阅读说明：本技术 一种提高司美鲁肽前体产量及菌体密度的方法 (Method for improving yield and thallus density of semaglutide precursor ) 是由 孙剑峰 孔凡楼 张颖 张立志 杨雨 闫飞 于 2020-06-18 设计创作，主要内容包括：本发明公开了用于大肠杆菌发酵生产司美鲁肽前体所需的基础培养基和补料培养基,以及利用该培养基发酵产生司美鲁肽前体的方法,针对大肠杆菌发酵过程中溶氧难控,补料速度慢,从而发酵密度低,表达量低等的不足,在基础培养中添加柠檬酸铁铵和全氟化碳乳液,并在补料培养基中添加全氟化碳乳液,从而使整个发酵过程溶氧可以控制在30％以上,菌体密度和产量显著提高。(The invention discloses a basic culture medium and a supplemented culture medium required by producing a semaglutide precursor by fermenting escherichia coli, and a method for producing the semaglutide precursor by fermenting the culture medium, aiming at the defects that dissolved oxygen is difficult to control and the supplemented culture speed is slow in the fermentation process of the escherichia coli, so that the fermentation density is low, the expression quantity is low and the like, ferric ammonium citrate and perfluorocarbon emulsion are added in the basic culture, and perfluorocarbon emulsion is added in the supplemented culture medium, so that the dissolved oxygen can be controlled to be more than 30% in the whole fermentation process, and the thallus density and the yield are obviously improved.)

1. A basic culture medium for producing semaglutide precursor by fermenting escherichia coli is characterized by comprising the following components in parts by weight: 0.5-10 g/L potassium dihydrogen phosphate, 1-15 g/L yeast extract powder, 0.5-4 g/L citric acid monohydrate, 0.05-0.3 g/L ferric ammonium citrate, 1-20 g/L glucose, 0.5-5 g/L magnesium sulfate heptahydrate, 10-30 ml/L trace element liquid, 1-10 ml/L perfluorocarbon emulsion and 6.80-7.20 of pH value.

2. The basal medium of claim 1, which comprises the following components in parts by weight: 3.5g/L of monopotassium phosphate, 6g/L of yeast extract powder, 1.4g/L of citric acid monohydrate, 0.13g/L of ferric ammonium citrate, 8g/L of glucose, 2.12g/L of magnesium sulfate heptahydrate, 16.7ml/L of trace element liquid, 8ml/L of perfluorocarbon emulsion and 7.00 of pH value.

3. The basal medium of any of claims 1 or 2, wherein the perfluorocarbon emulsion is prepared by:

a) preparation of the base emulsion: carrying out low-temperature ultrasonic emulsification on the emulsified components and the Tyrode solution to obtain a basic emulsion;

b) preparation of perfluorocarbon emulsion: adding the perfluorocarbon component into the basic emulsion, and further processing by a low-temperature ultrasonic and high-pressure homogenizer to obtain the perfluorocarbon emulsion.

4. The basal medium of any one of claims 1 or 2, wherein the trace element liquid comprises the following components in parts by weight: 2.9g/L of zinc sulfate heptahydrate, 3.7g/L of ammonium heptamolybdate tetrahydrate, 24.7g/L of boric acid, 2.5g/L of copper sulfate pentahydrate and 15.8g/L of manganese chloride tetrahydrate.

5. The basal medium of claim 3, wherein the emulsified component is one of egg yolk lecithin, lysophosphatidylcholine, cholesterol, sphingomyelin, and the like; the perfluorocarbon component is one or a mixture of perfluorotributylamine, perfluorotridecylamine, perfluorooctyl bromide, perfluoro-n-butyl furan, perfluorodecalin, perfluoromethyldecalin and perfluorotripropylamine.

6. A supplemented medium containing a perfluorinated carbonized emulsion for producing a semaglutide precursor by fermentation of escherichia coli is characterized by comprising the following components in parts by weight: 30-60 g/L of peptone, 50-200 g/L of yeast extract powder, 400-500 g/L of glucose, 0.1-1 g/L of magnesium sulfate heptahydrate and 5-20 ml/L of perfluorocarbon emulsion.

7. The feed medium of claim 6, wherein the formulation is: 45g/L of peptone, 150g/L of yeast extract powder, 500g/L of glucose, 0.5g/L of magnesium sulfate heptahydrate and 15ml/L of perfluorocarbon emulsion.

8. The method for producing the semaglutide precursor by utilizing escherichia coli fermentation is characterized by using the basic culture medium and the supplementary culture medium, and specifically comprises the following steps of:

(1) preparing a seed solution: inoculating escherichia coli glycerol strain stored in an ultra-low temperature refrigerator into a 500ml shake flask containing 100ml LB culture medium, and performing shake culture at 37 ℃ and 220rpm for 5-7 h to obtain seed liquid;

(2) batch culture: inoculating 0.5-2L of seed liquid into 50L fermentation tank containing 20L of fermentation basic culture medium, and performing initial cultureThe conditions were 37 ℃, pH7.0, rotation speed 300rpm, and aeration volume 0.5m³H, increasing aeration amount to 5.0m as the culture progresses³Increasing the rotation speed to 780rpm, controlling the dissolved oxygen to be 30% or more, and culturing for 6-7 h;

(3) fed-batch culture: after the batch culture is finished, feeding a fed-batch culture medium at the speed of 400-900 ml/h for 1-3 h;

(4) and (3) induction culture: after the fed batch is finished, controlling the temperature to be 22-28 ℃ and the pH to be 6.00-7.50, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1-1.0 mM to start induction, and controlling the dissolved oxygen to be 20-50% in the induction process;

(5) sampling detection and fermentation end: and after the induction culture is started, taking a fermentation liquid sample once every 3 hours, centrifuging at 3000-12000 rpm for 5-10 min, measuring the expression quantity of the semaglutide precursor in the supernatant, and ending the fermentation when the expression quantity is slowly increased or reduced.

9. The method according to claim 8, wherein the LB medium used in step (1) has a formulation of: 10g/L of peptone, 5g/L of yeast extract powder, 10g/L of sodium chloride and 7.00 of pH value; in the step (2), 1L of seed liquid is obtained; in the step (3), the feeding mode is constant-speed feeding, and the speed is 700 ml/h; in the step (4), the induction temperature is 23 ℃, the pH is 7.20, the concentration of IPTG is 0.3mM, and the dissolved oxygen is controlled at 30-40%.

Technical Field

The invention belongs to the field of microbial engineering, and particularly relates to a semaglutide precursor and a method for improving the thallus density of a semaglutide precursor production strain.

Background

Diabetes is considered to be a disease of dual hormonal abnormalities-absolute or relative insulin deficiency and relative glucagon excess. Insulin deficiency leads to abnormal glucose utilization and glucagon excess leads to increased glucose production, both of which can increase blood glucose levels. Glucagon-like peptide-1 (GLP-1) is an incretin with a variety of physiological effects of promoting insulin secretion, lowering glucagon levels, reducing gastric emptying rate, enhancing satiety, and stimulating islet cell proliferation and differentiation [ host J, groada J.

The semaglutide is a long-acting GLP-1 analogue and has an action mechanism similar to that of GLP-1. GLP-1 is a peptide hormone secreted by intestinal L cells, and after being specifically bound with a receptor, the GLP-1 mainly exerts a blood sugar-dependent incretin secretion effect through a cyclic adenosine monophosphate signal pathway. Researches find that semaglutide not only can greatly improve the blood sugar of a type 2 diabetic, but also can reduce appetite, reduce food intake and induce weight loss. In addition, the risk of major cardiovascular events (MACE) in type 2 diabetes patients can be significantly reduced.

The current research on the escherichia coli of semaglutide mainly focuses on purification or preparation, and reports on the improvement of the density of fermentation thalli and the improvement of the yield of precursors are less. An approved patent CN 101643764B (supplemented medium for fermentation production of proinsulin and supplemented culture optimization method) develops a novel supplemented method, but the basic culture medium lacks a trace element of ammonium ferric citrate and an enzyme auxiliary factor, the glycerol content is only 1.5ml/L, the thallus grows slowly before supplement, pure oxygen needs to be introduced in the supplementing process, the cost is high, and the thallus density at the fermentation end point is too low (OD of thallus cultured for 6h is adopted)₆₀₀The absorbance is only 15.0, the fermentation end point is 19h, and the bacterial body OD₆₀₀Absorbance of 69.0) resulting in no further increase in yield. The invention discloses a method for optimizing a fermentation culture medium for producing geraniol recombinant escherichia coli by using an improved M9 culture medium in the process of optimizing a forest chemical and industry (2015, 8 months), and the likeThe activity of iron components (e.g., succinate dehydrogenase, cytochrome reductase, etc.) decreases, resulting in a decrease in the electron transport flux of respiratory volume. As NADH can not be effectively oxidized and supply of ATP is insufficient, Escherichia coli can generate ATP for bacteria through an acetic acid generation path, and finally, the Escherichia coli generates a large amount of acetic acid. But the fermentation tank is not supplemented with materials, and the final thallus density of the fermentation liquor only reaches OD₆₀₀45。

The invention content is as follows:

in order to solve the problems of low density and low yield of target protein of the escherichia coli fermented thalli of the semaglutide, the invention provides a novel basic improved culture medium and a novel supplemented culture medium so as to achieve the aims of high-density fermentation of thalli and improvement of the yield of the protein.

In order to achieve the aim of the invention, the invention discloses a basic culture medium suitable for fermenting semaglutide by escherichia coli, wherein ferric ammonium citrate is added into the culture medium to promote the expression of a product, and perfluorocarbon emulsion is innovatively introduced to improve the problem of oxygen transfer in fermentation liquor in the whole fermentation process.

The adopted technical scheme is as follows:

a basic culture medium for producing semaglutide precursor by fermenting escherichia coli comprises the following formula: 0.5-10 g/L potassium dihydrogen phosphate, 1-15 g/L yeast extract powder, 0.5-4 g/L citric acid monohydrate, 0.05-0.3 g/L ferric ammonium citrate, 1-20 g/L glucose, 0.5-5 g/L magnesium sulfate heptahydrate, 10-30 ml/L trace element liquid, 1-10 ml/L perfluorocarbon emulsion and 6.80-7.20 of pH value;

preferably, the basic culture medium formula is as follows: 3.5g/L of monopotassium phosphate, 6g/L of yeast extract powder, 1.4g/L of citric acid monohydrate, 0.13g/L of ferric ammonium citrate, 8g/L of glucose, 2.12g/L of magnesium sulfate heptahydrate, 16.7ml/L of trace element liquid, 8ml/L of perfluorocarbon emulsion and 7.00 of pH value.

Preferably, the formula of the trace element liquid is as follows: 2.9g/L of zinc sulfate heptahydrate, 3.7g/L of ammonium heptamolybdate tetrahydrate, 24.7g/L of boric acid, 2.5g/L of copper sulfate pentahydrate and 15.8g/L of manganese chloride tetrahydrate.

Preferably, the perfluorocarbon emulsion is prepared by a process comprising:

a) preparation of the base emulsion: carrying out low-temperature ultrasonic emulsification on the emulsified components and the Tyrode solution to obtain a basic emulsion;

b) preparation of perfluorocarbon emulsion: adding the perfluorocarbon component into the basic emulsion, and further processing by a low-temperature ultrasonic and high-pressure homogenizer to obtain perfluorocarbon emulsion;

preferably, the emulsifying component is one of common egg yolk lecithin, lysophosphatidylcholine, cholesterol, sphingomyelin, and the like; the perfluorocarbon component is one or a mixture of perfluorotributylamine, perfluorotridecylamine, perfluorooctyl bromide, perfluoro-n-butyl furan, perfluorodecalin, perfluoromethyldecalin and perfluorotripropylamine.

Aiming at the problems of extremely high oxygen consumption, slow growth of thalli and poor expression level of target protein in the later fermentation stage of escherichia coli, the invention also provides a novel supplemented medium, and perfluorocarbon emulsion and other components are added, wherein the perfluorocarbon emulsion and the other components comprise glucose, yeast extract powder, peptone and magnesium sulfate.

Preferably, the formula of the feed medium is as follows: 30-60 g/L of peptone, 50-200 g/L of yeast extract powder, 400-500 g/L of glucose, 0.1-1 g/L of magnesium sulfate heptahydrate and 5-20 ml/L of perfluorocarbon emulsion;

preferably, the formula of the feed medium is as follows: 45g/L of peptone, 150g/L of yeast extract powder, 500g/L of glucose, 0.5g/L of magnesium sulfate heptahydrate and 15ml/L of perfluorocarbon emulsion.

The invention also provides a method for producing the semaglutide precursor by fermenting escherichia coli based on the basic culture medium and the supplementary culture medium, which specifically comprises the following steps:

(1) preparing a seed solution: inoculating escherichia coli glycerol strain stored in an ultra-low temperature refrigerator into a 500ml shake flask containing 100ml LB culture medium, and performing shake culture at 37 ℃ and 220rpm for 5-7 h to obtain seed liquid;

(2) batch culture: inoculating 0.5-2L of seed liquid into 50L fermentation tank containing 20L of fermentation basic culture medium, wherein the initial culture condition is 37 deg.C, pH7.0, rotation speed 300rpm, and ventilation amount 0.5m³H is used as the reference value. As the culture proceeded, the aeration amount was increased to 5.0m³Increasing the rotation speed to 780rpm, controlling the dissolved oxygen to be 30% or more, and culturing for 6-7h；

(3) Fed-batch culture: after the batch culture is finished, feeding a fed-batch culture medium at the speed of 400-900 ml/h for 1-3 h;

(4) and (3) induction culture: after the fed batch is finished, controlling the temperature to be 22-28 ℃ and the pH to be 6.00-7.50, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1-1.0 mM to start induction, and controlling the dissolved oxygen to be 20-50% in the induction process;

(5) sampling detection and fermentation end: and after the induction culture is started, taking a fermentation liquid sample once every 3 hours, centrifuging at 3000-12000 rpm for 5-10 min, measuring the expression quantity of the semaglutide precursor in the supernatant, and ending the fermentation when the expression quantity is slowly increased or reduced.

Preferably, the formulation of the LB medium in step (1) is: 10g/L of peptone, 5g/L of yeast extract powder, 10g/L of sodium chloride and 7.00 of pH value;

in the step (2), the preferable amount of the seed liquid is 1L;

in the step (3), the feeding mode is constant-speed feeding, the speed is preferably 700ml/h, and the feeding culture medium needs to be stirred to fully and uniformly mix the perfluorocarbon emulsion;

in the step (4), the induction temperature is preferably 23 ℃, the pH is preferably 7.20, the concentration of IPTG is preferably 0.3mM, and the dissolved oxygen is preferably controlled at 30-40%.

Has the advantages that:

1. the perfluorocarbon compound of the present invention is an organic compound formed by replacing all hydrogen atoms in a hydrocarbon molecule with fluorine atoms, and has high oxygen solubility due to the specific structure of perfluorocarbons, so that the perfluorocarbon compound is widely used in the biomedical field.

2. The invention adds the perfluorocarbon emulsion into the fermentation basal culture medium for the first time, can improve the oxygen transfer in the early batch culture fermentation stage, does not need to introduce pure oxygen, can control the dissolved oxygen DO to be more than 30 percent, and can control the optical density OD of the thallus before the batch feeding stage₆₀₀More than 50 is reached.

3. The invention adds perfluorocarbon into the fed-batch culture medium in the fed-batch culture and induction culture stagesThe compound emulsion greatly increases the oxygen transfer, so that the dissolved oxygen DO can be maintained at more than 30 percent on the premise of not introducing pure oxygen even if the feeding speed is increased to 700ml/h, the thallus density is increased rapidly, the fermentation is carried out for 20h, and the thallus optical density OD₆₀₀The wet weight of the thallus can reach 300g/L, the dry weight reaches 120g/L, the thallus density is improved by 29.4%, and the yield is improved by 73.6%.

4. The invention adds ferric ammonium citrate in the fermentation basic culture, wherein iron is a composition component of cytochrome, cytochrome oxidase and catalase, so that the iron is an essential element for aerobic oxidation of thalli, and the invention discovers that the addition of ferric ammonium citrate in the fermentation medium can improve the yield of the semaglutide precursor in the fermentation liquid by 131.5%.

Drawings

FIG. 1 is a graph comparing dissolved oxygen DO for an emulsion without and with a perfluorocarbon emulsion.

The specific implementation mode is as follows:

the present invention is further described in the following detailed description, which is for the purpose of illustration only, and the scope of the invention is not limited to these examples, and it will be understood by those skilled in the art that various equivalent substitutions and modifications may be made within the scope of the invention.