Strain for producing 3-hydroxybutyric acid, construction method therefor, and use thereof

Abstract

Disclosed in the present invention are a strain for producing 3-hydroxybutyric acid, a construction method therefor, and the use thereof. The construction method comprises the following steps: S1, amplifying sequences of phaA, phaB and tesB genes in vitro, linking the sequences of the genes into a single fragment, and introducing the single fragment into a plasmid; and S2, introducing the plasmid obtained in the step S1 into a strain. The present invention uses an engineered strain for fermentation to produce 3-HB; by means of further improving a combined metabolic pathway, and adding sodium salt of a short-chain fatty acid during fermentation as a substrate, the present invention reduces the influence of intermediate metabolite acetic acid on the yield of 3-HB of the strain, thereby further improving the 3-HB yield. In addition, the present invention involves a simple production process, achieves high production efficiency, and is suitable for continuous industrial production.

Description

A strain producing 3-hydroxybutyric acid, its construction method and application Technical Field

[0001] The present invention relates to the technical field of industrial microorganisms, and in particular to a strain for producing 3-hydroxybutyric acid, a construction method thereof, and an application thereof. Background Art

[0002] 3-Hydroxybutyric acid (3-HB) is an important chemical with broad application potential. It can participate in the synthesis of biodegradable plastics and, as a monomer in the biodegradable plastic poly(3-hydroxybutyrate) (PHB), is produced through microbial fermentation or chemical synthesis. PHB exhibits excellent biodegradability and biocompatibility and is used in packaging materials, medical devices, agricultural mulches, and other applications. It can also be copolymerized with other biopolymers to improve their physical properties, such as poly(3-hydroxybutyrate-4-hydroxybutyrate) and poly(3-hydroxybutyrate-6-hydroxyhexanoate).

[0003] 3-HB monomers, after polymerization to form PHB, can be used in a variety of applications, particularly in environmental protection. With increasing environmental protection requirements, biodegradable materials are gaining increasing attention in the packaging sector and are gradually replacing traditional non-degradable plastics. Polyhydroxybutyrate (PHB) exhibits physical and mechanical properties similar to those of polypropylene and is biodegradable. Its degradation products in nature are carbon dioxide and water, which are environmentally friendly, making it a promising candidate for broad application in the packaging sector. Therefore, the development and utilization of efficient methods for producing 3-HB is crucial for promoting the development of bio-based chemicals, reducing reliance on limited resources, and minimizing environmental impact. In recent years, biosynthesis of 3-HB has garnered significant attention. However, current 3-HB production faces a major challenge: the high yield of the intermediate acetic acid, which significantly reduces the amount of acetic acid available, thus reducing production efficiency and becoming the primary challenge hindering 3-HB production. Therefore, a method to increase 3-HB yield is urgently needed to improve industrial production efficiency and reduce production costs. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention proposes a strain for producing 3-hydroxybutyric acid, a construction method thereof, and an application thereof.

[0005] The present invention provides a method for constructing a strain for producing 3-hydroxybutyric acid, comprising the following steps:

[0006] S1: Amplify the sequences of phaA, phaB, and tesB genes in vitro, connect the gene sequences into a single fragment, and introduce the single fragment into a plasmid; the nucleotide sequence of tesB is shown in SEQ ID No. 63.

[0007] S2: Introduce the plasmid described in S1 into the strain.

[0008] Further, the strain in step S2 is various Halomonas, various probiotics, Pseudomonas, Escherichia coli, etc., such as Escherichia coli Nissle 1917, Escherichia coli JM109, Halomonas sp.LY01, Halomonas sp.LY02, Halomonas sp.LY03, Halomonas sp.LY04, Lactobacillus gasseri ATCC 33323, Halonoas campaniensis LS21, Clostridium ljungdahlii ATCC 55383, Burkholderia cepacia ATCC 17795, Pseudomonas sp, Alcaligenes latus, E.coli MG1655, E.coli BL21, Bifidobacterium, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, Ralstonia eutropha, Halomonas sp.OITC1261, Aeromonas hydrophila, Pseudomonas entomophila, Pseudomonas putida KT2440, Akkermansia muciniphila, Bacillus coagulans DSM1, Halomonas sp.TD01.

[0009] The deposit number of Halomonas sp. LY01 is GDMCC No. 62635, which is disclosed in patent CN116396886A; the deposit number of Halomonas sp. LY02 is GDMCC No. 63381, which is disclosed in patent CN116925981A; the deposit number of Halomonas sp. LY03 is GDMCC No. 63382, which is disclosed in patent CN116970538A; the deposit number of Halomonas sp. LY04 is GDMCC No. 63383, which is disclosed in patent CN117551585A; and the deposit number of Halomonas sp. TD01 is CGMCC No. 4353, which is disclosed in patent CN102120973A. Other probiotic strains, Pseudomonas, and Escherichia coli are all existing strains.

[0010] Furthermore, the phaA in step S1 can be derived from various species, for example, the phaA can be derived from Halomonas sp.TD01. TD , species origin is phaA of Ralstonia eutropha RE , phaA of Alcaligenes latus AL , phaA of H.campaniensis LS21 LS , phaA of Pseudomonas entomophila PE , phaA of species origin Pseudomonas putida KT2440 PP Any one of the phaA TD The nucleotide sequence of phaA is shown in SEQ ID No.61. RE The nucleotide sequence is shown in SEQ ID No.64; the phaA AL The nucleotide sequence is shown in SEQ ID No.66; the phaA LS The nucleotide sequence is shown in SEQ ID No.68; the phaA PE The nucleotide sequence is shown in SEQ ID No.70; the phaA PP The nucleotide sequence is shown as SEQ ID No.72.

[0011] Furthermore, the phaB in step S1 can be derived from various species, for example, the phaB can be derived from Halomonas sp.TD01. TD, phaB of Ralstonia eutropha RE , phaB from Alcaligenes latus AL , phaB from H.campaniensis LS21 LS , phaB from Pseudomonas entomophila PE , phaB from Pseudomonas putida KT2440 PP Any one of the phaB TD The nucleotide sequence of phaB is shown in SEQ ID No.62. RE The nucleotide sequence of phaB is shown in SEQ ID No.65; AL The nucleotide sequence is shown in SEQ ID No.67; the phaB LS The nucleotide sequence is shown in SEQ ID No.69; the phaB PE The nucleotide sequence is shown in SEQ ID No.71; the phaB PP The nucleotide sequence is shown as SEQ ID No.73.

[0012] Furthermore, the single fragment in step S1 is a free combination of tesB, phaA, and phaB from different sources, such as tesB-phaA TD -phaB TD tesB-phaA RE -phaB TD tesB-phaA RE -phaB RE tesB-phaB TD -phaA TD , phaA TD -tesB-phaB TD , phaB TD -tesB-phaA TD , phaA TD -phaB TD -tesB、phaB TD -phaA TD -tesB, tesB-pha AL -phaB AL ,tesB-pha LS -phaB LS ,tesB-pha PE -phaB PE ,tesB-phaPP -phaB PP Any one of .

[0013] tesB-phaA TD -phaB TD The nucleotide sequence of tesB-phaA is shown in SEQ ID No.53; RE -phaB TD The nucleotide sequence of tesB-phaA is shown in SEQ ID No.54; RE -phaB RE The nucleotide sequence of tesB-phaB is shown in SEQ ID No.55; TD -phaA TD The nucleotide sequence of phaA is shown in SEQ ID No.56; TD -tesB-phaB TD The nucleotide sequence of phaB is shown in SEQ ID No.57; TD -tesB-phaA TD The nucleotide sequence of phaA is shown in SEQ ID No.58; TD -phaB TD -tesB nucleotide sequence is shown in SEQ ID No.59; said phaB TD -phaA TD -tesB nucleotide sequence is shown in SEQ ID No.60;

[0014] The present invention also provides a strain for producing 3-hydroxybutyric acid, which is obtained by the construction method.

[0015] The present invention also provides a method for producing 3-hydroxybutyric acid using the strain, comprising the following steps: activating the strain, performing shake flask seed culture, and then performing fermentation culture in a fermentation medium.

[0016] Furthermore, the fermentation medium includes short-chain fatty acids and sodium salts thereof, preferably any one of sodium acetate, sodium propionate, sodium butyrate, sodium valerate, sodium pyruvate, sodium lactate, sodium citrate, sodium malate, and sodium succinate.

[0017] Furthermore, the addition amount of the short-chain fatty acid is 0-30 g / L, preferably 3-12 g / L, and the carbon source mixed with glucose can be adjusted according to the content of glucose.

[0018] Furthermore, the fermentation temperature is 36-42° C., the pH is 6-11, and the fermentation time is 12-48 hours.

[0019] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0020] (1) The combined metabolic pathway strain constructed by the present invention increases the production of 3-HB.

[0021] (2) The present invention further optimizes the combined metabolic pathway and adds sodium acetate substrate during fermentation, thereby reducing the impact of the intermediate metabolite acetic acid on the bacterial 3-HB production and further increasing the 3-HB production.

[0022] (3) The present invention utilizes engineered bacteria to ferment and produce 3-HB, which has a simple production process and high production efficiency and is suitable for industrial continuous production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] FIG1 is a pathway diagram of the present invention for synthesizing 3-HB from glucose;

[0025] FIG2 is a map of the pSEVA321tesB-phaA-phaB plasmid used in Example 1 of the present invention;

[0026] FIG3 is an electrophoresis diagram of the tesB-phaA-phaB target fragment in Example 1 of the present invention;

[0027] FIG4 is a map of the pSEVA321tesB-phaARE-phaBTD plasmid used in Example 2 of the present invention;

[0028] FIG5 is a map of the pSEVA321tesB-phaARE-phaBRE plasmid used in Example 2 of the present invention;

[0029] Figure 6 shows tesB-phaA in Example 2 of the present invention. RE -phaB TD Electropherogram of target fragment;

[0030] Figure 7 shows tesB-phaA in Example 2 of the present invention. RE -phaB RE Electropherogram of target fragment;

[0031] Figure 8 is a map of the pSEVA321tesB-phaARE-phaBTD plasmid used in Example 3 of the present invention; wherein Figure a is a map of the 2-pSEVA321tesB-phaB-phaA plasmid, Figure b is a map of the 3-pSEVA321phaA-tesB-phaB plasmid, Figure c is a map of the 4-pSEVA321phaB-tesB-phaA plasmid, Figure d is a map of the 5-pSEVA321phaA-phaB-tesB plasmid, and Figure e is a map of the 6-pSEVA321phaB-phaA-tesB plasmid;

[0032] FIG9 is an electrophoresis diagram of the tesB-phaB-phaA target fragment in Example 3 of the present invention;

[0033] Figure 10 is an electrophoretic diagram of the phaA-tesB-phaB target fragment in Example 3 of the present invention;

[0034] Figure 11 is an electrophoretic diagram of the phaB-tesB-phaA target fragment in Example 3 of the present invention;

[0035] FIG12 is an electrophoresis diagram of the phaA-phaB-tesB target fragment in Example 3 of the present invention;

[0036] FIG13 is an electrophoresis diagram of the target fragment phaB-phaA-tesB in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the embodiments described are only 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 making creative efforts should fall within the scope of protection of the present invention.

[0038] Escherichia coli Nissle 1917 itself does not have the ability to utilize 3-HB and does not have the ability to produce 3-HB. Therefore, the first part of the present invention introduces a synthetic pathway for generating 3-HB from glucose. By overexpressing three genes, phaA, phaB, and tesB, from different sources, the metabolic pathway is promoted to flow to 3-HB.

[0039] In order to increase the production of 3-HB, the second part of the present invention also explored the position effect of the three genes phaA, phaB, and tesB to determine the better expression level of metabolic pathway genes.

[0040] The third part of the present invention introduces sodium acetate into the fermentation medium, which, on the one hand, serves as a carbon source to enhance the synthesis of acetyl-CoA, and on the other hand, can be used to balance the redox level in the cell, thereby further increasing the yield of 3-HB.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0042] Example 1 Construction of a metabolic pathway for de novo synthesis of 3-HB

[0043] 1. tesB, phaA, and phaB gene expression

[0044] (1) Plasmid construction

[0045] PCR amplified tesB from E. coli MG1655, phaA and phaB from Halomonas sp. TD01, and the pSEVA321 backbone. Using Gibson ligase, the phaA, phaB, and tesB fragments were recombined with the pSEVA321 backbone to form a new plasmid, named pSEVA321tesB-phaA-phaB. tesB-phaA-phaB was generated by PCR using pSEVA321tesB-phaA-phaB as a template. A portion of the product was sent to a biotechnology company for sequencing. The plasmid information is shown in Figure 2.

[0046] The primer sequences (5'-3') for PCR amplification of tesB, phaA, phaB, and the plasmid pSEVA321 backbone are as follows:

[0047] tesB-F: see SEQ ID No. 1;

[0048] tesB-R: see SEQ ID No. 2;

[0049] phaA-F: see SEQ ID No. 3;

[0050] phaA-R: see SEQ ID No. 4;

[0051] phaB-F: see SEQ ID No. 5;

[0052] phaB-R: see SEQ ID No. 6;

[0053] pSEVA321-F: see SEQ ID No. 7;

[0054] pSEVA321-R: see SEQ ID No.8.

[0055] The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0056] Table 1 Amplification system

[0057] Table 2 Amplification program

[0058] After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light and quickly cut off the gel of the target DNA fragment, cutting off as much excess gel as possible.

[0059] (2) Gibson Assembly method connection

[0060] The recovered DNA was tested for concentration, and the DNA addition ratio was calculated based on the length and concentration of the target fragment and the pSEVA321 backbone. The Gibson enzyme mixture was then used for ligation. The Gibson Assembly ligation system and procedure are shown in Tables 3 and 4:

[0061] Table 3 Gibson Assembly connection system

[0062] Table 4 Gibson Assembly connection procedure

[0063] (3) Nissle 1917 Escherichia coli transformation

[0064] Step 1: Take out the prepared Nissle 1917 E. coli competent cells from -80℃ and thaw them on ice. After 5 minutes, wait for the cells to thaw.

[0065] Step 2: Add 5 μL of ligation product to the competent cells and gently tap the tube to mix the reaction mixture (do not oscillate). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0066] Step 3: Place on ice for 30 minutes, heat shock in a 42°C water bath for 2 minutes, and immediately cool on ice for 2 minutes. Note: shaking will reduce transformation efficiency;

[0067] Step 4: Add 400 μL of LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a 37°C shaker at 200 rpm for 60 min.

[0068] Step 5: Centrifuge at 5000 rpm for 5 min to harvest the bacteria. Discard 350 μL of the supernatant and retain 100 μL of the suspension. Gently pipette to resuspend the bacteria and spread the suspension onto LB medium containing the corresponding antibiotics.

[0069] Step 6: Invert the culture medium and place it in a 37°C incubator for 12 to 16 hours.

[0070] (4) Monoclonal colony positive verification

[0071] Pick out colonies on the corresponding resistance LB plate and perform colony PCR verification. Send the PCR products with the correct band size to a biological company for sequencing.

[0072] (5) Select a single colony with the correct sequence for expansion. After 12 to 16 hours, streak it on a resistance LB plate. Once a single clone has grown, verify the single clone colony again.

[0073] (6) Product identification

[0074] The colony verification results showed that the tesB-phaA-phaB gene sequence was successfully transferred into the Escherichia coli Nissle 1917 strain, as shown in Figure 3. The target fragment was 3667 bp, which was in line with the expected results. The strain was named Nissle-1.

[0075] Example 2 Expression of phaA and phaB genes from different sources

[0076] Plasmid construction: PCR amplification was performed as follows:

[0077] (1) tesB from E. coli MG1655;

[0078] (2) phaA derived from Halomonas sp.TD01, Ralstonia eutropha, Alcaligenes latus, H.campaniensis LS21, Pseudomonas entomophila, Pseudomonas putida KT2440;

[0079] (3) phaB derived from Halomonas sp.TD01, Ralstonia eutropha, Alcaligenes latus, H.campaniensis LS21, Pseudomonas entomophila, Pseudomonas putida KT2440;

[0080] (4) pSEVA321-porin58 backbone plasmid construction;

[0081] Please refer to Example 1 for the specific steps.

[0082] Plasmids containing the phaA and phaB genes from the various sources described above were constructed, including pSEVA321tesB-phaA-phaB (plasmid shown in Figure 2 ), pSEVA321tesB-phaARE-phaBRE (plasmid information shown in Figure 5 ), pSEVA321tesB-phaARE-phaBTD (plasmid information shown in Figure 6 ), pSEVA321tesB-phaAL-phaBAL, pSEVA321tesB-phaALS-phaBLS, pSEVA321tesB-phaAPE-phaBPE, and pSEVA321tesB-phaAPP-phaBPP. The products were sent to a biotechnology company for sequencing.

[0083] The primer sequences (5'-3') used for PCR amplification are as follows:

[0084] pSEVA321-porin58-F: see SEQ ID No. 9; pSEVA321-porin58-R: see SEQ ID No. 10;

[0085] tesB-F: see SEQ ID No. 11; tesB-R: see SEQ ID No. 12;

[0086] phaAB RE -F: see SEQ ID No. 13; phaAB RE -R: see SEQ ID No. 14;

[0087] phaA RE -F: see SEQ ID No.15; phaA RE -R: see SEQ ID No. 16;

[0088] phaB TD -F: see SEQ ID No. 17; phaB TD -R: see SEQ ID No. 18;

[0089] phaAB AL -F: see SEQ ID No.74; phaAB AL -R: see SEQ ID No.75;

[0090] phaAB LS -F: see SEQ ID No.76; phaAB LS -R: see SEQ ID No.77;

[0091] phaAB PE-F: see SEQ ID No.78; phaAB PE -R: see SEQ ID No.79;

[0092] phaAB PP -F: see SEQ ID No.80; phaAB PP -R: See SEQ ID No.81.

[0093] The pSEVA321tesB-phaA-phaB sequences from different sources constructed above were transformed into Escherichia coli Nissle 1917. The size of the target product was used to verify that the tesB-phaA-phaB sequences from different sources had been successfully transformed into Escherichia coli Nissle 1917. For example, tesB-phaA RE -phaB TD The gene sequence is shown in Figure 6; tesB-phaA was transferred RE -phaB RE The gene sequences are shown in Figure 7. The recombinant bacteria were named Nissle-ARE-BTD (tesB-phaA RE -phaB TD fragment), Nissle-RE (transferred into tesB-phaA RE -phaB RE fragment), Nissle-AL (transferred into tesB-phaA AL -phaB AL fragment), Nissle-LS (transferred into tesB-phaA LS -phaB LS fragment), Nissle-PE (transferred into tesB-phaA PE -phaB PE fragment), Nissle-PP (transferred into tesB-phaA PP -phaB PP snippet).

[0094] Example 3: Position Effects of phaA, phaB, and tesB Genes

[0095] Plasmid construction: Referring to the operation of Example 1, tesB from E. coli MG1655, phaA and phaB from Halomonas bluephagenesis, and the plasmid pSEVA321 backbone were amplified by PCR. Under the action of Gibson ligase, the phaA, phaB, and tesB fragments were ligated in different orders and recombined with the pSEVA321 backbone to form new plasmids, named 2-pSEVA321tesB-phaB-phaA, the plasmid map is shown in Figure 8a; 3-pSEVA321phaA-tesB-phaB, the plasmid map is shown in Figure 8b; 4-pSEVA321phaB-tesB-phaA, the plasmid map is shown in Figure 8c; 5-pSEVA321phaA-phaB-tesB, the plasmid map is shown in Figure 8d; 6-pSEVA321phaB-phaA-tesB, the plasmid map is shown in Figure 8e. Part of the product was sent to a biological company for sequencing.

[0096] The primer sequences (5'-3') for PCR amplification are as follows:

[0097] 2-pSEVA321-F: see SEQ ID No. 19; 2-pSEVA321-R: see SEQ ID No. 20;

[0098] 2-phaA-F: see SEQ ID No. 21; 2-phaA-R: see SEQ ID No. 22;

[0099] 2-phaB-F: see SEQ ID No. 23; 2-phaB-R: see SEQ ID No. 24;

[0100] 3-pSEVA321-F: see SEQ ID No. 25; 3-pSEVA321-R: see SEQ ID No. 26;

[0101] 3-phaA-F: see SEQ ID No. 27; 3-phaA-R: see SEQ ID No. 28;

[0102] 3-tesB-F: see SEQ ID No. 29; 3-tesB-R: see SEQ ID No. 30;

[0103] 4-pSEVA321-F: see SEQ ID No. 31; 4-pSEVA321-R: see SEQ ID No. 32;

[0104] 4-phaB-F: see SEQ ID No. 33; 4-phaB-R: see SEQ ID No. 34;

[0105] 4-tesB-F: see SEQ ID No. 35; 4-tesB-R: see SEQ ID No. 36;

[0106] 4-phaA-F: see SEQ ID No. 37; 4-phaA-R: see SEQ ID No. 38;

[0107] 5-pSEVA321-F: see SEQ ID No. 39; 5-pSEVA321-R: see SEQ ID No. 40;

[0108] 5-phaAB-F: see SEQ ID No. 41; 5-phaAB-R: see SEQ ID No. 42;

[0109] 5-tesB-F: see SEQ ID No. 43; 5-tesB-R: see SEQ ID No. 44;

[0110] 6-pSEVA321-F: see SEQ ID No. 45; 6-pSEVA321-R: see SEQ ID No. 46;

[0111] 6-phaB-F: see SEQ ID No. 47; 6-phaB-R: see SEQ ID No. 48;

[0112] 6-phaA-F: see SEQ ID No. 49; 6-phaA-R: see SEQ ID No. 50;

[0113] 6-tesB-F: see SEQ ID No.51; 6-tesB-R: see SEQ ID No.52.

[0114] The plasmids constructed above, 2-pSEVA321tesB-phaB-phaA, 3-pSEVA321phaA-tesB-phaB, 4-pSEVA321phaB-tesB-phaA, 5-pSEVA321phaA-phaB-tesB and 6-pSEVA321phaB-phaA-tesB, were transformed into Escherichia coli Nissle 1917, respectively.

[0115] The size of the target product was used for verification, and the results showed that the tesB-phaB-phaA gene sequence was successfully transferred into Escherichia coli Nissle 1917, as shown in FIG9 ; the phaA-tesB-phaB gene sequence was successfully transferred into Escherichia coli Nissle 1917, as shown in FIG10 ; the phaB-tesB-phaA gene sequence was successfully transferred into Escherichia coli Nissle 1917, as shown in FIG11 ; the phaA-phaB-tesB gene sequence was successfully transferred into Escherichia coli Nissle 1917, as shown in FIG12 ; and the phaB-phaA-tesB gene sequence was successfully transferred into Escherichia coli Nissle 1917, as shown in FIG13 ; the five recombinant bacteria were named Nissle-2, Nissle-3, Nissle-4, Nissle-5, and Nissle-6, respectively.

[0116] Example 4 Production of 3-HB using different modified bacteria

[0117] Referring to the operation of Example 1, the plasmid pSEVA321tesB-phaA-phaB constructed in Example 1 was introduced into Escherichia coli S17-1 and Escherichia coli JM109, and the Nissle-1, S17-1 and JM109 strains in Example 1 were used as fermentation base strains.

[0118] (1) Culture medium

[0119] LB plate medium: yeast extract powder 0.5 g / L, tryptone 1 g / L, sodium chloride 1 g / L, agar powder 1.8 g / 100 mL, pH = 7.

[0120] Fermentation medium (LBG): sodium chloride 10 g / L, yeast powder 5 g / L, tryptone 10 g / L, glucose 10 g / L.

[0121] (2) Seed solution preparation

[0122] ① Bacteria activation:

[0123] The recombinant strain was streaked on LB plate medium and cultured at 37°C for 24 h until a single colony was grown.

[0124] ②First-level seed cultivation:

[0125] A single colony was picked and inoculated into a 12 mL shaking tube (5 ml LB medium), the corresponding antibiotics were added, and the culture was placed in a shaking incubator at 37°C and 220 rpm for 12 h.

[0126] (3) Fermentation culture:

[0127] The seed solution was inoculated at 5% (1 mL) into a 150 mL conical flask and cultured in a shaker at 37°C and 220 rpm for 48 h.

[0128] (4) After the fermentation is completed, the cells are collected for testing:

[0129] (a)OD 600 Determination: Dilute 200 μL of fermented bacteria at appropriate dilution multiples and measure the OD value in a spectrophotometer.

[0130] (b) Determination of 3-HB Content: 200 μL of fermented culture was diluted to appropriate multiples and centrifuged at 12,000 rpm at 4°C. The diluted fermentation broth was then filtered through a 0.22 μm water filter and injected into a sample vial. The 3-HB content was then determined by liquid chromatography using an HPX-87h column with a mobile phase of 0.5 mM sulfuric acid at 60°C. The injection volume was 10 μL. 3-HB was quantified using an external standard method, and 3-HB yield was calculated based on peak area.

[0131] (c) Acetic acid determination: 200 μL of fermented culture was diluted to appropriate multiples and centrifuged at 12,000 rpm at 4°C. The diluted fermentation broth was then filtered through a 0.22 μm water filter and injected into a sample vial. Acetic acid content was determined by liquid chromatography using an HPX-87h column with a mobile phase of 0.5 mM sulfuric acid at 60°C. The injection volume was 10 μL. Acetic acid was quantified using an external standard method, and acetic acid yield was calculated based on peak area.

[0132] (5) The fermentation results are shown in Table 5.

[0133] Table 5 Fermentation effects of three modified bacteria

[0134] These results demonstrate that the 3-HB pathway can be constructed using the chassis strains S17-1, JM109, and Nissle-1. S17-1 cannot produce 3-HB, while JM109 can produce 3-HB, but at a lower yield than Nissle-1. Nissle-1 produces a maximum 3-HB yield of 5.12 g / L, with an acetate content of only 1.92 g / L. This suggests that both Escherichia coli Nissle 1917 and Escherichia coli JM109 are suitable as chassis strains for 3-HB synthesis.

[0135] Example 5 Comparison of phaA and phaB fermentation effects from different sources

[0136] Fermentation was performed using the Nissle-1 strain of Example 1 and the Nissle-ARE-BTD, Nissle-RE, Nissle-AL, Nissle-LS, Nissle-PE, and Nissle-PP strains of Example 2. The specific fermentation medium, steps, and parameter determination methods used were similar to those of Example 4. The fermentation results are shown in Table 6.

[0137] Table 6 Fermentation effects of phaA and phaB from different sources

[0138] The results showed that the Nissle-1 strain overexpressing phaA and phaB from Halomonas sp. TD01 had the best fermentation effect, with a 3-HB yield of up to 5.05 g / L.

[0139] Example 6 Production of 3-HB using modified bacteria with different gene connection sequences

[0140] Fermentation was performed using Nissle-1 from Example 1 and the five modified bacteria from Example 3. The specific fermentation medium, steps, and parameter determination methods were as described in Example 4. The fermentation results are shown in Table 7.

[0141] Table 7 Gene position effect fermentation effect

[0142] The above results show that the Nissle-3 strain has the best fermentation effect, with a 3-HB yield of up to 5.83 g / L, indicating that inserting the phaA, tesB, and phaB genes into the plasmid in the connection order of phaA-tesB-phaB and introducing them into the strain can further increase the 3-HB yield.

[0143] Example 7 Fermentation effect of adding sodium acetate on glucose

[0144] Fermentation was performed using the Nissle-3 strain according to the procedures of Example 4, with the exception of the carbon source. The carbon source consisted of 20 g / L glucose and varying concentrations of sodium acetate (0, 3, 6, 9, and 12 g / L). The configuration and operation were the same as in Example 4. The fermentation results are shown in Table 8.

[0145] Table 8 Fermentation effects of adding different concentrations of sodium acetate

[0146] The results showed that the fermentation effect was best when 12 g / L of sodium acetate was additionally added to the fermentation medium, which could promote the strain to utilize more glucose and thus increase the yield, with the yield reaching as high as 18.73 g / L.

[0147] Example 8 Fermentation of phaAB from different sources using glucose and sodium acetate as carbon sources

[0148] Fermentation was performed using Nissle-1, Nissle-ARE-BTD, Nissle-RE, Nissle-AL, Nissle-LS, Nissle-PE, and Nissle-PP according to the procedures of Example 4. Except for the carbon source, all other components of the fermentation medium remained unchanged. The carbon source was 20 g / L glucose and 12 g / L sodium acetate. The preparation and operation procedures were the same as in Example 4. The fermentation results are shown in Table 9.

[0149] Table 9 Gene position effect fermentation effect

[0150] Example 9 Fermentation effect of adding other short-chain fatty acids on glucose

[0151] Fermentation was performed using the Nissle-3 strain according to the procedures of Example 4, with the exception of the carbon source. The carbon source consisted of 20 g / L glucose and 12 g / L other short-chain fatty acids, with the same configuration and operation as in Example 4. The fermentation results are shown in Table 10.

[0152] Table 10 Fermentation effect of adding other short-chain fatty acids

[0153] The results showed that adding an additional 12g / L of other short-chain fatty acids to the fermentation medium could promote the strain to utilize more glucose and thus increase the yield, which was as high as 10.2g / L.

[0154] Example 10 Production of 3-hydroxybutyric acid using recombinant Halomonas (LY01-04, TD01), recombinant probiotics, recombinant Pseudomonas, recombinant Escherichia coli, etc.

[0155] Recombinant Halomonas LY01-3, LY02-3, LY03-3, LY04-3, TD01-3, recombinant probiotic Lactobacillus gasseri ATCC 33323 (Lg), recombinant bacteria Halonoas campaniensis LS21, Clostridium ljungdahlii ATCC 55383, Burkholderia cepacia ATCC 17795, Pseudomonas sp, Alcaligenes latus, E. coli MG1655, E. coli BL21, Bifidobacterium, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, Ralstonia eutropha, Halomonas sp.OITC1261, Aeromonas hydrophila, Pseudomonas entomophila, Pseudomonas putida KT2440, Akkermansia muciniphila and recombinant probiotic Bacillus coagulans DSM1 (Bc) produced 3-hydroxybutyrate.

[0156] (1) Construction of recombinant bacteria

[0157] The plasmid 3-pSEVA321phaA-tesB-phaB constructed in Example 3 was transformed into Halomonas sp. LY01, Halomonas sp LY02, Halomonas sp LY03, Halomonas sp LY04, Halomonas sp TD01, Lactobacillus gasseri ATCC 33323, Halonoas campaniensis LS21, Clostridium ljungdahlii ATCC 55383, Burkholderia cepacia ATCC 17795, Pseudomonas sp, Alcaligenes latus, E. coli MG1655, E. coli BL21, Bifidobacterium, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, Ralstonia eutropha, Halomonas sp. sp.OITC1261, Aeromonas hydrophila, Pseudomonas entomophila, Pseudomonas putida KT2440, Akkermansia muciniphila and Bacillus coagulans DSM1 were transferred into probiotic lactobacilli using the same method as in Example 1. The resulting recombinant bacteria were named LY01-3, LY02-3, LY03-3, LY04-3, TD01-3, Lg-3, LS-3, CL-3, BC-3, PS-3, AL-3, MG-3, BL-3, Bi-3, FP-3, LR-3, RE-3, HO-3, AH-3, PE-3, PP-3, Ak-3 and Bc-3, respectively.

[0158] (2) Application Reorganization

[0159] 3-Hydroxybutyrate production by LY01-3, LY02-3, LY03-3, LY04-3, TD01-3, Lg-3, LS-3, CL-3, BC-3, PS-3, AL-3, MG-3, BL-3, Bi-3, FP-3, LR-3, RE-3, HO-3, AH-3, PE-3, PP-3, Ak-3, and Bc-3

[0160] Activation of the strain: Streak the recombinant strain on LB plate medium and culture at 37°C for 24 hours until a single colony grows.

[0161] Primary seed culture: Pick a single colony and inoculate it into a 12 mL shake tube (5 mL LB medium), add the corresponding antibiotics, and culture the culture in a shaker at 37°C and 220 rpm for 12 h.

[0162] (3) Fermentation culture:

[0163] 1 mL of seed solution was inoculated at 5% in a 150 mL Erlenmeyer flask. The appropriate antibiotic, 20 g / L glucose, and 12 g / L sodium acetate were added and incubated on a shaker at 37°C and 220 rpm for 48 hours. After incubation, 3-HB content was determined. Two replicates were performed, each with three biological replicates, and the results were averaged.

[0164] The results are shown in Table 11.

[0165] Table 11 Recombinant bacteria 3HB production results

[0166] The results in Table 11 show that LY01-3, LY02-3, LY03-3, LY04-3, TD01-3, Lg-3, LS-3, CL-3, BC-3, PS-3, AL-3, MG-3, BL-3, Bi-3, FP-3, LR-3, RE-3, HO-3, AH-3, PE-3, PP-3, Ak-3 and Bc-3 can all successfully produce 3-HB. The results of this example show that the method of the present application is also applicable to recombinant Halomonas LY01-3, LY02-3, LY03-3, LY04-3, TD01-3, recombinant probiotic Lactobacillus gasseri ATCC 33323 (Lg), recombinant bacteria Halonoas campaniensis LS21, Clostridium ljungdahlii ATCC 55383, Burkholderia cepacia ATCC 17795, Pseudomonas sp, Alcaligenes latus, E. coli MG1655, E. coli BL21, Bifidobacterium, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, Ralstonia eutropha, Halomonas sp.OITC1261, Aeromonas hydrophila, Pseudomonas entomophila, Pseudomonas putida KT2440s, Akkermansia muciniphila and recombinant probiotic Bacillus coagulans DSM1 produced 3-hydroxybutyrate.

[0167] In summary, the present invention proposes a method for constructing and applying two metabolic pathways for producing 3-HB using glucose and sodium acetate. Using synthetic biology techniques, a new metabolic pathway for synthesizing 3-HB using glucose as a substrate was introduced, and a strain with a combined metabolic pathway was constructed. This reduces the impact of the intermediate metabolite acetic acid on bacterial 3-HB production, and further optimizes the metabolic pathway to achieve high 3-HB yields. This method demonstrates strong application potential in the fields of chemical materials and medical treatment.

[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a strain for producing 3-hydroxybutyric acid, characterized in that: The steps include: S1: Amplify the sequences of phaA, phaB, and tesB genes in vitro, connect the gene sequences into a single fragment, and introduce the single fragment into the plasmid; S2: Introduce the plasmid described in S1 into the strain.

2. The construction method according to claim 1, characterized in that The strains described in step S2 are Escherichia coli Nissle 1917, Escherichia coli JM109, Halomonas sp.LY01, Halomonas sp.LY02, Halomonas sp.LY03, Halomonas sp.LY04, Lactobacillus gasseri ATCC 33323, Halonoas campaniensis LS21, Clostridium ljungdahlii ATCC 55383, Burkholderia cepacia ATCC 17795, Pseudomonas sp, Alcaligenes latus, E.coli MG1655, E.coli BL21, Bifidobacterium, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, Ralstonia eutropha, Halomonas sp.OITC1261, Aeromonas hydrophila, Pseudomonas entomophila, Pseudomonas putida KT2440, Akkermansia muciniphila, Bacillus coagulans DSM1, Halomonas sp.TD01.

3. The construction method according to claim 1, characterized in that The phaA described in step S1 is the phaA of species Halomonas sp.TD01 TD , species origin is phaA of Ralstonia eutropha RE , phaA of Alcaligenes latus AL , phaA of H.campaniensis LS21 LS , phaA of Pseudomonas entomophila PE , phaA from Pseudomonas putida KT2440 PP Any one of .

4. The construction method according to claim 1, characterized in that The phaB described in step S1 is the phaB of species Halomonas sp.TD01 TD , phaB of Ralstonia eutropha RE , phaB from Alcaligenes latus AL , phaB from H.campaniensis LS21 LS , phaB from Pseudomonas entomophila PE , phaB from Pseudomonas putida KT2440 PP Any one of .

5. The construction method according to claim 1, characterized in that The single fragment in step S1 is tesB-phaA TD -phaB TD tesB-phaA RE -phaB TD tesB-phaA RE -phaB RE tesB-phaB TD -phaA TD , phaA TD -tesB-phaB TD , phaB TD -tesB-phaA TD , phaA TD -phaB TD -tesB、phaB TD -phaA TD -tesB, tesB-pha AL -phaB AL tesB-pha LS -phaB LS ,tesB-pha PE -phaB PE tesB-pha PP -phaB PP Any one of .

6. A strain producing 3-hydroxybutyric acid, characterized in that Obtained by the construction method according to any one of claims 1 to 5.

7. A method for producing 3-hydroxybutyric acid using the strain according to claim 6, characterized in that: The method comprises the following steps: activating the strain, carrying out shake flask seed culture and then carrying out fermentation culture in a fermentation medium.

8. The method according to claim 7, characterized in that The fermentation medium includes sodium salts of short-chain fatty acids, preferably any one of sodium acetate, sodium propionate, sodium butyrate, sodium valerate, sodium pyruvate, sodium lactate, sodium citrate, sodium malate, and sodium succinate.

9. The method according to claim 8, characterized in that The added amount of the sodium salt of the short-chain fatty acid is 3 to 12 g / L.

10. The method according to claim 7, characterized in that The fermentation temperature is 36-42° C., the fermentation pH is 6-11, and the fermentation time is 12-48 hours.

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