Recombinant strain of Ralstonia for high production of poly 3-hydroxybutyrate using formic acid and its construction method and application

Abstract

The present invention discloses a recombinant strain of Ralstonia that produces high-yield poly 3-hydroxybutyrate using formic acid, and a construction method and application thereof. The construction method comprises the following steps: knocking out the genes phaZ1, phaZ2, and phaZ3 of Ralstonia that degrade PHB to obtain a recombinant strain PHB01. Genes RS7405 and RS7400 encoding Rubisco enzymes from cyanobacteria are introduced into the phaZ1 site of the PHB01 strain genome, and gene expression cassettes of the pyruvate synthase gene pyk and the α-ketoglutarate synthase gene Idh from desulfurization bacteria are introduced into the above strain to construct Ralstonia PHB02 that produces high-yield poly 3-hydroxybutyrate using formic acid. The recombinant Ralstonia strain constructed by the present invention can synthesize poly 3-hydroxybutyrate from scratch using formic acid as a carbon source. The method of the present invention is simple to operate and can obtain poly 3-hydroxybutyrate with high yield.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and more particularly to a Ralstonia strain for synthesizing poly 3-hydroxybutyrate, a construction method and an application thereof. Background Art

[0002] Poly (3-hydroxybutyrate) (PHB) is a bioplastic synthesized by microorganisms under nutrient-restricted conditions. It is biodegradable and compatible, so it has great development potential in replacing petrochemical-derived plastics and has broad application prospects in the fields of medicine, packaging industry, nanotechnology and agriculture. At present, the commercial application of PHB is mainly limited by its high production cost. Ralstonia eutropha is a facultative chemoautotrophic Gram-negative bacterium. It has flexible and diverse metabolism. It can not only use fructose, lipids or other organic carbon sources for heterotrophic growth, but also use a mixture of H2, CO2 and O2 for autotrophic growth. However, regardless of the nutritional mode, when the necessary nutrients such as nitrogen, oxygen or phosphorus in the growth environment are limited, R. eutropha can naturally synthesize PHB, and its content can reach up to 90% of the cell dry weight. Therefore, R. eutropha is considered to be one of the ideal chassis for producing PHB.

[0003] At present, the industrial production of PHB is mainly limited by its high production cost. The main reason is that expensive fructose and other substances need to be used as substrates to produce PHB. If a mixed gas of H2, CO2 and O2 is used as a substrate, its industrial development is greatly restricted due to gas safety issues. Formic acid is a cheap and readily available chemical, which is often prepared using industrial waste gases such as CO and CO2. If formic acid is used as a substrate to produce PHB, it can not only reduce production costs, but also make some contributions to solving environmental problems caused by CO2. Summary of the invention

[0004] The object of the present invention is to overcome the shortcomings of the prior art and provide a Ralstonia strain that can produce poly 3-hydroxybutyrate in high yield using formic acid.

[0005] The second object of the present invention is to provide a method for constructing a Ralstonia strain that can produce high amounts of poly (3-hydroxybutyrate) using formic acid.

[0006] The third object of the present invention is to use the constructed recombinant Ralstonia strain to produce high-quality poly (3-hydroxybutyrate) using formic acid.

[0007] The technical solution of the present invention is summarized as follows:

[0008] The method for constructing a Ralstonia eutropha strain that can produce high PHB using formic acid comprises the following steps:

[0009] The genes phaZ1, phaZ2, and phaZ3 that degrade PHB in Ralstoniaeutropha H16 were knocked out to obtain the recombinant strain PHB01;

[0010] Genes RS7405 and RS7400 encoding Rubisco enzymes from Synechococcuse longatus PCC7942 were introduced into the phaZ1 site of the PHB01 strain genome, and gene expression cassettes of pyruvate synthase gene pyk and α-ketoglutarate synthase gene Idh from Sufurimonas autotrpphica DSM 16294 were introduced into the above strain to construct Ralstonia sp. PHB02, which can produce high-quality poly 3-hydroxybutyrate using formate.

[0011] The nucleotide sequence of the phaZ1 gene is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the phaZ2 gene is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the phaZ3 gene is shown in SEQ ID NO. 3;

[0014] The nucleotide sequence of the Rubisco large subunit gene sequence RS7400 is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of the Rubisco small subunit gene sequence RS7405 is shown in SEQ ID NO.5;

[0016] The nucleotide sequence of the pyk gene is shown in SEQ ID NO.6;

[0017] The nucleotide sequence of the Idh gene is shown in SEQ ID NO.7 and SEQ ID NO.8.

[0018] The Ralstonia strain that utilizes formic acid to produce high amounts of poly 3-hydroxybutyrate is constructed using the above method.

[0019] The invention discloses an application of producing poly 3-hydroxybutyrate by using a formic acid high-yielding poly 3-hydroxybutyrate Ralstonia strain, comprising the following steps: culturing and fermenting the poly 3-hydroxybutyrate synthesizing Ralstonia strain PHB02 in a fermentation medium to obtain poly 3-hydroxybutyrate, wherein the formic acid fermentation medium comprises 3.57 g / L Na2 HPO4, 1.5 g / L K H2PO4, 0.167 g / L ammonium sulfate, 0.02 g / L sodium bicarbonate, 20 g / L sodium formate, and the remainder water.

[0020] Beneficial effects of the present invention:

[0021] R.eutropha is considered to be one of the ideal bases for producing PHB, but the industrial production of PHB is mainly limited by its high production cost. The main reason is that it is necessary to use expensive fructose and other substances as substrates to produce PHB. The present invention constructs a Ralstonia strain that uses a cheap substrate formic acid to produce high-yield poly 3-hydroxybutyrate, which can synthesize poly 3-hydroxybutyrate from scratch using formic acid as a carbon source. This method of using microorganisms to ferment formic acid to produce poly 3-hydroxybutyrate is simple to operate and can obtain high-yield poly 3-hydroxybutyrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the poly 3-hydroxybutyrate synthesis pathway in the recombinant Ralstonia strain;

[0023] Figure 2 The yield-time diagram of the high-yield poly (3-hydroxybutyrate) Ralstonia strain PHB01 using formic acid;

[0024] Figure 3 The yield-time diagram of the high-yield poly (3-hydroxybutyrate) Ralstonia strain PHB02 using formic acid;

[0025] Figure 4 This is a comparison chart of the production of poly (3-hydroxybutyrate) produced by Ralstonia strains PHB01 and PHB02 using formic acid. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0027] The original wild type Ralstonia eutropha H16 was purchased from the ATCC official website in October 2016 ( https: / / www.atcc.org / products / 17699 ).

[0028] Origin of genes:

[0029] phaZ1 gene: from Ralstonia eutropha H16

[0030] phaZ2 gene: from Ralstonia eutropha H16

[0031] phaZ3 gene: from Ralstonia eutropha H16

[0032] Rubisco enzyme encoding genes RS7405 and RS7400: from cyanobacteria (Synechococcuse longatus PCC7942)

[0033] Pyruvate synthase pyk gene: from desulfurizing bacteria (Sufurimonas autotrpphica DSM 16294)

[0034] α-Ketoglutarate synthase Idh gene: from desulfurizing bacteria (Sufurimonas autotrpphica DSM16294)

[0035] Example

[0036] 1 Module Construction

[0037] The method for constructing a Ralstonia eutropha strain that produces high PHB using formic acid comprises the following steps: using a site-specific homologous recombination method to knock out PHB degradation genes phaZ1, phaZ2, and phaZ3 to obtain a PHB production strain PHB01.

[0038] Using the site-specific homologous recombination method, the expression cassettes of the two genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylation / oxygenase (hereinafter referred to as Rubisco), the Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405, were integrated into the phaZ1 position in the genome of the PHB production strain PHB01. Then, using the conjugative transfer method, the gene expression plasmids encoding the gene pyk of pyruvate synthase and the gene Idh of α-ketoglutarate synthase were introduced into the production strain PHB01 to obtain the recombinant strain PHB02.

[0039] The nucleotide sequence of the phaZ1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the phaZ2 gene is shown in SEQ ID NO.2; the nucleotide sequence of the phaZ3 gene is shown in SEQ ID NO.3;

[0040] The nucleotide sequence of the Rubisco large subunit gene sequence RS7400 is shown in SEQ ID NO.4; the nucleotide sequence of the Rubisco small subunit gene sequence RS7405 is shown in SEQ ID NO.5; the nucleotide sequence of the pyk gene is shown in SEQ ID NO.6; the nucleotide sequence of the Idh gene is shown in SEQ ID NO.7 and SEQ ID NO.8

[0041] The specific steps are:

[0042] (1) A method for constructing a Ralstonia eutropha strain PHB01 that produces high PHB yield using formic acid comprises the following steps:

[0043] The screening resistance gene KanR comes from the plasmid pK18mobSacB (purchased from Addgene, Inc. WWW.addgene.org).

[0044] The gene sequences of phaZ1, phaZ2, and phaZ3 from Ralstonia eutropha H16 were obtained from the NCBI database, and knockout plasmids of these three genes were constructed.

[0045] The knockout plasmid of phaZ1 gene used the wild-type Ralstoniaeutropha H16 genome as template, phaZ1-up and phaZ1-down as homologous upper arm and homologous lower arm, respectively (SEQ ID NO.9 and SEQ ID NO.10), and phaZ1up-F (SEQ ID NO.15), phaZ1up-R (SEQ ID NO.16) and phaZ1down-F (SEQ ID NO.17), phaZ1down-R (SEQ ID NO.18) as primers to amplify homologous arms phaZ1up (SEQ ID NO.9) and phaZ1down (SEQ ID NO.10), respectively;

[0046] The knockout plasmid of phaZ2 gene uses the wild-type Ralstoniaeutropha H16 genome as a template, uses phaZ2-up and phaZ2-down as the homologous upper arm and homologous lower arm, respectively (SEQ ID NO.11 and SEQ ID NO.12), and uses phaZ2up-F (SEQ ID NO.19), phaZ2up-R (SEQ ID NO.20) and phaZ2down-F (SEQ ID NO.21), phaZ2down-R (SEQ ID NO.22) as primers to amplify the homologous arms phaZ2up (SEQ ID NO.11) and phaZ2down (SEQ ID NO.12), respectively;

[0047] The knockout plasmid of phaZ3 gene used the wild-type Ralstoniaeutropha H16 genome as a template, phaZ3-up and phaZ3-down as the homologous upper arm and homologous lower arm, respectively (SEQ ID NO.13 and SEQ ID NO.14), and phaZ3up-F (SEQ ID NO.23), phaZ3up-R (SEQ ID NO.24) and phaZ3down-F (SEQ ID NO.25), phaZ3down-R (SEQ ID NO.26) as primers to amplify the homologous arms phaZ3up (SEQ ID NO.13) and phaZ3down (SEQ ID NO.14), respectively;

[0048] The DNA PCR enzyme used in the present invention is Fast Pfu polymerase from Quanshijin Biotechnology Co., Ltd. The 50 μL PCR amplification system is as follows: DNA template, 1 μL; 2.5 μL each of the front primer (10 μM) and the rear primer (10 μM); 5 μL of dNTP (10 mM); 10 μL of 5×PfuBuffer; 1 μL of Fast Pfu polymerase; and finally, double distilled water is used to make up to 50 μL.

[0049] The amplification program was set on the PCR instrument. The amplification conditions were 95°C pre-denaturation for 2 min (1 cycle); 95°C denaturation for 20 sec, 55°C annealing for 20 sec, 72°C extension for 30 s (35 cycles); 72°C extension for 5 min (1 cycle).

[0050] Each pair of homologous upper and lower arm fragments obtained by PCR amplification was mixed with the pK18mobSacB linearized suicide plasmid vector cut with HindIII and EcoRI restriction endonucleases, and the three fragments were spliced ​​into a complete recombinant vector using the BM Seamless Connection Kit (Bomed Biotechnology Co., Ltd.). The molar ratio of each fragment was 1:1, and the total amount of fragments reached 1μg. The connection volume was as follows: DNA fragment 5μL, 2× Seamless Cloning Mix 5μL. The connection program was set on the PCR instrument. The connection conditions were 50℃15min, 16℃10min.

[0051] The recombinant vectors pK18mobSacB-phaZ1, pK18mobSacB-phaZ2, and pK18mobSacB-phaZ3 obtained by the above method were introduced into Trans1-T1 Phage Resistant Chemically Competent Cell produced by Quanshijin Company by heat shock method, and single colonies were grown on LB-Kan50 agar plates (10 g / L NaCl, 10 g / L tryptone and 50 g / L yeast extract, 50 g / L kanamycin was added at 1:1000). ℃ After incubation at 220 rpm for 12 h, pick an appropriate number of single colonies and culture them in LB-Kan50 liquid medium at 37 ℃ After incubation at 220 rpm for 12 h, the samples were sent to GeneWeichi for sequencing.

[0052] After the sequencing results were confirmed, the knockout plasmid was extracted using the kit of Quanshijin Company, and the heat shock method was still used to introduce it into the competent state of the s17-1 strain prepared by chemical methods in advance. The single colony grew on LB-Kan50 agar plates (10g / L NaCl, 10g / L tryptone and 50g / L yeast extract, 50g / L kanamycin was added at 1:1000). ℃ After incubation at 220 rpm for 12 h, pick an appropriate number of single colonies and culture them in LB-Kan50 liquid medium at 37 ℃ After incubation at 220 rpm for 12 h, the samples were sent to GeneWeichi for sequencing.

[0053] After the sequencing results were confirmed, the cells were cultured in LB-Kan50 liquid medium at 37 ℃ The sequenced s17-1 strain was cultured at 220 rpm until OD 600 =0.6-0.8, 1 mL of wild-type Ralstonia eutropha H16 of the same volume and concentration was centrifuged and resuspended in 200 μL of sterile water, and finally mixed at a ratio of 2:3, and in NB agarose medium (0.5 g / L (NH4)2SO4, 10 g / L tryptone, 10 g / L yeast extract, 0.6 / L beef extract) for 30 ℃ Incubate at 230 rpm for 36-48 h to allow the constructed knockout plasmid to be introduced into the wild-type Ralstonia eutropha H16.

[0054] After the mixed strains grew to a suitable state, scrape an appropriate amount of the mixed strains and resuspend them in 50 μL of sterile water. After diluting them to an appropriate multiple, culture them on NB-Kan200+Gm10 agarose medium (NB medium with 200 g / L penicillin resistance and 10 g / L gentamicin resistance added at a dilution ratio of 1:1000) at 30 °C. ℃ The single exchange was initially screened at 230 rpm. The single colonies grown were used for initial verification of the single exchange results using colony PCR.

[0055] The colony PCR enzyme used in the present invention is Fastaq polymerase from Quanshijin Biotechnology Co., Ltd. The 15 μL PCR amplification system is as follows: 0.3 μL each of the front primer (10 μM) and the back primer (10 μM); 1.2 μL of dNTP (10 mM); 1.5 μL of 10×Fastaq Buffer; 0.15 μL of Fastaq polymerase; 10.15 μL of double distilled water, and finally pick an appropriate amount of colonies.

[0056] The amplification program was set on the PCR instrument. The amplification conditions were 94°C pre-denaturation for 5 min (1 cycle); 95°C denaturation for 30 sec, 58°C annealing for 20 sec, 72°C extension for 30 s (30 cycles); 72°C extension for 5 min (1 cycle).

[0057] After successful verification, single colonies were selected and cultured in NB liquid medium at 30°C and 280 rpm for 6 h. Then, they were cultured in NB-15% sucrose solid medium (10 g / L tryptone, 5 g / L yeast extract, 150 g / L sucrose, 15 g / L agar powder) for 30 ℃ A single colony was cultured at 230 rpm to an appropriate size, and the result of the second exchange was verified by colony PCR to obtain the desired knockout bacteria.

[0058] After knocking out phaZ1, phaZ2, and phaZ3 at once using the above method, the Ralstonia eutropha strain PHB01 that produces high PHB using formic acid was obtained.

[0059] (2) A method for constructing a Ralstonia eutropha strain PHB02 that produces high PHB yield using formic acid comprises the following steps:

[0060] The gene sequence of Rubisco large subunit RS7400 from Synechococcus elongatus PCC7942 was obtained from the NCBI database, and the nucleotide sequence (SEQ ID NO. 4) was obtained by artificial synthesis;

[0061] The gene sequence of the small subunit of Rubisco RS7405 from Synechococcus elongatus PCC7942 was obtained from the NCBI database, and the nucleotide sequence (SEQ ID NO.5) was obtained by artificial synthesis;

[0062] Construct the expression cassettes for these two genes:

[0063] The expression cassettes of the Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405 used the PBAD promoter and the rrnB T1 terminator; the expression cassettes of the Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405 were introduced into the phaZ1 site of the Ralstonia eutropha strain PHB01 that produces PHB in high yield using formic acid by site-specific homologous recombination;

[0064] Using the wild-type Ralstoniaeutropha H16 genome as a template, phaZ1up-F (SEQ ID NO.15), phaZ1up-Rubisco-R (SEQ ID NO.27) and phaZ1downRubisco-F (SEQ ID NO.28), phaZ1down-R (SEQ ID NO.18) as primers, homology arms phaZ1up (SEQ ID NO.9) and phaZ1down (SEQ ID NO.10) were amplified respectively;

[0065] Using the artificially synthesized ribulose-1,5-bisphosphate carboxylation / oxygenase expression plasmid PHG12-pBAD-Rubisco large subunit gene sequence RS7400-Rubisco small subunit gene sequence RS7405 as a template, phaZ1-Rubisco-F (SEQ ID NO.29) and phaZ1-Rubisco-R (SEQ ID NO.30) primers were used to amplify the ribulose-1,5-bisphosphate carboxylation / oxygenase gene expression cassette pBAD-Rubisco large subunit gene sequence RS7400-Rubisco small subunit gene sequence RS7405 containing the pBAD promoter and the rrnB T1 terminator.

[0066] The phaZ1up, phaZ1down, pBAD-Rubisco large subunit gene sequence RS7400-Rubisco small subunit gene sequence RS7405 amplified by the above PCR and the pK18mobSacB linearized suicide plasmid vector cut by HindIII and EcoRI restriction endonucleases were mixed and spliced ​​into a complete recombinant vector using the BM seamless ligation kit (Bomed Biotechnology Co., Ltd.). The molar ratio of each fragment was 1:1, the total amount of fragments reached 1μg, and the ligation volume was as follows: DNA fragment 5μL, 2×Seamless Cloning Mix 5μL. The ligation program was set on the PCR instrument. The ligation conditions were 50℃15min, 16℃10min.

[0067] The obtained recombinant vector was introduced into the Ralstonia eutropha strain PHB01 which uses formic acid to produce high PHB. The Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405 were introduced into the phaZ1 site of the PHB01 genome using the above-mentioned homologous recombination principle.

[0068] The pyk gene from Sulfurimonas autotrophica DSM 16294 was obtained from the NCBI database, and the nucleotide sequence (SEQ ID NO. 6) was obtained by artificial synthesis;

[0069] The gene Idh from Sulfurimonas autotrophica DSM 16294 was obtained from the NCBI database, and the nucleotide sequence (SEQ ID NO.8, SEQ ID NO.9) was obtained by artificial synthesis;

[0070] Construct the expression cassettes for these two genes:

[0071] The pyk gene expression cassette uses the PBAD promoter and the rrnB T1 terminator;

[0072] The Idh gene expression cassette uses the PBAD promoter and the rrnB T1 terminator);

[0073] By combining the transfer method, the pyk gene expression cassette and the idh gene expression cassette were introduced into the Ralstonia eutropha strain PHB01 for high production of PHB using formic acid, into which the Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405 expression cassette were introduced, thereby obtaining the Ralstonia eutropha strain PHB02 for high production of PHB using formic acid (see Figure 1 ).

[0074] The gene expression plasmid PHG-pBAD-pyk-Idh of the artificially synthesized pyruvate synthase gene pyk and α-ketoglutarate synthase gene Idh was introduced into Trans1-T1 PhageResistant Chemically Competent Cell produced by Quanshijin Company by heat shock method, and a single colony was grown on LB-Kan50 agar plate (10g / L NaCl, 10g / L tryptone and 50g / L yeast extract, 50g / L kanamycin was added at 1:1000). ℃ After incubation at 220 rpm for 12 h, pick an appropriate number of single colonies and culture them in LB-Kan50 liquid medium at 37 ℃ After incubation at 220 rpm for 12 h, the samples were sent to GeneWeichi for sequencing.

[0075] After the sequencing results were confirmed, the constructed expression plasmid was extracted using the kit of Quanshijin Company, and the heat shock method was still used to introduce it into the competent state of the s17-1 strain prepared in advance by chemical methods. Single colonies grew on LB-Kan50 agar plates (10g / L NaCl, 10g / L tryptone and 50g / L yeast extract, 50g / L kanamycin was added at 1:1000). After incubation at 37℃ and 220rpm for 12h. Pick an appropriate number of single colonies and culture them in LB-Kan50 liquid medium at 37℃. ℃ After incubation at 220 rpm for 12 h, the samples were sent to GeneWeichi for sequencing.

[0076] After the sequencing results were confirmed, the s17-1 strain with the correct sequencing was cultured in LB-Kan50 liquid medium at 37°C and 220 rpm until OD 600 =0.6-0.8, take 1mL and the same volume and concentration of wild-type Ralstonia eutropha H16, centrifuge and resuspend in 200μL sterile water, finally mix at a ratio of 2:3, in NB agarose medium (0.5g / L (NH4)2SO4, 10g / L tryptone, 10g / L yeast extract, 0.6g / L beef extract), incubate at 30℃ and 230rpm for 36-48h, so that the constructed expression plasmid is introduced into the constructed PHB01 strain introduced with the Rubisco large subunit gene sequence RS7400 and the Rubisco small subunit gene sequence RS7405, and the Ralstonia eutropha strain PHB02 with high PHB production using formic acid is obtained.

[0077] 2. Fermentation method

[0078] Seed culture medium: yeast extract 10g / L, trypsin 10g / L, beef extract powder 6g / L, ammonium sulfate 5g / L, and the rest is water.

[0079] Fructose culture medium: fructose 10g / L, Na2HPO4 3.57g / L, KH2PO4 1.5g / L, ammonium sulfate 0.167g / L, sodium bicarbonate 0.02g / L, and the rest is water.

[0080] Formic acid fermentation medium: Na2 HPO4 3.57 g / L, K H2PO4 1.5 g / L, ammonium sulfate 0.167 g / L, sodium bicarbonate 0.02 g / L, sodium formate 20 g / L, and the rest is water.

[0081] Control group: The recombinant strain PHB01 was inoculated into 4 mL of seed culture medium at 30°C and 230 rpm for 24 h, and then 1 mL of the bacterial solution was transferred to 10 mL of fresh fructose culture medium at 30°C and 230 rpm for 24 h. Then the bacterial solution was transferred to 30 mL of fresh formic acid fermentation medium and the bacterial density was adjusted so that the final bacterial density was between OD 600 The temperature was 2, 30°C and 230 rpm, and the culture was cultured for 96 h. The culture medium was used for product analysis.

[0082] Experimental group: The recombinant strain PHB02 was inoculated into 4 mL of seed culture medium at 30°C and 230 rpm for 24 h, and then 1 mL of the bacterial solution was transferred to 10 mL of fresh fructose culture medium at 30°C and 230 rpm for 24 h. Then the bacterial solution was transferred to 30 mL of fresh formic acid fermentation medium and the bacterial density was adjusted so that the final bacterial density was between OD 600 The culture was carried out at 2, 30°C and 230 rpm for 96 h, and the culture medium was used for product analysis.

[0083] 3. Product analysis

[0084] PHB is the target product of this experiment. For the content detection of organic polymers, PHB is first hydrolyzed into monomeric hydroxybutyric acid by a mixture of esterification liquid and chloroform, and then hydroxybutyric acid is extracted into the aqueous phase by an extraction method. The sample is drawn using a 1mL syringe and tested by gas chromatography. The PHB detection process mainly includes the following steps: bacterial collection, freeze drying, weighing and cracking, extraction product, sample detection and content calculation.

[0085] 1) Bacteria collection: Use a 5mL disposable syringe to insert from the bottom sampling port of the photoreactor, draw several times to ensure uniform sampling, and then slowly draw 5mL of reaction solution and transfer it to a centrifuge tube. Use a pipette to accurately draw 4mL of reaction solution and transfer it to a centrifuge tube, place it in a small high-speed centrifuge and centrifuge it at 12000rpm for 2min. After taking it out, pour out the supernatant and resuspend it with sterile water for washing, and collect the bacteria by centrifugation. Repeat the operation 2-3 times to clean the bacteria. Then remove the excess water and store the bacterial precipitate in a -80℃ refrigerator waiting for the next step.

[0086] 2) Freeze drying: Take out the frozen bacteria in the -80℃ refrigerator, open the centrifuge tube lid and wrap it with tin foil. Use a needle to poke holes in the tin foil and transfer it to a freeze-drying vacuum machine precooled to -55℃. After sealing, vacuum freeze-dry for 24 hours. After completion, take it out and place it at room temperature, waiting for the next step. 3. Weighing and lysis: Weigh the mass of the dried sample in 2, and then transfer the dried bacteria in the centrifuge tube to a 5mL vial. Add 500μL of esterification solution and 500μL of chloroform, and use a capping device to fix the headspace cover to the vial. The polytetrafluoroethylene coating in the rubber gasket in the headspace cover is required to be at the bottom and in direct contact with the vial. After completion, use 509 glue to glue the edge of the bottle cap to prevent the organic solvent from volatilizing. Place the sealed vial in a 90°C oven for lysis for 4 hours. During this time, take out the vial and shake it with a vortex oscillator to break up the lumps of bacteria in the bottle to ensure sufficient lysis. After the lysis is completed, take out the vial and cool it to room temperature before proceeding to the next step.

[0087] 3) Weighing and lysis: Weigh the mass of the dried sample in step 2, and then transfer the dried bacteria in the centrifuge tube to a 5mL vial. Add 500μL of esterification solution and 500μL of chloroform, and use a capping device to fix the headspace cover to the vial. The polytetrafluoroethylene coating in the rubber gasket in the headspace cover is required to be at the bottom and in direct contact with the vial. After completion, use 509 glue to glue the edge of the bottle cap to prevent the organic solvent from volatilizing. Put the sealed vial into a 90°C oven for lysis for 4 hours. During this period, take out the vial and shake it with a vortex oscillator to break up the blocky bacteria in the bottle to ensure sufficient lysis. After the lysis is completed, take out the vial and cool it to room temperature before proceeding to the next step.

[0088] 4) Extraction product: After the vial is cooled to room temperature, remove the seal and open the cap. Use a pipette to aspirate the solution in the bottle and transfer it to a 10mL centrifuge tube. Add 250μL of deionized water to the centrifuge tube in advance. After the organic phase and the aqueous phase are evenly mixed, place the centrifuge tube in a sealed container at 4℃ and let it stand overnight. After the extraction process is completed, the sample is extracted into the lower aqueous solution for sampling and testing.

[0089] 5) Sample detection: Use a disposable syringe with a capacity of 1mL to absorb the liquid in the lower layer of the 4 centrifuge tubes, and then transfer it to the gas injection bottle. After covering the lid, put it into the gas chromatograph and prepare for injection. The detection parameters of the instrument are as follows: the chromatographic column is initially set to 140℃, stay for 6min, increase the temperature to 250℃ at a rate of 40℃ / min, and stay for 14min. The temperature of AuxI is 250℃ throughout the process, and the temperature of Det is 250℃ throughout the process. The solvent is acetone, and it is cleaned 5 times before and after the injection. It is cleaned with the sample 3 times during the injection, and the amount of each cleaning is 3uL. The gas chromatography detection column was a RB-lonWax capillary column (0.32 mm × 30 m, 0.25 mm film thickness), the detector was 240 nm (2998 PDA detector, Waters), nitrogen was the carrier gas, the pressure was 0.06 MPa, the split ratio was 8:1, the injection column length was 2.5 cm, the detector column length was 10.7 cm, the injection volume was 1 μL, and the running time was 27 min.

[0090] 6) Content calculation: Draw a standard curve based on the test results of the PHB standard, and substitute the test results into the standard curve to calculate the PHB content in the sample.

[0091] 4. Results

[0092] In this study, we first used the homologous recombination method to knock out three genes related to PHB degradation, phaZ1, phaZ2, and phaZ3, to obtain the strain PHB01. This strain can produce 1.51 g / L PHB after 120 hours of formic acid fermentation. In order to further improve the strain's ability to produce PHB, the two genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylation / oxygenase (hereinafter referred to as Rubisco) were integrated into the phaZ1 position in the genome of the PHB-producing strain PHB01 by using the site-specific homologous recombination method. Then, the gene expression plasmids encoding the gene pyk of pyruvate synthase and the gene Idh of α-ketoglutarate synthase were introduced into the production strain PHB01 by using the conjugative transfer method to obtain the recombinant strain PHB02. After 120 h of fermentation in the fermentation medium, the recombinant strain PHB02 finally obtained 2.36 g / L of PHB, which was about 1.56 times higher than that of the previous culture medium.

Claims

1. A method for constructing a formic acid high-yielding poly (3-hydroxybutyrate) Ralstonia strain, characterized in that: The steps include: (1) The genes phaZ1, phaZ2, and phaZ3 that degrade PHB in Ralstoniaeutropha H16 were knocked out to obtain the recombinant strain PHB01; (2) Genes RS7405 and RS7400 encoding Rubisco enzymes from Synechococcuse longatus PCC7942 were introduced into the phaZ1 locus of the PHB01 strain genome; (3) introducing the gene expression cassette of the pyruvate synthase gene pyk and the α-ketoglutarate synthase gene Idh from Sufurimonas autotrpphica DSM 16294 into the above strain to construct Ralstonia sp. PHB02, which is a high-yield poly (3-hydroxybutyrate) strain utilizing formate; The nucleotide sequence of the phaZ1 gene is shown in SEQ ID NO.1; The nucleotide sequence of the phaZ2 gene is shown in SEQ ID NO.2; The nucleotide sequence of the phaZ3 gene is shown in SEQ ID NO. 3; The nucleotide sequence of Rubisco large subunit gene RS7400 is shown in SEQ ID NO.4; The nucleotide sequence of Rubisco small subunit gene RS7405 is shown in SEQ ID NO.5; The nucleotide sequence of the pyk gene is shown in SEQ ID NO.6; The nucleotide sequence of the Idh gene is shown in SEQ ID NO.7 and SEQ ID NO.

8.

2. The Ralstonia strain that produces high amounts of poly (3-hydroxybutyrate) using formic acid obtained by the method of claim 1.

3. Use of the Ralstonia strain with high formic acid production in producing poly 3-hydroxybutyrate according to claim 2.

4. The use according to claim 3, characterized in that: The method comprises the following steps: fermenting the synthetic poly 3-hydroxybutyrate Ralstonia strain PHB02 of claim 2 using formic acid in a fermentation medium to obtain poly 3-hydroxybutyrate, wherein the formic acid fermentation medium comprises 3.57 g / L Na2 HPO4, 1.5 g / L K H2PO4, 0.167 g / L ammonium sulfate, 0.02 g / L sodium bicarbonate, 20 g / L sodium formate, and the remainder is water.

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