Culture medium for improving utilization rate of pichia pastoris xylose and application of culture medium

Abstract

The invention discloses a culture medium for improving the utilization rate of pichia pastoris xylose and application of the culture medium. The culture medium comprises a YNB culture medium, xylose, methanol and histidine, wherein the content of the YNB culture medium is 13.4 g / L, the content of the xylose is 1-10 g / L, the content of the methanol is 7.91-31.64 g / L, and the content of the histidine is 0.04 g / L. Based on the problem that the xylose metabolism capability of a wild type K.phaffi.GS115 strain is not high, and culture medium optimization is needed to improve the xylose metabolism capability of the strain, methanol is added into a xylose culture medium to relieve the inhibition effect of high-concentration xylose on the strain and improve the xylose utilization rate of the strain; according to the method, the inhibition effect of high-concentration xylose on the wild type K.phaffi.GS115 strain is solved, the xylose consumption rate of the wild type K.phaffi.GS115 strain is increased, and the method has positive promotion significance on popularization of xylose microorganism application.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and in particular relates to a culture medium for improving xylose utilization rate of Pichia pastoris and application thereof. Background Art

[0002] K.phaffi.GS115 is a model strain that has gradually become used in the production of various biological products. The wild-type K.phaffi.GS115 strain has a low xylose utilization rate and a certain xylose inhibition effect.

[0003] In order to further improve the xylose utilization rate of K.phaffi.GS115 strain, the culture method of the strain in xylose medium was improved; Chinese patent document CN110511949A disclosed a method of improving the ability of E. coli to metabolize xylose by overexpressing E. coli xylose isomerase; Chinese patent document CN118667892A disclosed a method of using nitrosoguanidine to induce yeast to increase the conversion rate of xylose and xylitol after adding functional materials to the fermentation medium. Chinese patent document CN106701605B disclosed a method of improving the xylose utilization rate of Saccharomyces cerevisiae strains by constructing fusion proteins (xylose reductase XYL1, xylitol oxidase XYL2).

[0004] Currently, there is no report on improving the xylose tolerance of Pichia pastoris by medium optimization. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a culture medium for improving the xylose utilization rate of Pichia pastoris.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a culture medium for improving xylose utilization of Pichia pastoris, comprising YNB culture medium, xylose, methanol and histidine;

[0009] Wherein, in terms of g / L, the YNB medium is 13.4, the xylose is 1-10, the methanol is 7.91-31.64, and the histidine is 0.04.

[0010] As a preferred embodiment of the culture medium of the present invention, the YNB culture medium contains ammonium sulfate but does not contain amino acids.

[0011] As a preferred embodiment of the culture medium of the present invention, in which: in g / L, the YNB culture medium is 13.4, the xylose is 2.5-10, the methanol is 7.91-31.64, and the histidine is 0.04.

[0012] As a preferred embodiment of the culture medium of the present invention, in which: in g / L, the YNB culture medium is 13.4, the xylose is 5-10, the methanol is 7.91-31.64, and the histidine is 0.04.

[0013] As a preferred embodiment of the culture medium of the present invention, in which: in g / L, the YNB culture medium is 13.4, the xylose is 7.5, the methanol is 7.91, and the histidine is 0.04.

[0014] As a preferred embodiment of the culture medium of the present invention, in which: in g / L, the YNB culture medium is 13.4, the xylose is 10, the methanol is 31.64, and the histidine is 0.04.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a culture medium for improving xylose utilization of Pichia pastoris, comprising: dissolving YNB in ​​distilled water and mixing thoroughly for standby use;

[0016] Dissolve xylose in distilled water and mix thoroughly for later use;

[0017] Dissolve histidine in distilled water and mix thoroughly for later use;

[0018] In a sterile environment, the YNB solution, the xylose solution, the histidine solution and the pure methanol were filtered into sterile containers using a sterile filter membrane, and stored at 4° C. in the dark to prepare the culture medium.

[0019] As a preferred embodiment of the preparation method of the present invention, the pore size of the sterilizing filter membrane is 0.22 μm.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide a culture medium for use in improving xylose utilization in Pichia pastoris.

[0021] As a preferred embodiment of the application of the present invention, the Pichia pastoris includes the K.phaffi.GS115 strain.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention is based on the problem that "the xylose metabolism ability of the wild-type K.phaffi.GS115 strain is not high, and the culture medium optimization is needed to improve the xylose metabolism ability of the strain". By adding methanol to the xylose culture medium, the inhibitory effect of high-concentration xylose on the strain is relieved and the xylose utilization rate of the strain is improved. The final improved xylose high-utilization culture medium is as follows: (g / L): YNB (containing ammonium sulfate, not containing amino acids) 13.4, xylose 1-10, methanol 7.91-31.64, histidine 0.04, which solves the inhibitory effect of high-concentration xylose on the wild-type K.phaffi.GS115 strain and improves the xylose consumption rate of the wild-type K.phaffi.GS115 strain, which has a positive promoting significance for the promotion of xylose microbial application.

[0024] (2) The present invention has carried out culture optimization based on the K.phaffi.GS115 strain, resulting in improved xylose tolerance and xylose utilization of Pichia pastoris, which will further promote the microbial application of xylose and promote the application of non-food carbon sources in biomanufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0026] Figure 1 Graph showing the growth curve of K.phaffi GS115 strain in MXH medium in the embodiment of the present invention.

[0027] Figure 2 This is a graph showing changes in xylose concentration of K.phaffi GS115 strain in MXH medium in an example of the present invention.

[0028] Figure 3 This is a graph showing the growth trend and xylose concentration data of the K. phaffi GS115 strain in MMXH (methanol 7.91 g / L, xylose 7.5 g / L) medium in an example of the present invention.

[0029] Figure 4 This is a graph showing the growth trend and xylose concentration data of the K. phaffi GS115 strain in MMXH (methanol 31.64 g / L, xylose 7.5 g / L) medium in an example of the present invention.

[0030] Figure 5This is a graph showing the growth trend and xylose concentration data of the K. phaffi GS115 strain in MMXH (methanol 31.64 g / L, xylose 10 g / L) medium in an example of the present invention.

[0031] Figure 6 This is a graph showing the xylose consumption rate of the K. phaffi GS115 strain in MXH (7.5 g / L) and MMXH (methanol 31.64 g / L, xylose 7.5 g / L) culture media in the examples of the present invention. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] See Table 1 for the sources of biological materials in the examples of the present invention.

[0036] Table 1

[0037]

[0038] Culture medium:

[0039] (1) YPDS liquid medium (g / L): yeast extract powder 10, peptone 20, glucose 20, sorbitol 182, pH 7.0;

[0040] Preparation method: Weigh 10g yeast extract powder, 20g peptone, 20g glucose, and 182g sorbitol and add to 1L sterile water, then divide into 250mL Erlenmeyer flasks and sterilize at 115℃ for 20min.

[0041] (2) MXH liquid medium (g / L): YNB (containing ammonium sulfate, without amino acids) 13.4, xylose 1-10 (1, 2.5, 5, 7.5, 10, respectively), histidine 0.04;

[0042] Preparation method: Dissolve 67g YNB in ​​0.5L distilled water and mix thoroughly for use; dissolve 50g xylose in 0.5L distilled water and mix thoroughly for use; dissolve 2g histidine in 0.5L distilled water and mix thoroughly for use; then use a 0.22μm sterile filter membrane in a sterile environment to filter the YNB solution, xylose solution, and histidine solution, and store them at 4℃ away from light;

[0043] During the experiment, the corresponding volume was added according to the different final concentrations of xylose.

[0044] (3) MMXH liquid medium (g / L): YNB (with ammonium sulfate, without amino acids) 13.4, methanol 7.91-31.64, xylose 0.1-10, histidine 0.04;

[0045] Preparation method: Dissolve 67g YNB in ​​0.5L distilled water and mix thoroughly for use; dissolve 50g xylose in 0.5L distilled water and mix thoroughly for use; dissolve 2g histidine in 0.5L distilled water and mix thoroughly for use; then use a 0.22μm sterile filter membrane to filter the YNB solution, xylose solution, histidine solution and pure methanol in a sterile environment and store them at 4℃ away from light;

[0046] During the experiment, the corresponding volumes were added according to the different final concentrations of xylose and methanol.

[0047] (4) YNB medium (containing ammonium sulfate but not amino acids) was purchased from Yisheng Biotechnology Co., Ltd. The product name is Yeast Nitrogen base and the product number is 60901ES60.

[0048] Example 1

[0049] Growth curve of K.phaffiGS115 strain in MXH medium:

[0050] 1) The K.phaffiGS115 strain was inoculated into a 100 mL Erlenmeyer shake flask containing 20 mL YPDS liquid culture medium and cultured in a constant temperature shaker at 30°C and 200 rpm for 2 days.

[0051] 2) In a sterile environment, place the bacterial solution in (1) above in a 50 mL sterile centrifuge tube and centrifuge at 6000 rpm for 10 min. Remove the supernatant and resuspend in 10 mL sterile water. Repeat this process 3 times.

[0052] 3) Resuspend the bacterial precipitate with 1 mL of sterile water; then take the bacterial solution and inoculate it into MXH medium to make its initial OD 600 The values ​​were between 0.04 and 0.1 (three replicates were performed for each group of samples).

[0053] 4) Place the shake flask at 30°C, 200 rpm, and take samples every 6-8 days to measure OD 600 Values, such as Figure 1 As shown, it can be seen that GS115 grows best in MXH medium with a xylose concentration of 1 g / L, and other xylose concentrations higher than 1 g / L will inhibit the growth of the strain.

[0054] 0.5 mL of bacterial liquid was sampled and placed in a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was then filtered through a 0.22 μm membrane into a liquid phase bottle. After sampling, the xylose concentration was detected using a high performance liquid chromatograph. The xylose concentration changes at xylose concentrations of 1 and 7.5 were as follows: Figure 2 As shown, it can be seen that the strain showed an obvious xylose consumption trend in 1 g / L MXH medium; while the strain had almost no xylose consumption in 7.5 g / L MXH medium; this indicates that high concentrations of xylose inhibit the growth of the strain, so the strain will not consume xylose in MXH medium with high xylose concentrations.

[0055] Example 2

[0056] Growth detection and xylose concentration detection of K.phaffiGS115 strain in MMXH medium.

[0057] 1) Refer to step (1) of Example 1.

[0058] 2) Refer to step (2) of Example 1.

[0059] 3) The bacterial precipitate was resuspended with 1 mL of sterile water; then the bacterial solution was inoculated into MMXH (methanol 7.91 g / L, 31.64 g / L; xylose 7.5 g / L, 10 g / L) medium to an initial OD 600 The values ​​were between 0.04 and 0.1 (three replicates were performed for each group of samples).

[0060] 4) Place the shake flask at 30°C, 200 rpm, and take samples every 1-2 days to measure OD 600 value,

[0061] Figure 3 The medium is MMXH containing 7.91 g / L methanol and 7.5 g / L xylose;

[0062] Figure 4 The medium is MMXH containing 31.64 g / L methanol and 7.5 g / L xylose;

[0063] Figure 5 It is an MMXH medium containing 31.64 g / L methanol and 10 g / L xylose.

[0064] from Figure 3 , Figure 4 and Figure 5 It can be seen that 7.5 g / L xylose has no inhibitory effect on the strain after adding methanol.

[0065] Example 3

[0066] The xylose consumption rate of K. phaffi GS115 strain changed in MXH and MMXH.

[0067] The final xylose consumption rate was calculated based on the changes in xylose concentration in the two culture media, MXH in Example 1 (xylose concentration of 7.5 g / L) and MMXH in Example 2 (xylose concentration of 7.5 g / L, methanol concentration of 4%).

[0068] The xylose consumption rate of the strain was calculated based on the detected xylose concentration. The calculation method is as shown in formula (1). The result is as follows: Figure 6 shown.

[0069] V 木糖消耗速率 =(C1-C2) / T (Formula 1)

[0070] V 木糖消耗速率 : g / L / d;

[0071] C1: xylose concentration of the nth sampling (g / L);

[0072] C2: xylose concentration of the n+1th sampling (g / L);

[0073] T: time interval (d).

[0074] It can be seen that adding methanol to the xylose medium can relieve the inhibitory effect of high concentration xylose on the strain and improve the xylose utilization rate of the strain.

[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A culture medium for improving xylose utilization of Pichia pastoris, characterized in that: include, YNB medium, xylose, methanol, and histidine; Wherein, in terms of g / L, the YNB medium is 13.4, the xylose is 1-10, the methanol is 7.91-31.64, and the histidine is 0.

04.

2. The culture medium according to claim 1, characterized in that: The YNB medium contains ammonium sulfate but no amino acids.

3. The culture medium according to claim 1 or 2, characterized in that: In terms of g / L, the YNB medium is 13.4, the xylose is 2.5-10, the methanol is 7.91-31.64, and the histidine is 0.

04.

4. The culture medium according to claim 3, characterized in that: In g / L, the YNB medium is 13.4, the xylose is 5-10, the methanol is 7.91-31.64, and the histidine is 0.

04.

5. The culture medium according to claim 4, characterized in that: In g / L, the YNB medium is 13.4, the xylose is 7.5, the methanol is 7.91, and the histidine is 0.

04.

6. The culture medium according to claim 4, characterized in that: In g / L, the YNB medium is 13.4, the xylose is 10, the methanol is 31.64, and the histidine is 0.

04.

7. The method for preparing the culture medium according to any one of claims 1 to 6, characterized in that: include, Dissolve YNB in ​​distilled water and mix thoroughly for later use; Dissolve xylose in distilled water and mix thoroughly for later use; Dissolve histidine in distilled water and mix thoroughly for later use; In a sterile environment, the YNB solution, the xylose solution, the histidine solution and the pure methanol were filtered into sterile containers using a sterile filter membrane, and stored at 4° C. in the dark to prepare the culture medium.

8. The preparation method according to claim 7, characterized in that: The pore size of the sterilizing filter membrane is 0.22 μm.

9. Use of the culture medium according to any one of claims 1 to 6 for improving xylose utilization of Pichia pastoris.

10. The use according to claim 9, characterized in that: The Pichia pastoris includes K. phaffi. GS115 strain.

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