Halomonas sp. for producing pha using co2 electrochemical derivatives, and construction method and application thereof

Abstract

The application discloses Halomonas for producing PHA by using CO2 electrochemical derivatives, a construction method and application of the Halomonas, and the Halomonas expresses Rhs644 genes and Rhs647 genes from Halomonas sp.TD01. The application realizes production of poly-3-hydroxybutyrate by using CO2 electrochemical derivatives as a single carbon source in the Halomonas, without additional adding of antibiotics and inducers, greatly reduces production cost, and improves single batch fermentation income. Meanwhile, efficient utilization of a cheap carbon source is realized, and the process of low-carbon biological manufacturing can be promoted. The poly-3-hydroxybutyrate is produced by using the application, and the time period is short, the production process is simple, the environment is green and pollution-free, and the application is beneficial to industrialized large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Halomonas sp. for producing PHA by using CO2 electrochemical derivatives, and a construction method and application thereof. BACKGROUND

[0002] Carbon dioxide (CO2) is both a global challenge and an opportunity focus. The large amount of CO2 emitted by human activities threatens the balance of the earth's climate and may lead to a catastrophic rise in global temperature. However, CO2 also has the potential to replace fossil fuels as the main raw material for the production of carbon-based value-added chemicals (such as fuels, plastics, solvents, feed and food). But from the current technical means, it still faces great challenges to achieve efficient utilization of CO2. Plants and algae can naturally fix CO2 on a large scale through photosynthesis, but this ability is limited by various factors, including competition with agricultural resources affecting food security, land use endangering biodiversity, difficulty in processing lignocellulosic biomass, and low efficiency of sunlight utilization by phototrophic organisms. In addition, CO2 can also be fixed by pure chemical means, such as synthesis gas for the production of complex hydrocarbons. However, these processes rely on extreme conditions, have limited operational flexibility, a narrow range of products and low selectivity. Currently, some researches have tried to convert CO2 into formic acid, acetic acid and other products by electrochemical method, and then use these electrochemical products as fermentation carbon sources. This method provides a new way for the efficient utilization of CO2.

[0003] The products of CO2 electrochemical reduction usually include formic acid (HCOOH), methanol (CH3OH), formaldehyde (HCHO), methane (CH4), ethylene (CH2CH2), ethanol (CH3CH2OH), acetic acid (CH3COO - ) and the like. Research teams have tried to combine CO2 electrolysis with microbial fermentation to effectively produce high-value multi-carbon products such as poly-3-hydroxybutyrate (PHB) from gaseous CO2. However, in traditional industrial production, commonly used microbial chassis strains such as Escherichia coli, Alcaligenes odorans and Pseudomonas can only grow under neutral pH, sterile and low osmotic pressure conditions. This makes it difficult for them to adapt to the high-salt environment of CO2 electrochemical derivatives, and they are easily contaminated, requiring a large amount of energy to maintain a sterile environment. Halomonas sp. can naturally grow rapidly under high-salt, high-alkaline and non-sterile conditions, and has a PHA synthesis pathway in the body. This strain can directly utilize CO2 electrochemical reduction products. However, wild-type Halomonas sp. does not grow well in high-concentration CO2 electrochemical derivatives and the product production effect is poor. Therefore, it is of great significance to construct a recombinant Halomonas sp. strain that can tolerate CO2 electrochemical derivatives (mainly acetic acid) for industrial large-scale utilization of CO2 electrochemical derivatives. SUMMARY

[0004] In view of the defects in the prior art, the application provides a Halomonas sp. for producing polyhydroxyalkanoate (PHA) by using CO2 electrochemical derivatives, and a construction method and application thereof.

[0005] The application provides a Halomonas sp. for producing polyhydroxyalkanoate by using CO2 electrochemical derivatives, wherein the Halomonas sp. expresses Rhs644 and Rhs647 genes from Halomonas sp. TD01, and the polyhydroxyalkanoate is preferably poly-3-hydroxybutyrate (PHB). The nucleotide sequence of the Rhs644 gene is shown as SEQ ID No. 31, and the nucleotide sequence of the Rhs647 gene is shown as SEQ ID No. 32.

[0006] Further, the Halomonas sp. further expresses a moy gene. The nucleotide sequence of the moy gene is shown as SEQ ID No. 33.

[0007] Further, the Halomonas sp. further expresses an aceE1 gene. The nucleotide sequence of the aceE1 gene is shown as SEQ ID No. 34.

[0008] Further, the Halomonas sp. includes Halomonas sp., preferably Halomonas sp. LY01, Halomonas sp. LY02, Halomonas sp. LY03, Halomonas sp. LY04 or Halomonas sp. LY08.

[0009] The Halomonas sp. LY01, Halomonas sp. LY02, Halomonas sp. LY03, Halomonas sp. LY04 and Halomonas sp. LY08 used in the application are all preserved in the Guangdong Microbial Culture Collection Center (GDMCC, address: 5th Floor, Building 59, Xianlie Middle Road, Guangzhou, Guangdong Microbial Institute, postcode: 510070), and are classified and named as Halomonas sp.

[0010] Halomonas sp. LY01 was deposited on July 19, 2022, and the deposit number is GDMCC NO: 62635; Halomonas sp. LY02 was deposited on April 23, 2023, and the deposit number is GDMCC NO: 63381; Halomonas sp. LY03 was deposited on April 23, 2023, and the deposit number is GDMCC NO: 63382; Halomonas sp. LY04 was deposited on April 23, 2023, and the deposit number is GDMCC NO: 63383; Halomonas sp. LY08 was deposited on May 21, 2024, and the deposit number is GDMCC NO: 64665.

[0011] Further, the expression of the Rhs644 gene and the Rhs647 gene uses a pRE112 vector.

[0012] Further, the vector comprises a promoter, and the promoter is porin226.

[0013] Further, the CO2 electrochemical derivative is mainly acetic acid or a sodium salt thereof.

[0014] The application also provides the use of the Halomonas sp. in any one of the following:

[0015] (i) in the metabolism of CO2 electrochemical derivatives;

[0016] (ii) in the production of polyhydroxyalkanoates.

[0017] The application also provides a construction method of Halomonas sp. grown by CO2 electrochemical derivatives, comprising the following steps: introducing any one of the following into the Halomonas sp.:

[0018] (1) Rhs644-Rhs647 nucleotide sequence;

[0019] (2) Rhs644-Rhs647 nucleotide sequence and moy nucleotide sequence;

[0020] (3) Rhs644-Rhs647 nucleotide sequence, moy nucleotide sequence and aceE1 nucleotide sequence;

[0021] The Rhs644-Rhs647 nucleotide sequence is shown in SEQ ID No. 35; the moy nucleotide sequence is shown in SEQ ID No. 33; and the aceE1 nucleotide sequence is shown in SEQ ID No. 34.

[0022] The application also provides a method for producing polyhydroxyalkanoate by fermentation of the salt bacterium, comprising the following steps: activating the salt bacterium, carrying out shake flask seed culture, and then carrying out fermentation culture in a fermentation medium with CO2 electrochemical derivatives as the sole carbon source.

[0023] Further, the fermentation temperature is 30-40°C.

[0024] Further, the fermentation pH is 7.0-11.0.

[0025] Further, the fermentation time is 28-52h.

[0026] Compared with the prior art, the application has the following technical effects:

[0027] 1. The application realizes production of poly-3-hydroxybutyrate by using CO2 electrochemical derivatives as the sole carbon source in the salt bacterium, without additional addition of antibiotics and inducers, greatly reduces the production cost, and improves the single batch fermentation income. Meanwhile, the application realizes efficient utilization of cheap carbon source, and can promote the process of low-carbon biological manufacturing.

[0028] 2. The application has a short production cycle of poly-3-hydroxybutyrate, a simple production process, and green and non-polluted environment, and is beneficial to industrialized large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation to the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0030] Figure 1 pSEVA321 constructed for the embodiment 1 of the application Mmp1-Rhs644-647 Plasmid map

[0031] Figure 2 Electrophoretogram of the target fragment for the embodiment 1 of the application

[0032] Figure 3 pSEVA321 constructed for the embodiment 3 of the application porin226-Rhs644-Rhs647 Plasmid map

[0033] Figure 4 pRE112 constructed for the embodiment 3 of the application porin226-Rhs644-Rhs647 Plasmid map

[0034] Figure 5 Electrophoretogram of the target fragment for the embodiment 3 of the application

[0035] Figure 6 pSEVA321 constructed for the embodiment 4 of the present application porin58-moy Plasmid map;

[0036] Figure 7 pRE112 constructed for the embodiment 4 of the present application G49-porin226-moy Plasmid map;

[0037] Figure 8 Electrophoretogram of the target fragment for the embodiment 4 of the present application

[0038] Figure 9 pSEVA321 constructed for the embodiment 4 of the present application porin141-aceE1 Plasmid map;

[0039] Figure 10 pRE112 constructed for the embodiment 5 of the present application G4-porin141-aceE1 Plasmid map;

[0040] Figure 11 Electrophoretogram of the target fragment for the embodiment 5 of the present application DETAILED DESCRIPTION

[0041] In order to make the personnel in the art better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the personnel in the art without creative labor should belong to the protection scope of the present application.

[0042] The wild-type Halomonas sp. grows and product production effect is poor in high-concentration carbon dioxide electrochemical derivatives, in order to realize the efficient use of cheap carbon source, the T6SS effector protein which plays an important role in the process of bacteria contact-dependent growth inhibition is overexpressed in the Halomonas sp., the stress resistance of the Halomonas sp. is enhanced, and then the acetic acid salt metabolic capacity of the Halomonas sp. is improved. By introducing the gene (Pyruvate dehydrogenase, aceE1) for acetic acid salt conversion, the conversion of acetic acid salt to acetyl coenzyme A is strengthened, the conversion of short-chain fatty acid salt to growth intermediates is promoted by expressing serine protease (moy), the acetic acid salt metabolism is further improved, and the PHB yield is improved.

[0043] The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified.

[0044] Halomonas sp. TD01 was obtained from Tsinghua University.

[0045] Example 1 Overexpression of T6SS effector proteins (RHs)

[0046] 1. Construction of Rhs644, Rhs647 gene expression plasmid

[0047] Plasmid pSEVA321 Mmp1 -Backbone as a template to amplify the backbone pSEVA321 Mmp1 Rhs644 and Rhs647 gene fragments were amplified from Halomonas sp. TD01 as a template, respectively, to construct pSEVA321 Mmp1-Rhs644-647 plasmid. The plasmid information is shown in Figure 1

[0048] (1) The target genes Rhs644 and Rhs647 and pSEVA321 Mmp1 The primer sequences (5'-3') of the backbone are as follows:

[0049] Rhs644-F: SEQ ID NO. 1;

[0050] Rhs644-R: SEQ ID NO. 2;

[0051] Rhs647-F: SEQ ID NO. 3;

[0052] Rhs647-R: SEQ ID NO. 4;

[0053] pSEVA321 Mmp1 -F: SEQ ID NO. 5;

[0054] pSEVA321 Mmp1 -R: SEQ ID NO. 6.

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

[0056] Table 1 Amplification system table

[0057]

[0058] Table 2 Amplification procedure table

[0059]

[0060] ​After the PCR reaction is completed, prepare the corresponding concentration of agarose gel, and perform electrophoresis to observe the size of the DNA band. Place the gel under the ultraviolet lamp, quickly cut the gel of the target DNA fragment, and cut off as much excess gel as possible.

[0061] (2) Gibson Assembly method connection

[0062] The recovered DNA is detected for its concentration, and the addition ratio of DNA is calculated according to the length and concentration of the target fragment and the skeleton, and Gibson mixed enzyme is used for connection. The Gibson Assembly connection system and procedure are shown in Tables 3 and 4.

[0063] Table 3 Gibson Assembly connection system table

[0064]

[0065] Table 4 Gibson Assembly connection procedure

[0066]

[0067] (3) S17-1 E. coli transformation

[0068] Step 1: Take the prepared S17-1 E. coli competent cells out of -80°C, thaw on ice, and after 5 min, melt the bacterial block;

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

[0070] Step 3: Ice bath for 30 min, 42°C water bath heat shock for 2 min, then immediately place on ice for 2 min. Note: Shaking will reduce the transformation efficiency;

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

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

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

[0074] (4) Single colony positive verification

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

[0076] (5) The single colonies with correct sequences were selected for expansion. After 12-16 h, the recombinant bacteria were mated with Halomonas sp. LY01 on 20 LB plates. After 8 h, a small amount of the mated bacteria were inoculated on 60 LB plates with corresponding resistance. After 36-48 h, the single colonies were verified again.

[0077] (6) Product identification

[0078] The size of the target product was verified by PCR. The results showed that the Rhs644 and Rhs647 gene sequences were successfully introduced into the Halomonas sp. LY01 strain. As shown in FIG. 1, the target fragment was 7400 bp, which was consistent with the expected result. Figure 2

[0079] Example 2 Test of the growth ability of the recombinant bacteria in a shake flask

[0080] Halomonas is an excellent strain for producing PHB (poly-3-hydroxybutyrate). The utilization ability of the strain for carbon source can be reflected to some extent by determining the cell dry weight and PHB yield of the strain.

[0081] The recombinant strain of Example 1 was used for fermentation culture.

[0082] (1) Preparation of seed liquid

[0083] ① Activation of bacteria

[0084] The recombinant strain was streaked on a 60 LB plate and activated at 37°C for 24 h until single colonies grew.

[0085] ② First-stage seed culture:

[0086] A single colony was picked and inoculated in a shake flask containing 5 mL of seed culture medium (60 LB+Cm), and incubated at 37°C, 220 rpm for 12 h.

[0087] ③ Second-stage seed culture:

[0088] The first-stage bacteria were inoculated in a 150 mL conical flask containing 20 mL of seed culture medium (60 LB+Cm) at an inoculation amount of 1%, and incubated at 37°C, 220 rpm for 12 h.

[0089] (2) Shake flask fermentation production

[0090] Medium preparation: 50 MM medium system:

[0091] ​Base medium: NaCl 50 g / L, yeast extract 1 g / L;

[0092] Component I: MgSO4 20 g / L, CO(NH2)2 30 g / L;

[0093] Component II: KH2PO4 175 g / L;

[0094] Component III: 5 g / L Fe(III)-NH4-Citrate, 2 g / L CaCl2 2H2O and 41.7 mL of 12 mol / L HCl were mixed thoroughly and made up to 1 L;

[0095] Component IV: ZnSO4 7H2O 0.1 g / L, MnCl2 4H2O 0.03 g / L, H3BO3 0.3 g / L, CoCl2 6H2O 0.2 g / L, CuSO4 5H2O 0.01 g / L, NiCl2 6H2O 0.02 g / L and NaMoO4 2H2O 0.03 g / L;

[0096] Component III & IV: 100 mL of component III and 10 mL of component IV were mixed with 90 mL of deionized water, and finally the pH was adjusted to 4.5-5.5 using 5 M NaOH;

[0097] Carbon source (g / L): sodium acetate 500 g / L.

[0098] Fermentation medium: 18 mL of base medium, 0.4 mL of components I, II, III & IV, 1.4 mL of carbon source, and finally the pH was adjusted to 8.5-9.0 using sodium hydroxide.

[0099] (3) Fermentation parameter determination:

[0100] (a) OD 600 determination:

[0101] 1 mL of the fermented bacterial solution was centrifuged at 12000 r / min for 10 min, the supernatant was discarded, and the same volume of deionized water was added to resuspend the bacterial cells. The absorbance at a wavelength of 600 nm was detected using a spectrophotometer (if necessary, the bacterial solution was diluted to ensure that the reading of the spectrophotometer at a wavelength of 600 nm was within the range of 0.3-0.8).

[0102] (b) Cell dry weight (DCM) determination:

[0103] Take 15 mL of fermented bacteria, centrifuge at 9000 r / min for 5 min, discard the supernatant, add an equal volume of deionized water, resuspend the bacteria, centrifuge again at 9000 r / min for 5 min, discard the supernatant and store in a -80°C refrigerator for 30 min, then freeze dry in a freeze dryer for 12 h. Weigh the weight of the freeze-dried bacteria on an analytical balance to obtain the dry weight.

[0104] (c) PHB determination:

[0105] Take 30-40 mg of bacteria and place it in an esterification tube, add 2 mL of esterification liquid (485 mL of methanol + 15 mL of concentrated sulfuric acid) and 2 mL of chloroform, and esterify at 100°C for 4 h. After completion, take 1 mL of deionized water and place it in the esterification tube, shake at 1500 rpm for 3 min, then let it stand for 40 min. Take 1 mL of the lower chloroform layer for GC analysis.

[0106] (4) Fermentation results:

[0107] Halomonas sp. LY01 and recombinant LY01 were tested for growth in shake flasks under different IPTG induction concentrations (0 mg / L, 5 mg / L, 10 mg / L):

[0108] Table 5 Fermentation production results of recombinant bacteria

[0109]

[0110] The results are shown in Table 5. Under an IPTG induction concentration of 5 mg / L, the recombinant LY01 strain had an 8.5% increase in cell dry weight and a 6.3% increase in PHB production compared to Halomonas sp. LY01.

[0111] Example 3 Constitutive promoter replacement and gene integration

[0112] 1. Construction of promoter replacement plasmid

[0113] The Rhs644-Rhs647 sequence was amplified using the plasmid pSEVA321 Mmp1-Rhs644-647 as a template. The backbone pSEVA321 porin58 -GFP was amplified using the plasmid pSEVA321 porin58 as a template. The plasmid pSEVA321 porin226-Rhs644-Rhs647 was constructed according to the method of Example 1. The plasmid information is shown in Figure 3 .

[0114] The primer sequences (5'-3') for amplifying the target fragment and the pSEVA321 porin226 backbone are as follows:

[0115] Porin226-Rhs-644-647-F: SEQ ID NO. 7;

[0116] Porin226-Rhs-644-647-R: SEQ ID NO. 8;

[0117] pSEVA321 porin226 -F: SEQ ID NO. 9;

[0118] pSEVA321 porin226 -R: SEQ ID NO. 10.

[0119] 2. Construction of insertion plasmid

[0120] Refer to the plasmid construction method of Example 1, using plasmid pSEVA321 porin226-Rhs644-Rhs647 as the template to amplify porin226-Rhs644-Rhs647, and using plasmid pRE112-G49-Backbone as the template to amplify the backbone pRE112, the plasmid pRE112 porin226-Rhs644-Rhs647 was constructed. Figure 4

[0121] The primer sequences (5'-3') for amplifying the porin226-Rhs644-Rhs647 gene sequence and the backbone pRE112 by PCR are as follows:

[0122] porin226-Rhs644-Rhs647-F: SEQ ID NO. 11;

[0123] porin226-Rhs644-Rhs647-R: SEQ ID NO. 12;

[0124] pRE112-G49-F: SEQ ID NO. 13;

[0125] pRE112-G49-R: SEQ ID NO. 14.

[0126] 3. Gene integration

[0127] (1) The integration plasmid constructed above was expanded, and after 12-16 h, it was conjugated with Halomonas sp. LY01 in a 20 LB plate, and after 8 h, a small amount of conjugated bacterial body was picked and plated on a 60 LB plate with corresponding resistance. After 36-48 h, the single colony was verified again.

[0128] (2) After the single colony was grown on the plate, it could be verified for resistance.

[0129] ​(3) The colonies with successful deantibiotic were selected for double verification (universal F1 / R and specific F2 / R), and the bands were verified by PCR cloning. The results are shown in Figure 5 , the length of the amplified fragment with universal primer was 7600 bp, and there was no band with specific primer. The PCR reaction liquid with successful double verification was selected for sequencing, and the strain was named LY01-A1 after successful sequencing.

[0130] Example 4 Overexpression of Serine Protease (moy)

[0131] 1. Referring to the plasmid construction steps of Example 1, the Halomonas sp. TD01 was used as a template to amplify the fragment moy, and the sequence of the target gene was obtained. The plasmid pSEVA321 porin58 -GFP was used as a template to amplify the backbone pSEVA321 porin226 , and the plasmid pSEVA321 porin58-moy was constructed by referring to the method of Example 1. The plasmid information is shown in Figure 6 .

[0132] The primer sequences (5'-3') for amplifying the moy gene sequence and the backbone pRE112 are as follows:

[0133] moy-F: SEQ ID NO. 15;

[0134] moy-R: SEQ ID NO. 16;

[0135] pSEVA321 porin58 -F: SEQ ID NO. 17;

[0136] pSEVA321 porin58 -R: SEQ ID NO. 18.

[0137] 2. Construction of Insertion Plasmid

[0138] Referring to the plasmid construction method of Example 1, the porin58-moy was amplified with the plasmid pSEVA321 porin58-moy as a template, and the backbone pRE112 was amplified with the plasmid pRE112-G7-Backbone as a template, and the plasmid pRE112 G49-porin226-moy was constructed as shown in Figure 7 .

[0139] The primer sequences (5'-3') for amplifying the porin226-moy gene sequence and the backbone pRE112 are as follows:

[0140] porin226-moy-F: SEQ ID NO. 19;

[0141] porin226-moy-R: SEQ ID NO. 20;

[0142] pRE112-G7-F: SEQ ID NO. 21;

[0143] pRE112-G7-R: SEQ ID NO. 22.

[0144] 3. Gene integration:

[0145] Gene integration was carried out with LY01-A1 as the chassis strain, following the procedure of Example 3. After PCR cloning verification, the results are shown in Figure 8 , the length of the amplified fragment is 5000 bp, there is no specific primer band, and the PCR reaction liquid selected by double verification is sent for testing. After successful sequencing, the strain is named LY01-A2.

[0146] Example 5 Overexpression of pyruvate dehydrogenase E1 (aceE1)

[0147] 1. Refer to the plasmid construction steps of Example 1, and use Halomonas sp. TD01 as the template to amplify the aceE1 target gene sequence. Use plasmid pSEVA321 porin141 -GFP as the template to amplify the backbone pSEVA321 porin141 , and the pSEVA321 porin141-aceE1 plasmid information is shown in Figure 9 .

[0148] The primer sequences (5'-3') of PCR amplification of aceE1 and pSEVA321 porin141 backbone are as follows:

[0149] aceE1-F: SEQ ID NO. 23;

[0150] aceE1-R: SEQ ID NO. 24;

[0151] pSEVA321 porin141 -F: SEQ ID NO. 25;

[0152] pSEVA321 porin141 -R: SEQ ID NO. 26.

[0153] 2. Construction of insertion plasmid

[0154] Refer to the plasmid construction method of Example 1, and use plasmid pSEVA321 porin141-aceE1Porin141-aceE1 was amplified using the template of pRE112-G4-Backbone, and the backbone pRE112 was amplified using the template of plasmid pRE112-G4-Backbone, to construct plasmid pRE112-G4 porin141-aceE1 As shown in Figure 10 .

[0155] The primer sequences for amplifying porin68-aceE1 gene sequence and the backbone pRE112 (5'-3') were as follows:

[0156] Porin141-aceE1-F: SEQ ID NO. 27;

[0157] Porin141-aceE1-R: SEQ ID NO. 28;

[0158] pRE112-F: SEQ ID NO. 29;

[0159] pRE112-R: SEQ ID NO. 30.

[0160] 3. Gene integration:

[0161] Gene integration was performed according to the procedure of Example 3 using LY01-A2 as the chassis. The band was verified to be correct by PCR cloning, and the results are shown in Figure 11 . The length of the amplified fragment was 4800 bp, and there was no specific primer band. The PCR reaction liquid that was successfully verified twice was sent for testing, and the strain was named LY01-A3 after successful sequencing.

[0162] Example 6: Fermentation production capacity test using recombinant bacteria LY01-A1-A3

[0163] Fermentation was performed according to the procedure of Example 2, and the fermentation results are shown in Table 6.

[0164] Table 6: Fermentation production results of three recombinant bacteria

[0165]

[0166] According to the fermentation results, LY01 and LY01-A1-3 both successfully grew with sodium acetate as the sole carbon source, and the maximum cell dry weight of LY01-A3 was 9.5 g / L, and the PHB content reached 82.5%, indicating that overexpression of several modules of exogenous genes on the chromosome level can effectively improve the ability of bacteria to tolerate sodium acetate.

[0167] Example 7: Fermentation growth capacity test using other Halomonas

[0168] The above genetic modification strategies are introduced into Halomonas sp. LY02, Halomonas sp. LY03, Halomonas sp. LY04 and Halomonas sp. LY08, respectively, and are named as TD01-A3, LY02-A3, LY03-A3, LY04-A3 and LY08-A3, respectively.

[0169] The constructed recombinant strains and the original strain are subjected to shake flask fermentation growth ability test, and the specific implementation method is the same as in Example 6.

[0170] Table 7 Fermentation results of four recombinant strains

[0171]

[0172] According to the fermentation results, the cell dry weight and PHB content of LY02-A, LY03-A, LY04-A and LY08-A strains are significantly increased compared with the original strain. It is shown that the overexpression of several modules of exogenous genes at the chromosome level can effectively improve the strain's ability to resist carbon dioxide derivatives. The results of this example show that the method of the present application is not only suitable for Halomonas sp. LY01, but also suitable for other Halomonas sp. The introduction of metabolic genes in other Halomonas sp. can achieve the ability to strengthen the metabolism of sodium acetate.

[0173] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Halomonas bacterium that produces polyhydroxy fatty acid esters using CO2 electrochemical derivatives, characterized in that, The *Halomonas* strain overexpresses the Rhs644 and Rhs647 genes derived from *Halomonas* sp. TD01; the nucleotide sequence of the Rhs644 gene is shown in SEQ ID No. 31; the nucleotide sequence of the Rhs647 gene is shown in SEQ ID No.

32. The electrochemical derivative of CO2 is sodium acetate; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate; The halomonas are Halomonas sp.LY01, Halomonas sp.LY02, Halomonas sp.LY03, or Halomonas sp.LY04.

2. The Halomonas bacterium according to claim 1, characterized in that, The Halomonas strain also overexpresses the moy gene; The nucleotide sequence of the moy gene is shown in SEQ ID No.

33.

3. The Halomonas bacterium according to claim 2, characterized in that, The Halomonas strain also overexpresses the aceE1 gene; The nucleotide sequence of the aceE1 gene is shown in SEQ ID No.

34.

4. The Halomonas bacterium according to claim 1, characterized in that, The overexpression of the Rhs644 and Rhs647 genes was performed using the pRE112 vector.

5. The Halomonas bacterium according to claim 4, characterized in that, The pRE112 vector contains a promoter, which is porin226.

6. The use of *Haloxylon ammodendron* as described in any one of claims 1 to 5 in the production of polyhydroxy fatty acid esters; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate.

7. A method for constructing *Halomonas* according to any one of claims 1 to 5, characterized in that, The steps include: introducing any of the following into *Haloxylon ammodendron*: (1) Rhs644-Rhs647 nucleotide sequence; (2) Rhs644-Rhs647 nucleotide sequence and moy nucleotide sequence; (3) Rhs644-Rhs647 nucleotide sequence, moy nucleotide sequence and aceE1 nucleotide sequence; The nucleotide sequences of Rhs644-Rhs647 are shown in SEQ ID No. 35; the nucleotide sequence of moy is shown in SEQ ID No. 33; and the nucleotide sequence of aceE1 is shown in SEQ ID No.

34. The electrochemical derivative of CO2 is sodium acetate; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate.

8. A method for producing polyhydroxy fatty acid esters by fermentation using *Halomycium spp.* according to any one of claims 1 to 5, characterized in that, The process includes the following steps: activating the Halomonas bacteria, performing shake-flask seed culture, and then fermenting the bacteria in a fermentation medium with CO2 electrochemical derivatives as the sole carbon source. The electrochemical derivative of CO2 is sodium acetate; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate.

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