of this invention has high production efficiency, is easy to operate, and has industrialization potential. Attached Figure Description
[0029] Figure 1 The metabolic pathways and related enzymes for the synthesis of 5-hydroxyvalerate and 6-hydroxyhexanoate were constructed.
[0030] Figure 2 This is a graph showing the online parameter curves of the fermentation process in Scheme 3 of Example 1;
[0031] Figure 3 This is a graph showing the microbial growth, changes in 5-hydroxyvalerate content, and 5-hydroxyvalerate synthesis rate during the fermentation process in Example 2.
[0032] Figure 4 This is a graph showing the online parameters of the fermentation process in Example 2;
[0033] Figure 5 This is a graph showing the online parameter curves of the fermentation process in Scheme 6 of Example 4. Detailed Implementation
[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0035] The contents of 5-hydroxyvalerate and 6-hydroxyhexanoic acid in the fermentation broth were determined by gas chromatography with external standard method. Specifically, the supernatant of the fermentation broth was centrifuged and analyzed using a GC2010Pro (Shimadzu) microscope. The inlet temperature was set to 280℃, and the nitrogen flow rate was 2.6 mL / min. Autosampler was used with an injection volume of 0.5 μL, a split ratio of 30:1, an FID detector temperature of 300℃, an air flow rate of 400 mL / min, a nitrogen flow rate of 25 mL / min, and a hydrogen flow rate of 30 mL / min. The column temperature program was set as follows: initial temperature 50℃, hold for 5 min, then increase to 210℃ and hold for 5 min. The column type was Wondacap-530m×0.25mm×0.25µm.
[0036] Example 1
[0037] The effect of different ammonium sulfate feeding strategies on the synthesis of 5-hydroxyvalerate during fermentation
[0038] The strains used in fermentation include, but are not limited to, one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast. Specifically, in this embodiment, a genetically engineered strain is used, specifically Escherichia coli W3110 as the chassis strain. This strain overexpresses lysine 2-monooxygenase and δ-aminovaleramidinase from Pseudomonas putida, γ-aminobutyric acid transaminase and aldehyde reductase genes from Escherichia coli, as well as aspartate kinase, phosphoenolpyruvate carboxylase, dihydropyridine dicarboxylic acid synthase, and diaminopimelic acid decarboxylase, thus constructing the genetically engineered strain.
[0039] The specific fermentation process is as follows:
[0040] The strain activation process is as follows: After the glycerol tube seed is naturally thawed, 300 μL of seed solution is pipetted onto a plate culture medium, then spread onto LB agar plates using a spreader, and incubated upside down in a 37℃ constant temperature incubator for 24 h. The corresponding antibiotics are added to the LB agar as needed.
[0041] Seed culture preparation: When bacterial colonies form on the plate, pick up the bacterial cells from the plate with an inoculation loop and place them in a seed shake flask. Incubate at 37°C and 200 rpm for 8-12 h to obtain a seed culture containing the bacterial strain. Inoculate the seed culture into a fermenter.
[0042] The fermentation process parameters were as follows: inoculum size 0.5%, aeration rate 0.6 vvm, tank pressure 0.05-0.1 MPa, and addition of 10-40% (w / v) sodium hydroxide solution to maintain the pH of the fermentation broth at 6.7. The initial fermentation speed was controlled at 300 rpm, and the stirring speed during fermentation was correlated with dissolved oxygen levels, which were maintained at 20-40%. The initial fermentation temperature was maintained at 37℃. After 9 hours of fermentation, IPTG was added to achieve a final IPTG concentration of 0.05 mM in the fermentation broth, and the fermentation temperature was maintained at 30℃ to induce the expression of related genes and the synthesis of 5-hydroxyvalerate. During fermentation, 40-70% (w / v) glucose solution was added to maintain a glucose concentration of 0.5-1.0% in the fermentation broth.
[0043] Three ammonium sulfate feeding strategies were employed during the fermentation process:
[0044] Option 1: When the ammonia nitrogen content in the fermentation broth is 0.15%, start adding ammonium sulfate solution to make the ammonia nitrogen content in the fermentation broth between 0.1% and 0.15%.
[0045] Option 2: When the ammonia nitrogen content in the fermentation broth is 0.1%, start adding ammonium sulfate solution to bring the ammonia nitrogen content in the fermentation broth to 0.05-0.1%.
[0046] Option 3: When the ammonia nitrogen content in the fermentation broth is 0.05%, start adding ammonium sulfate solution to keep the ammonia nitrogen content in the fermentation broth between 0-0.05%.
[0047] Fermentation results: As shown in Table 1, 5-hydroxyvalerate was produced using three ammonium sulfate feedstock strategies. Scheme 3 yielded the highest 5-hydroxyvalerate, reaching 19.59 g / L.
[0048] Table 1
[0049]
[0050] Example 2
[0051] Effects of different dissolved oxygen control strategies on 5-hydroxyvalerate
[0052] The process parameters and methods of the third scheme in Example 1 were followed, with the difference being that the fermentation speed was related to dissolved oxygen. Dissolved oxygen was maintained at 20-40% in the early and middle stages of fermentation, while the RQ gradually decreased to 0.7-0.8 in the later stages. By adjusting the fermentation speed to maintain dissolved oxygen at 50-80%, the RQ was increased to 0.9-1.0. The final yield of 5-hydroxyvalerate in the fermentation broth reached 28.69 g / L.
[0053] Example 3
[0054] Following the process parameters and methods of Example 2, 10 g / L of 6-aminohexanoic acid was added to the fermentation medium for fermentation, resulting in the co-production of 5-hydroxyvalerate and 6-hydroxyhexanoic acid. Ultimately, the yield of 5-hydroxyvalerate in the fermentation broth was 19.37 g / L, and the yield of 6-hydroxyhexanoic acid was 1.71 g / L.
[0055] Example 4
[0056] Effects of different 6-aminohexanoic acid addition strategies on the synthesis of 5-hydroxyvalerate and 6-hydroxyhexanoic acid
[0057] Based on the process parameters and methods of Example 2, four schemes were implemented to supplement 6-aminohexanoic acid during fermentation, based on the formation rate of 5-hydroxyvalerate in Example 2:
[0058] Option 4: Based on Example 2, after 24-36 h of fermentation, the synthesis rate of 5-hydroxyvalerate was approximately 0.5-0.7 g·L⁻¹. -1 ·h -1 Based on the 5-hydroxyvalerate synthesis rate at this stage, 1160 mL of a solution containing 6-aminohexanoic acid was added at a flow rate of 96 mL / h.
[0059] Option 5: Based on Example 2, after 36-54 h of fermentation, the synthesis rate of 5-hydroxyvalerate was approximately 0.4-0.6 g·L. -1 ·h -1 Based on the 5-hydroxyvalerate synthesis rate at this stage, 1420 mL of 6-aminohexanoic acid solution was added at a flow rate of 79 mL / h.
[0060] Option 6: Based on the fermentation method of Example 2, after 24-60 h, the synthesis rate of 5-hydroxyvalerate was approximately 0.4-0.7 g·L. -1 ·h -1 Based on the 5-hydroxyvalerate synthesis rate at this stage, 3070 mL of 6-aminohexanoic acid solution was added at a flow rate of 85 mL / h. The fermentation speed was correlated with dissolved oxygen. Dissolved oxygen was maintained at 20-40% in the early and middle stages of fermentation. In the later stages, the RQ gradually decreased to 0.7-0.8, and the fermentation speed was reduced to maintain dissolved oxygen at 10-20%, thus lowering the RQ to 0.6-0.7.
[0061] Option 7: Based on Option 6, feed 6-aminocaproic acid without feeding glucose during fermentation.
[0062] Table 2. Fermentation results of 5-hydroxyvalerate and 6-hydroxyhexanoate under different schemes.
[0063]
Claims
1. A method for fermenting to produce 5-hydroxyvalerate and 6-hydroxyhexanoic acid, comprising: The seed culture containing the bacterial strain was inoculated into a fermentation medium, and the target product was obtained through batch fermentation. During batch fermentation, one or more of glucose, ammonium sulfate, and 6-aminocaproic acid are added.
2. The method according to claim 1, characterized in that, The substrate for the fermentation production of 5-hydroxyvalerate is glucose; the substrate for the fermentation production of 6-hydroxyhexanoate is 6-aminohexanoate.
3. The method according to claim 1, characterized in that, The process of adding glucose is as follows: during fermentation, a glucose solution is added to maintain a glucose concentration of 0.5-1.0% in the fermentation broth; The process of adding ammonium sulfate is as follows: ammonium sulfate is added during fermentation to make the ammonia nitrogen content in the fermentation broth 0-0.15%.
4. The method according to claim 1, characterized in that, The process of adding 6-aminohexanoic acid is as follows: the amount and rate of 6-aminohexanoic acid added are implemented according to the generation rate and amount of 5-hydroxyvalerate. The flow acceleration of the 6-aminohexanoic acid is the flow velocity. Where M refers to the relative molecular mass of 6-aminohexanoic acid, Mo refers to the relative molecular mass of 5-hydroxyvalerate, and C2V2-C1V1 refers to... The amount of 5-hydroxyvalerate produced by T, T represents the time period, and C refers to the concentration of the added 6-aminocaproic acid aqueous solution.
5. The method according to claim 1, characterized in that, During fermentation, the respiratory quotient (RQ) was monitored. When the yield of 5-hydroxyvalerate was increased, the RQ decreased to 0.7-0.8 during fermentation. Through process control, dissolved oxygen was maintained at 50-80%, and the RQ was maintained at 0.9-1.
0. When the yield of 6-hydroxyhexanoic acid is increased, the RQ during fermentation decreases to 0.7-0.
8. Through process control, dissolved oxygen is maintained at 10-20%, and the RQ value is maintained at 0.6-0.
7.
6. The method according to claim 1, characterized in that, Fermentation process parameters include: inoculum size 0.2-15%, aeration rate 0.4-0.6 vvm, tank pressure 0.05-0.1 MPa, pH of fermentation broth controlled at 6.7-7.2 by adding sodium hydroxide; initial fermentation speed controlled at 300-400 rpm, the speed is related to dissolved oxygen during fermentation, initial fermentation temperature maintained at 37℃, after 9-12 h of fermentation, IPTG is added to make the final IPTG concentration in fermentation broth 0.05 mM, and then the fermentation temperature is controlled at 30℃ until fermentation ends.
7. The method according to claim 1, characterized in that, The strains include one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast.
8. The method according to claim 1, characterized in that, The fermentation medium comprises inorganic salts, nitrogen source, glucose, amino acids, and vitamins.