Pichia pastoris and application thereof

Abstract

The invention discloses pichia pastoris and application thereof, and belongs to the field of microbial fermentation. According to the present invention, the lactic acid producing type pichia pastoris special for companies is subjected to mutagenesis through the ARTP mutagenesis technology, such that the pichia pastoris mutant strain Pichia pastoris PLS006 capable of maintaining the lactic acid producing performance under the low ventilation condition (0.33 vvm) is obtained, and the preservation number is CCTCC M 20251051; the fermentation ventilation quantity of the strain is remarkably reduced to 0.33 vvm from 1 vvm in the traditional process, the energy consumption is reduced, meanwhile, the lactic acid production efficiency is not remarkably reduced, and the strain is suitable for industrial fermentation production. The invention also provides application of the strain in the fields of lactic acid preparation, food fermentation and biosynthesis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a Pichia pastoris and application thereof. BACKGROUND

[0002] Pichia pastoris is a host strain widely used in industrial fermentation, which has high protein expression capacity and high-density culture resistance. In recent years, its application in metabolic product synthesis (such as organic acids and enzyme preparations) has gradually increased. Lactic acid, as an important organic acid, is widely used in food, medicine and degradable material fields, and its biosynthesis depends on the sugar metabolic pathway of microorganisms. The traditional Pichia pastoris fermentation process usually needs to maintain a high ventilation rate (more than 1vvm) to ensure metabolic activity, but high ventilation rate leads to increased energy consumption and equipment cost, which limits the economic efficiency of industrial application.

[0003] In the prior art, there are few reports on reducing ventilation demand through strain improvement, and most researches focus on improving product yield rather than optimizing process parameters. Therefore, breeding a Pichia pastoris strain that can efficiently produce lactic acid under low ventilation conditions is of great significance to reduce production cost and promote green manufacturing. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a Pichia pastoris and application thereof.

[0005] The object of the present application can be achieved by the following technical solutions:

[0006] In a first aspect of the present application, a Pichia pastoris strain is provided, which is mutant Pichia pastoris PLS006, and the preservation number is CCTCC M 20251051.

[0007] In a second aspect of the present application, a preparation method of the Pichia pastoris strain is provided, which comprises the following steps:

[0008] The genetically engineered strain preserved in glycerol tube is revived in liquid YPD medium, and then screened and purified to obtain colonies, which are then inoculated into liquid YPD medium again and then inoculated into fermentation medium for fermentation to obtain the starting strain.

[0009] The starting strain obtained by screening is subjected to mutagenesis by using normal pressure room temperature plasma mutagenesis technology.

[0010] The mutant strain Pichia pastoris PLS006 is obtained by screening and culturing the mutagenized strain in a limited oxygen medium containing lactic acid under ventilation conditions of 0.33-1vvm.

[0011] Optionally, the fermentation condition is 180-220 rpm for 45-60 hours, the ventilation amount is 1vvm, and the bottom fungus is confirmed as a lactic acid production.

[0012] Optionally, the fermentation medium formula is (w / v): 0.1%-0.5% yeast extract, 0.5%-1% peptone, 5% glucose, and 0.1% inorganic salt.

[0013] Optionally, the atmospheric room temperature plasma mutagenesis comprises the following steps: taking the starting fungus liquid in the logarithmic growth phase, resuspending with physiological saline after centrifugation, uniformly coating on a slide, and using an atmospheric room temperature plasma mutagenesis device to obtain a mutagenesis fungus group.

[0014] In a third aspect, the present application relates to the Pichia pastoris fungus obtained by the preparation method of the Pichia pastoris fungus.

[0015] In a fourth aspect, the present application relates to the application of the Pichia pastoris fungus and the Pichia pastoris fungus obtained by the preparation method of the Pichia pastoris fungus in reducing the ventilation requirement in the lactic acid production fermentation process.

[0016] The present application has the following beneficial effects:

[0017] The Pichia pastoris PLS006 provided by the present application can greatly reduce the ventilation requirement in the fermentation process, is suitable for the fields of dairy product processing and biological plastic raw material production, and has significant energy saving and consumption reducing benefits. BRIEF DESCRIPTION OF DRAWINGS

[0018] The present application will be further described below in combination with the drawings.

[0019] Figure 1 : PLS006 colony morphology in the embodiments of the present application;

[0020] Figure 2 : Lactic acid production comparison chart under different ventilation amounts in Embodiment 2 of the present application;

[0021] Figure 3 : Lactic acid production comparison chart under the same ventilation amount in Embodiment 3 of the present application. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0023] The application provides a Pichia pastoris strain, and the Pichia pastoris is obtained by the following method.

[0024] (1) Taking wild type Pichia pastoris as a starting strain, a wild strain capable of tolerating low pH is obtained through screening, and then the strain is treated by ARTP mutagenesis, and the mutagenesis conditions are as follows: power 100-120 W, helium flow rate 12 SLM, and treatment time 10-20 s; after mutagenesis, the PLS006 strain is found and screened out;

[0025] (2) A low ventilation dependent mutant strain is obtained through high-throughput screening, wherein the lactic acid production efficiency of the PLS006 is 98% of that of the starting strain under 0.33 VVM ventilation, and the fermentation cycle is unchanged, and the ventilation is reduced by nearly 70%.

[0026] Example 1: Screening of Pichia pastoris strain PLS006

[0027] 1. Isolation and purification of the strain

[0028] 1) The strain preserved in a glycerol tube is taken out and then inoculated in a liquid YPD medium for recovery, wherein the YPD medium is a general-purpose culture medium.

[0029] 2) The liquid activated strain is streaked on a solid YPD medium with a coating rod for isolation and purification, and a microscope is used for microscopic examination to ensure the single nature of the colony.

[0030] 3) Shake flask acid production fermentation verification of the starting strain

[0031] The single colony screened above is inoculated in a liquid YPD medium again, and then inoculated in a fermentation medium at an inoculation amount of 2%, and then subjected to fermentation in a 5L tank, and the fermentation conditions are as follows: rotation speed 180-220 rpm, culture time 45-60 hours, ventilation amount 1 VVM, and the strain is confirmed as a lactic acid production chassis.

[0032] The fermentation medium formula is as follows (w / v): 0.1%-0.5% yeast extract, 0.5%-1% peptone, 5% glucose, 0.1% inorganic salt, and the rest is water.

[0033] 1. Strain mutagenesis and screening

[0034] The starting strain screened is subjected to mutagenesis by using atmospheric room temperature plasma (ARTP) mutagenesis technology.

[0035] Mutagenesis equipment: ARTP, Wuxi Source Tianmu Biotechnology Co., Ltd., and the equipment is operated according to the equipment instruction manual.

[0036] The experimental operation is as follows:

[0037] 1) ARTP mutagenesis: take the log growth of Pichia yeast liquid, after centrifugation, resuspended with 0.9% saline to OD600 = 1.0, evenly coated on the slide, set power 110W, helium flow 12SLM, processing time 15s, obtain mutagenesis bacteria.

[0038] 2) Low ventilation adaptive screening: the mutagenesis bacteria are inoculated in 96-well plates, each well is added with oxygen-limited medium containing lactic acid, and cultured for 48 hours under 0.33vvm ventilation condition, the lactic acid content is detected, and the dominant strain PLS006 is screened.

[0039] Example 2: Fermentation performance verification

[0040] 5L fermenter test is respectively carried out with the starting strain and PLS006, the ventilation amount is 1vvm and 0.33vvm respectively, the inoculation amount is 2%, and the culture is carried out at 32℃ for 60 hours. The results show that the lactic acid production of PLS006 is 48g / L, which has no significant difference with the starting strain (50g / L), but the ventilation amount is reduced by 67%. It can be seen that the strain of the present application can obtain the same lactic acid production as the starting strain under high ventilation amount, which reveals that the strain of the present application has lower ventilation requirement.

[0041] Example 3:

[0042] 5L fermenter test is respectively carried out with the starting strain and PLS006, the ventilation amount is 1vvm and 0.33vvm respectively, the inoculation amount is 2%, and the culture is carried out at 32℃ for 60 hours. The results show that the lactic acid production of PLS006 is 48g / L, which has no significant difference with the starting strain (50g / L), but the ventilation amount is reduced by 67%. It can be seen that the strain of the present application can obtain the same lactic acid production as the starting strain under high ventilation amount, which reveals that the strain of the present application has lower ventilation requirement.

[0043] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0044] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. A Pichia pastoris strain, characterized in that, The Pichia pastoris mutant strain is Pichia pastoris PLS006, with accession number CCTCC M 20251051.

2. A method for preparing Pichia pastoris, characterized in that, Includes the following steps: Genetically engineered bacterial strains preserved in glycerol tubes were revived in liquid YPD medium. After screening and purification, the obtained colonies are transferred to liquid YPD medium again and then inoculated into fermentation medium for fermentation to obtain the starting bacteria. The selected starting bacteria were mutagenized using atmospheric pressure and room temperature plasma mutagenesis technology. The mutated strain was cultured in a lactic acid-containing oxygen-limited medium under aeration conditions of 0.33–1 vvm, and the mutant strain Pichia pastoris PLS006 was obtained through screening.

3. The method for preparing Pichia pastoris according to claim 2, characterized in that, The fermentation conditions were: culture at 180-220 rpm for 45-60 hours, with an aeration rate of 1 vvm, and the bacteria were confirmed to be lactic acid-producing substrate bacteria.

4. The method for preparing Pichia pastoris according to claim 2, characterized in that, The fermentation medium formulation is (w / v): 0.1%-0.5% yeast extract, 0.5%-1% peptone, 5% glucose, and 0.1% inorganic salts.

5. The method for preparing Pichia pastoris according to claim 2, characterized in that, The method of atmospheric pressure and room temperature plasma mutagenesis includes the following steps: take the starting bacterial culture in the logarithmic growth phase, centrifuge it, resuspend it in physiological saline, spread it evenly on a slide, and process it with an atmospheric pressure and room temperature plasma mutagenesis device to obtain the mutagenic bacterial population.

6. Pichia pastoris obtained by the preparation method of any one of claims 2 to 5.

7. The application of the Pichia pastoris according to claim 1 and the Pichia pastoris obtained by the preparation method of any one of claims 2 to 5 in reducing the ventilation requirements during lactic acid fermentation.

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