Kluyveromyces lactis and its use

Abstract

The present invention belongs to the field of biological fermentation technology, and specifically relates to Kluyveromyces lactis and its use. The present invention provides a Kluyveromyces lactis, which is deposited in the China Center for Type Culture Collection, and its deposit number is CCTCC NO: M 20241460. The Kluyveromyces lactis significantly improves the problem of poor extracellular protein secretion and is a good host choice for protein expression.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to Kluyveromyces lactis and uses thereof. Background Art

[0002] Kluyveromyces lactis ( Kluyeromyces lactis ) is a non-pathogenic yeast widely found in nature. It has good biological characteristics of many types of degrading enzymes and strong decomposition ability. It is an important source of lactase and inulinase in dairy products. It is widely used in agricultural production with good safety. Kluyveromyces lactis has a high ability to secrete proteins and a high biomass. It can be used to produce enzyme preparations and can be used to construct recombinant protein expression systems. Kluyveromyces lactis chassis cells also have many advantages as heterologous expression hosts: first, it has been certified as a food-safe (generally recognized as safe, GRAS) microorganism by the US Food and Drug Administration; second, its genetic background is clear, easy to genetically manipulate, and the stability of both free and integrated gene expression is high; third, glycosylation is moderate; fourth, it can grow in standard yeast culture medium, and large-scale fermentation does not require the explosion-proof fermentation equipment required by methylotrophic yeasts (such as Pichia pastoris and Hansenula polymorpha).

[0003] The existing lactic acid Kluyveromyces has a low secretion of extracellular proteins, which may be a limitation of the current application of lactic acid Kluyveromyces in recombinant protein expression. Therefore, it is urgent to find new lactic acid Kluyveromyces to solve problems such as extracellular protein secretion. Summary of the invention

[0004] In order to overcome the defects of the prior art, the object of the present invention is to provide a new Kluyveromyces lactis to improve the problem of poor secretion of extracellular proteins.

[0005] The first aspect of the present invention provides a Kluyveromyces lactis ( Kluyveromyces lactis ), which is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241460.

[0006] The second aspect of the present invention provides a fermentation method of the aforementioned Kluyveromyces lactis, which comprises the following steps:

[0007] The lactic acid Kluyveromyces is fermented in the presence of a nitrogen source and a carbon source to collect extracellular proteins.

[0008] In some embodiments, the fermentation method further comprises isolating and purifying the extracellular protein.

[0009] In some embodiments, the carbon source includes at least glucose.

[0010] In some embodiments, the nitrogen source includes at least ammonium sulfate.

[0011] In some embodiments, the mass ratio of the carbon source to the nitrogen source is (1-5): 1.

[0012] In some embodiments, the fermentation time is 32-80 hours.

[0013] In some embodiments, the fermentation temperature is 25-40°C.

[0014] In some embodiments, the inoculation amount of Kluyveromyces lactis is 0.05%-5%.

[0015] In some preferred embodiments, the Kluyveromyces lactis is fermented in an ammonium sulfate-dextrose medium or a corn steep liquor medium.

[0016] The third aspect of the present invention provides use of the aforementioned Kluyveromyces lactis in expressing heterologous proteins.

[0017] The fourth aspect of the present invention provides use of the aforementioned Kluyveromyces lactis in preparing food or feed additives.

[0018] The fifth aspect of the present invention provides a food composition or a feed composition, which comprises the aforementioned Kluyveromyces lactis or a culture thereof.

[0019] The lactic acid Kluyveromyces provided by the present invention overcomes the defects of the prior art and significantly improves the problem of poor extracellular protein secretion. Fermentation in a 100 mL shake flask detected that the total extracellular protein secretion was about 0.58 ± 0.06 mg / mL, and the obtained total extracellular protein secretion amount far exceeded the level of the prior art.

[0020] Collection information:

[0021] Classification and naming: Kluyveromyces lactis YN2-1 Kluyveromyces lactis YN2-1

[0022] Date of deposit: July 3, 2024

[0023] Deposit number: CCTCC NO: M 20241460

[0024] Collection Unit: China Center for Type Culture Collection

[0025] Deposit address: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphology of Kluyveromyces lactis grown in KDM solid medium is shown.

[0027] Figure 2 The cell morphology of Kluyveromyces lactis in YPD liquid medium is shown.

[0028] Figure 3 The electrophoresis diagram of the amplified ITS gene fragment of Kluyveromyces lactis is shown.

[0029] Figure 4 A growth curve of amplified Kluyveromyces lactis YN2-1 is shown.

[0030] Figure 5 A comparison of amplified extracellular secretory proteins of Kluyveromyces lactis YN2-1 with those of other strains is shown. DETAILED DESCRIPTION

[0031] The first aspect of the present invention provides a lactic acid Kluyveromyces, which is classified and named as lactic acid Kluyveromyces ( Kluyveromyces lactis ) YN2-1, whose deposit number is CCTCC NO: M 20241460. In the present invention, the lactic acid Kluyveromyces can also be referred to as YN2-1.

[0032] When the present invention screens Kluyveromyces lactis, the total extracellular protein is used as the screening index, the fermentation broth cultured for 72 hours is taken, the supernatant is taken by centrifugation, and the protein concentration of the fermentation broth is determined by the Bradford method, and a strain of Kluyveromyces lactis YN2-1 is screened, and the extracellular protein content detected after 72 hours is 0.58 mg / mL, which is significantly higher than that of other strains. The Kluyveromyces lactis YN2-1 of the present invention has good extracellular protein secretion ability and is suitable for the expression of exogenous proteins.

[0033] The second aspect of the present invention provides a fermentation method of the aforementioned Kluyveromyces lactis, comprising the following steps: fermenting the Kluyveromyces lactis in the presence of a nitrogen source and a carbon source, and collecting the bacteria; optionally, further comprising collecting total extracellular protein.

[0034] The term "fermentation" as used in the present invention refers to the process in which Kluyveromyces lactis produces the yeast itself, or direct metabolites or secondary metabolites through life activities under aerobic conditions.

[0035] The term "fermentation product" or "culture" described in the present invention refers to a product produced by a method including a fermentation step using a fermenting organism, and may include the bacteria of Kluyveromyces lactis YN2-1 of the present invention and / or the extracellular proteins produced thereby.

[0036] The present invention does not limit the method for collecting the extracellular supernatant, and conventional methods in the art, such as centrifugation and filtration, can be used. The present invention does not limit the method for separation and purification, and conventional methods in the art, such as precipitation, membrane separation, filtration, flocculation, etc. can be used.

[0037] In some embodiments, the carbon source includes at least glucose. In some embodiments, the carbon source may also include at least one of glycerol, sucrose, lactose, xylose, and molasses.

[0038] In some embodiments, the nitrogen source includes at least ammonium sulfate. In some embodiments, the nitrogen source may also include at least one of corn steep liquor, whey powder, and tryptone.

[0039] In some embodiments, the mass ratio of the carbon source to the nitrogen source is (1-5): 1. The present invention finds that under the mass ratio, it is conducive to rapid proliferation of bacteria and obtains a fermentation product with better taste.

[0040] In some embodiments, the fermentation time is 32-80 hours. The present invention finds that if the fermentation time is too short, the yield of the fermentation product is low, and if the fermentation time is too long, the bacteria will age, affecting the quality of the fermentation product.

[0041] In some embodiments, the fermentation temperature is 25-40° C. If the fermentation temperature is too low, the fermentation speed is slow, and if the fermentation temperature is too high, the activity of the bacteria is affected. The present invention finds that when the fermentation temperature is within the temperature range, it is beneficial to improve the fermentation speed and the quality of the fermentation product.

[0042] In some embodiments, the inoculation amount of Kluyveromyces lactis is 0.05%-5%. If the inoculation amount is too low, it is not conducive to the rapid proliferation of the bacteria. If the inoculation amount is too high, the proliferation rate is too fast, the bacteria are prone to aging, and the quality of the fermentation product is affected.

[0043] In some embodiments, the lactic acid Kluyveromyces is fermented in an ammonium sulfate glucose medium (basal medium). The present invention finds that the fermentation of the lactic acid Kluyveromyces in the above medium is beneficial to the growth, reproduction and product synthesis of the strain. In the present invention, the formulas of the culture medium used are all conventional formulas in the art. Taking YPD medium as an example, it can contain 1 wt% yeast powder, 2 wt% peptone, and 2 wt% glucose. If it is a solid culture medium, it can also contain 2 wt% agar; the culture medium can be obtained by sterilizing at 121°C for 15 min.

[0044] The third aspect of the present invention provides use of the aforementioned Kluyveromyces lactis in preparing food or feed additives.

[0045] In some embodiments of the present invention, the YN2-1 strain can be used to ferment the corresponding substrate for preparing food; in other embodiments of the present invention, the YN2-1 strain or the heterologous protein it produces can be used as a fermentation substrate for other strains, such as lactic acid bacteria, to prepare food products; in other embodiments of the present invention, the YN2-1 strain can be directly added to food.

[0046] The fourth aspect of the present invention provides a food composition or a feed composition, which comprises the aforementioned Kluyveromyces lactis or a culture thereof.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0048] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0049] Example 1: Screening and identification of Kluyveromyces lactis

[0050] 1.1: Strain Isolation

[0051] (1) Add 5 g of the sample (yogurt) collected from Shangri-La, Yunnan in May 2023 to 45 mL of sterile saline, sonicate for 30 min, and dilute the solution by 10 -5 , 10 -6 , 10 -7 , 10 -8 The dilution was spread on the solid separation medium KDM and cultured at 30℃ for 48 h.

[0052] (2) Streak a single blue colony that resembles a yeast strain onto solid culture medium KDM and culture it. Select a single colony and repeat the streaking.

[0053] (3) The purified single colony was inoculated into YPD liquid culture medium and cultured at 30°C for 18-28 h. The extracellular protein content was screened (the protein concentration of the fermentation broth was determined by the Bradford method). A strain with strong extracellular protein secretion ability was obtained and named YN2-1. The obtained bacterial liquid was stored in a glycerol tube.

[0054] In the above separation step, the components of the solid separation medium KDM are: yeast extract 5 g / L; peptone 3 g / L; malt extract 3 g / L; glucose 10 g / L; agar 20 g / L, sterilized at 115℃, cooled to 45-50℃, added with XGal 0.08 mg / L; IPTG 0.1 mg / L; chloramphenicol 0.5 mg / L. The YPD liquid medium is: yeast powder 1%, peptone 2%, glucose 2%, sterilized at 121℃ for 15 min.

[0055] 1.2: Identification of YN2-1 strain

[0056] (1) Morphology of YN2-1 bacteria

[0057] Figure 1 This is the morphology of YN2-1 bacteria in KDM solid culture medium. The colonies are round, light blue, convex in the middle, smooth and moist on the surface, and with neat edges. Figure 2 This is a microscopic magnification (40 times) of YN2-1 bacteria in YPD liquid culture medium.

[0058] (2) Sequencing analysis of YN2-1 strain

[0059] a. ITS sequence amplification

[0060] Before sequencing the YN2-1 strain, the genomic DNA of the YN2-1 strain needs to be extracted. The present invention uses a yeast genomic DNA extraction kit (Solebo, item number D1900) to extract DNA. In order to study the taxonomy of unknown yeast strains, the above method is usually used for the detection and identification of yeast by amplifying the ITS gene fragment.

[0061] The ITS gene fragment was amplified by PCR using the genomic DNA of the YN2-1 strain as a template. The primers used are shown in Table 1. The PCR reaction system included 10 × buffer 2.5 μL, Mg 2+(25 mmol / L) 1.5 μL, Taq enzyme 0.25 μL, dNTP (25 mmol / L) 0.3 μL, forward primer (10 mmol / L) 0.5 μL, reverse primer (10 mmol / L) 0.5 μL, template DNA 0.1 μL, deionized water 19.35 μL. PCR amplification conditions are: denaturation at 95°C, annealing at 55°C, extension at 72°C, this process is cycled 30 times, then extended at 72°C for 10 min, after the PCR reaction is completed, the product is stored at 4°C. Use 1% agarose and add appropriate amount of nucleic acid dye GelRed to prepare gel blocks. After adding appropriate amount of PCR product and DNA markers (DNA Maker) containing fragments of various lengths to the gel blocks, place them in an electrophoresis instrument filled with TBE (Tris boric acid) buffer. After working at a certain voltage for 20 minutes, take them out and place them in a gel imaging system for observation to check whether the PCR amplification reaction is successful. The electrophoresis diagram of the amplification results is shown in the figure below. Figure 4 The PCR product is then sent to a sequencing company for sequencing, and the primers required for sequencing are the same as those used for amplification.

[0062] Table 1

[0063]

[0064] Figure 3 The results showed that the ITS sequence was amplified from the YN2-1 chromosome and can be used for sequencing comparison. The ITS sequencing results are as follows:

[0065] SEQ ID NO: 3:

[0066] .

[0067] Comparison analysis showed that the homology between YN2-1 and Kluyveromyces lactis H1-3 was 99.85%.

[0068] Based on the above identification results, the yeast strain YN2-1 of the present invention is Kluyveromyces lactis ( Kluyveromyces lactis ).

[0069] The YN2-1 strain of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC, deposit address is Wuhan University, Wuhan, China) on July 3, 2024, with the deposit number of CCTCC NO: M 20241460, and is classified as Kluyveromyces lactis ( Kluyveromyces lactis ) YN2-1.

[0070] Example 2: Fermentation of YN2-1 strain

[0071] 1) Growth assay

[0072] 1.1) Culture medium

[0073] Inorganic salt medium + 0.2% CaCO 3 (Glucose 30 g / L, ammonium sulfate 5 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 3 g / L, disodium edetate 0.015 g / L, zinc sulfate 0.0045 g / L, manganese chloride 0.001 g / L, sodium molybdate 0.0004 g / L, copper sulfate 0.0003 g / L, calcium chloride 0.0045 g / L, ferrous sulfate 0.003 g / L, potassium iodide 0.0001g / L, cobalt chloride 0.0003 g / L, boric acid 0.001 g / L, calcium pantothenate 0.001 g / L, niacin 0.001 g / L, biotin 0.0001 g / L, calcium carbonate 2 g / L).

[0074] 1.2) Culture conditions

[0075] The activated YN2-1 strain seed solution was adjusted to OD 600 =5, inoculated with 1% inoculation into a 250 mL conical flask containing 50 mL of inorganic salt medium, cultured at 30°C, 200 rpm for 32 hours, and the growth of the strain was determined.

[0076] 1.3) Strain growth

[0077] Growth curve Figure 4 As shown, the OD 600 OD of 1.8 at 24 hours 600 The value is 14, 32 hours plateau OD 600 The value is 16.5.

[0078] Example 3: Total extracellular protein content of YG medium

[0079] 3.1 Culture medium

[0080] YG medium: glucose 40 g / L, yeast extract 20 g / L.

[0081] 3.2 YN2-1 culture conditions

[0082] Incubate at 30°C, 200 rpm for 72 hours.

[0083] 3.3 Detection of extracellular protein content

[0084] The fermentation broth cultured for 72 hours was centrifuged to obtain the supernatant, and the protein concentration of the fermentation broth was determined using the Bradford method.

[0085] 3.4 Extracellular protein determination results

[0086] The extracellular protein content of the YN2-1 strain cultured in the test tube system gradually increased from 24 to 72 hours, reaching the highest level at 72 hours, which was 0.63 ± 0.05 mg / mL ( Figure 5 The YN2-1 strain was cultured in a shake flask system, and the extracellular protein content was 0.58 ± 0.06 mg / mL after 72 hours ( Figure 5 ).

[0087] The results showed that the total extracellular protein of the YN2-1 strain of the present invention was significantly better than that of other Kluyveromyces lactis, achieving an increase in the extracellular protein content ( Figure 5 ). The lactic acid Kluyveromyces YN2-1 of the present invention has good heterologous protein expression ability.

[0088] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Kluyveromyces lactis, characterized in that It is deposited in China Center for Type Culture Collection, and its deposit number is CCTCC NO: M 20241460.

2. A fermentation method of Kluyveromyces lactis according to claim 1, characterized in that: It includes the following steps: The lactic acid Kluyveromyces is fermented in the presence of a nitrogen source and a carbon source to collect extracellular proteins.

3. The fermentation method according to claim 2, characterized in that The fermentation method also includes separating and purifying the extracellular protein.

4. The fermentation method according to claim 2 or 3, characterized in that: The carbon source includes at least glucose.

5. The fermentation method according to claim 4, characterized in that The nitrogen source includes at least ammonium sulfate.

6. The fermentation method according to claim 2, characterized in that The mass ratio of the carbon source to the nitrogen source is (1-5):

1.

7. The fermentation method according to claim 2, characterized in that The fermentation time is 32-80 hours.

8. The fermentation method according to claim 2, characterized in that The fermentation temperature is 25-40°C.

9. The fermentation method according to claim 2, characterized in that: The inoculation amount of the lactic acid Kluyveromyces is 0.05%-5%.

10. The fermentation method according to claim 2, characterized in that: The lactic acid Kluyveromyces is fermented in an ammonium sulfate glucose medium or a corn steep liquor medium.

11. Use of Kluyveromyces lactis according to claim 1 in expressing foreign proteins.

12. Use of Kluyveromyces lactis according to claim 1 in preparing food or feed additives.

13. A food composition or feed composition, characterized in that: The method comprises the Kluyveromyces lactis according to claim 1.

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