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

Abstract

The present invention discloses a strain for producing 3-hydroxybutyric acid, a construction method and an application thereof. The construction method comprises the following steps: S1: amplifying the sequences of phaA, phaB and tesB genes in vitro, connecting the sequences of the genes into a single fragment, and introducing the single fragment into a plasmid; S2: introducing the plasmid described in S1 into the strain. The present invention utilizes engineered bacteria to ferment and produce 3-HB, and further optimizes the combined metabolic pathway and adds sodium acetate substrate during fermentation, thereby reducing the effect of the intermediate metabolite acetic acid on the bacterial 3-HB yield, and further increasing the 3-HB yield. The production process is simple and the production efficiency is high, and it is suitable for industrial continuous production.

Description

Technical Field

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

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

[0003] After 3-HB monomer is polymerized to form PHB, it can be used in many applications, especially in environmental protection. With the improvement of environmental protection requirements, biodegradable materials in the packaging field have received more and more attention and are gradually replacing traditional non-degradable plastics. The physical and mechanical properties of polyhydroxybutyrate are similar to those of polypropylene, and it is biodegradable. Its degradation products in nature are carbon dioxide and water, which are pollution-free to the environment, so it has broad application prospects in the packaging field. Therefore, researching, developing and utilizing methods for efficient production of 3-HB is of great significance for promoting the development of bio-based chemicals, reducing dependence on limited resources and reducing environmental impact. In recent years, methods for synthesizing 3-HB using biological methods have received widespread attention. However, the current production of 3-HB faces a major problem: the output of the intermediate product acetic acid is large, resulting in a significant reduction in the amount flowing to 3-HB, thereby reducing production efficiency and becoming the biggest challenge restricting 3-HB production. Therefore, there is an urgent need for a method that can increase the output of 3-HB to improve the efficiency of industrial production and reduce production costs. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a strain for producing 3-hydroxybutyric acid and a construction method and 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 sequences of the genes 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] Furthermore, the strain in step S2 is any one of Escherichia coli Nissle 1917, Escherichiacoli JM109, Halomonas sp.LY03, Lactobacillus gasseri ATCC 33323 and Bacilluscoagulans DSM1.

[0009] The Halomonas sp.LY03 strain used in this application has been deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postal Code 510070) on April 23, 2023. The deposit number is GDMCC NO: 63382. The strain name is Halomonas sp.LY03, and the classification name is Halomonas sp.

[0010] Furthermore, the phaA in step S1 is phaA of species origin Halomonas sp.TD01 TD , phaA from Ralstonia eutropha RE 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;

[0011] Furthermore, the phaB in step S1 is phaB from Halomonas sp.TD01 TD , phaB from Ralstonia eutropha RE Any one of the following. TD The nucleotide sequence of phaB is shown in SEQ ID No.62. RE The nucleotide sequence is shown in SEQ ID No.65;

[0012] Furthermore, 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, preferably phaA TD -tesB-phaB TD .

[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 -The nucleotide sequence of tesB 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, preferably any one of sodium acetate, sodium propionate, sodium butyrate and sodium valerate.

[0017] Furthermore, the added amount of the short-chain fatty acid is 3 to 12 g / L.

[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 effect 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 drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The present invention is a pathway diagram for synthesizing 3-HB using glucose;

[0025] Figure 2 pSEVA321 used in Example 1 of the present invention tesB-phaA-phaB Plasmid map;

[0026] Figure 3 This is the electrophoresis diagram of the tesB-phaA-phaB target fragment in Example 1 of the present invention;

[0027] Figure 4 pSEVA321 used in Example 2 of the present invention tesB-phaARE-phaBTD Plasmid map;

[0028] Figure 5 pSEVA321 used in Example 2 of the present invention tesB-phaARE-phaBRE Plasmid map;

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

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

[0031] Figure 8 pSEVA321 used in Example 3 of the present invention tesB-phaARE-phaBTD Plasmid map; Figure a is 2-pSEVA321 tesB-phaB-phaA Plasmid map, Figure b is 3-pSEVA321 phaA-tesB-phaB Plasmid map, Figure c is 4-pSEVA321 phaB-tesB-phaA Plasmid map, Figure d is 5-pSEVA321 phaA-phaB-tesB Plasmid map, Figure e is 6-pSEVA321 phaB-phaA-tesB Plasmid map;

[0032] Fig. 9 This is the electrophoresis diagram of the tesB-phaB-phaA target fragment in Example 3 of the present invention;

[0033] Fig.10 This is the electrophoresis diagram of the phaA-tesB-phaB target fragment in Example 3 of the present invention;

[0034] Fig.11 This is the electrophoresis diagram of the phaB-tesB-phaA target fragment in Example 3 of the present invention;

[0035] Fig.12 This is the electrophoresis diagram of the phaA-phaB-tesB target fragment in Example 3 of the present invention;

[0036] Fig.13 This is the electrophoresis diagram of the phaB-phaA-tesB target fragment in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work 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, and promotes the metabolic pathway to flow to 3-HB by overexpressing three genes, phaA, phaB, and tesB, from different sources.

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

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

[0041] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial sources unless otherwise specified. Halomonas sp. TD01 was obtained from Tsinghua University.

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

[0043] 1. Expression of tesB, phaA, and phaB genes

[0044] (1) Plasmid construction

[0045] PCR amplified tesB from E. coli MG1655, phaA, phaB from Halomonas sp. TD01 and the plasmid pSEVA321 backbone. Under the action of Gibson ligase, phaA, phaB, tesB fragments and the pSEVA321 backbone were recombined to form a new plasmid named pSEVA321. tesB-phaA-phaB . The pSEVA321 tesB-phaA-phaB tesB-phaA-phaB was obtained as a template, and part of the product was sent to a biological company for sequencing. The plasmid information is as follows Figure 2 shown.

[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]

[0058] Table 2 Amplification program

[0059]

[0060] 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, quickly cut out the gel of the target DNA fragment, and cut off as much excess gel as possible.

[0061] (2) Gibson Assembly method connection

[0062] The concentration of the recovered DNA was tested, and the proportion of DNA added was calculated according to the length and concentration of the target fragment and the pSEVA321 backbone, and the Gibson mixed enzyme was used for ligation. The Gibson Assembly ligation system and procedure are shown in Tables 3 and 4:

[0063] Table 3 Gibson Assembly connection system

[0064]

[0065] Table 4 Gibson Assembly connection procedure

[0066]

[0067] (3) Nissle 1917 Escherichia coli transformation

[0068] Step 1: Take out the prepared Nissle 1917 E. coli competent cells from -80℃, thaw on ice, and wait for the bacteria to melt after 5 minutes;

[0069] Step 2: Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not oscillate to mix). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0070] Step 3: Place on ice for 30 minutes, heat shock at 42°C for 2 minutes, and immediately place on ice for 2 minutes. Note: shaking will reduce transformation efficiency.

[0071] Step 4: Add 400 μL LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a 37°C shaker at 200 rpm for 60 min.

[0072] Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of the supernatant, keep 100 μL of the suspension, gently blow to resuspend the bacteria, and spread it on LB medium containing the corresponding antibiotics;

[0073] Step 6: Invert the culture medium into a 37°C incubator and culture for 12 to 16 hours.

[0074] (4) Monoclonal colony positive verification

[0075] Colonies were picked out on the corresponding resistance LB plates and verified by colony PCR. PCR products with the correct band size were sent to a biological company for sequencing.

[0076] (5) Select a single colony with the correct sequence for expansion. After 12 to 16 hours, streak it on a resistant LB plate. After a single clone grows out, verify the single clone colony again.

[0077] (6) Product identification

[0078] The colony verification results showed that the tesB-phaA-phaB gene sequence was successfully transferred into the Escherichia coli Nissle 1917 strain. Figure 3 As shown, the target fragment was 3667 bp, which was consistent with the expected result, and the strain was named Nissle-1.

[0079] Example 2 Expression of phaA and phaB genes from different sources

[0080] Plasmid construction: Plasmid pSEVA321 was constructed by PCR amplification of tesB from E. coli MG1655, phaB from Halomonas sp. TD01, phaA and phaB from Ralstonia eutropha, and the pSEVA321-porin58 backbone. tesB-phaA-phaBTD Specific steps refer to Example 1; plasmid pSEVA321 containing phaA and phaB genes from Ralstonia eutropha tesB-phaARE-phaBRE , plasmid information such as Figure 4 As shown; construct plasmid pSEVA321 containing phaA from Ralstonia eutropha and phaB from Halomonas bluephagenesis tesB -phaARE-phaBTD, template is pSEVA321 tesB-phaARE-phaBRE , plasmid information such as Figure 5 As shown. tesB-phaARE-phaBTD tesB-phaA was obtained as a template RE -phaB TD pSEVA321 tesB-phaARE-phaBRE tesB-phaA was obtained as a template RE -phaB RE Sequence, and take part of the product to send to a biological company for sequencing.

[0081] The primer sequences (5'-3') used for PCR amplification are as follows:

[0082] pSEVA321-porin58-F: see SEQ ID No.9;

[0083] pSEVA321-porin58-R: see SEQ ID No.10;

[0084] tesB-F: see SEQ ID No.11;

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

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

[0087] phaAB RE -R: see SEQ ID No.14;

[0088] phaA RE -F: see SEQ ID No.15;

[0089] phaARE -R: see SEQ ID No.16;

[0090] phaB TD -F: see SEQ ID No.17;

[0091] phaB TD -R: see SEQ ID No.18.

[0092] pSEVA321 tesB-phaARE-phaBTD and pSEVA321 tesB-phaARE-phaBRE The results were verified by the size of the target product, and the results showed that tesB-phaA was successfully transferred into Escherichia coli Nissle 1917. RE -phaB TD Gene sequences, such as Figure 6 As shown; tesB-phaA was successfully transferred into Escherichia coli Nissle1917 RE -phaB RE Gene sequences, such as Figure 7 As shown, the two recombinant bacteria were named Nissle-ARE-BTD and Nissle-RE, respectively.

[0093] Example 3: Effect of phaA, phaB, tesB gene position

[0094] 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, phaA, phaB, and tesB fragments were connected in different orders and recombined with the pSEVA321 backbone to form a new plasmid named 2-pSEVA321. tesB-phaB-phaA , plasmid map Figure 8 a, 3-pSEVA321 phaA-tesB-phaB , plasmid map Figure 8 b, 4-pSEVA321 phaB-tesB-phaA , plasmid map Figure 8 c, 5-pSEVA321 phaA-phaB-tesB , plasmid map Figure 8 d, 6-pSEVA321 phaB-phaA-tesB , plasmid map Figure 8 As shown in e, part of the product was sent to a biological company for sequencing.

[0095] The primer sequences (5'-3') used for PCR amplification are as follows:

[0096] 2-pSEVA321-F: see SEQ ID No. 19; 2-pSEVA321-R: see SEQ ID No. 20;

[0097] 2-phaA-F: see SEQ ID No.21; 2-phaA-R: see SEQ ID No.22;

[0098] 2-phaB-F: see SEQ ID No. 23; 2-phaB-R: see SEQ ID No. 24;

[0099] 3-pSEVA321-F: see SEQ ID No.25; 3-pSEVA321-R: see SEQ ID No.26;

[0100] 3-phaA-F: see SEQ ID No. 27; 3-phaA-R: see SEQ ID No. 28;

[0101] 3-tesB-F: see SEQ ID No.29; 3-tesB-R: see SEQ ID No.30;

[0102] 4-pSEVA321-F: see SEQ ID No.31; 4-pSEVA321-R: see SEQ ID No.32;

[0103] 4-phaB-F: see SEQ ID No. 33;

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

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

[0106] 4-tesB-R: see SEQ ID No.36;

[0107] 4-phaA-F: see SEQ ID No.37;

[0108] 4-phaA-R: see SEQ ID No.38;

[0109] 5-pSEVA321-F: see SEQ ID No. 39;

[0110] 5-pSEVA321-R: see SEQ ID No.40;

[0111] 5-phaAB-F: see SEQ ID No.41;

[0112] 5-phaAB-R: see SEQ ID No.42;

[0113] 5-tesB-F: see SEQ ID No.43;

[0114] 5-tesB-R: see SEQ ID No.44;

[0115] 6-pSEVA321-F: see SEQ ID No.45;

[0116] 6-pSEVA321-R: see SEQ ID No.46;

[0117] 6-phaB-F: see SEQ ID No.47;

[0118] 6-phaB-R: see SEQ ID No.48;

[0119] 6-phaA-F: see SEQ ID No.49;

[0120] 6-phaA-R: see SEQ ID No.50;

[0121] 6-tesB-F: see SEQ ID No.51;

[0122] 6-tesB-R: see SEQ ID No.52.

[0123] The plasmid 2-pSEVA321 constructed above tesB-phaB-phaA , 3-pSEVA321 phaA-tesB-phaB , 4-pSEVA321 phaB-tesB-phaA , 5-pSEVA321 phaA-phaB-tesB and 6-pSEVA321 phaB-phaA-tesB , respectively transferred into Escherichia coli Nissle 1917.

[0124] The size of the target product was verified, and the results showed that the tesB-phaB-phaA gene sequence was successfully transferred into Escherichia coli Nissle 1917. Fig. 9 As shown; the phaA-tesB-phaB gene sequence was successfully introduced into Escherichia coli Nissle 1917, as shown Fig.10 As shown; the phaB-tesB-phaA gene sequence was successfully introduced into Escherichia coli Nissle 1917, as shown Fig.11As shown; the phaA-phaB-tesB gene sequence was successfully introduced into Escherichia coli Nissle 1917, as shown Fig.12 As shown; the phaB-phaA-tesB gene sequence was successfully introduced into Escherichia coli Nissle 1917, as shown Fig.13 As shown; the five recombinant bacteria were named Nissle-2, Nissle-3, Nissle-4, Nissle-5, and Nissle-6.

[0125] Example 4 Production of 3-HB using different modified bacteria

[0126] Referring to the operation of Example 1, the plasmid pSEVA321 constructed in Example 1 was tesB-phaA-phaB The mixture was introduced into Escherichia coli S17-1 and Escherichia coli JM109, and the Nissle-1, S17-1 and JM109 bacteria of Example 1 were used as fermentation base bacteria.

[0127] (1) Culture medium

[0128] LB plate culture 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.

[0129] Fermentation medium (LBG): sodium chloride 10 g / L, yeast powder 5 g / L, tryptone 10 g / L, glucose 10 g / L.

[0130] (2) Seed solution preparation

[0131] ① Bacteria activation:

[0132] The recombinant strain was streaked on LB plate medium and cultured at 37°C for 24 h until a single clone grew out.

[0133] ②First-level seed cultivation:

[0134] Pick a single colony and inoculate it into a 12 mL shaking 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.

[0135] (3) Fermentation culture:

[0136] 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.

[0137] (4) After the fermentation is completed, the cells are collected for testing:

[0138] (a)OD 600 Determination: Dilute 200 μL of fermented bacteria at appropriate multiples and measure the OD value in a spectrophotometer.

[0139] (b) Determination of 3-HB content: 200 μL of fermented bacteria was diluted by appropriate multiples, centrifuged at 12000 rpm under 4 degrees, and then filtered with a 0.22 μm water filter. After injecting it into the sample bottle, it was placed in the liquid phase to test the 3-HB content. The liquid phase detection used HPX-87h column, the mobile phase was 0.5 mM sulfuric acid, and the temperature was 60°C. The injection volume was 10 μL, and the external standard method was used for quantitative analysis of 3-HB, and the yield of 3-HB was calculated based on the peak area.

[0140] (c) Determination of acetic acid: 200 μL of fermented bacteria was diluted in appropriate multiples, centrifuged at 12000 rpm at 4 degrees, and then filtered with a 0.22 μm water filter. After injecting it into a sample bottle, it was placed in the liquid phase to test the acetic acid content. The liquid phase detection used an HPX-87h column, the mobile phase was 0.5 mM sulfuric acid, and the temperature was 60°C. The injection volume was 10 μL, and the acetic acid was quantitatively analyzed by the external standard method, and the acetic acid yield was calculated based on the peak area.

[0141] (5) The fermentation results are shown in Table 5.

[0142] Table 5 Fermentation effects of three modified bacteria

[0143]

[0144] The above results show that the 3-HB pathway was constructed using the Escherichia coli S17-1, JM109, and Nissle-1 strains. Among them, S17-1 cannot produce 3-HB, and JM109 can produce 3-HB, but the yield is not as high as Nissle-1. The 3-HB yield produced by Nissle-1 can reach up to 5.12 g / L, and the acetic acid content is only 1.92 g / L. This shows that the Escherichia coli Nissle 1917 strain is suitable as a chassis strain for synthesizing 3-HB.

[0145] Example 5 Comparison of the fermentation effects of phaA and phaB from different sources

[0146] Fermentation was performed using the Nissle-1 of Example 1, the Nissle-ARE-BTD and the Nissle-RE strains of Example 2. The specific culture medium, steps and parameter determination methods used in the fermentation were referred to Example 4.

[0147] The fermentation results are shown in Table 6.

[0148] Table 6 Fermentation effects of phaA and phaB from different sources

[0149]

[0150] The results showed that the Nissle-1 strain overexpressing phaA and phaB from Halomonas sp. TD01 had the best fermentation effect, and the production of 3-HB could reach 5.05 g / L.

[0151] Example 6 Production of 3-HB using modified bacteria with different gene connection sequences

[0152] Fermentation was performed using Nissle-1 of Example 1 and the five modified bacteria of Example 3. The specific culture medium, steps and parameter determination methods used in the fermentation were referred to Example 4.

[0153] The fermentation results are shown in Table 7.

[0154] Table 7 Gene position effect fermentation effect

[0155]

[0156] The above results show that the Nissle-3 strain has the best fermentation effect, and the production of 3-HB is as high as 5.83g / L, which means that inserting the three genes phaA, tesB, and phaB into the plasmid in the connection order of phaA-tesB-phaB and introducing them into the strain can further increase the production of 3-HB.

[0157] Example 7 Fermentation effect of adding sodium acetate to glucose

[0158] The Nissle-3 strain was used for fermentation. According to the steps of Example 4, except for the carbon source, the other components in the fermentation medium remained unchanged. The carbon source was 20 g / L glucose and different concentrations of sodium acetate (0, 3, 6, 9, 12 g / L), and the configuration and operation method were the same as those in Example 4.

[0159] The fermentation results are shown in Table 8.

[0160] Table 8 Fermentation effect of adding different concentrations of sodium acetate

[0161]

[0162] 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.

[0163] Example 8 Fermentation effect of adding other short-chain fatty acids to glucose

[0164] The Nissle-3 strain was used for fermentation. According to the steps of Example 4, except for the carbon source, the other components in the fermentation medium remained unchanged. The carbon source was 20 g / L glucose and 12 g / L other short-chain fatty acids, and the configuration and operation method were the same as those in Example 4. The fermentation results are shown in Table 8.

[0165] Table 9 Fermentation effect of adding other short-chain fatty acids

[0166]

[0167] The results showed that adding an additional 12 g / L of other short-chain fatty acids to the fermentation medium could promote the strain to utilize more glucose and thus increase the yield, with the yield reaching as high as 10.2 g / L.

[0168] Example 9 Production of 3-hydroxybutyric acid using recombinant bacteria LY03-3, Lg-3 and Bc-3

[0169] Recombinant Halomonas LY03, recombinant probiotic Lactobacillus gasseri ATCC 33323 and recombinant probiotic Bacillus coagulans DSM1 produced 3-hydroxybutyrate.

[0170] (1) Construction of recombinant bacteria

[0171] The plasmid 3-pSEVA321 constructed in Example 3 phaA-tesB-phaB The recombinant bacteria were respectively named LY03-3, Lg-3 and Bc-3 by conjugation transformation, in the same manner as in Example 1.

[0172] (2) Production of 3-hydroxybutyrate using recombinant LY03-3, Lg-3 and Bc-3

[0173] Activation of strains: Streak the recombinant strain on LB plate medium and culture at 37°C for 24 hours until a single clone grows.

[0174] Primary seed culture: Pick a single colony and inoculate it into a 12 mL shaking 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.

[0175] (3) Fermentation culture:

[0176] The seed solution was inoculated at 5% (1 mL) in a 150 mL conical flask, and the corresponding antibiotics, 20 g / L glucose, and 12 g / L sodium acetate were added. The flask was placed in a shaker at 37°C and 220 rpm for 48 hours. After the culture was completed, the 3-HB content was determined. Two repeated experiments were performed, and three biological parallel samples were set for each repeated experiment, and the results were averaged.

[0177] The results are shown in Table 10.

[0178] Table 10 Recombinant bacteria 3HB production results

[0179]

[0180] The results in Table 10 show that LY03-3, Lg-3 and Bc-3 can all successfully produce 3-HB, with yields of 10.2 g / L, 5.67 g / L and 6.42 g / L, respectively. The results of this example show that the method of the present application is also applicable to the production of 3-hydroxybutyric acid by recombinant Halomonas LY03, recombinant probiotic Lactobacillus gasseri ATCC 33323 and recombinant probiotic Bacillus coagulans DSM1.

[0181] In summary, the present invention proposes a strain for synthesizing 3-HB using glucose and a method for constructing two metabolic pathways for producing 3-HB using sodium acetate and their applications. Synthetic biology technology is used to introduce a new metabolic pathway for synthesizing 3-HB using glucose as a substrate, a combined metabolic pathway strain is constructed, the effect of the intermediate metabolite acetic acid on the bacterial 3-HB yield is reduced, and its metabolic pathway is further optimized to achieve the goal of high 3-HB production, thereby showing strong application potential in the fields of chemical materials and medical treatment.

[0182] 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 principle of the present invention should be included in the protection scope 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; The phaA is the phaA of Halomonas sp.TD01 TD ; The phaB is the phaB of Halomonas sp.TD01 TD ; The single fragment in step S1 is tesB-phaA TD -phaB TD tesB-phaB TD -phaA TD , phaA TD -tesB-phaB TD , phaB TD -tesB-phaA TD , phaA TD -phaB TD - Any one of tesB; tesB-phaA TD -phaB TD The nucleotide sequence of tesB-phaB is shown in SEQ ID No.53; 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; The strain in step S2 is any one of Escherichia coli Nissle 1917 and Escherichia coli JM109.

2. A strain producing 3-hydroxybutyric acid, characterized in that: Obtained by the construction method described in claim 1.

3. A method for producing 3-hydroxybutyric acid using the strain according to claim 2, characterized in that: The method comprises the following steps: activating the strain, carrying out shaking bottle seed culture, and then carrying out fermentation culture in a fermentation medium.

4. The method according to claim 3, characterized in that The fermentation medium comprises sodium salt, and the sodium salt is sodium acetate.

5. The method according to claim 4, characterized in that The added amount of the sodium salt is 3 to 12 g / L.

6. The method according to claim 3, 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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