Construction method and application of pichia pastoris engineering bacteria for synthesizing functional fatty acid

Abstract

The invention provides a construction method and application of pichia pastoris engineering bacteria for synthesizing functional fatty acid, the pichia pastoris engineering bacteria are obtained by introducing recombinant plasmids into host pichia pastoris, exogenous gene segments BkdB, LpdV and BkdAA are integrated in the recombinant plasmids, the nucleotide sequence of the exogenous gene segment BkdB is as shown in SEQ ID NO.1, the nucleotide sequence of the LpdV is as shown in SEQ ID NO.2, and the nucleotide sequence of the BkdAA is as shown in SEQ ID NO.3. The nucleotide sequence of the exogenous gene segment LpdV is as shown in SEQ ID NO.2, the nucleotide sequence of the exogenous gene segment BkdAA is as shown in SEQ ID NO.3, and the nucleotide sequence of the exogenous gene segment BkdAB is as shown in SEQ ID NO.4; the branched-chain fatty acid produced by fermentation of the engineering bacteria can reach 187mg / L, and the production activity of the branched-chain fatty acid is greatly improved, so that the production cost of industrial production of the branched-chain fatty acid is remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a method for constructing a Pichia pastoris engineering bacterium capable of synthesizing functional fatty acids and its application. Background Art

[0002] Pichia pastoris has significant metabolic system advantages in expressing foreign proteins. Not only because of its strong methanol metabolic pathway, it can strongly express methanol-related enzymes through a unique methanol utilization pathway, but also has good protein folding and secretion capabilities, and can effectively perform post-translational modifications of eukaryotic proteins, such as glycosylation, correct protein folding, and formation of disulfide bonds, etc.

[0003] Currently, the main strains for the biological fermentation of branched-chain fatty acids are Escherichia coli and Bacillus, and there is temporarily a lack of corresponding technologies for constructing Pichia pastoris engineering bacteria as production vectors, and the great advantages of Pichia pastoris in expressing foreign genes cannot be exerted.

[0004] In addition, peanut meal is a by-product with high protein content, economy and efficiency. According to FAO data, the global peanut production is approximately 46 million tons, and peanut meal in the by-products of peanut oil production accounts for a quite large proportion. The annual global output of peanut meal is between 6 million and 8 million tons. Peanut meal contains rich essential amino acids, such as lysine, arginine, etc., and is suitable as a protein supplement source in animal feed.

[0005] In summary, there is an urgent need to develop a technology for producing branched-chain fatty acids using Pichia pastoris as a fermentation strain and peanut meal as a fermentation raw material. Summary of the Invention

[0006] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, the abstract of the specification and the title of the invention of this application to avoid obscuring the purpose of this part, the abstract of the specification and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a Pichia pastoris engineering bacterium.

[0009] To solve the above technical problems, the present invention provides the following technical solution: a Pichia pastoris engineering bacterium, which is obtained by introducing a recombinant plasmid into a host Pichia pastoris, and the recombinant plasmid integrates foreign gene fragments BkdB, LpdV, BkdAA, and BkdAB;

[0010] Among them, the nucleotide sequence of the exogenous gene fragment BkdB is as shown in SEQ ID NO.1, the nucleotide sequence of the exogenous gene fragment LpdV is as shown in SEQ ID NO.2, the nucleotide sequence of the exogenous gene fragment BkdAA is as shown in SEQ ID NO.3, and the nucleotide sequence of the exogenous gene fragment BkdAB is as shown in SEQ ID NO.4.

[0011] As a preferred embodiment of the Pichia pastoris engineering bacteria of the present invention, wherein: the expression vector is pPIC9K or pPICZA.

[0012] As a preferred embodiment of the Pichia pastoris engineering bacteria of the present invention, wherein: the expression vector contains an autonomous replication sequence, and the nucleotide sequence of the autonomous replication sequence is as shown in SEQ ID NO.5.

[0013] As a preferred embodiment of the Pichia pastoris engineering bacteria of the present invention, wherein: the host cell is Pichia pastoris GS115.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for increasing gene expression or protein synthesis.

[0015] As a preferred embodiment of the method of the present invention, wherein: introducing a recombinant plasmid containing the exogenous protein genes BkdB, LpdV, BkdAA, and BkdAB as claimed in claim 1.

[0016] As a preferred embodiment of the method of the present invention, wherein: inserting the gene fragments BkdAA and BkdAB encoding the protein into the pPICZA expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant cell, and then culturing the recombinant cell.

[0017] As a preferred embodiment of the method of the present invention, wherein: inserting the gene fragments BkdB and LpdV encoding the protein into the pPIC9K expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant cell, and then culturing the recombinant cell.

[0018] As a preferred embodiment of the method of the present invention, wherein: including,

[0019] Using the Pichia pastoris recombinant strain GS115AK as the production strain, after activation, culturing at 28-31°C and 200-220 rpm for 30-48 h to obtain a primary seed fermentation broth;

[0020] Inoculating the primary seed fermentation broth into the seed medium at an inoculation amount of 2.5%, and culturing at 28-31°C and 200-220 rpm for 30-48 h to obtain a secondary seed fermentation broth;

[0021] Inoculate the secondary seed fermentation broth into the fermentation medium at an inoculum size of 10%, and culture it at 28 - 31°C and 200 - 220 rpm for 30 - 48 h.

[0022] As a preferred embodiment of the method of the present invention, wherein: the seed medium is YPD medium, and the fermentation medium is BSM medium.

[0023] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of a Pichia pastoris engineering bacterium in the preparation of branched-chain fatty acids.

[0024] Advantages of the present invention:

[0025] 1. In the present invention, a free plasmid of the 2-oxoacid dehydrogenase (bkd) gene derived from Bacillus licheniformis is transferred into Pichia pastoris containing an integrative expression vector to obtain a free heterologous expression Pichia pastoris strain, which secretes 2-oxoacid dehydrogenase. Compared with the wild-type expression strain GS115, the yield of branched-chain fatty acids expressed by the free heterologous expression recombinant bacterium is 1.8 times that of the wild-type expression strain GS115, thus significantly reducing the production cost of industrial production of branched-chain fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0027] Figure 1 It is the construction process of the recombinant plasmid pPICZA-Bkd in Example 1.

[0028] Figure 2 It is the construction process of the recombinant plasmid pPIC9K-BkdV in Example 1.

[0029] Figure 3 It is the electrophoresis pattern of the recombinant plasmid pPICZA-Bkd and the recombinant plasmid pPIC9K-BkdV in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the following will provide a detailed description of the specific embodiments of the present invention in conjunction with the embodiments of the specification.

[0031] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways than those specifically described herein. Those skilled in the art can make similar generalizations without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0032] Secondly, as used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other.

[0033] Unless otherwise specified, the raw materials and reagents used in the present invention are all commercially available.

[0034] The formula of the BSM medium is: 0.17 g / L CaSO 4 ×2H 2 O, 2.86 g / L K 2 SO 4 , 0.64 g / L KOH, 2.32 g / L MgSO 4 ×7H 2 O, 4.25 g / L H 3 PO 4 、0.22 g / L NaCl, 33 g / L glucose monohydrate, 10 g / L NH 4 Cl, 4.35 mL / L Pichia pastoris trace metal solution (PTM1), 80 mg / L L-histidine, and 0.1 g / L antifoaming agent 204.

[0035] The methods used in the present invention are all conventional methods in the art unless otherwise specified.

[0036] Example 1 Construction of a free vector

[0037] 1. Construction of the free vector pPICZA-Bkd

[0038] The gene fragments BkdAA (nucleotide sequence as shown in SEQ ID NO.3) and BkdAB (nucleotide sequence as shown in SEQ ID NO.4) were introduced into a plasmid to obtain a recombinant plasmid, and the recombinant plasmid contained an autonomous replication sequence with a nucleotide sequence as shown in SEQ ID NO.5;

[0039] According to the gene sequence of the commercial vector pPICZA, a recombinant plasmid with a sequence as shown in SEQ ID NO.6 was designed, as Figure 1 shown. For reference, this recombinant plasmid was synthesized by Genewiz (Suzhou) Inc. and the deposited strain Escherichia coli DH5α of this vector was provided;

[0040] 2. Construction of Free Vector pPIC9K-BkdV

[0041] The gene fragment BkdB (nucleotide sequence as shown in SEQ ID NO.1) was introduced into a plasmid to obtain a recombinant plasmid expressing the E2 subunit;

[0042] The gene fragment LpdV (nucleotide sequence as shown in SEQ ID NO.2) was introduced into the recombinant plasmid of the E2 subunit to obtain a recombinant plasmid expressing the E3 subunit, as Figure 2 shown;

[0043] According to the gene sequence of the commercial vector pPIC9k, a recombinant plasmid with the sequence shown in SEQ ID NO.7 was designed. For reference, this recombinant plasmid was synthesized by Genewiz (Suzhou) Inc. and the storage strain Escherichia coli DH5a of this vector was provided.

[0044] Example 2 Enzymatic Digestion and Ligation of Recombinant Plasmids pPICZA-Bkd and pPIC9K-BkdV

[0045] The system for enzymatic digestion and ligation of pPICZA-Bkd is shown in Table 1, and the system for enzymatic digestion and ligation of pPIC9K-BkdV is shown in Table 2.

[0046] The prepared enzymatic digestion system was placed in a 37 °C constant temperature metal bath for reaction for 2 h. Take 5 μL of the linearized reaction mixture and perform electrophoresis in 1% agarose gel as Figure 3 shown to identify whether the linearization was successful and recover the product.

[0047] Table 1

[0048]

[0049]

[0050] Table 2

[0051]

[0052] Example 3 Preparation and Transformation of Pichia pastoris GS115 Competent Cells

[0053] 1. Preparation of Pichia pastoris GS115 Competent Cells

[0054] Take 50 μL of GS115 glycerol bacteria and inoculate them into 5 mL of YPD liquid medium. Incubate overnight at 37 °C and 200 rpm for 24 h;

[0055] Take 200 μL of the bacterial solution and spread it on a YPD plate. Incubate at 30 °C for 18 - 24 h;

[0056] Mix 1 mL of liquid YPD with 40 μL of DTT (dithiothreitol) and 40 μL of HEPES buffer;

[0057] Centrifuge to collect cells after culturing at 200 rpm and 30 °C for 15 min;

[0058] Resuspend the cells in 25 mL of sterile water, centrifuge and discard the supernatant, repeat once, centrifuge at 3000 rpm for 3 min, and discard the supernatant;

[0059] Resuspend the cells in 1 mL of sterile water, transfer to a 1.5 mL EP tube, centrifuge at 3000 rpm for 3 min, and discard the supernatant;

[0060] Wash the cells with 1 mL of sterile water twice, let it stand to separate the solid and liquid;

[0061] Resuspend the cells in 1 mL of 1 M LiCl, incubate at 30 °C for 1 h, centrifuge at 3000 rpm for 3 min, and discard the supernatant;

[0062] Resuspend the cells in 400 μL of 1 M LiCl, dispense 50 μL into 1.5 mL EP tubes to obtain competent cells, store at -80 °C or prepare for plasmid transduction

[0063] 2. Transformation of plasmid

[0064] Thaw the Pichia pastoris competent cells on ice, add 1 μg of linear or circular plasmid, mix and place in an electroporation cuvette, and place on ice for 15 min;

[0065] Place the electroporation cuvette in the electroporator and shock twice in Sc2 mode;

[0066] Quickly add 1 mL of 1 M sorbitol and mix well;

[0067] Place the electroporation cuvette at 30 °C and culture for 2 h;

[0068] Centrifuge at 4500 rpm for 5 min, discard the supernatant, coat on MD or Bleo-resistant YTD plates, and culture at 30 °C for screening;

[0069] Colonies will form after 36 - 48 h;

[0070] Use a sterile toothpick to pick a single colony on the resistant plate in Example 3 and inoculate it into the YPD medium to obtain a recombinant single colony GS115A containing pPICZA - Bkd and pPIC9K - BkdV, namely the Pichia pastoris strain GS115 / pPICZA - Bkd—pPIC9K - BkdV.

[0071] Flask fermentation of the strain in Example 4

[0072] The free heterologous expression Pichia pastoris strain GS115 / pPICZA-Bkd—pPIC9K-BkdV in Example 3 was subjected to shake flask fermentation. The specific method was as follows:

[0073] Strain activation: Streak the preserved Pichia pastoris strain on YPD solid medium and culture it at 28 °C for 48 h;

[0074] Seed culture: Pick a single colony and inoculate it into 10 mL of liquid YPD medium. Culture it at 28 °C and 130 rpm until the OD600 of the seed liquid reaches 4.0 - 6.0;

[0075] Shake flask fermentation: Pipette 1 mL of the seed liquid into a triangular flask containing 20 mL of fermentation medium, and add 10.8 mL of basal salt medium (0.17 g / L CaSO 4 ×2H 2 O, 2.86 g / L K 2 SO 4 、0.64 g / L KOH, 2.32 g / L MgSO 4 ×7H 2 O, 4.25 g / L H 3 PO 4 、0.22 g / L NaCl, 33 g / L glucose monohydrate, 10 g / L NH 4 Cl, 4.35 mL / L Pichia pastoris trace metal solution (PTM1), 80 mg / L L-histidine, and 0.1 g / L antifoaming agent 204). Ferment at 130 rpm and 28 °C for 48 h; when OD600 reaches 0.8 - 1.0, add the branched-chain α-keto acid precursor (1 g / L) during induction. Harvest the cells 48 hours after induction and analyze the production of fatty acids.

[0076] Fermentation conditions:

[0077] 30 °C, 10.8 mL BSM medium + 1 mL seed liquid (OD 600 = 4.0 - 6.0); Comparative fermentation was carried out in two groups (engineered bacteria group and wild-type bacteria group), with 3 samples in each group.

[0078] There were mainly 40 kinds of total fatty acids in the fermentation. The fatty acid concentration in the engineered bacteria group was 3 g / L, and the fatty acid concentration in the wild-type group (g / L) was 2 g / L. The product concentrations of the three main branched-chain fatty acids are shown in Table 3

[0079] Table 3 Product Concentrations of Three Main Branched-Chain Fatty Acids

[0080]

[0081]

[0082] The results show that the three main branched-chain fatty acids in the engineered bacteria group are approximately 1.8 times that of the wild-type group.

[0083] Comparative Example 1

[0084] Except for the types of enzymes, other conditions are the same as in Example 2.

[0085] The systems for restriction enzyme digestion and ligation of pPICZA-Bkd are shown in Table 4, and the systems for restriction enzyme digestion and ligation of pPIC9K-BkdV are shown in Table 5.

[0086] Table 4

[0087]

[0088] Table 5

[0089]

[0090] Place the prepared restriction enzyme digestion system in a 37°C constant-temperature metal bath for reaction for 2 h. Take 5 μL of the linearized reaction mixture and perform electrophoresis in 1% agarose gel to identify whether the linearization is successful and recover the product.

[0091] The gel electrophoresis results show that there is no linearized plasmid product with sufficient molecular weight.

[0092] The transformation of the recombinant plasmid pPIC9K-BkdV also fails.

[0093] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A Pichia pastoris engineered bacterium, characterized in that: The Pichia pastoris engineered bacteria are obtained by introducing a recombinant plasmid into a host Pichia pastoris, wherein the recombinant plasmid is integrated with foreign gene fragments BkdB, LpdV, BkdAA and BkdAB; Among them, the nucleotide sequence of the exogenous gene fragment BkdB is shown as SEQ ID NO.1, the nucleotide sequence of the exogenous gene fragment LpdV is shown as SEQ ID NO.2, the nucleotide sequence of the exogenous gene fragment BkdAA is shown as SEQ ID NO.3, and the nucleotide sequence of the exogenous gene fragment BkdAB is shown as SEQ ID NO.

4.

2. The Pichia pastoris engineered bacterium according to claim 1, characterized in that: The expression vector is pPIC9K or pPICZA.

3. The Pichia pastoris engineered bacterium according to claim 1 or 2, characterized in that: The expression vector contains an autonomous replication sequence, and the nucleotide sequence of the autonomous replication sequence is shown in SEQ ID NO.

5.

4. The Pichia pastoris engineered bacterium according to claim 1, characterized in that: The host cell is Pichia pastoris GS115.

5. A method for increasing gene expression or protein synthesis, characterized in that: A recombinant plasmid containing the foreign protein genes BkdB, LpdV, BkdAA and BkdAB according to claim 1 is introduced.

6. The method according to claim 5, characterized in that: The protein encoding gene fragments BkdAA and BkdAB are inserted into the pPICZA expression vector to obtain a recombinant expression vector; the recombinant expression vector is transformed into a host cell to obtain a recombinant cell, and then the recombinant cell is cultured.

7. The method according to claim 5, characterized in that: The gene fragments BkdB and LpdV encoding proteins are inserted into the pPIC9K expression vector to obtain a recombinant expression vector; the recombinant expression vector is transformed into a host cell to obtain a recombinant cell, and then the recombinant cell is cultured.

8. A method for increasing branched-chain fatty acids, characterized in that: include, The recombinant Pichia pastoris GS115AK was used as a production strain, and after activation, it was cultured at 28-31° C. and 200-220 rpm for 30-48 hours to obtain a first-level seed fermentation liquid; Inoculate the first-level seed fermentation liquid into the seed culture medium at an inoculum of 2.5%, and culture at 28-31° C. and 200-220 rpm for 30-48 hours to obtain the second-level seed fermentation liquid; The secondary seed fermentation liquid was inoculated into the fermentation medium at an inoculation rate of 10% and cultured at 28-31° C. and 200-220 rpm for 30-48 hours.

9. The method according to claim 8, characterized in that: The seed culture medium is YPD culture medium, and the fermentation culture medium is BSM culture medium.

10. Use of the Pichia pastoris engineered bacteria according to any one of claims 1 to 4 in the preparation of branched-chain fatty acids.

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