阅读说明：本技术 半连续发酵培养毕赤酵母工程菌的方法 (Method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation ) 是由 赵枭阳 柴丹丹 李婷 李新乐 张晓雷 徐延伟 赵巧辉 李桂林 付光宇 吴学炜 杨 于 2020-12-21 设计创作，主要内容包括：本发明涉及生物工程领域,具体涉及半连续发酵培养毕赤酵母工程菌的方法。该方法以BSM为基础培养基,甲醇为诱导剂,进行毕赤酵母工程菌的连续发酵及外源蛋白的连续表达,接种后菌体重复利用,连续性强,工艺流程较批次发酵有了简化,大大减少了工作强度及工作量,外源蛋白收率较批式发酵提升了40％,本发明流程简单,成本较低,适用于工业化生产。(The invention relates to the field of bioengineering, in particular to a method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation. The method uses BSM as a basic culture medium and methanol as an inducer to perform continuous fermentation of pichia pastoris engineering bacteria and continuous expression of foreign protein, the inoculated bacteria are recycled, the continuity is strong, the process flow is simplified compared with batch fermentation, the working intensity and the working amount are greatly reduced, the yield of the foreign protein is improved by 40% compared with batch fermentation, the process is simple, the cost is low, and the method is suitable for industrial production.)

1. The method for culturing the pichia pastoris engineering bacteria by semi-continuous fermentation is characterized by comprising the following steps:

step 1: preparing a seed solution;

step 2: putting the mixture into a tank for fermentation; inoculating the seed liquid prepared in the step 1 to a fermentation culture medium, performing first round fermentation, feeding glycerol containing PTM1, adjusting the feeding rate to ensure that the dissolved oxygen is not lower than 20%, and sampling to obtain wet weight; adding methanol to induce the expression of the foreign protein, and adjusting the adding rate according to the dissolved oxygen to ensure that the dissolved oxygen is not lower than 20 percent;

and step 3: continuous fermentation; collecting part of fermentation liquor obtained in the step 2, supplementing a fermentation culture medium without glycerol to the wet weight before induction, and performing second round fermentation;

and 4, step 4: and (5) repeating the step (3) until the expression quantity of the foreign protein does not reach the standard.

2. The method of claim 1, wherein the seed solution is prepared in step 1 by: inoculating pichia pastoris into a seed liquid culture medium, and culturing at 30 ℃ and 230rpm until the OD is 5.0-8.0;

the seed liquid culture medium (BMGY) comprises: yeast extract 1%, Peptone 2%, potassium phosphate buffer (pH6.0)100mmol/L, YNB 1.34.34%, Biotin (4X 10-5)%, Glycerol 1%.

3. The method according to claim 1 or 2, wherein the fermentation medium (BSM) in step 2 comprises: 85% H₃PO₄ 26.7ml/L、CaSO₄·2H₂O 0.93g/L、K₂SO₄ 18.2g/L、MgSO₄·2H₂O14.9 g/L, KOH 4.13.13 g/L, glycerin 40g/L, 100% ammonia water to adjust pH to 5.0, sterilizing, and adding PMT14.0 ml/L;

the PMT1 includes: CuSO₄·5H₂O 6.0g/L、KI 0.088g/L、MnSO₄·H₂O 3.0g/L、Na₂MoO₄·2H₂O 0.2g/L、H₃BO₃ 0.02g/L、CoCl₂·6H₂O 0.5g/L、ZnCl₂ 20.0g/L、FeSO₄·7H₂O65.0 g/L, Biotin 0.2.2 g/L, concentrated H₂SO₄ 5.0ml。

4. A method according to any one of claims 1 to 3, wherein the inoculum size of the inoculation in step 2 is 5%, and the first fermentation run is in particular: and (3) fermenting at the temperature of 30 ℃, at the stirring speed of 300-600rpm, at the ventilation volume of 1vvm and at the pH of 5.0 until the nutrient in the culture medium is exhausted.

5. The method according to any one of claims 1 to 4, wherein the time for the first fermentation run is 20 h.

6. The process according to any one of claims 1 to 5, wherein the glycerol containing PTM1 is glycerol containing PTM1 at a volume concentration of 50% and 12ml/L, and the feed of the glycerol containing PTM1 is 80ml of the glycerol containing PTM1 per 1L of the fermentation medium; the time for feeding the glycerol containing PTM1 was 8 h.

7. The process according to any one of claims 1 to 6, wherein the methanol is methanol containing 12ml/L PTM1, and the time for feeding methanol is 120 h.

8. A process according to any one of claims 1 to 7 wherein the portion of the fermentation broth in step 3 is 50% v/v broth.

9. The method of any one of claims 1 to 8, wherein the number of repetitions in step 4 is at least 2.

10. The method of any one of claims 1 to 9, wherein the pichia pastoris engineered bacterium is pichia pastoris GS 115.

Technical Field

The invention relates to the field of bioengineering, in particular to a method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation.

Background

The fermentation engineering refers to the production of useful products for human beings by using certain specific functions of microorganisms by adopting modern engineering technical means, and is generally divided into batch fermentation, fed-batch fermentation, continuous fermentation and the like.

Pichia pastoris has been used in the eighties for the production of Single Cell Protein (SCP), with a good fermentation base and a cell density of up to 100g/L dry weight. The components of the growth culture solution comprise inorganic salt, trace elements, biotin, a nitrogen source and a carbon source, and are cheap and nontoxic. It can grow fast in culture medium with methanol as the only carbon source, and the key enzyme of alcohol oxidase AOX-methanol metabolic pathway can reach 30% of soluble protein in cell. And AOX can not be detected in cultured cells using glucose, glycerol or ethanol as a carbon source, and the gene promoter has an obvious regulation function and can be used for regulating the expression of an exogenous gene.

The recombinant pichia pastoris is utilized to ferment and produce exogenous protein, the expression system is a set of very complete expression systems, thousands of proteins are successfully expressed in the system, and a plurality of proteins such as insulin, albumin, hepatitis B antigen and the like are used in the pharmaceutical field by utilizing the pichia pastoris expression system and are commercialized, so that the research on efficient and industrialized fermentation process is always the direction of fermentation human effort. The pichia pastoris belongs to induction type fermentation, and the current situation of the industry is observed, the fermentation form of batch or fed-batch is mainly used, the consumption of human and material resources is huge, the operations of dilute preparation, sterilization, inoculation, emptying, cleaning and the like are required to be carried out in each batch of fermentation, the working procedures are complicated, the production efficiency is low, and therefore how to design a fermentation mode with high fermentation efficiency is a technical point which needs to be solved urgently in the field.

Disclosure of Invention

In view of this, the invention provides a method for semi-continuous fermentation culture of pichia pastoris engineering bacteria. The semi-continuous fermentation technology is applied to the culture of pichia pastoris engineering bacteria, new nutrient substances are supplemented, metabolic waste is diluted, product inhibition is relieved by means of discharging part of fermentation liquor and supplementing fresh culture medium, meanwhile, operation procedures such as sterilization, inoculation and cleaning are reduced, and production efficiency is improved.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for culturing pichia pastoris engineering bacteria by semi-continuous fermentation, which comprises the following steps:

step 1: preparing a seed solution;

step 2: putting the mixture into a tank for fermentation; inoculating the seed liquid prepared in the step 1 to a fermentation culture medium, performing first round fermentation, feeding glycerol containing PTM1, adjusting the feeding rate to ensure that the dissolved oxygen is not lower than 20%, and sampling to obtain wet weight; adding methanol to induce the expression of the foreign protein, and adjusting the adding rate according to the dissolved oxygen to ensure that the dissolved oxygen is not lower than 20 percent;

and step 3: continuous fermentation; collecting part of fermentation liquor obtained in the step 2, supplementing a fermentation culture medium without glycerol to the wet weight before induction, and performing second round fermentation;

and 4, step 4: and (5) repeating the step (3) until the expression quantity of the foreign protein does not reach the standard.

In some embodiments of the present invention, the seed solution in step 1 is prepared by: inoculating pichia pastoris into a seed liquid culture medium, and culturing at 30 ℃ and 230rpm until the OD is 5.0-8.0;

the seed liquid culture medium (BMGY) comprises: yeast extract 1%, Peptone 2%, potassium phosphate buffer (pH6.0)100mmol/L, YNB 1.34.34%, Biotin (4X 10-5)%, Glycerol 1%.

In some embodiments of the invention, the fermentation medium (BSM) in step 2 comprises: 85% H₃PO₄26.7ml/L、CaSO₄·2H₂O 0.93g/L、K₂SO₄ 18.2g/L、MgSO₄·2H₂O14.9 g/L, KOH 4.13.13 g/L, glycerin 40g/L, 100% ammonia water to adjust pH to 5.0, sterilizing, and adding PMT14.0 ml/L;

the PMT1 includes: CuSO₄·5H₂O 6.0g/L、KI 0.088g/L、MnSO₄·H₂O 3.0g/L、Na₂MoO₄·2H₂O 0.2g/L、H₃BO₃ 0.02g/L、CoCl₂·6H₂O 0.5g/L、ZnCl₂ 20.0g/L、FeSO₄·7H₂O65.0 g/L, Biotin 0.2.2 g/L, concentrated H₂SO₄ 5.0ml。

In some embodiments of the invention, the inoculum size of the inoculation in step 2 is 5%, and the first fermentation run is in particular: and (3) fermenting at the temperature of 30 ℃, at the stirring speed of 300-600rpm, at the ventilation volume of 1vvm and at the pH of 5.0 until the nutrient in the culture medium is exhausted.

In some embodiments of the invention, the time for the first round of fermentation is 20 hours.

In some embodiments of the invention, the glycerol containing PTM1 is 50% glycerol containing 12ml/L PTM1 by volume, and the PTM 1-containing glycerol is fed in an amount of 80ml of the PTM1 per 1L fermentation medium; the time for feeding the glycerol containing PTM1 was 8 h.

In some embodiments of the invention, the methanol is methanol containing 12ml/L PTM1, and the feeding time of the methanol is 120 h.

In some embodiments of the invention, the portion of the fermentation broth in step 3 is 50% v/v fermentation broth.

In some embodiments of the invention, the number of repetitions in step 4 is at least 2.

In some embodiments of the invention, the pichia engineering bacterium is pichia GS 115.

The semi-continuous fermentation technology is applied to the culture of pichia pastoris engineering bacteria, new nutrient substances are supplemented, metabolic waste is diluted and product inhibition is relieved by discharging part of fermentation liquor and supplementing a fresh culture medium, and the used supplemented culture medium is a BSM basic fermentation culture medium (not containing glycerol), so that the aim of the supplemented culture medium is to maintain the relative stability of the cell growth state and the cell density among semi-continuous fermentation batches without using the glycerol as a carbon source to improve the cell density and directly using methanol for induced expression is fulfilled. Meanwhile, the operation procedures of sterilization, inoculation, cleaning and the like are reduced, and the production efficiency is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a comparison of the wet weight change trend between semi-continuous fermentation stages;

FIG. 2 shows a comparison of the change in supernatant protein concentration over the semi-continuous fermentation stage;

FIG. 3 shows a comparison of the trend of the amount of the desired protein between the semi-continuous fermentation stages.

Detailed Description

The invention discloses a method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation.

The invention adopts the following technical scheme:

preparation of seed liquid

Inoculating pichia pastoris into a seed liquid culture medium, wherein the culture method comprises the following steps: the temperature is 30 ℃, and the stirring speed is 230 rpm; seed liquid medium composition (BMGY): yeast extract 1%, Peptone 2%, potassium phosphate buffer (pH6.0)100mmol/L, YNB 1.34.34%, Biotin (4X 10-5)%, Glycerol 1%.

② fermenting in a tank

Basal fermentation medium component (BSM): 85% H₃PO₄ 26.7ml/L、CaSO₄·2H₂O 0.93g/L、K₂SO₄18.2g/L、MgSO4·2H₂O14.9 g/L, KOH 4.13.13 g/L, glycerin 40g/L, 100% ammonia adjusted pH to 5.0, sterilized and PMT14.0ml/L added.

PMT1 composition: CuSO₄·5H₂O 6.0g/L、KI 0.088g/L、MnSO₄·H₂O 3.0g/L、Na₂MoO₄·2H₂O 0.2g/L、H₃BO₃ 0.02g/L、CoCl₂·6H₂O 0.5g/L、ZnCl₂ 20.0g/L、FeSO₄·7H₂O65.0 g/L, Biotin 0.2.2 g/L, concentrated H₂SO₄ 5.0ml。

Inoculating the seed solution prepared in the step I into a culture medium for fermentation, wherein the inoculation amount is 5%, and the culture method comprises the following steps: the temperature is 30 ℃, the stirring speed is 300-. After the glycerol feeding is finished, sampling and recording the wet weight of the thalli (wet weight before induction), feeding methanol (containing 12ml/L PTM1) for carrying out induction expression of the exogenous protein, and adjusting the feeding rate according to dissolved oxygen to ensure that the dissolved oxygen is not less than 20 percent.

③ continuous fermentation

After fermenting for 120 hours, discharging 50% of feed liquid for harvesting, supplementing a basic fermentation culture medium (without glycerol) into the fermentation tank until the wet weight is equal to that before induction, starting a new round of fermentation, wherein the fermentation conditions are the same, and continuously repeating the above operations in the next batch until the expression quantity of the exogenous protein does not reach the standard.

The glycerol is a carbon source and is used in a culture stage to play a role in improving cell density, the methanol is a carbon source and an inducer and is mainly used in an induction stage to induce the expression of the foreign protein and can be used as a carbon source for cell metabolism to perform one-time feeding and supplementing, after the completion of the supplementing, the wet weight is at a node after the culture stage and before the induction stage, and the methanol induction is considered to be directly performed, and the fermentation liquor does not contain the glycerol which is not consumed, so that the BSM which does not contain the glycerol is added. In summary, the feed medium used was BSM basal fermentation medium (without glycerol) aiming at the feed medium not using glycerol as carbon source to increase cell density, but using methanol to induce expression, meaning that the cell growth status and density were relatively stable between the semi-continuous fermentation batches.

The method provided by the invention uses BSM as a basic culture medium and methanol as an inducer to perform continuous fermentation of pichia pastoris engineering bacteria and continuous expression of foreign protein, the inoculated bacteria are recycled, the continuity is strong, the process flow is simplified compared with batch fermentation, the working intensity and the working amount are greatly reduced, the yield of the foreign protein is improved by 40% compared with batch fermentation, the process is simple, the cost is lower, and the method is suitable for industrial production.

In the method for culturing the pichia pastoris engineering bacteria through semi-continuous fermentation, the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

Inoculating 500ml of seed liquid into a 10LBSM culture medium according to an inoculation proportion of 5% by adopting a 30L fermentation tank, culturing until dissolved oxygen rises suddenly at a stirring speed of 300rpm, an air flow of 1vvm, a tank pressure of 0.05MP and a pH value of 5.0 at 30 ℃, and gradually increasing the rotation speed to 700rpm when the dissolved oxygen is below 20% in the culture process.

② adding 800ml of 50 percent glycerin (containing 12ml/L PTM1), controlling the rate to ensure that the dissolved oxygen is not less than 20 percent, sampling after the completion to determine the wet weight of about 180g/L, and the whole process is about 8 hours.

③ adding methanol (containing 12ml/L PTM1) for induction, controlling the speed to ensure that the dissolved oxygen is not less than 20 percent, and continuing for 120 hours.

Fourthly, after 120 hours of induction, 50 percent of feed liquid is discharged for harvesting, and sterile BSM basic fermentation medium (without glycerol) is supplemented into the fermentation tank until the wet weight is equal to the wet weight before induction.

Fifthly, the methanol (containing 12ml/L PTM1) is fed again for induction, the rate is controlled to ensure that the dissolved oxygen is not less than 20 percent, and the process is continued for 120 hours.

Sixthly, discharging 50 percent of feed liquid after 120 hours of induction for harvesting, and supplementing a sterile BSM basic fermentation medium (without glycerol) into the fermentation tank until the wet weight is equal to the wet weight before induction.

Seventhly, the reciprocating process is carried out for three times, the whole reciprocating process is described as four stages, the first stage refers to feeding methanol from the beginning to the first discharging, the second stage refers to feeding the methanol from the first feeding to the second discharging, the third stage refers to feeding the methanol from the second feeding to the third discharging, and the fourth stage refers to feeding the methanol from the third feeding to the end of fermentation, and the results are as follows:

table 1: semi-continuous fermentation result carding

Example 2

And (4) carrying out wet weight, concentration and electrophoresis analysis on the fermentation process sample. The determination method comprises the following steps: taking 30ml of fermentation liquor from a sampling port of the fermentation tank, centrifuging for 10 minutes by using 10000 revolutions of a centrifugal tube with known weight, and carrying out wet weight: weighing the supernatant after discarding, and subtracting the weight of the centrifugal tube from the weight to obtain the wet weight; concentration: taking the supernatant, and determining the protein concentration by a G250 method; electrophoresis: the electrophoretic analysis was performed by SDS-PAGE with Coomassie blue staining. The results are shown in FIGS. 1 to 3 and tables 2 to 3.

As can be seen from fig. 1, 2 and 3, the wet weight of the cells, the concentration of the supernatant protein and the content of the target protein in the 4 stages have the same trend of change, and the fermentation time from the second fermentation is greatly shortened compared with the first fermentation time by continuously circulating the three fermentations, and the wet weight of the cells, the concentration of the supernatant protein and the content of the target protein have the same trend of change, so that the activity of the yeast does not obviously show a trend of decrease.

Table 2: comparison data of wet weight change trend between semi-continuous fermentation stages

Table 3: comparison data of change trend of supernatant protein concentration between semi-continuous fermentation stages

Example 3

After harvest of the fermentation broth, the fermentation supernatant was purified using a nickel column with the following results:

table 4: purified result carding

And (3) comparing the production efficiency of semi-continuous fermentation with batch fermentation:

TABLE 5

The ratio of manual energy consumption to batch energy consumption of semi-continuous fermentation:

TABLE 6

Type of fermentation	Mean of artifacts	Average energy consumption	Average water consumption
				Semi-continuous fermentation	1.25 persons/day	25 degree/day	2.5L/day
Batch fermentation	1.8 persons/day	32 degree/day	10L/day
				Ratio of	0.69:1	0.78:1	0.25:1

The results show that the semi-continuous fermentation thalli is recycled, the continuity is strong, the process flow is simplified compared with batch fermentation, the working intensity and the workload are greatly reduced, and in addition, the production efficiency of the semi-continuous fermentation is improved by 40 percent compared with the batch fermentation.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.