Recombinant halophilic microorganism and method for preparing corn protein by using same

Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant halophilic microorganism and a method for preparing corn protein by using the same. The recombinant halophilic microorganism expresses or overexpresses a corn protein subunit Zein1c2. According to the present invention, the halophilic microorganism is used to express the corn protein subunit Zein1c2 so as to achieve the efficient microorganism synthesis of the corn protein, the yield of the corn protein is significantly improved, the recombinant halophilic microorganism is used to produce the corn protein through fermentation, the open type fermentation can be performed without additional sterilization, and the production cost is low. Various air filter membranes and complicated sterile operation are not needed in the fermentation process to reduce the possibility of contamination, so that the production cost, manpower and material resources are greatly saved, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a recombinant halophilic microorganism and a method for preparing corn protein using the recombinant halophilic microorganism. Background Art

[0002] Halophilic microorganisms are a type of microorganism that can grow in high-salt environments. They have unique adaptation mechanisms and biological functions and are widely used in fermentation production. Halomonas ) bacteria are a type of moderately halophilic bacteria isolated from the Aidin Salt Lake in Xinjiang. Their optimal growth salt concentration is 6%, and their optimal growth pH is 9.0. During their growth, they consume a large amount of glucose to synthesize the dominant product polyhydroxyalkanoates (PHA).

[0003] Zein is a mixture of proteins with an average molecular weight of 25,000 to 45,000. It is affected by the hydrogen bonds between the hydroxyl group and the imino group on the peptide main chain to form an α-helix. Zein has low surface energy and unique film-forming properties. Zein forms an irregular coil structure in an alcohol-water solution, but after the solvent evaporates, it can form a transparent, glossy film with moisture-proof, oxygen-proof, UV-resistant, fragrance-preserving, oil-proof, and anti-static properties. Zein is divided into two categories: α-Zein and β-Zein, which are composed of many subunits. The most important subunit, Zein1c2, is encoded by the gene z1C2 and is rich in amino acids such as glutamine, leucine, proline, and alanine. By expressing the z1C2 gene in microorganisms through synthetic biology technology, the subunit can be synthesized in large quantities. After industrial fermentation, an extract with an amino acid composition (i.e., nutritional components) similar to that of corn can be obtained, which can be added as a substitute for corn in existing corn feed.

[0004] The current fermentation scheme often adds a certain amount of urea to the fermentation base, and then adds a small amount of urea and a large amount of glucose during the fermentation process to promote the growth of microorganisms. Under the existing process conditions, the corn protein in the fermentation process mainly relies on the accumulation of endogenous proteins. The yield of corn protein is low (<1 g / L), and the types of proteins in the solution are complex, making it difficult to purify the downstream to obtain protein products, which cannot meet the production needs. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a recombinant halophilic microorganism, which expresses the zein subunit Zein1c2.

[0006] The present invention finds that the production of zein by fermentation can be greatly increased by expressing the zein subunit Zein1c2 using halophilic microorganisms, and the zein product can be easily purified.

[0007] Preferably, the recombinant halophilic microorganism overexpresses the zein subunit Zein1c2.

[0008] In the present invention, there is no special limitation on the technical means for achieving the expression or overexpression. For example, the gene, encoded protein, regulatory element or regulatory protein of the corn protein subunit Zein1c2 can be transformed or modified by commonly used genetic engineering means and gene editing methods to achieve expression or overexpression.

[0009] Preferably, the overexpression techniques include: (1) Introducing a plasmid containing the Zein1c2 gene, a subunit of zein protein; (2) Increasing the copy number of the Zein1c2 gene, a subunit of zein protein; (3) Changing the promoter sequence of the Zein1c2 gene; (4) operably linking a strong promoter to the Zein1c2 gene; (5) Import enhancers.

[0010] Preferably, the halophilic microorganism is of the genus Halomonas ( Halomonas )bacteria.

[0011] Preferably, the Halomonas genus ( Halomonas ) Bacteria Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194 or Halomonas bluephagenesis TDH4, preferably Halomonas bluephagenesis WZY254.

[0012] Preferably, the amino acid sequence of the zein subunit Zein1c2 is shown in SEQ ID NO.1.

[0013] Preferably, the nucleotide sequence of the gene encoding the zein subunit Zein1c2 is shown in SEQ ID NO.2.

[0014] It is well known in the art that due to the degeneracy of codons, the above nucleotide sequence is only one of the nucleotide sequences encoding the zein subunit Zein1c2, and other nucleotide sequences encoding the sequence shown in SEQ ID NO.1 can also realize the present invention.

[0015] Furthermore, the present invention provides a method for constructing the recombinant halophilic microorganism, comprising: introducing a gene encoding the zein subunit Zein1c2 into the halophilic microorganism.

[0016] In the present invention, the introduction method includes but is not limited to genetic engineering methods such as transformation, electroporation, microinjection, gene gun, Agrobacterium transformation, viral vector, gene editing system (such as CRISPR / Cas system, etc.), transposon, etc.

[0017] In the present invention, a constitutive promoter or an inducible promoter can be used to drive the expression or overexpression of the gene encoding the zein subunit Zein1c2.

[0018] Preferably, porin58 promoter (shown in SEQ ID NO.3) is used to drive the expression or overexpression of the gene encoding the zein subunit Zein1c2. The fluorescence intensity (FI) of the porin58 promoter is 80291.

[0019] Furthermore, the present invention provides the use of the recombinant halophilic microorganism or the construction method in the preparation of zein.

[0020] Furthermore, the present invention provides a method for preparing zein, comprising: utilizing the recombinant halophilic microorganism to ferment and produce zein.

[0021] Preferably, the fermentation temperature is 35°C to 40°C, most preferably 36°C to 38°C.

[0022] Preferably, the culture medium used for the fermentation is MM culture medium as the base culture medium, glucose with a final concentration of 10-30 g / L and sodium chloride of 5-100 g / L is added, and the pH is 8.5-9.5 (preferably 8.8-9.2).

[0023] The beneficial effects of the present invention include at least: the present invention utilizes halophilic microorganisms to express the zein subunit Zein1c2 to achieve efficient microbial synthesis of zein, significantly improving the yield of zein, and utilizing the recombinant halophilic microorganisms of the present invention to ferment and produce zein, which can be open fermented without the need for additional sterilization. At the same time, during the fermentation process, it is not necessary to add various air filter membranes and complex aseptic operations to reduce the possibility of contamination, which greatly saves production costs and manpower and material resources, and has broad application prospects. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The comparison of the English abbreviations and the Chinese full names used in the present invention is shown in Table 1.

[0026] Table 1

[0027] The following examples of Halomonas Halomonas bluephagenesis TD01 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on November 19, 2010, with the deposit registration number of CGMCC No.4353 and the classification name of Halomonas Halomonas sp. TD01, also known as Halomonas bluephagenesis TD01; it is recorded in the patent application with publication number CN102120973A, and the public can obtain the bacterium from Tsinghua University. Halomonas campanian LS21 is disclosed in "Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation" (Chen, Ling, Guan-Qing, et al. Biotechnology and Bioengineering, 2018. DOI: 10.1002 / bit.26897.). Halomonas Halomonas bluephagenesis WZY254 is disclosed in "Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors" (Wang Z, Zheng Y, Ji M, et al. Microbial Biotechnology. DOI: 10.1111 / 1751-7915.13999.).

[0028] The specific formula of the MM medium used in the following examples is as follows: Urea 0.7 g / L; MgSO4 0.4 g / L; KH2PO4 1.75 g / L; and a total of 0.05 g / L Fe(III)-NH4-Citrate, 0.02 g / L CaCl2·2H2O, 0.1 mg / L ZnSO4·7H2O, 0.03 mg / L MnCl2·4H2O, 0.3 mg / LH3BO3, 0.2 mg / L CoCl2·6H2O, 0.01 mg / L CuSO4·5H2O, 0.02 mg / L NiCl2·6H2O, and 0.03 mg / LNaMoO4·2H2O.

[0029] The specific formula of the LB60 liquid culture medium used in the following examples is as follows: 60 g / L sodium chloride, 10 g / L peptone (OXID, UK, product catalog number LP0042), 5 g / L yeast extract (OXID, UK, product catalog number LP0021).

[0030] Example 1

[0031] This embodiment provides a recombinant halophilic microorganism and a method for producing zein by fermentation, the steps are as follows: 1. Construction of plasmid pKD-zein

[0032] The backbone of the pKD-zein plasmid is pSEVA341 (for plasmid, see the literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41.D1 (2012): D666-D675), the plasmid sequence without the porin58 promoter and z1C2 gene sequence inserted is SEQ ID No.4, and the arrangement order of the modules of the plasmid after insertion is sgRNA expression module (nucleotides 1-152), upstream homology arm (nucleotides 153-1152), porin58 promoter sequence (shown in SEQ ID NO.3, nucleotides 1153-1395), z1C2 gene sequence (shown in SEQ ID NO.2, nucleotides 1396-2038), and downstream homology arm (nucleotides 2039-3039).

[0033] 2. Construction of recombinant halophilic microorganism TD+z1C2

[0034] The plasmid pKD-zein and pQ08 plasmid (see Qin Q, Ling C, ZhaoY, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp[J]. Metabolic engineering, 2018, 47: 219-229.) constructed in step 1 were respectively transformed into the host Halomonas by conjugation transformation. Halomonas bluephagenesis In TD01, CRISPR / Cas9 technology was used to generate Halomonas bluephagenesis The z1C2 gene was overexpressed in the TD01 chromosome. For the CRISPR / Cas9 genome editing method and editing sites, please refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0035] Verification by PCR and sequencing z1C2 Genetic success Halomonas bluephagenesis The CRISPR plasmid was inserted into the TD01 genome to obtain the corresponding recombinant halophilic microorganism TD+z1C2. Then, the recombinant halophilic microorganism after successful editing was continuously and multiple times subcultured in LB60 liquid culture medium, and streaked on spectinomycin-resistant, chloramphenicol-resistant and non-resistant plates, respectively, to identify the recombinant halophilic microorganism TD+z1C2 strain with lost CRISPR plasmid.

[0036] 3. Fermentation tank production of corn protein

[0037] The recombinant halophilic microorganism TD+z1C2 strain was used as the fermentation strain, MM medium was used as the basal medium, and 30 g / L of glucose was added as the carbon source and 10 g / L of sodium chloride was added to provide the osmotic pressure environment required for the growth of halophilic bacteria. A fermentation tank experiment was conducted. The fermentation tank used this time was a 7L fermentation tank, the fermentation liquid volume was 3 L, the pH was set at 9.0, and the temperature was maintained at 37°C. This embodiment adopted open fermentation without additional sterilization. At the same time, various air filter membranes and complex aseptic operations were not required during the fermentation process to reduce the possibility of contamination. The salt monosaccharides before transformation Halomonas bluephagenesis TD01 was used as the fermentation strain as the control group under the same fermentation conditions for 48 h. After the fermentation, the concentration of zein1c2 subunit in the fermentation broth was detected.

[0038] The concentration of zein1c2 subunit of zein in the fermentation broth was determined by near infrared method according to GB / T 24901-2010. The results showed that the concentration of zein1c2 subunit of zein in the control group was 0 mg / L, while the concentration of zein1c2 subunit of zein in the fermentation broth of the recombinant halophilic microorganism TD+z1C2 strain was 59 mg / L, and the method was easy to purify and obtain zein products.

[0039] Example 2

[0040] This example provides a method for producing zein by fermentation using a recombinant halophilic microorganism. The steps are different from those in Example 1 only in that: Halomonas Halomonas bluephagenesis TD01 replaced by Halomonas Halomonas campanian LS21, obtained the recombinant halophilic microorganism LS+z1C2 strain.

[0041] The recombinant halophilic microorganism LS+z1C2 strain constructed in this example was further used to ferment and produce zein. The fermentation process was the same as in Example 1. Halomonas campaniensis LS21 was used as a fermentation strain as the control group under the same fermentation conditions. The results showed that the concentration of zein1c2 subunit of corn protein in the control group was 0 mg / L, while the concentration of zein1c2 subunit of corn protein in the fermentation broth of the recombinant halophilic microorganism LS+z1C2 strain was 57 mg / L.

[0042] Example 3

[0043] This example provides a recombinant halophilic microorganism and a method for producing zein by fermentation. The steps are different from those in Example 1 only in that: Halomonas Halomonas bluephagenesis TD01 replaced by Halomonas Halomonas bluephagenesis WZY254, obtained the recombinant halophilic microorganism WZY+z1C2 strain.

[0044] The recombinant halophilic microorganism WZY+z1C2 strain constructed in this example was further used to ferment and produce zein. The fermentation process was the same as in Example 1. Halomonas bluephagenesis WZY254 was used as a fermentation strain as the control group under the same fermentation conditions. The results showed that the concentration of zein1c2 subunit of corn protein in the control group was 0 mg / L, while the concentration of zein1c2 subunit of corn protein in the fermentation broth of the recombinant halophilic microorganism WZY+z1C2 strain was 81 mg / L.

[0045] Finally, 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant halophilic microorganism, characterized in that The recombinant halophilic microorganism expresses the zein subunit Zein1c2.

2. A recombinant halophilic microorganism, characterized in that The recombinant halophilic microorganism overexpresses the zein subunit Zein1c2.

3. The recombinant halophilic microorganism according to claim 1 or 2, characterized in that The halophilic microorganism is a member of the genus Halomonas ( Halomonas )bacteria.

4. The recombinant halophilic microorganism according to claim 3, characterized in that The genus Halomonas ( Halomonas ) Bacteria Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194 or Halomonas bluephagenesis TDH4, preferably Halomonas bluephagenesis WZY254.

5. The recombinant halophilic microorganism according to any one of claims 1 to 4, characterized in that The amino acid sequence of the zein subunit Zein1c2 is shown in SEQ ID NO.

1.

6. The recombinant halophilic microorganism according to claim 5, characterized in that The nucleotide sequence of the gene encoding the zein subunit Zein1c2 is shown in SEQ ID NO.

2.

7. The method for constructing a recombinant halophilic microorganism according to any one of claims 1 to 6, characterized in that: include: The gene encoding the zein subunit Zein1c2 was introduced into the halophilic microorganism; Preferably, porin58 promoter is used to drive the expression or overexpression of the gene encoding the zein subunit Zein1c2.

8. Use of the recombinant halophilic microorganism according to any one of claims 1 to 6 or the construction method according to claim 7 in the preparation of zein.

9. A method for preparing zein, characterized in that: include: The recombinant halophilic microorganism according to any one of claims 1 to 6 is used to ferment and produce zein.

10. The preparation method according to claim 9, characterized in that: The fermentation temperature is 35°C to 40°C; And / or, the culture medium used for the fermentation is MM culture medium as the basic culture medium, glucose with a final concentration of 10-30 g / L and sodium chloride with a pH of 8.5-9.5 is added.

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