Fermentation process capable of shortening fermentation period and efficiently synthesizing Ectoine

Abstract

The invention discloses a fermentation process for efficiently synthesizing Ectoine by shortening a fermentation period, which is characterized in that the fermentation process for producing Ectoine by fermenting engineering escherichia coli taking glucose, xylose and arabinose as a mixed carbon source is adopted, so that the fermentation time is shortened to 40.5 h, the yield of Ectoine can reach 112.2 g / L, the production efficiency of Ectoine reaches 2.77 g / L / h, and the yield of Ectoine is greatly improved. And compared with fermentation production of Ectoin by taking glucose as a carbon source, the efficiency is improved by 132%.

Description

Technical Field

[0001] The invention belongs to the field of fermentation engineering and relates to a fermentation process for efficiently synthesizing ectoine by shortening the fermentation cycle. Background Art

[0002] Ecdoin, also called tetrahydropyrimidine, is a derivative of cyclic amino acids and an important compatible solute. It is the most common compatible solute in salt-tolerant microorganisms and can help organisms resist the pressure of high osmotic pressure. Among the known compatible solutes, such as trehalose, glutamic acid and betaine, Ecdoin has a strong cell protection ability.

[0003] Due to its long-lasting moisturizing effect, Ecdoin is widely used in the cosmetic and pharmaceutical fields. It is a more effective long-lasting moisturizer than glycerin, and can reduce the damage of ultraviolet and visible light to cells in many ways, protect the skin from oxidative damage, and resist aging. Ecdoin is also an excellent biofunctional stabilizer, skin protectant, and potential drug. Due to its wide application in the cosmetic and pharmaceutical fields, the annual demand for Ecdoin is about 15,000 tons, the market size is billions of dollars, and the retail price is about $1,000 / kg.

[0004] At present, there are two paths for the fermentation process of Ecdoin: fermentation with wild bacteria and fermentation with engineered bacteria. Wild bacteria fermentation usually requires a high-salt environment, which corrodes the fermentation equipment more seriously and has a low yield. The engineered bacteria based on Escherichia coli and Corynebacterium glutamicum have higher production potential, but in the fermentation process of high-yield Ecdoin, there are still problems of long production cycle and low production efficiency (generally less than 1.5g / L / h), which leads to higher production costs. Therefore, there is a need for a fermentation method that can shorten the production cycle and provide production efficiency. The present invention is to achieve the efficient synthesis of Ecdoin by using a mixed carbon source fermentation strategy in the fermentation process of recombinant Escherichia coli to synthesize Ecdoin. By adding a mixed carbon source, the metabolism and growth balance inside the Ecdoin synthesis strain are adjusted, and the efficiency of fermentation is improved without adding expensive growth stimulating factors or by metabolic engineering of the strain. Finally, a mixed carbon source fermentation process for efficiently producing Ecdoin is obtained, reaching the highest production rate reported in the current high-yield strain. Summary of the invention

[0005] The purpose of the present invention is to provide a mixed carbon source fermentation process for efficiently synthesizing ectoine by shortening the fermentation cycle, thereby improving the efficiency of synthesizing ectoine by engineering Escherichia coli and reducing the fermentation cycle.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The invention discloses a mixed carbon source fermentation process for efficiently synthesizing ectoine, wherein an ectoine synthesis strain is cultured and then inoculated into a mixed carbon source culture medium for fermentation.

[0008] The ectoine synthetic strain is an engineered Escherichia coli;

[0009] The engineered Escherichia coli is an Escherichia coli MG1655 engineering strain expressing the ectABC gene cluster, knocking out the crr gene, and over-expressing the lysC gene.

[0010] The carbon source is a mixed carbon source in which glucose, xylose and arabinose are mixed in a mass ratio of (1-10):(0-10):(0-1.5).

[0011] The carbon source in the fermentation medium is a mixture of at least two of glucose, xylose and arabinose;

[0012] The carbon source is a mixture of glucose and xylose in a mass ratio of 2:1 to 1:2, and arabinose is added, and the mass ratio of arabinose to the mixed carbon source is 5 to 8%.

[0013] More preferably, glucose, xylose and arabinose are added to the mixed carbon source in a mass ratio of 1:1:0.15.

[0014] The fermentation medium is composed of: 3-8 g / L nitrogen source, 0.5-2 g / L sodium citrate dihydrate, 1-4 g / L potassium dihydrogen phosphate, 0.5-2 g / L magnesium sulfate, 0.1-0.5 g / L iron sulfate, 0.1-0.5 g / L manganese sulfate, 6.6-35 g / L carbon source, including 6.6-20 g / L glucose, 0-13.4 g / L xylose and 0-1.5 g / L arabinose.

[0015] The specific method of producing ectoine by using engineered Escherichia coli in the mixed carbon source fermentation process is as follows:

[0016] Streak the ectoine synthetic strain onto solid LB medium and culture at 35-39° C. for 8-16 h;

[0017] Pick the cultured bacteria, inoculate into liquid LB medium, and culture at 35-39°C for 8-16 hours to obtain seed liquid;

[0018] The seed liquid is inoculated into the fermentation medium at an inoculation amount of 1-10%, ampicillin, streptomycin and inducer IPTG are added, and the fermentation culture is carried out at 35-39° C. for 36-100 hours.

[0019] The preferred fermentation time is 36 to 60 hours; the more preferred fermentation time is 40 to 50 hours;

[0020] More preferably, the fermentation time is 40.5 h.

[0021] The addition amount of ampicillin is 50-100 mg / L, the addition amount of streptomycin is 30-50 mg / L, and the addition amount of inducer IPTG is 0.05-0.2 mol / L.

[0022] The fermentation medium composition for strain fermentation is preferably: 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L iron sulfate, 0.1 g / L manganese sulfate, and the carbon source is a mixture of two or three of glucose (6.6-20 g / L), xylose (0-13.4 g / L) and arabinose (0-1.5 g / L).

[0023] The fermentation medium composition is preferably: 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L iron sulfate, 0.1 g / L manganese sulfate, 10 g / L glucose, 10 g / L xylose, and 1.5 g / L arabinose.

[0024] The carbon source is preferably glucose and xylose added in a mass ratio of 2:1 to 1:2, and preferably 1.5 g / L of arabinose is additionally added (when the total concentration of glucose and xylose is 20 g / L, the final concentration of arabinose is 1.5 g / L; when the concentration of the culture medium is increased, it is increased proportionally). As a preferred embodiment, the carbon source is glucose, xylose, and arabinose added in a mixing ratio of 1:1:0.15, and the concentrations of glucose, xylose, and arabinose in the fermentation medium are preferably 10 g / L, 10 g / L, and 1.5 g / L, respectively; the concentrations of glucose, xylose, and arabinose in the carbon source mother liquor of the feed are 300 g / L, 300 g / L, and 45 g / L, respectively;

[0025] When fermenting in a fermenter, the carbon source is added by feeding, and the glucose concentration in the fermenter is maintained at 1-5 g / L.

[0026] In the process of fed-batch fermentation, the carbon source added is a mixed carbon source, and the proportion is consistent with that of the fermentation medium.

[0027] When using a fermentation tank for fermentation, the OD 600 When the pH value reached 8, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mmol.

[0028] In the present invention, by using glucose, xylose and arabinose as a mixed carbon source, the common utilization of multiple carbon sources can regulate the metabolic flow within the strain, adjust the balance between strain growth and product synthesis, accelerate the synthesis of ectoine, and shorten the fermentation cycle.

[0029] The present invention has the following beneficial effects:

[0030] The present invention realizes a process that can improve the fermentation efficiency of engineering Escherichia coli in producing ectoine and shorten the fermentation cycle. By using mixed carbon source fermentation, the metabolism and growth balance inside the ectoine synthesis strain are adjusted, so that the fermentation cycle of engineering Escherichia coli synthesizing ectoine is shortened to 40.5h, and the production rate can reach 2.77g / L / h, which is the highest production rate reported so far, and the output is also maintained at a high level of 112.2g / L. The present invention provides a more market-competitive fermentation process method for synthesizing ectoine by fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the fermentation result of Example 10;

[0032] Figure 2 It is the fermentation result of Example 11. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0034] The detection method of Ecdoin in the embodiment is as follows:

[0035] Take 2mL of sample every 4-12h, take a part and dilute to the appropriate multiple, use spectrophotometer to detect the absorbance at 600nm to reflect the amount of bacteria. The remaining samples are centrifuged at 12000rpm for 2min, the supernatant is aspirated, and a part of it is detected by biosensor analyzer to detect the remaining sugar concentration; a part is diluted to the appropriate multiple and filtered with a 0.22 micron pore size filter membrane, and added to a liquid sample bottle, and detected by high performance liquid chromatography (HPLC): liquid chromatography uses a C18 column, and the mobile phase is 20% acetonitrile and 80% water. The concentration of ectoine is calculated by comparing the peak area with that of the standard sample.

[0036] Production rate calculation method: yield (g / L) / fermentation time (h).

[0037] The strain used for fermentation in the following examples is the Escherichia coli MG1655 engineered strain Fect3 constructed in patent CN118028204A. The crr gene of Escherichia coli MG1655 is knocked out by CRISPR-Cas9 technology to obtain a gene-knockout chassis strain; the ectABC gene sequence is amplified and inserted into a vector plasmid by enzyme cutting and one-step cloning to obtain an expression plasmid; the expression plasmid is introduced into a host bacterium; the lysC gene sequence is amplified and inserted into a vector plasmid by enzyme cutting and one-step cloning to obtain another expression plasmid; the expression plasmid is introduced into the host bacterium to obtain the ectoine synthetic strain.

[0038] Example 1

[0039] In this embodiment, the strain is fermented and cultured, and the steps are as follows:

[0040] (1) Plate culture: The strain was inoculated onto LB solid medium (5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar sterilized at 115°C for 20 min) by streaking at 37°C for 12-18 h.

[0041] (2) Seed culture: Pick the cultured bacteria, inoculate them into a test tube containing 5 mL of LB medium, place it in a shaker at 37°C and culture it at 180 rpm for 12 h as the seed solution;

[0042] (3) Shake flask fermentation: The activated seed liquid was inoculated into a shake flask with an inoculation amount of 1% (v / v). The fermentation medium was 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L iron sulfate, 0.1 g / L manganese sulfate, 100 mg / L ampicillin, 50 mg / L streptomycin, and mixed sugar solution was added as a carbon source. Place in a 37°C shaker and ferment at 180 rpm for 48 hours. After culturing for 5 hours, add 0.1 mmol / L IPTG. Add ammonia water every 12 hours to adjust the pH to 7.

[0043] Other conditions were the same as those in Example 1. Examples 2-9 were the results of shake flask fermentation using strains with different mixed sugar ratios. The effects of mixed sugar and mixed sugar ratio on the fermentation results were studied. The results are shown in Table 1:

[0044] Table 1 Fermentation results of Examples 2-9

[0045]

[0046] From the results in Table 1, it can be seen that the ratio of glucose to xylose has an effect on the fermentation of ectoine. When glucose and xylose are used as mixed carbon sources, the total sugar is 20g / L. With the decrease of glucose concentration and the increase of xylose concentration, the yield of ectoine also shows a trend of increasing and then decreasing. When the concentrations of glucose and xylose are both 10g / L, the yield of ectoine reaches a maximum of 6.21g / L, which is 43.8% higher than that of glucose alone. At the same time, adding 1.5g / L of arabinose to the culture medium can further increase the yield of ectoine. When both glucose and xylose are 10g / L and 1.5g / L of arabinose is added at the same time, the yield of ectoine can reach the maximum, which can reach 5.73g / L, which is 102.1% higher than that of glucose alone.

[0047] Embodiment 10-11

[0048] Example 10 studied the ability to produce ectoin by fermentation under conditions close to actual fermentation conditions using the mixed sugar conditions optimized in the previous example (glucose 10 g / L, xylose 10 g / L, arabinose 1.5 g / L). A fermentation tank fermentation experiment was conducted to verify the yield of the conditions.

[0049] (1) Plate culture: Streak the strain onto LB solid medium and culture at 37°C for 12-18 h.

[0050] (2) Seed culture: Pick a spot from the plate containing the bacterial strain or take the bacterial strain from the seed tube, inoculate it into a test tube containing 5 mL of LB medium, place it in a shaking incubator at 37°C and culture it at 180 rpm for 12 h as the seed solution;

[0051] (3) Fermentation tank culture: The activated seed liquid is inoculated into the fermentation tank, the inoculation amount is 10% (v / v), the stirring speed is 600-800rpm, the dissolved oxygen is controlled at 40%, the fermentation is cultured at 37°C, the pH is controlled to 7.0 with ammonia water throughout the process, and the fermentation is carried out for 60 hours. The fermentation medium is 6g / L ammonium chloride, 1g / L sodium citrate dihydrate, 2g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 0.1g / L iron sulfate, and 0.1g / L manganese sulfate. When the fermentation starts, add ampicillin with a final concentration of 100mg / L and streptomycin with a final concentration of 50mg / L. The initial mixed sugar concentration is 20g / L. When the glucose in the fermentation tank is used up, start adding mixed sugar to keep the glucose concentration in the fermentation tank at around 1g / L, OD 600 When the pH reaches 8, add 0.1 mmol / L IPTG.

[0052] Example 11 is a control group using glucose for fermentation. The initial plate culture and seed culture conditions are the same. The fermentation culture process and method are also consistent. The initial glucose concentration in the fermenter is 20g / L. When the glucose in the fermenter is exhausted, glucose is added to maintain the glucose concentration in the fermenter at about 1g / L. The fermentation time is 96h.

[0053] Table 2 Fermentation results of Examples 10-11

[0054]

[0055] The results of Example 10 are as follows Figure 1 As shown, E. coli OD 600 It can reach 76.3, the ectoine yield can reach 112.2g / L at 40.5h, the production rate can reach 2.77g / L / h, and the highest can reach 117.5g / L at 60h.

[0056] The results of Example 11 are as follows Figure 2 As shown, E. coli OD600 It can reach 69.1, the ectoine yield reaches 114.7g / L at 96h, and its production rate reaches 1.19g / L / h.

[0057] This result shows that mixed sugar fermentation can significantly shorten the fermentation time while maintaining similar yields, increasing the production rate by 132%. This method further improves production efficiency and can bring considerable economic benefits to the industrial production of ectoine by engineered Escherichia coli.

Claims

1. A mixed carbon source fermentation process for synthesizing ectoine, characterized in that: The ectoine synthetic strain is cultured and inoculated into a fermentation medium for fermentation, wherein the carbon source in the fermentation medium is a mixed carbon source formed by mixing glucose, xylose and arabinose in a mass ratio of (1-10): (0-10): (0-1.5).

2. The mixed carbon source fermentation process according to claim 1, characterized in that: The ectoine synthetic strain is an engineered strain obtained by knocking out the crr gene of Escherichia coli MG1655 and then over-expressing the ectABC gene cluster and the lysC gene.

3. The mixed carbon source fermentation process according to claim 1, characterized in that: The fermentation medium composition is: nitrogen source 3-8 g / L, sodium citrate dihydrate 0.5-2 g / L, potassium dihydrogen phosphate 1-4 g / L, magnesium sulfate 0.5-2 g / L, iron sulfate 0.1-0.5 g / L, manganese sulfate 0.1-0.5 g / L, carbon source 6.6-35 g / L, carbon source 6.6-20 g / L glucose, 0-13.4 g / L xylose, 0-1.5 g / L arabinose.

4. The mixed carbon source fermentation process according to claim 1, characterized in that: The fermentation temperature is 35-39°C, and the fermentation culture time is 36-100 h.

5. The mixed carbon source fermentation process according to claim 4, characterized in that: The fermentation temperature is 37℃ and the fermentation time is 36~60 h.

6. The mixed carbon source fermentation process according to claim 1, characterized in that: Glucose, xylose and arabinose were added to the mixed carbon source at a mass ratio of 1:1:0.

15.

7. The mixed carbon source fermentation process according to claim 1, characterized in that: When the fermentation is carried out by fed-batch fermentation, the carbon source mother liquor is added by feeding to maintain the glucose concentration in the fermenter at 1-5 g / L.

8. The mixed carbon source fermentation process according to claim 7, characterized in that: In the process of fed-batch fermentation, the carbon source added is a mixed carbon source, and the proportion is consistent with that of the fermentation medium.

9. The mixed carbon source fermentation process according to claim 3, characterized in that: The fermentation medium comprises 10 g / L glucose, 10 g / L xylose, 1.5 g / L arabinose, 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L iron sulfate and 0.1 g / L manganese sulfate.

10. The mixed carbon source fermentation process according to claim 1, characterized in that: The seed liquid is inoculated into the fermentation medium, and ampicillin, streptomycin and inducer IPTG are added. The amount of ampicillin added is 50-100 mg / L, the amount of streptomycin added is 30-50 mg / L, and the amount of inducer IPTG added is 0.05-0.2 mol / L.

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