Halomonas for producing PHA (polyhydroxyalkanoate) by using CO2 electrochemical derivative as well as construction method and application of halomonas

Abstract

The invention discloses halomonas for producing PHA (polyhydroxyalkanoate) by using a CO2 electrochemical derivative as well as a construction method and application of the halomonas. The halomonas expresses an Rhs644 gene and an Rhs647 gene from Halomonas sp.TD01. According to the method disclosed by the invention, the poly-3-hydroxybutyrate is produced by taking the CO2 electrochemical derivative as a single carbon source in the halomonas, and no additional antibiotics or inducers are needed, so that the production cost is greatly reduced, and the single-batch fermentation income is increased. Meanwhile, efficient utilization of a cheap carbon source is achieved, and the process of low-carbon biological manufacturing can be promoted. The time period for producing the poly-3-hydroxybutyrate is short, the production process is simple, the environment is green and pollution-free, and industrial large-scale production is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to utilizing CO 2 Halomonas bacteria producing PHA by electrochemical derivatives and their construction method and application. Background Art

[0002] Carbon dioxide (CO 2 ) is both a global challenge and a focus of opportunity. Large amounts of anthropogenic carbon dioxide emissions threaten the balance of the Earth's climate and could lead to a catastrophic rise in global temperatures. However, carbon dioxide also has the potential to replace fossil fuels and become the main raw material for the production of carbon-based value-added chemicals (such as fuels, plastics, solvents, feed and food). However, from the current technical means, achieving efficient use of carbon dioxide still faces huge challenges. Plants and algae naturally fix CO2 on a large scale through photosynthesis. 2 However, this capacity is limited by multiple factors, including competition with agricultural resources affecting food security, land use endangering biodiversity, difficulties in processing lignocellulosic biomass, and inefficient use of sunlight by phototrophic organisms. 2 It can also be fixed by purely chemical means, such as synthesis gas for the production of complex hydrocarbons. However, these processes rely on extreme conditions, have limited operating flexibility, a narrow product range and low selectivity. 2 The electrochemical products are then converted into formic acid, acetic acid, etc., and then used as fermentation carbon sources. This method provides a new idea for the efficient utilization of carbon dioxide.

[0003] The products of electrochemical reduction of carbon dioxide usually include formic acid (HCOOH), methanol (CH 3 OH), formaldehyde (HCHO), methane (CH 4 ), ethylene (CH 2 CH 2 ), ethanol (CH 3 CH 2 OH), acetate (CH 3 COO - ) etc. The research team has tried to convert CO 2 Electrolysis combined with microbial fermentation can effectively convert gaseous CO 2 Produce high value-added multi-carbon products such as poly-3-hydroxybutyrate (PHB). However, in traditional industrial production, commonly used microbial chassis strains such as Escherichia coli, Alcaligenes eutropha and Pseudomonas can usually only grow under neutral pH, sterile and low osmotic pressure conditions. This makes it difficult for them to adapt to CO 2The high-salt environment of electrochemical derivatives is easily contaminated and requires a lot of energy to maintain a sterile environment. Halomonas can naturally grow rapidly under high-salt, high-alkali and non-sterile conditions, and has a PHA synthesis pathway in the body. This strain can directly use CO 2 However, wild-type Halomonas do not grow well in high-concentration carbon dioxide electrochemical derivatives and do not produce products well. Therefore, constructing a recombinant Halomonas strain that is resistant to carbon dioxide electrochemical derivatives (mainly acetate) is of great significance for the large-scale industrial utilization of CO 2 Electrochemical derivatives are of great significance. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a method of using CO 2 Halomonas for producing polyhydroxyalkanoates (PHA) through electrochemical derivatives, and its construction method and application.

[0005] The present invention provides a method for utilizing CO 2 Halomonas bacteria that produce polyhydroxyalkanoates by electrochemical derivatives, the Halomonas bacteria express 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 in SEQ ID No. 31; the nucleotide sequence of the Rhs647 gene is shown in SEQ ID No. 32.

[0006] Furthermore, the Halomonas also expresses the moy gene, and the moy nucleotide sequence is shown in SEQ ID No.33.

[0007] Furthermore, the Halomonas also expresses the aceE1 gene. The aceE1 nucleotide sequence is shown in SEQ ID No.34.

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

[0009] Halomonas sp.LY01, Halomonas sp.LY02, Halomonas sp.LY03, Halomonas sp.LY04, and Halomonas sp.LY08 used in the present invention are all preserved in Guangdong 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), and are classified and named Halomonas sp.

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

[0011] Furthermore, the expression of the Rhs644 gene and the Rhs647 gene used the pRE112 vector.

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

[0013] Furthermore, the CO 2 The electrochemical derivative is mainly acetic acid or its sodium salt.

[0014] The present invention also provides the use of the Halomonas in any of the following:

[0015] (i) In the metabolism of CO 2 Applications in electrochemical derivatives;

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

[0017] The present invention also provides a method for utilizing CO 2 A method for constructing a Halomonas grown by electrochemical derivatives comprises the following steps: introducing any one of the following into the Halomonas:

[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] Among them, the nucleotide sequence of Rhs644-Rhs647 is 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.

[0022] The present invention also provides a method for producing polyhydroxyalkanoates by fermentation using the Halomonas bacteria, comprising the following steps: activating the Halomonas bacteria, performing shake flask seed culture, and then culturing the bacteria in a CO 2 Fermentation culture is carried out in a fermentation medium with the electrochemical derivative as the sole carbon source.

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

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

[0025] Furthermore, the fermentation time is 28 to 52 hours.

[0026] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0027] 1. The present invention realizes the use of CO in Halomonas 2 Electrochemical derivatives produce poly-3-hydroxybutyrate as a single carbon source, without the need to add antibiotics and inducers, which greatly reduces production costs and increases the yield of single-batch fermentation. At the same time, it achieves efficient utilization of cheap carbon sources and can promote the process of low-carbon biomanufacturing.

[0028] 2. The time period for producing poly-3-hydroxybutyrate by the present invention is short, the production process is simple, the environment is green and pollution-free, and it is conducive to industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 pSEVA321 constructed in Example 1 of the present invention Mmp1-Rhs644-647 Plasmid map;

[0031] Figure 2 This is the electrophoresis diagram of the target fragment of Example 1 of the present invention;

[0032] Figure 3 pSEVA321 constructed in Example 3 of the present invention porin226-Rhs644-Rhs647 Plasmid map;

[0033] Figure 4 pRE112 constructed in Example 3 of the present invention porin226-Rhs644-Rhs647 Plasmid map;

[0034] Figure 5 This is the electrophoresis diagram of the target fragment of Example 3 of the present invention;

[0035] Figure 6 pSEVA321 constructed in Example 4 of the present invention porin58-moy Plasmid map;

[0036] Figure 7 pRE112 constructed in Example 4 of the present invention G49-porin226-moy Plasmid map;

[0037] Figure 8 This is the electrophoresis diagram of the target fragment of Example 4 of the present invention;

[0038] Fig. 9 pSEVA321 constructed in Example 4 of the present invention porin141-aceE1 Plasmid map;

[0039] Fig.10 pRE112 constructed in Example 5 of the present invention G4-porin141-aceE1 Plasmid map;

[0040] Fig.11 This is the electrophoresis diagram of the target fragment of Example 5 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] Wild-type Halomonas has poor growth and product production effects in high-concentration carbon dioxide electrochemical derivatives. In order to achieve efficient utilization of cheap carbon sources, the present invention overexpresses T6SS effector proteins that play an important role in bacterial contact-dependent growth inhibition in Halomonas, enhances the stress resistance of Halomonas, and thus improves the acetate metabolism capacity of Halomonas. By introducing the gene for acetate conversion (Pyruvate dehydrogenase, aceE1), the conversion of acetate to acetyl-CoA is strengthened, and by expressing serine protease (moy), the conversion of short-chain fatty acid salts into growth intermediates is promoted, the metabolism of acetate is further improved, and the yield of PHB is increased.

[0043] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all 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 and Rhs647 gene expression plasmids

[0047] plasmid pSEVA321 Mmp1 -Backbone was used as a template to amplify the backbone pSEVA321 Mmp1 , Halomonas sp.TD01 was used as template to amplify the target gene fragments of Rhs644 and Rhs647, respectively, and construct pSEVA321 Mmp1-Rhs644-647 Plasmid, plasmid information such as Figure 1 shown.

[0048] (1) PCR amplification of target genes Rhs644 and Rhs647, 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

[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 backbone, and the Gibson mixed enzyme was used for connection. The Gibson Assembly connection 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) S17-1 Escherichia coli transformation

[0068] Step 1: Take out the prepared S17-1 E. coli competent cells from -80℃, thaw them 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 cool 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, combine it with Halomonas sp.LY01 in a 20LB plate. After 8 hours, pick a small amount of the combined bacteria and spread it on a 60LB plate with corresponding resistance. After 36 to 48 hours, perform another single clone colony verification.

[0077] (6) Product identification

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

[0079] Example 2 Shake flask growth ability test of recombinant bacteria

[0080] Halomonas itself is an excellent strain for producing PHB (poly-3-hydroxybutyrate). The ability of the strain to utilize carbon sources can be reflected to a certain extent by measuring the dry cell weight and PHB production of the strain.

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

[0082] (1) Seed solution preparation

[0083] ① Bacteria activation

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

[0085] ②First-level seed cultivation:

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

[0087] ③Secondary seed cultivation:

[0088] The primary bacteria were aspirated and inoculated into a 150 mL conical flask containing 20 mL seed culture medium (60 LB + Cm) at a 1% inoculation rate, and then placed in a shaker at 37°C and 220 rpm for 12 h.

[0089] (2) Shake flask fermentation production

[0090] Culture medium preparation: 50MM culture medium system:

[0091] Base material: NaCl 50g / L, yeast powder 1g / L;

[0092] Component I: MgSO 4 20g / L, CO(NH 2 ) 2 30g / L;

[0093] Component II: KH 2 PO 4 175g / L;

[0094] Component III: 5 g / L Fe(III)-NH 4 -Citrate and 2g / L CaCl 2 ·2H 2 O and (12mol / L) concentrated hydrochloric acid 41.7mL are thoroughly mixed and the volume is adjusted to 1L;

[0095] Component IV: ZnSO 4 7H 2 O 0.1g / L, MnCl 2 ·4H 2 O 0.03g / L, H 3 BO 3 0.3g / L, CoCl 2 6H 2 O0.2g / L, CuSO 4 ·5H 2 O 0.01g / L, NiCl 2 6H 2 O 0.02g / L and NaMoO 4 ·2H 2 O 0.03 g / L;

[0096] Component III & IV: Take 100 mL of component III and 10 mL of component IV, add 90 mL of deionized water and mix, and finally adjust the pH value to 4.5-5.5 with 5M NaOH;

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

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

[0099] (3) Fermentation parameter determination:

[0100] (a)OD 600 Determination:

[0101] Take 1 mL of the fermented bacterial solution, centrifuge it at 12000 r / min for 10 min, discard the supernatant, add an equal volume of deionized water, resuspend the bacteria, and use a spectrophotometer to detect the absorbance at a wavelength of 600 nm (if necessary, dilute the bacterial solution to ensure that the reading of the spectrophotometer at a wavelength of 600 nm is in the range of 0.3 to 0.8).

[0102] (b) Determination of dry cell weight (DCM):

[0103] Take 15mL of the fermented bacterial liquid, centrifuge it at 9000r / min for 5min, discard the supernatant, add an equal volume of deionized water, resuspend the bacteria, centrifuge it again at 9000r / min for 5min, discard the supernatant and store it in a -80℃ refrigerator for 30min, then place it in a freeze dryer and freeze-dry it for 12h. The weight of the freeze-dried bacteria is weighed on an analytical balance as the dry weight.

[0104] (c) PHB determination:

[0105] Take 30-40 mg of bacteria and place them in an esterification tube. Add 2 mL of esterification solution (485 mL of methanol + 15 mL of concentrated sulfuric acid) and 2 mL of chloroform. Esterify at 100 degrees Celsius for 4 hours. After the end, take 1 mL of deionized water and place it in the esterification tube. Oscillate at 1500 rpm for 3 minutes and let it stand for 40 minutes. Take 1 mL of the lower chloroform layer for GC analysis.

[0106] (4) Fermentation results:

[0107] The growth test of Halomonas sp.LY01 and recombinant LY01 in shake flasks was carried out under different IPTG induction concentrations (0 mg / L, 5 mg / L, 10 mg / L):

[0108] Table 5 Recombinant bacteria fermentation production results

[0109]

[0110] The results are shown in Table 5. When the IPTG induction concentration was 5 mg / L, the cell dry weight of the recombinant LY01 strain increased by 8.5% and the PHB production increased by 6.3% compared with Halomonas p.LY01.

[0111] Example 3 Constitutive promoter replacement and gene integration

[0112] 1. Promoter replacement plasmid construction

[0113] plasmid pSEVA321 Mmp1-Rhs644-647 The fragment Rhs644-Rhs647 sequence was obtained using plasmid pSEVA321 porin58 -GFP was used as a template to amplify the backbone pSEVA321 porin58 , construct plasmid pSEVA321 according to the method of Example 1 porin226-Rhs644-Rhs647 , plasmid information such as Figure 3 shown.

[0114] PCR amplification of target fragment, pSEVA321 porin226 The primer sequences (5'-3') of the 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] Referring to the plasmid construction method in Example 1, plasmid pSEVA321 porin226-Rhs644-Rhs647 porin226-Rhs644-Rhs647 was amplified using plasmid pRE112-G49-Backbone as a template, and the backbone pRE112 was amplified using plasmid pRE112 to construct plasmid pRE112 porin226-Rhs644-Rhs647 ,like Figure 4 shown.

[0121] The primer sequences (5'-3') for PCR amplification of the porin226-Rhs644-Rhs647 gene sequence and the backbone pRE112 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 integrated plasmid constructed above was cultured and conjugated with Halomonas sp.LY01 in a 20LB plate after 12 to 16 hours. After 8 hours, a small amount of conjugated bacteria was selected and spread on a 60LB plate with corresponding resistance. After 36 to 48 hours, single clone colony verification was performed again.

[0128] (2) After a single colony grows on the plate, resistance verification can be performed.

[0129] (3) Select the colonies that have successfully removed the antibodies for double verification (universal F1 / R and specific F2 / R). Verify the correct bands by PCR cloning. The results are as follows: Figure 5 As shown, the length of the fragment amplified by the universal primer band was 7600bp, and there was no specific primer band. The PCR reaction solution that successfully passed the double verification was selected for testing, and the strain obtained after successful sequencing was named LY01-A1.

[0130] Example 4 Overexpression of serine protease (moy)

[0131] 1. Referring to the plasmid construction steps of Example 1, Halomonas sp. TD01 was used as a template for amplification to obtain the target gene sequence of the fragment moy. porin58 -GFP was used as a template to amplify and obtain the backbone pSEVA321 porin226 , construct plasmid pSEVA321 according to the method of Example 1 porin58-moy , plasmid information such as Figure 6 shown.

[0132] The primer sequences (5'-3') for PCR amplification of 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 in Example 1, plasmid pSEVA321 porin58-moy porin58-moy was amplified as a template, and the backbone pRE112 was amplified using the plasmid pRE112-G7-Backbone as a template to construct plasmid pRE112 G49-porin226-moy ,like Figure 7 shown.

[0139] The primer sequences (5'-3') for PCR amplification of 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] Using LY01-A1 as the base strain, gene integration was performed according to the steps in Example 3. The bands were verified by PCR cloning, and the results were as follows Figure 8 As shown, the length of the amplified fragment was 5000 bp, and there was no specific primer band. The PCR reaction solution that successfully passed the double verification was selected for testing, and the strain obtained after successful sequencing was named LY01-A2.

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

[0147] 1. Referring to the plasmid construction steps of Example 1, Halomonas sp. TD01 was used as a template for amplification to obtain the aceE1 target gene sequence, and the plasmid pSEVA321 porin141 -GFP was used as a template to amplify the backbone pSEVA321 porin141 , pSEVA321 porin141-aceE1 Plasmid information such as Fig. 9shown.

[0148] PCR amplification of aceE1 and pSEVA321 porin141 The primer sequences (5'-3') of the 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] Referring to the plasmid construction method in Example 1, plasmid pSEVA321 porin141-aceE1 porin141-aceE1 was amplified using plasmid pRE112-G4-Backbone as a template, and the backbone pRE112 was amplified using plasmid pRE112-G4-Backbone as a template. porin141-aceE1 ,like Fig.10 shown.

[0155] The primer sequences (5'-3') for PCR amplification of the porin68-aceE1 gene sequence and the backbone pRE112 are 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] Using LY01-A2 as the base strain, gene integration was performed according to the steps in Example 3. The bands were verified by PCR cloning, and the results were as follows Fig.11 As shown, the length of the amplified fragment was 4800 bp, and there was no specific primer band. The PCR reaction solution that successfully passed the double verification was selected for testing, and the strain obtained after successful sequencing was named LY01-A3.

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

[0163] Fermentation was carried out according to the fermentation steps of Example 2. 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, with the maximum cell dry weight being 9.5 g / L for LY01-A3, and the PHB content reaching 82.5%, indicating that the overexpression of several module exogenous genes at the chromosome level can effectively improve the bacterial resistance to sodium acetate.

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

[0168] The above gene modification strategy was introduced into Halomonas sp. LY02, Halomonas sp LY03, Halomonas sp LY04, and Halomonas sp. LY08 and named TD01-A3, LY02-A3, LY03-A3, LY04-A3, and LY08-A3, respectively.

[0169] The constructed recombinant strain and the original strain were tested for their growth ability by shake flask fermentation, and the specific implementation method was the same as that 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 were significantly increased compared with the original strains. This shows that the overexpression of several module exogenous genes at the chromosome level can effectively improve the strain's ability to resist carbon dioxide derivatives. The results of this embodiment show that the method of the present application is not only applicable to Halomonas sp.LY01, but also to other Halomonas. Introducing metabolic genes in other Halomonas can enhance the ability to metabolize sodium acetate.

[0173] 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 of producing polyhydroxyalkanoates using CO2 electrochemical derivatives by using Halomonas, characterized in that: The Halomonas sp. expresses Rhs644 gene and Rhs647 gene 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.

2. The Halomonas according to claim 1, characterized in that The Halomonas also expresses the moy gene.

3. The Halomonas according to claim 2, characterized in that The Halomonas also expresses the aceE1 gene.

4. The Halomonas according to claim 1, characterized in that The Halomonas include Halomonas sp.

5. The Halomonas according to claim 1, characterized in that The pRE112 vector was used to express the Rhs644 gene and the Rhs647 gene.

6. The Halomonas according to claim 3, characterized in that The vector comprises a promoter, and the promoter is porin226.

7. The Halomonas according to claim 1, characterized in that The CO2 electrochemical derivative is acetic acid or its sodium salt.

8. Use of the Halomonas according to any one of claims 1 to 7 in any one of the following: (i) Application in the electrochemical derivatization of CO2; (ii) Application in the production of polyhydroxyalkanoates.

9. A method for constructing Halomonas grown using CO2 electrochemical derivatives, characterized in that: The method comprises the following steps: introducing any one of the following into Halomonas: (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; Among them, the nucleotide sequence of Rhs644-Rhs647 is 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.

10. A method for producing polyhydroxyalkanoates by fermentation using the Halomonas bacteria described in any one of claims 1 to 7, characterized in that: The method comprises the following steps: activating the Halomonas, carrying out shake flask seed culture, and then carrying out fermentation culture in a fermentation medium with a CO2 electrochemical derivative as the sole carbon source.

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