An engineered halophilic bacterium and a method for producing 3-hydroxybutyric acid using the same

Abstract

The present invention relates to the field of microbial technology, and in particular to an engineered halophilic bacterium and a method for producing 3-hydroxybutyric acid using the same. The engineered halophilic bacterium does not express a poly 3-hydroxybutyrate synthase gene phaC Overexpression of thioesterase gene tesB . The present invention uses halophilic bacteria through engineering transformation to obtain an engineered halophilic bacteria that can efficiently synthesize 3-hydroxybutyric acid, achieving a breakthrough in the synthesis of 3-hydroxybutyric acid from zero to one in halophilic bacteria. The invention provides an excellent strain for the industrial production of 3-hydroxybutyric acid, and the production process of the invention is simple and low-cost, providing more options for the industrial fermentation production of 3-hydroxybutyric acid, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an engineered halophilic bacterium and a method for producing 3-hydroxybutyric acid using the engineered halophilic bacterium. Background Art

[0002] 3-Hydroxybutyrate (3HB), also known as β-hydroxybutyrate, is a ketone body. 3HB is one of the components of human ketone bodies and is often used as an energy source during hunger or exercise. In addition, 3HB is also considered a therapeutic agent. Experiments have shown that oral 3HB drugs have therapeutic effects on a variety of diseases such as epilepsy, hypertension, NLRP3-mediated inflammation or neurodegenerative diseases. After administration of 3HB in mice, it was also observed that bone formation was promoted, learning and memory were improved, and glial cells were protected. In animal models, 3HB has been shown to improve colitis induced by dextran sulfate sodium (DSS). EcN is usually used to treat inflammatory bowel disease (IBD), so the combined use of therapeutic compound 3HB and probiotic EcN will show synergistic effects. The in situ synthesis of therapeutic compound 3HB can also reduce the off-target effect of the drug and effectively deliver 3HB over a longer period of time, thereby significantly improving the therapeutic effect of colitis.

[0003] In the prior art, CN 117736958 A discloses an Escherichia coli producing (R)-3-hydroxybutyric acid and its construction method and application, which is achieved by knocking out the 6-phosphogluconate dehydratase gene in Escherichia coli. edd 6-phosphokinase I gene pfkA, And overexpress the following genes: acetoacetyl-CoA thiolactonase gene phaA , (R) -3-hydroxybutyrate CoA dehydrogenase gene phaB , thioesterase gene tesB and bifunctional phosphoketolase gene xfpK and diphosphatase genes fbp Recombinant Escherichia coli capable of producing (R)-3-hydroxybutyric acid was obtained. However, this engineering transformation method is complicated and is only applicable to laboratory shake flask fermentation, making it difficult to promote and apply it industrially. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention obtains an engineered halophilic bacterium capable of efficiently synthesizing 3-hydroxybutyric acid by engineering transformation using halophilic bacteria.

[0005] Based on this, the following technical solution is proposed.

[0006] First, the present invention provides an engineered halophilic bacterium, wherein the engineered halophilic bacterium does not express a poly 3-hydroxybutyrate synthase gene. phaC Overexpression of thioesterase gene tesB .

[0007] Halophiles 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. Halophiles can naturally synthesize poly ((R)-3-hydroxybutyrate], P3HB) endogenously, but cannot synthesize small molecules of 3-hydroxybutyrate.

[0008] The present invention allows the poly 3-hydroxybutyrate synthase gene to phaC thioesterase gene tesB Overexpression allows halophilic bacteria to efficiently synthesize 3-hydroxybutyric acid small molecules. The schematic diagram of the engineering transformation route is shown in Figure 1 shown.

[0009] Preferably, the engineered halophilic bacteria are engineered Halomonas.

[0010] Halomonas is a type of moderately halophilic bacteria isolated from Aidin Salt Lake in Xinjiang Uygur Autonomous Region. As a widely used type of halophilic bacteria, it has broad prospects for industrial application after engineering transformation.

[0011] Preferably, the Halomonas is Halomonas bluephagenesis TD1.0, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194 or Halomonas bluephagenesis TDH4.

[0012] Preferably, the poly 3-hydroxybutyrate synthase gene phaC The nucleotide sequence is shown in SEQ ID NO.1; the thioesterase gene tesB The nucleotide sequence is shown in SEQ ID NO.2.

[0013] In the specific implementation process, the engineered halophilic bacteria are not allowed to express the poly 3-hydroxybutyrate synthase gene. phaC The means include but are not limited to: knocking out the poly 3-hydroxybutyrate synthase gene phaC , Silencing of poly 3-hydroxybutyrate synthase gene phaC Expression of poly 3-hydroxybutyrate synthase gene phaC expression of poly 3-hydroxybutyrate synthase gene phaC The transcription or translation of poly 3-hydroxybutyrate synthase genephaC Inactivation of poly 3-hydroxybutyrate synthase gene phaC mutation (e.g., insertion, deletion or substitution of one or more nucleotides in the gene), etc.

[0014] In the specific implementation process, the overexpression means include but are not limited to:

[0015] (1) Introduction of thioesterase gene tesB Plasmids;

[0016] (2) Increase of thioesterase gene tesB The number of copies;

[0017] (3) Modification of thioesterase gene tesB promoter sequence;

[0018] (4) Integrate a strong promoter with the thioesterase gene tesB operatively connected;

[0019] (5) Import enhancers.

[0020] Preferably, the thioesterase gene is overexpressed tesB Use an inducible expression system or a constitutive expression system.

[0021] Preferably, the promoter used in the inducible expression system is mmp1 (SEQ ID No. 3).

[0022] When an inducible expression system is used, the amount of the inducer (eg, IPTG) is 0.5-500 mg / L, preferably 1-200 mg / L, more preferably 10-200 mg / L, more preferably 80-120 mg / L, and most preferably 90-110 mg / L.

[0023] Preferably, the promoter used in the constitutive expression system is Pporin221 (SEQ ID No.4), Pporin68 (SEQ ID No.5), Pporin42 (SEQ ID No.6) or Pporin58 (SEQ ID No.7); preferably Pporin58.

[0024] The present invention further found that different promoters were selected to overexpress the thioesterase gene tesB When the Pporin58 promoter was used, the ability of the engineered halophilic bacteria to synthesize 3-hydroxybutyrate was greatly improved.

[0025] Furthermore, the present invention provides the use of the engineered halophilic bacteria in the production of 3-hydroxybutyric acid or the breeding of 3-hydroxybutyric acid producing bacteria.

[0026] The selection and breeding of the above-mentioned production bacteria can adopt methods such as genetic engineering, mutagenesis, and adaptive evolution.

[0027] Furthermore, the present invention provides a method for producing 3-hydroxybutyric acid, comprising: fermenting and culturing the engineered halophilic bacteria.

[0028] Preferably, the culture medium used for the fermentation culture is MM culture medium as the base 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.

[0029] Preferably, the fermentation culture temperature is 35°C-39°C, most preferably 37°C.

[0030] Preferably, after the fermentation culture is completed, 3-hydroxybutyric acid in the culture is collected, and the bacteria are centrifuged and resuspended, and then pyruvic acid is added to further collect 3-hydroxybutyric acid in the resuspended liquid.

[0031] The present invention further finds that by utilizing pyruvate for whole-cell catalysis and optimizing the extraction process of 3-hydroxybutyrate, the yield of 3-hydroxybutyrate can be further increased.

[0032] Compared with the prior art, the beneficial effects of the present invention are at least:

[0033] The present invention uses halophiles to obtain an engineered halophile capable of efficiently synthesizing 3-hydroxybutyric acid through engineering transformation, achieving a breakthrough in the synthesis of 3-hydroxybutyric acid from zero to one in halophiles. The present invention provides an excellent strain for the industrial production of 3-hydroxybutyric acid, and the production process of the present invention is simple and low-cost, providing more options for industrial fermentation production of 3-hydroxybutyric acid, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the engineering transformation route of halophilic bacteria provided by the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Halomonas bluephagenesisTD1.0 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. TD1.0 (also known as Halomonas bluephagenesis TD1.0), which is recorded in patent application CN102120973A and can be obtained by the public from Tsinghua University.

[0037] The specific formula of the MM medium used in the following examples is as follows:

[0038] Urea 0.7g / L; MgSO4 0.4g / L; KH2PO4 1.75g / L; and a total of 0.05g / L Fe(III)-NH4-Citrate, 0.02g / L CaCl2·2H2O, 0.1mg / L ZnSO4·7H2O, 0.03mg / L MnCl2·4H2O, 0.3mg / LH3BO3, 0.2mg / LCoCl2·6H2O, 0.01mg / L CuSO4·5H2O, 0.02mg / L NiCl2·6H2O, 0.03mg / LNaMoO4·2H2O.

[0039] In the following examples, the poly 3-hydroxybutyrate synthase gene was knocked out phaC The conventional methods in the field of genetic engineering can be used.

[0040] In the following examples, the detection method of 3-hydroxybutyric acid in the fermentation broth is as follows:

[0041] Take 1 mL of fermentation broth and centrifuge at 12000 rpm for 10 min at room temperature to obtain the supernatant and precipitate. Use Agilent high performance liquid chromatograph (HPLC, Agilent1260), configure Bio-rad organic acid chromatographic column AMINEXHPX-87H (300×7.8 mm, 5 μm), configure differential detector (50°C): the mobile phase is 5 mM sulfuric acid solution, the column temperature is 60°C, and the flow rate is 0.5 ml min -1 The injection volume was 10 μL. A linear regression equation was constructed based on the measured HPLC peak area and the corresponding standard concentration, and the concentration of 3-hydroxybutyric acid in the supernatant was calculated using the obtained linear regression equation.

[0042] Example 1

[0043] This embodiment provides an engineered halophilic bacterium, and the construction method thereof is as follows:

[0044] Halomonas Halomonas bluephagenesis ) TD1.0 knockout phaCAfter the gene (sequence as shown in SEQ ID NO.1) was knocked out phaC The strain was named TD1.0Δ phaC , then in strain TD1.0Δ phaC Overexpression of thioesterase genes tesB , the specific steps are as follows:

[0045] (1) Construction of pSEVA321-mmp1- tesB Expression plasmid: promoter mmp1 (SEQ ID No.3) and tesB The gene (SEQ ID No. 2) was sequentially concatenated and inserted into the exogenous gene expression site of the pSEVA321 plasmid; the pSEVA321 plasmid was disclosed in Durante-Rodríguezg, de Lorenzo V, Martínez-García E. The Standard European Vector Architecture (SEVA) plasmid toolkit. Methods Mol Biol. 2014;1149:469-78. doi: 10.1007 / 978-1-4939-0473-0_36. PMID: 24818926.

[0046] (2) The plasmid was transformed into Halomonas by conjugation. Halomonas bluephagenesis In TD1.0 hosts, overexpression was identified by transcriptome sequencing tesB The strain was named TD1.0Δ phaC - tesB .

[0047] Furthermore, this embodiment adopts TD1.0Δ phaC - tesB The strain produced 3-hydroxybutyric acid by fermentation in a shake flask experiment. The specific steps are as follows:

[0048] MM medium was used as the basal medium, and glucose and sodium chloride were added at a final concentration of 30 g / L and 50 g / L, respectively. The pH was adjusted to 9.0, the fermentation temperature was 37 °C, and the TD was 1.0Δ phaC - tesB The inoculum volume of the strain was 1 mL, and IPTG with concentrations of 1, 10, 100, and 200 mg / L was added to induce TD1.0Δ phaC - tesB The strain expressed and produced 3-hydroxybutyric acid. After 48 h of fermentation, the yields of 3-hydroxybutyric acid in shake flasks were 5.1 g / L, 6.3 g / L, 12.2 g / L and 6.3 g / L, respectively.

[0049] Example 2

[0050] This embodiment provides an engineered halophilic bacterium, and the construction method thereof is different from that of embodiment 1 in that the promoter mmp1 is replaced by the promoter P porin221 (SEQ ID No.4), P porin68 (SEQ ID No.5), P porin42 (SEQ ID No. 6), P porin58 (SEQ ID No.7), that is, converting the inducible expression system into a constitutive expression system to obtain an engineered Halomonas.

[0051] Furthermore, in this example, the engineered Halomonas containing the above constitutive expression system was used to produce 3-hydroxybutyric acid by fermentation in a shake flask experiment. The specific steps were different from those in Example 1 in that IPTG was not added. In the shake flask, pSEVA321-P porin221 - tesB , pSEVA321-P porin68 - tesB , pSEVA321-P porin42 - tesB , pSEVA321-P porin58 - tesB The engineered Halomonas can achieve 3.1g / L, 4.3g / L, 7.2g / L and 13.3g / L of 3-hydroxybutyric acid, respectively.

[0052] Example 3

[0053] Furthermore, this example uses the engineered Halomonas containing the constitutive expression system in Example 2 to produce 3-hydroxybutyric acid through fermentation in a fermenter experiment, and the fermentation conditions are as follows:

[0054] MM medium was used as the basal medium, and glucose and sodium chloride were added at a final concentration of 30 g / L, and 50 g / L, respectively. The pH was adjusted to 9.0, the fermentation temperature was 37 °C, the inoculum volume of engineered Halomonas was 1 mL, and the pSEVA321-P porin58 -tesB The engineered Halomonas can achieve 20 g / L of 3-hydroxybutyrate in the fermenter.

[0055] Example 4

[0056] In order to further increase the accumulation of 3-hydroxybutyric acid produced by fermentation in Example 3, this example increases the accumulation of 3-hydroxybutyric acid by changing the extraction process. The specific steps are as follows:

[0057] After the fermentation culture in Example 3 is completed, the bacterial precipitate and the supernatant are obtained by centrifugation. The bacterial precipitate is resuspended in the fermentation medium and 10 g / L of pyruvate is added. The whole-cell catalysis is carried out for 12 hours, and the resuspension is collected by centrifugation. The resuspension is mixed with the supernatant, and the 3-hydroxybutyric acid content in the mixed solution is detected. The results show that the optimized extraction process can accumulate 25 g / L of 3-hydroxybutyric acid.

[0058] 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. An engineered halophilic bacterium, characterized in that: The engineered halophile does not express the poly 3-hydroxybutyrate synthase gene phaC Overexpression of thioesterase gene tesB ; The engineered halophilic bacteria are engineered Halomonas bluephagenesis .

2. The engineered halophilic bacteria according to claim 1, characterized in that The poly 3-hydroxybutyrate synthase gene phaC The nucleotide sequence is shown in SEQ ID NO.1; the thioesterase gene tesB The nucleotide sequence is shown in SEQ ID NO.

2.

3. The engineered halophilic bacteria according to claim 1 or 2, characterized in that Overexpression of thioesterase genes tesB Use an inducible expression system or a constitutive expression system.

4. The engineered halophilic bacteria according to claim 3, characterized in that The promoter used in the inducible expression system is mmp1.

5. The engineered halophilic bacteria according to claim 3, characterized in that The promoter used in the constitutive expression system is P porin221 , P porin68 , P porin42 or P porin58 .

6. Use of the engineered halophilic bacteria according to any one of claims 1 to 5 in the production of 3-hydroxybutyric acid or the breeding of 3-hydroxybutyric acid producing bacteria.

7. A method for producing 3-hydroxybutyric acid, characterized in that: include: Fermentation culture of the engineered halophilic bacteria according to any one of claims 1 to 5.

8. The production method according to claim 7, characterized in that: The culture medium used in the fermentation culture 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.

9. The production method according to claim 7 or 8, characterized in that: After the fermentation culture is completed, 3-hydroxybutyric acid in the culture is collected, and the bacteria are centrifuged and resuspended, and then pyruvic acid is added to further collect 3-hydroxybutyric acid in the resuspended liquid.

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