CN112029810B

CN112029810B - Culture medium for producing insulin glargine and fermentation method

Info

Publication number: CN112029810B
Application number: CN201910478456.1A
Authority: CN
Inventors: 张贵民; 冀成法; 王霞
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2023-08-22
Anticipated expiration: 2039-06-03
Also published as: CN112029810A

Abstract

The invention belongs to the field of fermentation engineering, and particularly relates to a culture medium for producing insulin glargine and a fermentation method, and the formula of the culture medium and control parameters of a fermentation process are disclosed. Amplifying and culturing engineering bacteria in seed culture medium step by step to obtain seed liquid; inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, and feeding the fed-batch culture medium in batches until the culture is finished. The invention obtains high-yield insulin glargine by optimizing the composition of a basic culture medium and a feed supplement culture medium and controlling the flow rate and the feed supplement amount of the feed supplement culture medium, and simultaneously controls the loss rate of plasmids to be within 10 percent and the electrophoretic expression amount to be above 34 percent. The high-density fermentation of insulin glargine improves the production efficiency and has good application prospect.

Description

Culture medium for producing insulin glargine and fermentation method

Technical Field

The invention belongs to the field of fermentation engineering, and particularly relates to a culture medium for producing insulin glargine and a fermentation method.

Background

The main objective of fermentation research is to produce good cost-effectiveness of the product of interest using high productivity techniques. Since most proteins are accumulated intracellularly in recombinant E.coli, productivity is directly proportional to final cell density and unit productivity. High-density fermentation not only can improve productivity, but also can reduce culture volume, production cost and equipment investment, and is convenient for downstream processing and wastewater discharge reduction, so that high-density culture becomes one of important targets and directions of fermentation industry in recent years.

Pan (Adv Biochem Eng Biotechno,2003; 85:43-93) and the like produce human growth hormone by constant-speed fed-batch culture, and the OD600 value of the thalli reaches 120; allen (Biophanmacology, 1987; 1:38-419) et al have used synthetic media to culture recombinant E.coli, with a cell density of 79g (DCW)/L, but no expression of foreign proteins; jung (Ann Inst Pasteur Microbio,1988; 139:129-146) and the like, which adopts the technology to produce the interferon, the concentration of the thalli reaches 46g (DCW)/L, and the specific productivity of the interferon is 17mg/g thalli; zhou Yuxun (journal of bioengineering, 2005, 21 (4): 615-625) and the like, the highest fermentation density of the antibacterial peptide genetically engineered bacteria reaches OD600 = 119, the concentration of the bacteria reaches 40g/L (DCW), the expression quantity of the target protein is only about 5%, and 40% of the engineering bacteria lose plasmids. The variable flow rate or gradient can promote the growth of cells by adding more nutrient substances under the condition of higher cell density, and is beneficial to the expression of products. Li Min (journal of bioengineering, 1998, 14 (3): 270-276) adopts a three-stage glucose feeding mode, recombinant bacteria YK537/pDH-B2m are cultured at high density to produce bone morphogenetic protein (BMP-2A), the fermentation density reaches OD600 = 53, and the yield of BMP-2A is 2.78g/L.

The recombinant plasmid of the genetically engineered bacterium has certain instability in the fermentation process, which leads to the failure to obtain the expected target gene product and yield. The instability of the plasmid is classified into structural instability in which recombination, deletion or insertion of a DNA fragment occurs and separation instability in which the plasmid does not enter a daughter cell at the time of cell division. The stability of a plasmid is affected by various genetic and environmental factors such as the host and plasmid genotypes, host and plasmid interactions, the degree of gene expression, culture temperature, nutrient limitations, and reactor operating modes. Genetically engineered fermentation typically requires high density cell culture to obtain more of the desired product, however, too high cell density can affect plasmid stability of the engineered bacteria.

Engineering bacteria high-density culture is an important means for obtaining exogenous gene expression products, but one of the main obstacles of high-density culture is accumulation of metabolic by-product acetic acid. With the increase of the fermentation culture density, the accumulation of acetic acid is increased, and the growth of thalli and the expression of exogenous proteins are directly influenced, so that the acetic acid is gradually an important factor for restricting the high-density culture of engineering bacteria. Jensen (Biotech Bioeng,1990; 36:1-11) et al reported that acetic acid significantly inhibited cell growth when the concentration of acetic acid in the culture broth was greater than 6 g/L; when the acetic acid concentration is more than 2.4g/L, the specific productivity is significantly lowered. The bacterial growth was completely stopped when the acetic acid concentration in the culture broth was more than 15g/L as reported by Konstan (Biotech Bioeng,1990;36 (1): 750-758). Boon (Biotechnol letters, 1992;14 (12): 1115-1118) et al have used three host bacteria derived from E.coli and the corresponding recombinant bacteria for acetic acid inhibition experiments and found that the recombinant bacteria are more susceptible to acetic acid inhibition than the host bacteria.

CN104726524a discloses a culture medium and a method for producing insulin glargine precursor by fermentation using the culture medium, which reduces accumulation of harmful metabolites (mainly acetic acid) by adding salts and microelements, improves cell growth, increases thallus yield, and although yield is improved, the method is still unsatisfactory; CN106282274a discloses a high-density fermentation method of pichia pastoris for insulin precursor protein, CN107022591B discloses a pichia pastoris fermentation method for improving expression of insulin and insulin analogue precursor, which belongs to high-density fermentation, but needs to be fermented and cultured for more than 130h, and the fermentation period is long.

The high-density fermentation of the genetically engineered bacteria not only needs to obtain high cell density, but also has the effects of acetic acid accumulation, plasmid stability and high-efficiency expression, and the four effects complement each other. As can be seen from the above patents and literature, the prior art generally only considers one or several aspects, and in particular, there are fewer documents to examine plasmid stability during high density culture. Therefore, the production of the genetically engineered bacteria is far from sufficient only according to the traditional fermentation process, and the factors influencing the high-density fermentation and the expression of the exogenous genes are required to be comprehensively analyzed, so that a set of high-density fermentation process suitable for the efficient expression of the exogenous genes is explored.

Disclosure of Invention

In view of the defects of the prior art, the invention provides a simple and feasible culture medium and fermentation method which can not only improve the insulin glargine yield, but also ensure the low plasmid loss rate.

A first object of the present invention is to provide a basal medium for producing insulin glargine, which comprises glycerol, yeast extract, tryptone, ammonium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride and trace elements.

Preferably, the trace elements comprise ferrous sulfate, cobalt chloride, copper sulfate, calcium chloride, zinc chloride, boric acid, manganese sulfate.

Further preferably, the basal medium comprises per liter the following components as shown in Table 1:

TABLE 1

Further preferably, the trace elements include the components shown in table 2 below:

TABLE 2

Component (A)	Concentration (g/L)	Component (A)	Concentration (g/L)
				FeSO ₄ ·7H ₂ O	10～20	ZnCl ₂	0.5～2.5
CoCl ₂ ·6H ₂ O	1～5	H ₃ BO ₃	0.2～1
				CuSO ₄ ·5H ₂ O	0.2～1	MnSO ₄ ·H ₂ O	0.2～1
CaCl ₂	5～15	—	—

A second object of the present invention is to provide a feed medium for producing insulin glargine. The feed medium comprises glycerol, yeast extract, tryptone and magnesium sulfate.

Preferably, the feed medium composition is: glycerol 400-600 g/L, yeast extract 40-80 g/L, tryptone 80-120 g/L, mgSO ₄ ·7H ₂ O 1～3g/L。

The third object of the present invention is to provide a fermentation method for producing insulin glargine, which comprises the following steps:

a. amplifying and culturing engineering bacteria in seed culture medium step by step to obtain seed liquid;

b. inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, and feeding a fed-batch culture medium in batches until the culture is finished.

Preferably, the engineering bacteria strain is pET-Glargine/BL21 (DE 3) PlysS, and the construction method refers to patent CN1663960B. The method is also suitable for the strains such as pET-Glargine/BL21 (DE 3), pET-Glargine/BL21star and the like for producing the insulin Glargine.

Preferably, the seed solution is inoculated into the basic culture medium of the fermentation tank in an inoculum size of: the volume of the seed liquid is 5-10% of the volume of the basic culture medium.

Preferably, the amount of feed medium required in the feed process is: the total feed amount is 23-27% of the volume of the basal medium.

Preferably, the first feeding and feeding culture medium controls the flow rate to be 30-35% of the total feeding amount fed in every 5-8 h, and the second feeding and feeding culture medium controls the flow rate to be 65-70% of the total feeding amount fed in every 15-18 h.

Preferably, in the high-density culture process, the dissolved oxygen content of the fermentation liquid is controlled to be not lower than 30%, the temperature is 36.0-38.0 ℃, and the pH value is 7.0-7.2.

Acetic acid is generated in the fermentation process, the generation of a large amount of acetic acid can reduce the pH value and inhibit the growth of thalli, and in order to reduce the influence of the acetic acid on the fermentation, the pH value of a fermentation system is required to be controlled and adjusted, and preferably, ammonia water is used for controlling the pH value of a reaction system; the amount of ammonia reflects the amount of acetic acid produced by the reaction system.

The following further details a fermentation process for producing insulin glargine comprising the steps of:

step a, seed liquid culture specifically comprises the following steps:

inoculating engineering bacteria strain to the inclined plane of the solid seed culture medium, culturing for 12-15 h at 37.0 ℃, taking out and storing in a refrigerator at 2-8 ℃; inoculating the cultured seed slant colony into a liquid seed culture medium, and culturing for 5-8 hours at 37.0 ℃ in a constant temperature oscillator to obtain first-stage shake flask seed liquid; inoculating the cultured first-stage shake flask seed liquid into a liquid seed culture medium, and culturing for 12-15 h at 37.0 ℃ in a constant-temperature oscillator to obtain a second-stage shake flask seed liquid.

The present invention is not limited to the seed liquid culture step, and any existing solid medium, liquid seed medium and culture method for seed liquid culture can be used in the present invention.

b. Inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, and feeding a fed-batch culture medium in batches until the culture is finished. The method comprises the following specific steps:

inoculating the second-stage shake flask seed liquid into a basic culture medium of a fermentation tank according to the inoculation amount of 5-10%, controlling the dissolved oxygen amount in the fermentation tank to be not lower than 30%, controlling the temperature to be 36-38 ℃, and automatically controlling the pH value to be 7.0-7.2 by using ammonia water. When dissolved oxygen rises and pH rises simultaneously, the first feeding of the feed medium is started, the flow rate is controlled to be 30-35% of the total feeding amount every 5-8 h, and after feeding is suspended, the pH and the dissolved oxygen value rise briefly. When the culture was carried out until the OD 600=50 to 60, induction was carried out by adding an inducer. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 65-70% of the total feeding amount fed in every 15-18 h, and finishing the fermentation after the feeding is finished, wherein the total feeding amount is the total amount of the feeding culture medium fed in the two times and is 23-27% of the volume of the basic culture medium.

Preferably, the rise of dissolved oxygen means that the dissolved oxygen reaches 90%.

Preferably, the inducer is IPTG with a final concentration of 0.1-0.3 mM.

In a preferred embodiment, a fermentation process for producing insulin glargine comprises the steps of:

a. Amplifying and culturing engineering bacteria in seed culture medium step by step: inoculating engineering bacteria strain to the inclined plane of solid seed culture medium (the composition of the solid seed culture medium is Yeast extract 5g/L, tryptone 10g/L, naCl 10g/L and Agar powder 20 g/L), culturing for 12-15 h at 37.0deg.C, taking out and storing at 2-8deg.C; inoculating the cultured seed inclined plane colony into a liquid seed culture medium (the liquid seed culture medium consists of Yeast extract 5g/L, tryptone 10g/L and NaCl 10 g/L), and culturing for 5-8 h at 37.0 ℃ in a constant-temperature oscillator to obtain first-stage shake flask seed liquid; inoculating the cultured first-stage shake flask seed liquid into a liquid seed culture medium, and culturing for 12-15 h at 37.0 ℃ in a constant-temperature oscillator to obtain a second-stage shake flask seed liquid.

b. High-density culture in a fermentation tank:

the basic culture medium comprises the following components:

TABLE 3 Table 3

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Yeast extract	1.5～5	FeSO ₄ ·7H ₂ O	4～12
Tryptone	2.5～10	CoCl ₂ ·6H ₂ O	0.4～3
				Glycerol	10～20	CuSO ₄ ·5H ₂ O	0.08～0.6
NH ₄ Cl	1～5	CaCl ₂	2～9
				Na ₂ HPO ₄ ·12H ₂ O	25～35	ZnCl ₂	0.2～1.5
KH ₂ PO ₄	1～3	H ₃ BO ₃	0.08～0.6
				MgSO ₄	0.2～2	MnSO ₄ ·H ₂ O	0.08～0.6
NaCl	0.2～2	—	—

The feed medium consists of: 80-120 g/L of Tryptone, 40-80 g/L of Yeast extract, 400-600 g/L of glycol and MgSO ₄ ·7H ₂ O 1～3g/L

Inoculating the second-stage shake flask seed liquid to a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen rises to 90% and the pH value rises simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30-35% of the total feeding amount for every 5-6 hours, 4.5-5.25L, and the pH value and the dissolved oxygen value rise briefly after feeding is stopped. When cultured until the OD 600=50 to 60, induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 65-70% of the total feeding amount to be 9.75-10.5L every 15-18 h, and fermenting and culturing for 25-38 h after the feeding is finished.

The invention has the beneficial effects that:

according to the invention, by optimizing the composition of the basic culture medium and the feeding culture medium and controlling the flow rate and the feeding amount of the feeding culture medium, the fermentation density of insulin glargine produced by recombinant escherichia coli fermentation can reach OD600 = 146, the yield of inclusion bodies is above 986 g/tank, and the bacterial density and the yield are both improved by more than 5 times compared with low-density fermentation. The carbon source of the culture medium uses glycerol to replace glucose, the production of acetic acid is obviously reduced by regulating and controlling the feeding speed, the consumption of ammonia water is reduced, and the high-density and high-expression of fermentation is facilitated. After fermentation, the loss rate of the plasmid is within 10%, and the electrophoretic expression level is above 34%. The high-density fermentation of insulin glargine improves the production efficiency and has good application prospect.

Detailed Description

Although the present invention has been described with reference to the preferred embodiments, it should be understood that the invention is not limited to the embodiments described, but various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention. The protection scope of the present invention is defined by the claims, and the protection scope includes equivalent alternatives to the technical features of the claims. I.e., equivalent replacement modifications within this range, and equivalent scale up or down tests are also within the scope of the invention.

The strain used in the following embodiments was constructed with reference to patent CN 1663960B. The reagents required in the fermentation process are commercially available (commercially available) without any specific explanation.

Example 1

a. Amplifying and culturing engineering bacteria in seed culture medium step by step

The solid seed culture medium consists of: yeast extract 5g/L, tryptone 10g/L, naCl 10g/L, agar powder20g/L;

the liquid seed culture medium consists of: yeast extract 5g/L, tryptone 10g/L, naCl 10g/L;

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 4 Table 4

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Yeast extract	3	FeSO ₄ ·7H ₂ O	7.5
Tryptone	5	CoCl ₂ ·6H ₂ O	1.5
				Glycerol	15	CuSO ₄ ·5H ₂ O	0.25
NH ₄ Cl	2	CaCl ₂	5
				Na ₂ HPO ₄ ·12H ₂ O	30	ZnCl ₂	0.75
KH ₂ PO ₄	2	H ₃ BO ₃	0.2
				MgSO ₄	1	MnSO ₄ ·H ₂ O	0.25
NaCl	1	—	—

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glycerol 500g/L, mgSO ₄ ·7H ₂ O 2g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 2

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 5

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Yeast extract	2	FeSO ₄ ·7H ₂ O	5
Tryptone	4	CoCl ₂ ·6H ₂ O	0.5
				Glycerol	12	CuSO ₄ ·5H ₂ O	0.1
NH ₄ Cl	3	CaCl ₂	2.5
				Na ₂ HPO ₄ ·12H ₂ O	28	ZnCl ₂	0.25
KH ₂ PO ₄	2	H ₃ BO ₃	0.1
				MgSO ₄	0.5	MnSO ₄ ·H ₂ O	0.1
NaCl	0.5	—	—

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L,Glycerol 500g/L，MgSO ₄ ·7H ₂ O 2g/L。

inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 7 hours, 4.8L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 70% of the total feeding amount to 11.2L every 17h, and fermenting and culturing for 32h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 3

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 6

Example 4

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 7

The feed medium consists of: 120g/L of Tryptone, 80g/L of Yeast extract, 600g/L of glycol and MgSO ₄ ·7H ₂ O 1g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to 10% of inoculation amount, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 35% of the total feeding amount for every 7 hours, 5.67L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.53L of 65% of the total feeding amount in every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 5

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 8

The feed medium consists of: tryptone 80g/L, yeast extract 40g/L, glycerol 400g/L, mgSO ₄ ·7H ₂ O3g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 7%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.14L, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and the feeding culture medium is added into the medium with the final concentration of 0.2mMInduction is performed by the inducer IPTG. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 70% of the total feeding amount to 9.66L every 17h, and fermenting and culturing for 32h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 6

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 9

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the set rotating speed is 200rpm, the tank pressure is 0.04-0.06 MPa, and ammonia is used in the fermentation processThe pH value of water is automatically controlled to 7.1+/-0.1, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in each 18h, and fermenting and culturing for 31h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 7

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 10

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Yeast extract	10	FeSO ₄ ·7H ₂ O	15
Tryptone	15	CoCl ₂ ·6H ₂ O	5
				Glycerol	25	CuSO ₄ ·5H ₂ O	1
NH ₄ Cl	10	CaCl ₂	15
				Na ₂ HPO ₄ ·12H ₂ O	40	ZnCl ₂	2.5
KH ₂ PO ₄	5	H ₃ BO ₃	1.2
				MgSO ₄	3	MnSO ₄ ·H ₂ O	1.5
NaCl	5	—	—

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 7 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then starting feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount every 16h, and finishing the fermentation after feeding the feeding. Fermenting and culturing for 30h. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 8

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 11

The feed medium consists of: tryptone 60g/L, yeast extract 20g/L, glycerol 300g/L, mgSO ₄ ·7H ₂ O1g/L。

Inoculating the secondary shake flask seed liquid to 100L for fermentation according to the inoculation amount of 7%In the tank, the volume of the basic culture medium is 60L, and the initial ventilation of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 7 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in every 16h, and fermenting and culturing for 31h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 9

The solid seed culture medium consists of: yeast extract 9g/L, tryptone 5g/L, naCl 6g/L, agar powder 15g/L;

the liquid seed culture medium consists of: glucose 5g/L KH ₂ PO ₄ 3g/L，K ₂ HPO ₄ 5g/L，Yeast extract 12g/L，NaCl 6g/L；

Culturing the strain on a solid seed culture medium, and culturing at 35 ℃ for 24 hours to obtain the bevel strain. Scraping the slant strain with inoculating needle, transferring into triangular flask (0.07 MP a10 min) containing liquid seed culture medium, and shake culturing at 37deg.C for 18 hr to obtain liquid seed.

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 12

The feed medium consists of: 150g/L of Tryptone, 100g/L of Yeast extract, 700g/L of glycol and MgSO ₄ ·7H ₂ O 5g/L。

Inoculating the seed solution into 100L fermentation tank according to 5% inoculum size, wherein the volume of basic culture medium is 60L, and the initial ventilation volume of fermentation tank is 4+ -0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 7 hours, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 70% of the total feeding amount to 9.8L every 17h, and fermenting and culturing for 28h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 10

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 13

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glycerol 500g/L, mgSO ₄ ·7H ₂ O 3g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 7%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 5 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in each 18h, and fermenting and culturing for 33h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 11

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 14

Inoculating the seed solution into 100L fermentation tank according to 5% inoculum size, wherein the volume of basic culture medium is 60L, and the initial ventilation volume of fermentation tank is 4+ -0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 35% of the total feeding amount for every 8 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then starting feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 65% of the total feeding amount every 15h, and fermenting and culturing for 28h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 12

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 15

Supplementary material cultureThe nutrient medium comprises the following components: tryptone 100g/L, yeast extract 60g/L, glycerol 500g/L, mgSO ₄ ·7H ₂ O 2g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 3 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then starting feeding the feeding culture medium for the second time, controlling the flow rate to be 70% of the total feeding amount to 10.5L every 14h, and fermenting and culturing for 25h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 13

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 16

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 8 hours, 4.5L of the total feeding amount is fed in, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. Then the feeding culture medium is fed for the second time, the flow rate is controlled to be 70 percent 1 of the total feeding amount of each 20hAnd 0.5L, and finishing fermentation after the feeding is finished. Fermenting and culturing for 38h. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 14

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 17

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen is increased to 90% and the pH value is increased simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 40% of the total feeding amount for every 6 hours, after feeding is suspended, the pH value and the dissolved oxygen value are increased briefly, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then starting feeding the feeding culture medium for the second time, controlling the flow rate to be 60%9L of the total feeding amount every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Example 15

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 18

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When dissolved oxygen rises to 90% and pH rises simultaneously, feeding the feed medium for the first time is started, the flow rate is controlled to be 20% of the total feed amount for every 6h, 3.0L of the total feed amount is fed in a feeding manner, and feeding is suspendedAfter the incubation, the pH and dissolved oxygen values were briefly raised and induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then starting feeding the feeding culture medium for the second time, controlling the flow rate to be 80% of the total feeding amount to be 12.0L every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 1

b. High density culture in fermenter

The basic culture medium comprises the following components: trptone 10g/L, yeast extract 5g/L, glucose 10g/L, naCl 10g/L.

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glycerol 600g/L.

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When dissolved oxygen rises to 90% and pH rises simultaneously, feeding the feed medium for the first time, and controlling the flow rate to flow every 6hAfter the addition of 30% of the total feed amount, 4.5L, and the suspension of the feed, the pH and the dissolved oxygen value were briefly increased, and the induction was performed by adding IPTG as an inducer with a final concentration of 0.2 mM. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in each 17h, and fermenting and culturing for 28h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 2

b. High density culture in fermenter

The basic culture medium comprises the following components:

TABLE 19

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Yeast extract	3	FeSO ₄ ·7H ₂ O	7.5
Tryptone	5	CoCl ₂ ·6H ₂ O	1.5
				Glucose	15	CuSO ₄ ·5H ₂ O	0.25
NH ₄ Cl	2	CaCl ₂	5
				Na ₂ HPO ₄ ·12H ₂ O	30	ZnCl ₂	0.75
KH ₂ PO ₄	2	H ₃ BO ₃	0.2
				MgSO ₄	1	MnSO ₄ ·H ₂ O	0.25
NaCl	1	—	—

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glucose 600g/L, mgSO ₄ ·7H ₂ O2g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen and the pH value rise simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.5L, the pH value and the dissolved oxygen value rise briefly after feeding is suspended, and an inducer IPTG with the final concentration of 0.2mM is added for induction. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in every 15h, and fermenting and culturing for 24h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 3

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 20

Component (A)	Concentration (g/L)	Component (A)	Concentration (g/L)
				Yeast extract	3	Na ₂ HPO ₄ ·12H ₂ O	30
Tryptone	5	KH ₂ PO ₄	2
				Glycerol	15	MgSO ₄	1
NH ₄ Cl	2	NaCl	1

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glycerol 500g/L, mgSO ₄ ·7H ₂ O2g/L。

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ Per h, oxygen flow rate of 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen rises and the pH rises simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.5L, and after feeding is suspended, the pH and the dissolved oxygen value rise briefly. When cultured until the OD 600=50 to 60, induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 4

the liquid seed culture medium consists of: yeast extract 9g/L, tryptone 5g/L and NaCl 6g/L;

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 21

Component (A)	Concentration (g/L)	Component (A)	Concentration (mg/L)
				Glucose	12	Ammonium molybdate	0.8
Glycerol	2	Copper sulfate	0.5
				Yeast extract	13	Boric acid	2
Citric acid	5	Potassium iodide	0.4
				Ferric sulfate	0.05	Manganese chloride	3
Diammonium phosphate	5	Zinc acetate	3
				Monopotassium phosphate	3	—	—
Magnesium sulfate	2	—	—
				Vitamin B10	0.1	—	—

Inoculating the second-stage shake flask seed liquid into a 100L fermentation tank according to an inoculum size of 5%, wherein the volume of a basic culture medium is 60L, and the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ /h, oxygenThe flow rate is 0-1 m ³ And/h, controlling the dissolved oxygen to be not lower than 30% under the pressure of 0.04-0.09 MPa. When the dissolved oxygen rises and the pH rises simultaneously, the feeding culture medium is fed in for the first time, the flow rate is controlled to be 30% of the total feeding amount for every 6 hours, 4.5L, and after feeding is suspended, the pH and the dissolved oxygen value rise briefly. When cultured until the OD 600=50 to 60, induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then feeding the feeding culture medium for the second time, controlling the flow rate to be 10.5L of 70% of the total feeding amount in every 16h, and fermenting and culturing for 30h after the feeding is finished. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 5

The recombinant E.coli strain was cultured on a solid slant medium (pH 6.7, peptone 5g, yeast powder 9g, sodium chloride 6g, agar 15g, dissolved in tap water and mixed to 1L), and cultured at 35℃for 24 hours to obtain a slant strain.

The inclined plane is placed by about 1cm by an inoculating needle ² Transferring to 500ml triangular flask (0.07 MP a10 min) containing 20ml liquid seed culture medium (pH 6.8, glucose 5g, potassium dihydrogen phosphate 3g, dipotassium hydrogen phosphate 5g, sodium chloride 6g, yeast powder 12g, and 1L) dissolved and mixed with tap water, and shake culturing at 37deg.C with reciprocating shaking table (amplitude 65cm, shaking table rotation speed 180 r/m) for 18 hr to obtain liquid seed.

Inoculating 3.0L liquid seed into 60L fermentation medium (comprising citric acid 3g, ferric sulfate 0.01g, diammonium phosphate 2g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, glucose 8g, glycerol 1g, yeast extract 10g, and vitamin B) ₁ 0.05g, trace elements ammonium molybdate 0.5mg, copper sulfate 0.1mg, boric acid 1mg, potassium iodide 0.2mg, manganese chloride 1mg and zinc acetate 1mg, and tap water was added to dissolve and mix them to 1L, and the ventilation rate (i.e., the volume ratio of air to be introduced per unit volume of fermentation broth per minute) was 1 in a 100L fermenter (sterilized at 120℃for 8 minutes before inoculation) in which the pH of the above solution was adjusted to 6.8 with 5% NaOH: 0.1 Fermenting and culturing at 37 ℃, and stopping fermentation when the fermentation is carried out for 13 hours. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 6

b. High density culture in fermenter

The basic culture medium comprises the following components:

table 22

The feed medium consists of: tryptone 50g/L, yeast extract 30g/L, glucose 300g/L，MgSO ₄ ·7H ₂ O2g/L。

Inoculating shake flask seed liquid into a fermentation tank according to an inoculum size of 5%, wherein the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ The pressure of the tank per hour is controlled to be 0.04-0.09 MPa, and the dissolved oxygen is controlled to be not lower than 30 percent. When dissolved oxygen was increased and the pH was increased simultaneously, induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then feeding a feeding culture medium, controlling the flow rate to feed 15L of the feeding culture medium every 4-5 h until the culture is finished, and fermenting and culturing for 20h. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

Comparative example 7

b. High density culture in fermenter

The basic culture medium comprises the following components:

Table 23

The feed medium consists of: tryptone 100g/L, yeast extract 60g/L, glucose 500g/L, mgSO ₄ ·7H ₂ O2g/L。

Inoculating shake flask seed liquid into a fermentation tank according to an inoculum size of 5%, wherein the initial ventilation volume of the fermentation tank is 4+/-0.5 m ³ And/h, the temperature is 37.0 ℃, the rotating speed is set to 200rpm, the tank pressure is 0.04-0.06 MPa, the pH value is automatically controlled to 7.1+/-0.1 by ammonia water in the fermentation process, the temperature is controlled to 37.0+/-1.0 ℃, and the air flow is controlled to 0.6-5.5 m by adjusting the rotating speed to 200-600 rpm ³ The pressure of the tank per hour is controlled to be 0.04-0.09 MPa, and the dissolved oxygen is controlled to be not lower than 30 percent. When dissolved oxygen was increased and the pH was increased simultaneously, induction was performed by adding the inducer IPTG at a final concentration of 0.2 mM. And then feeding a feeding culture medium, controlling the flow rate to feed 15L of the feeding culture medium every 16-20h until the culture is finished, and fermenting and culturing for 26h. And taking fermentation liquor to detect the cell density, the inclusion body yield and the like.

The test results of the examples and comparative examples are shown in Table 24.

TABLE 24 test results for different culture conditions

/>

Claims

1. A basal medium for producing insulin glargine, the basal medium comprising:

TABLE 1

The microelements comprise the following components:

TABLE 2

。

2. A fermentation process for producing insulin glargine using the basal medium of claim 1, comprising the steps of:

a. Amplifying and culturing recombinant escherichia coli strains in a seed culture medium step by step to obtain seed liquid;

b. inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, controlling the dissolved oxygen content of the fermentation liquid to be not lower than 30%, controlling the temperature to be 36.0-38.0 ℃, controlling the pH value to be 7.0-7.2, adding ammonia water as a substance for controlling the pH value, feeding the fed-batch culture medium in batches, and when the dissolved oxygen content reaches more than 90% and the pH value rises, carrying out first feeding of the fed-batch culture medium, and controlling the flow rate to be 30-35% of the fed-batch total feed amount every 5-8 h; and adding an inducer for induction, then adding a feed supplement culture medium for the second time, and controlling the flow speed to be 65-70% of the total feed supplement amount in each 15-18 h till the end of culture.

3. The fermentation process of claim 2, wherein the feed medium composition is: 80-120 g/L of Tryptone, 40-80 g/L of Yeast extract, 400-600 g/L of glycol and MgSO (MgSO) ₄ ·7H ₂ O 1~3g/L。

4. The fermentation process of claim 2, wherein the seed liquid is inoculated into the fermenter basal medium in an amount of: the volume of the seed liquid is 5-10% of the volume of the basic culture medium.

5. The fermentation process of claim 2, wherein the feed medium is used in an amount of 23 to 27% by volume of the basal medium.