An engineered bacterium for synthesizing pha by using carbon dioxide and a construction method and application thereof

Abstract

The application discloses an engineering bacterium for synthesizing PHA by using carbon dioxide and a construction method and application thereof. First, the Rubisco gene from Methanosaeta sp. GP6 is integrated on the genome, and the original promoter is replaced; then, the hydrogenase gene from Citrobacter freundii ATCC 29220 is integrated on the genome, the lactate dehydrogenase gene in the competitive pathway is knocked out, and the PHA synthesis gene is overexpressed, so as to further improve the PHA yield. The engineering bacterium provided by the application adopts a high-density fermentation culture method, the PHA yield in a 5L fermenter can reach 83.5% of the cell dry weight, and the cell dry weight reaches 22.14g / L. The application has important application significance for industrialized production of PHA.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and relates to an engineered bacterium that synthesizes PHA using carbon dioxide, its construction method, and its application. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of bio-based polymers that can be produced by various bacteria. When carbon sources are abundant but other nutrients such as nitrogen and phosphorus are insufficient, microorganisms convert excess carbon sources into carbon and energy storage substances within their cells, accumulating them and gradually transforming them from water-soluble small molecules into hydrophobic particulate PHA. The material properties of PHA are mainly determined by its monomer composition and ratio. Depending on the monomer composition, PHA exhibits a range of polymer properties, from hard and brittle crystals to soft elastomers. Short-chain PHAs mostly have high crystallinity and exhibit strong plastic properties; while medium- and long-chain PHAs, due to their low crystallinity and low hardness, exhibit soft but tough elastomer characteristics. In addition to thermoplasticity, PHA also possesses properties not found in traditional plastics, such as optical chirality, biocompatibility, biodegradability, gas barrier properties, piezoelectricity, and recyclability. Because PHA possesses some mechanical properties similar to those of plastics such as polyethylene and polypropylene, and can be degraded by microorganisms and enter the natural ecological cycle, it is considered a "biodegradable plastic" that can replace traditional petrochemical plastics. The development and utilization of PHA materials can alleviate environmental problems such as "white pollution" and reduce carbon emissions caused by plastics, thus attracting global attention and being widely studied and applied by society and scientists.

[0003] While PHA possesses numerous desirable properties that allow it to replace traditional petrochemical plastics, its production cost remains relatively high. Studies indicate that 30% of PHA's production cost comes from the carbon source in its substrate. Using CO2 as the carbon source not only effectively reduces CO2 emissions but also achieves the dual benefit of organic matter production.

[0004] *Cupriavidus necator*, formerly known as *Ralstonia eutropha*, is a Gram-negative bacterium belonging to the phylum Betaproteobacteria, isolated in Germany 55 years ago. It is a rod-shaped, non-pathogenic, non-spore-forming neutrophil with an optimal growth temperature of 30°C. *Cupriavidus necator* can achieve autotrophic growth by assimilating CO2 through the Calvin-Benson-Basham cycle (CBB) and can also achieve heterotrophic growth using fructose, gluconic acid, various organic acids, and even aromatic compounds as carbon and energy sources. For many years, *Cupriavidus necator* has been used as a model microorganism for studying autotrophic PHA metabolism using H2 and CO2. Therefore, providing a method for constructing an engineered bacterium that synthesizes PHA using carbon dioxide, achieving the conversion of carbon dioxide to PHA, and increasing PHA production is a problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an engineered strain of hookworm copper-loving bacterium (Cupriavidusnecator) that utilizes carbon dioxide to synthesize PHA.

[0006] In a first aspect, the present invention provides a method for constructing engineered bacteria that synthesize polyhydroxy fatty acid esters using carbon dioxide. The method uses *Cupriavidus necator* H16 as the starting strain, integrating the Rubisco (ribulose-1,2-bisphosphate carboxylase) gene from *Methanothrix soehngenii* GP6 (promoted by the Pj5 promoter) into the genome of *Cupriavidus necator* H16 to replace the original Rubisco gene, thus obtaining strain H16-1; and using *Citrobacter youngae* ATCC 29220. The hydrogenase gene from strain 29220 was integrated into the genome of strain H16-1 to replace the original hydrogenase gene, resulting in strain H16-2. The LDH (lactate dehydrogenase) gene was knocked out in the genome of strain H16-2 to obtain strain H16-3. Finally, a recombinant plasmid containing genes encoding acetyl-CoA-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway was transferred into strain H16-3 to overexpress these three enzymes, resulting in strain H16-4.

[0007] In some embodiments, in the above construction method, the sequence of the Rubisco gene derived from Methanophora spp. GP6 is shown as positions 476 to 1903 in SEQ ID NO:9, and the amino acid sequence of the ribulose diphosphate carboxylase it encodes is shown in SEQ ID NO:10.

[0008] In some implementations, in any of the above-described construction methods, the sequence of the Pj5 promoter is as shown in positions 401 to 475 of SEQ ID NO: 9.

[0009] In some embodiments, in any of the above-described construction methods, the Rubisco gene derived from *Methanophora somnifera* GP6 promoted by the Pj5 promoter is located on the Pj5-MsRubisco-Donor fragment (sequence as shown in SEQ ID). As shown in NO:9, this fragment was integrated into the genome of H16 hookworm copper-eating bacterium using a suicide plasmid-mediated target gene deletion strategy to replace the original Rubisco gene, resulting in strain H16-1. Specifically, the Pj5-MsRubisco-Donor fragment and the suicide pK19mobSacB plasmid were ligated to obtain the pK19-Pj5-MsRubisco-Donor plasmid. The pK19-Pj5-MsRubisco-Donor plasmid was then transferred into H16 hookworm copper-eating bacterium through conjugation. After two homologous single-exchange screenings, a strain was obtained in which the Rubisco gene from GP6 methanotherapeutic bacteria, promoted by the Pj5 promoter, was integrated into the genome of H16 hookworm copper-eating bacterium to replace the original Rubisco gene, i.e., strain H16-1.

[0010] In some embodiments, in any of the above-described construction methods, the sequence of the hydrogenase gene derived from *Citrobacter yangensis* ATCC29220 is shown from position 501 to position 1781 in SEQ ID NO:21, and the amino acid sequence of the hydrogenase it encodes is shown in SEQ ID NO:22.

[0011] In some embodiments, in any of the above-described construction methods, the hydrogenase gene fragment from *Citrobacter yangensis* ATCC29220 is located on the CyHydrogenase-Donor fragment (sequence shown in SEQ ID NO:21). This fragment is integrated into the H16-1 genome using a suicide plasmid-mediated target gene deletion strategy to replace the original hydrogenase gene, resulting in strain H16-2. Specifically, this can be achieved by: ligating the CyHydrogenase-Donor fragment and the suicide pK19mobSacB plasmid to obtain the pK19-CyHydrogenase-Donor plasmid; then, transferring the pK19-CyHydrogenase-Donor plasmid into strain H16-1 via conjugation; and finally, obtaining strain H16-2 by two homologous single-crossover screenings, in which the hydrogenase gene fragment from *Citrobacter yangensis* ATCC29220 is integrated into the H16-1 genome to replace the original hydrogenase gene.

[0012] In some embodiments, in any of the above-described construction methods, the LDH gene on the genome of strain H16-2 is replaced by an LDH-Donor fragment, the sequence of which is shown in SEQ ID NO:31. This fragment is integrated into the genome of H16-2 to knock out the LDH gene using a suicide plasmid-mediated target gene deletion strategy, resulting in strain H16-3. Specifically, the LDH gene knockout can be achieved by: ligating the LDH-Donor fragment and the suicide pK19mobSacB plasmid to obtain the pK19-LDH-Donor plasmid; then transferring the pK19-LDH-Donor plasmid into H16-2 via conjugation; and finally, obtaining the LDH gene knockout strain, i.e., strain H16-3, through two homologous single-exchange screenings.

[0013] In some embodiments, in any of the above-described construction methods, the integration of the Rubisco gene from *Methanophora sowii* GP6, the integration of the hydrogenase gene from *Citrobacter yangii* ATCC 29220, and the knockout of the LDH gene are all achieved through a suicide plasmid-mediated target gene deletion strategy.

[0014] A suicide plasmid-mediated target gene deletion strategy, with recombination primarily mediated by the endogenous RecA-dependent system. A sacB-based anti-selection method is employed. The sacB gene encodes levoglucosidase, which hydrolyzes sucrose to release glucose and synthesize fructose. Because fructose accumulates in the periplasm of Gram-negative bacteria, leading to cell lysis, it is highly toxic to many bacteria. After the first single crossover occurs during resistance selection, bacteria are placed on a reverse-selection medium containing a high concentration of sucrose. Under these conditions, mutants that have undergone one single crossover express the sacB gene; only cells reverting to wild-type or target mutants can grow. PCR can then be used to distinguish between wild-type and target mutants.

[0015] In some embodiments, in any of the above-described construction methods, the amino acid sequences of acetyl-CoA C-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway are shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively; the gene sequences of these three enzymes are shown in positions 1886 to 3067, 3142 to 3882, and 32 to 1801 of SEQ ID NO:38, respectively.

[0016] In some embodiments, in any of the above-described construction methods, the genes encoding acetyl-CoA C-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway are derived by P phaC Startup sub-boot.

[0017] In some implementations, in the above construction method, the P phaC The sequence of the promoter is shown in positions 1 to 31 of SEQ ID NO:38.

[0018] In some implementations, any of the above-described construction methods contains P pha The fragment of the gene for acetyl-CoA C-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway initiated by the promoter is the PphaC-phaCAB fragment, the sequence of which is shown in SEQ ID NO:38.

[0019] The PphaC-phaCAB fragment was transferred into strain H16-3 via a recombinant plasmid to obtain strain H16-4.

[0020] The recombinant plasmid can be pBBR1-PphaC-PHA plasmid, which is constructed by the following method: digesting pBBRMSC1 plasmid with XbaI to obtain the backbone; ligating the PphaC-phaCAB fragment and the backbone in the Gibson manner to obtain pBBR1-PphaC-PHA plasmid.

[0021] In a second aspect, the present invention provides engineered bacteria constructed by any of the methods described above.

[0022] In a third aspect, the present invention provides the application of the above-mentioned engineered bacteria in the synthesis of polyhydroxy fatty acid esters.

[0023] In a fourth aspect, the present invention provides a method for synthesizing polyhydroxy fatty acid esters using carbon dioxide, comprising the steps of fermenting the engineered bacteria to produce polyhydroxy fatty acid esters with a volume ratio of H2:CO2:O2 of (5-9):(1-3):1, pH of 6-8, temperature of 25℃-30℃, tank pressure of 0.03Mpa-0.08Mpa, aeration rate of 3-7L / min, and aeration for 1min every 4-8h;

[0024] Fermentation is preferably carried out under the following conditions: a volume ratio of H2:CO2:O2 of 7:1:1, pH 7.0, temperature of 30°C, tank pressure of 0.05 MPa, aeration rate of 5 L / min, aeration for 1 min every 6 hours, and a rotation speed of 220 rpm.

[0025] In some embodiments, the fermentation medium in the above method consists of 8-10 g / L Na2HPO4·12H2O, 1-2 g / L KH2PO4, 0.5-2 g / L (NH4)2SO4, 70-100 mg / L MgSO4·7H2O, 0.5-2 mg / L CaSO4·2H2O, 0.4-0.6 mg / L NiSO4·7H2O, 0.2-0.6 mg / L ferric citrate, 100-300 mg / L NaHCO3, and 0.5-1.5 mL / L trace elements.

[0026] Trace elements are commercially available and their components are ferric disodium ethylenediaminetetraacetate (EDTA-Na2Fe), boric acid (H3BO3), manganese sulfate (MnSO4·4H2O), zinc sulfate (ZnSO4·7H2O), copper sulfate (CuSO4·5H2O), and ammonium molybdate (NH4)6Mo7O4·4H2O.

[0027] In some embodiments, the method described above further includes a seed culture step prior to fermentation, inoculating the engineered bacteria into MM liquid medium with fructose as the carbon source and culturing to OD. 600 The concentration is approximately 1.5, resulting in seed liquid.

[0028] During fermentation, the inoculum volume of the seed liquid can be 5-15% (v / v), for example, 10% (v / v).

[0029] This invention provides a method for constructing an engineered bacterium that synthesizes PHA using carbon dioxide, thereby increasing PHA yield by enhancing carbon dioxide fixation efficiency. First, the exogenous Rubisco (ribulose-1,2-bisphosphate carboxylase) gene is integrated into the genome of H16 hookworm *Bacillus thuringiensis*, and the original promoter is replaced. Next, an exogenous hydrogenase gene is integrated into the genome, and the PHA synthesis gene is overexpressed. To further increase yield, the competing pathway lactate dehydrogenase gene is knocked out. The engineered bacterium H16-4 provided by this invention utilizes CO2 as a carbon source and H2 as an electron donor, achieving a PHA yield of 80.8% of cell dry weight and a cell dry weight of 1.56 g / L at the shake-flask level. Using a high-density fermentation culture method, the PHA yield in a 5L fermenter can reach 83.5% of cell dry weight and a cell dry weight of 22.14 g / L, representing a 4.2-fold increase in PHA yield compared to the starting strain. Furthermore, the modified strain grows faster than the starting strain. This invention lays the foundation for the industrial production of PHA by metabolically engineering hookworm copper-loving bacteria H16, and provides an effective solution for the production of biodegradable plastics. Attached Figure Description

[0030] Figure 1The image shows a gel electrophoresis diagram of nucleic acid in H16 hookworm copper-eating bacterium, where the Rubisco gene from Methanotherium GP6 has been integrated into the genome for PCR verification. In the image, M is the DNA Marker; lane C is the PCR product of the wild-type hookworm copper-eating bacterium H16 genome; and lane 1 is the PCR product of the H16-1 strain obtained by colony PCR screening.

[0031] Figure 2 The image shows a nucleic acid gel electrophoresis diagram of H16-1, in which the hydrogenase gene from Citrobacter yangi ATCC 29220 has been integrated into the genome for PCR verification. In the image, M is the DNA Marker; lane C is the PCR product of the wild-type hookworm copper scavenger H16; lanes 1 and 2 are the PCR products of the H16-2 strain obtained by colony PCR screening and the PCR product of another positive strain, respectively.

[0032] Figure 3 The image shows the gel electrophoresis results of the LDH gene knocked out in H16-2 for PCR verification; where M is the DNA Marker; lane C is the PCR product of the wild-type hookworm H16; lanes 1-4 are the PCR products of the H16-3 strain obtained by colony PCR screening and the PCR products of three other positive strains, respectively.

[0033] Figure 4 This is a plasmid map of the recombinant plasmid pBBR1-PphaC-PHA.

[0034] Figure 5 This is a gas chromatogram of PHA standard.

[0035] Figure 6 The image shows a gas chromatogram of the fermentation broth sample of strain H16-4. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0037] The pKRSF1010-Pj5-egfp plasmid was published in the literature “Gruber S, Hagen J, Schwab H, et al. Versatile and stable vectors for efficient gene expression in Ralstoniaeutropha H16[J]. New Biotechnology, 2014” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0038] Methanothrix soehngenii GP6 was disclosed in the literature “Boone D R.StrainGP6 Is Proposed as the Neotype Strain of Methanothrix soehngeniiVP prosynon. Methanothrix conciliiVP and Methanosaeta conciliiVP Request for an Opinion[J].Int.j.syst Bacteriol,1991,41(4):588-589” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0039] Cupriavidus necator H16 has been disclosed in the literature “Panich J, Fong B, Singer SW. Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2[J]. Trends in Biotechnology,2021” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0040] The pK19mobSacB plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P1363.

[0041] pET-28a(+)-CyHydrogenase plasmid is a product of Qingke Biotechnology.

[0042] The pBBRMSC1 plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P0305.

[0043] Example 1: Integration of exogenous Rubisco gene into Cupriavidus necator H16

[0044] 1. Obtaining the Pj5-MsRubisco fragment

[0045] (1) Using pKRSF1010-Pj5-egfp plasmid as a template, PCR amplification was performed with primers 1 and 2 to obtain the Pj5 promoter.

[0046] Primer 1: 5'-ggcgtcgcgccagggcaaggtcgatccattaggt-3' (SEQ ID NO: 1);

[0047] Primer 2: 5'-agagttcttcttgaatcatagcggatataaaaacc-3' (SEQ ID NO: 2).

[0048] (2) Using the genomic DNA of Methanothrix soehngenii GP6 as a template, the Rubisco gene was obtained by PCR amplification using primers 3 and 4.

[0049] Primer 3: 5'-ttttatatccgctatgattcaagaagaactctcaaa-3' (SEQ ID NO:3);

[0050] Primer 4: 5'-caaggagacaagctcacttctttttcttcttgctga-3' (SEQ ID NO: 4).

[0051] (3) Using the Pj5 promoter obtained in step (1) and the Rubisco gene obtained in step (2) as templates, fusion PCR was performed using primers 1 and 4 to obtain the Pj5-MsRubisco fragment.

[0052] 2. Obtaining the Pj5-MsRubisco-Donor fragment

[0053] (1) Using the genomic DNA of Cupriavidus necator H16 as a template, PCR amplification was performed using primers 5 and 6 to obtain the upstream fragment of the Rubisco gene.

[0054] Primer 5: 5'-aacgacaacaggagacaggcacctggcgcaccaggcggaa-3' (SEQ ID NO: 5);

[0055] Primer 6: 5'-taaacctaatggatcgaccttgccctggcgcgacgccacc-3' (SEQ ID NO: 6).

[0056] (2) Using the genomic DNA of Cupriavidus necator H16 as a template, PCR amplification was performed using primers 7 and 8 to obtain the downstream fragment of the Rubisco gene.

[0057] Primer 7: 5'-gcaagaagaaaaagaagtgagcttgtctccttgcgtggtt-3' (SEQ ID NO:7);

[0058] Primer 8: 5'-ttcttcagcagcgcgttgaagcgttccgtataccgcgcga-3' (SEQ ID NO: 8).

[0059] (3) Using the upstream fragment of the Rubisco gene obtained in step (1), the downstream fragment of the Rubisco gene obtained in step (2), and the above-mentioned Pj5-MsRubisco fragment as templates, fusion PCR was performed using primers 5 and 8 to obtain the Pj5-MsRubisco-Donor fragment. The sequence of this fragment is shown in SEQ ID NO:9, where positions 401 to 475 are the Pj5 promoter, and positions 476 to 1903 are the Rubisco gene from Methanophora GP6. The amino acid sequence of the protein (ribulose diphosphate carboxylase) encoded by this Rubisco gene is shown in SEQ ID NO:10.

[0060] 3. Construction of pK19-Pj5-MsRubisco-Donor plasmid

[0061] The pK19mobSacB plasmid was digested with BamHI to obtain the backbone; the Pj5-MsRubisco-Donor fragment and the backbone were ligated using Gibson ligation to obtain the pK19-Pj5-MsRubisco-Donor plasmid. The plasmid was then sequenced, and the results were correct.

[0062] 4. The constructed plasmid was transferred into H16 hookworm copper-loving bacteria via conjugation. The specific steps are as follows:

[0063] (1) The pK19-Pj5-MsRubisco-Donor plasmid was transformed into Escherichia coli S17 by chemical transformation to obtain a single colony of Escherichia coli S17 containing the target plasmid.

[0064] (2) Take out the H16 strain of hookworm copper-loving bacteria preserved in the -80℃ freezer, use a sterilized pipette tip to draw 10μL of bacterial solution, streak it on LB solid medium containing 10μg / L gentamicin, and incubate it overnight in a 30℃ incubator. The next day, pick a single colony and culture it in LB medium.

[0065] (3) Pick up a single colony of Escherichia coli S17 containing the target plasmid with a pipette tip and culture it in liquid LB containing 50 μg / L kanamycin at 37°C and 250 rpm for 12 h with shaking.

[0066] (4) Take 1 mL of each of the bacterial cultures of Escherichia coli S17 containing the target plasmid and H16 hookworm copper-loving bacteria, and centrifuge at 12000 rpm for 1 min; remove the supernatant, wash the cells with 1 mL LB, then resuspend the cells in 100 μl LB, spread them on LB plates without antibiotics, and incubate at 30℃ for 24 h.

[0067] (5) Genotype verification after the first homologous single crossover: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and culture for 24 h; then extract 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0068] Primers A1 and B1 are primers located on the pK19-MsRubisco-Donor plasmid:

[0069] Primer A1: 5'-tcccctgtacacctgcgctgtg-3' (SEQ ID NO:11);

[0070] Primer B1: 5'-cgcgcgcggcatcagccgcc-3' (SEQ ID NO:12);

[0071] Primers C1 and D1 are located upstream and downstream of the Rubisco gene in the H16 genome of hookworm copper-eating bacteria:

[0072] Primer C1: 5'-attcacgatgaacgacatcacc-3' (SEQ ID NO:13);

[0073] Primer D1: 5'-atcgagaccgctggctgggtca-3' (SEQ ID NO: 14);

[0074] To verify whether the first homologous single exchange was successful, colony PCR was performed using primers C1 and B1 or primers A1 and D1. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 minute, only one pair of primers can produce a band for the same clone.

[0075] Finally, a single clone that could amplify the band was obtained. During the first homologous single exchange, the entire plasmid pK19-MsRubisco-Donor was integrated into the H16 genome of hookworm copper worm through a single exchange of one homologous arm.

[0076] (6) Second homologous single exchange: The single clones obtained in step (5) were subjected to two sucrose inductions (i.e., one liquid induction and one plate induction), and then resistance screening was performed to obtain clones that grew on LB plates containing 10 μg / L gentamicin but did not grow on LB plates containing 50 μg / L kanamycin. Colony PCR verification was performed using primers C1 and D1 to obtain positive clones that could amplify the target band. In this positive clone, homologous recombination was performed between homologous regions within the integrated fragment through the second homologous single exchange, and the Pj5-MsRubisco fragment was integrated into the genome of H16 hookworm copper worm to replace the original Rubisco gene. This positive clone was named strain H16-1. In the colony PCR verification, H16 hookworm copper worm was used as a control.

[0077] The specific steps for the two sucrose inductions are as follows:

[0078] The single clone was liquid-induced for 72 h in LB agar plates without NaCl and with 100 g / L sucrose. After liquid induction, 1 μL of bacterial culture was diluted 1000 times, and 100 μL was spread on LB agar plates without NaCl and with 50 g / L sucrose. The plates were incubated for 24 h and then induced again.

[0079] Generally, the gene knockout efficiency using the suicide-type pK19mobSacB plasmid is up to 50%, so 16-24 single colonies are usually selected for PCR verification. In order to obtain a stable monoclonal strain, after PCR verification, it is generally necessary to perform subculture and PCR verification again.

[0080] Colony PCR validation results are as follows Figure 1 As shown, the results indicate that engineered bacteria H16-1, which integrates the exogenous Rubisco gene at the original Rubisco gene locus, were successfully screened.

[0081] Example 2: Integration of exogenous hydrogenase gene into engineered strain H16-1

[0082] 1. Obtaining the hydrogenase gene

[0083] Using pET-28a(+)-CyHydrogenase plasmid as a template, PCR amplification was performed using primers 9 and 10 to obtain the hydrogenase gene derived from Citrobacter youngae ATCC 29220.

[0084] Primer 9: 5'-atgcctcacaggacaacgcgatgaacagcgtacaactcgc-3' (SEQ ID NO: 15);

[0085] Primer 10: 5'-gcgccccatacctgcgtcgtttatttttcctcgccgtaca-3' (SEQ ID NO: 16).

[0086] 2. Obtaining the CyHydrogenase-Donor fragment

[0087] (1) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 11 and 12 to obtain the upstream fragment of the hydrogenase gene.

[0088] Primer 11: 5'-aacgacaacaggagacaggcagatgtttcttgcccccatc-3' (SEQ ID NO: 17);

[0089] Primer 12: 5'-gcgagttgtacgctgttcatcgcgttgtcctgtgaggcat-3' (SEQ ID NO: 18).

[0090] (2) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 13 and 14 to obtain the downstream fragment of the hydrogenase gene.

[0091] Primer 13: 5'-tgtacggcgaggaaaaataaacgacgcaggtatggggcgc-3' (SEQ ID NO: 19);

[0092] Primer 14: 5'-ttcttcagcagcgcgttgaaatcccgctctccgaggcatg-3' (SEQ ID NO:20).

[0093] (3) Using the upstream fragment of the hydrogenase gene obtained in step (1), the downstream fragment of the hydrogenase gene obtained in step (2), and the above-mentioned hydrogenase gene as templates, fusion PCR was performed using primers 11 and 14 to obtain the CyHydrogenase-Donor fragment. The sequence of this fragment is shown in SEQ ID NO:21, where positions 501 to 1781 are hydrogenase genes derived from Citrobacter yangensis ATCC 29220, and the amino acid sequence of the hydrogenase encoded by it is shown in SEQ ID NO:22.

[0094] 3. Construction of pK19-CyHydrogenase-Donor plasmid

[0095] The obtained CyHdrogenase-Donor fragment and pK19mobSacB plasmid backbone were ligated using Gibson to obtain the recombinant plasmid pK19-CyHydrogenase-Donor. The plasmid was then sequenced, and the results were correct.

[0096] 4. The constructed plasmid is transferred into H16-1 via conjugation. The specific steps are as follows:

[0097] (1)-(4) The specific steps are the same as (1)-(4) in step 4 of Example 1, except that the plasmid pK19-Pj5-MsRubisco-Donor is replaced with pK19-CyHydrogenase-Donor, and the H16 strain of Hookworm Copper-Loving Bacteria is replaced with H16-1.

[0098] (5) Genotype verification after the first homologous single crossover: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and culture for 24 h; then extract 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0099] Primers A2 and B2 are primers located on the pK19-Cydrogenase-Donor plasmid:

[0100] Primer A2: 5'-tgcaaatacgcctggcaaga-3' (SEQ ID NO:23);

[0101] Primer B2: 5'-cgcccagatgcttcttcagc-3' (SEQ ID NO:24);

[0102] Primers C2 and D2 are located upstream and downstream of the hydrogenase gene in the H16 genome of hookworm copper-loving bacteria:

[0103] Primer C2: 5'-cgacgacggacgcagtaccgcc-3' (SEQ ID NO:25);

[0104] Primer D2: 5'-gcatgggccgtatggcgcta-3' (SEQ ID NO:26);

[0105] To verify whether the first homologous single exchange was successful, primers C2 and B2 or primers A2 and D2 were selected for verification. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 min, only one pair of primers can produce a band for the same clone.

[0106] Finally, a single clone that could amplify the band was obtained. During the first homologous single crossover, the entire plasmid pK19-Cydrogenase-Donor was integrated into the H16-1 genome through a single crossover of one homologous arm.

[0107] (6) Second homologous single exchange: The specific steps are the same as step 4 (6) in Example 1, except that the single clone to be induced is replaced with the single clone obtained by screening in step (5) above, and colony PCR is performed using primer C2 and primer D2.

[0108] Finally, a positive clone capable of amplifying the target band was obtained. In this positive clone, homologous recombination occurred between homologous regions within the integrated fragment through a second homologous single exchange, achieving the integration of a hydrogenase gene fragment from *Citrobacter yangensis* ATCC29220 into the H16-1 genome, replacing the original hydrogenase gene. This positive clone was named strain H16-2. In colony PCR verification, *Hookworm Copper-Loving Bacteria* H16 was used as a control.

[0109] Colony PCR validation results are as follows Figure 2 As shown, the results indicate that engineered bacteria H16-2, which integrates an exogenous hydrogenase gene at the original hydrogenase gene locus, were successfully screened.

[0110] Example 3: Knockout of the LDH gene in engineered bacterium H16-2

[0111] 1. Obtaining LDH-Donor fragments

[0112] (1) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 15 and 16 to obtain the upstream fragment of the LDH gene.

[0113] Primer 15: 5'-gacatgattacaagcttggaattttctgccagcgccattac-3' (SEQ ID NO:27);

[0114] Primer 16: 5'-gaggtacgaaccctcgagaggttccgcctgcggcg-3' (SEQ ID NO: 28).

[0115] (2) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 17 and 18 to obtain the downstream fragment of the LDH gene.

[0116] Primer 17: 5'-cggaacctctcgagggttcgtacctcctcagacc-3' (SEQ ID NO: 29);

[0117] Primer 18: 5'-gagctcggtacccgggcgcaatgtcggtggtgctgt-3' (SEQ ID NO: 30).

[0118] (3) Using the upstream fragment of the LDH gene obtained in step (1) and the downstream fragment of the LDH gene obtained in step (2) as templates, fusion PCR was performed using primers 15 and 18 to obtain the LDH-Donor fragment, the sequence of which is shown in SEQ ID NO:31.

[0119] 2. Construction of pK19-LDH-Donor plasmid

[0120] The obtained LDH-Donor fragment and the pK19mobSacB plasmid backbone were ligated using Gibson to obtain the recombinant plasmid pK19-LDH-Donor. The plasmid was then sequenced, and the results were correct.

[0121] 3. The constructed plasmid was transferred into H16-2 via conjugation. The specific steps are as follows:

[0122] (1)-(4) The specific steps are the same as (1)-(4) in step 4 of Example 1, except that the plasmid pK19-Pj5-MsRubisco-Donor is replaced with pK19-LDH-Donor, and the H16 strain of hookworm copper-loving bacteria is replaced with H16-2.

[0123] (5) Genotype verification after the first homologous single crossover: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and culture for 24 h; then extract 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0124] Primers A3 and B3 are primers located on the pK19-LDH-Donor plasmid;

[0125] Primer A3: 5'-tgcaaatacgcctggcaaga-3' (SEQ ID NO:32);

[0126] Primer B3: 5'-cgcccagatgcttcttcagc-3' (SEQ ID NO:33);

[0127] Primers C3 and D3 are located upstream and downstream of the LDH gene in the H16 genome of hookworm copper-loving bacteria;

[0128] Primer C3: 5'-cgccattaccgacggcttac-3' (SEQ ID NO:34);

[0129] Primer D3: 5'-cctggtgcgcttgctgctgc-3' (SEQ ID NO:35);

[0130] To verify whether the first homologous single exchange was successful, primers C3 and B3 or primers A3 and D3 were selected for verification. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 min, only one pair of primers can produce a band for the same clone.

[0131] Finally, a single clone that could amplify the band was obtained. During the first homologous single crossover, the entire plasmid pK19-LDH-Donor was integrated into the H16-2 genome through a single crossover of one homologous arm.

[0132] (6) Second homologous single exchange: The specific steps are the same as step 4 (6) in Example 1, except that the single clone to be induced is replaced with the single clone obtained by screening in step (5) above, and colony PCR is performed using primer C3 and primer D3.

[0133] Finally, a positive clone capable of amplifying the target band was obtained. In this positive clone, homologous recombination occurred between homologous regions within the integrated fragment through a second homologous single crossover, resulting in the knockout of the LDH gene on the H16-2 genome. This positive clone was named strain H16-3. In colony PCR verification, H16 hookworm copper scavenger was used as a control.

[0134] Colony PCR validation results are as follows Figure 3 As shown, the results indicate that engineered bacteria H16-3 with the LDH gene knocked out were obtained through screening.

[0135] Example 4: Construction of PHA-producing strain

[0136] 1. Obtaining the PphaC-phaCAB fragment

[0137] Using the genomic DNA of H16 hookworm copper bacterium as a template, PCR amplification was performed with primers 19 and 20 to obtain the PphaC-phaCAB fragment.

[0138] Primer 19: 5'-tgagcctttcgttttatttgctcagcccatatgcaggc-3' (SEQ ID NO:36);

[0139] Primer 20: 5'-ccgggccccccctagatctagacgaatagtg-3' (SEQ ID NO:37).

[0140] The sequence of the PphaC-phaCAB fragment is shown in SEQ ID NO:38, wherein positions 1 to 31 are the PphaC promoter, positions 1886 to 3067 are the acetyl-CoA C-acetyltransferase gene, positions 3142 to 3882 are the acetyl-CoA reductase gene, and positions 32 to 1801 are the PHA synthase gene. The amino acid sequence of acetyl-CoA C-acetyltransferase is shown in SEQ ID NO:39, the amino acid sequence of acetyl-CoA reductase is shown in SEQ ID NO:40, and the amino acid sequence of the PHA synthase gene is shown in SEQ ID NO:41.

[0141] 2. Construction of pBBR1-PphaC-PHA plasmid

[0142] The pBBRMSC1 plasmid was digested with XbaI to obtain the backbone. The PphaC-phaCAB fragment and the backbone were ligated using the Gibson method to obtain the pBBR1-PphaC-PHA plasmid. This plasmid was sequenced, and the results were correct. The pBBR1-PphaC-PHA plasmid map is shown below. Figure 4 As shown.

[0143] 3. The pBBR1-PphaC-PHA plasmid was transferred into H16 and H16-3 of Hookworm Copper-Loving Bacteria by electroporation. The recombinant engineered bacteria were obtained by kanamycin screening and named H16-PHA and H16-4 strains, respectively.

[0144] Example 5: Shake-flask culture of engineered bacteria and PHA detection

[0145] Strains H16-PHA and H16-4 were selected and inoculated into 5 mL of LB medium, respectively, and cultured at 30℃ and 220 r / min for 16 h. Then, 1% (v / v) of the culture medium was inoculated into a 250 mL fermenter containing 100 mL of MM liquid medium (9 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 1 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L ferric citrate, 200 mg / L NaHCO3, 1 mL / L trace elements) for fermentation.

[0146] The fermentation system was placed inside a fume hood, utilizing a hydrogen generator and a 100mL fermenter to minimize the risk of explosion. The gas volume ratio of H2:CO2:O2 = 7:1:1 was used to achieve a low initial OD. 600 The inoculum level was adjusted to achieve rapid cell growth and product production in one step. The aeration rate was 5L / min, with aeration time of 1min every 6 hours. Fermentation was carried out at 30℃ and 220rpm.

[0147] After the strain completes the target fermentation, 1 ml of the sample is frozen and stored for OD measurement. 600 The remaining bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was removed, and the bacterial cells were washed three times with distilled water, centrifuged at 8000 rpm for 5 min after each wash. The cells were then dried in a 60℃ drying oven (approximately 5 h). The bacterial cells were crushed, and 20 mg of the dried culture was weighed into a heat-resistant black-capped test tube. 2 ml of chloroform and 2 ml of a 3% concentrated sulfuric acid methanol solution were added, the black-capped test tube was tightened, and the tube was dried in a forced-air drying oven at 100℃ for 4 h. After cooling, 1 ml of distilled water was added, the mixture was shaken, and the layers were allowed to separate. The lower chloroform layer was then collected for GC analysis.

[0148] The standard GC analysis procedure is as follows: An Agilent 7890B gas chromatograph equipped with an HP-5 column, a nitrogen flow rate of 19 ml / min as the carrier gas, an initial column temperature of 50°C for 5 min, followed by an increase to 300°C at a rate of 10°C per minute, and a holding time of 3 min.

[0149] The gas chromatogram of PHA standard is shown below. Figure 5 As shown. The gas chromatogram of the H16-4 strain fermentation broth sample is shown below. Figure 6 As shown.

[0150] The retention time of the PHA standard was 3.005 min, and the retention time of the PHA peak in the fermentation broth was 2.998 min.

[0151] The growth rate and maximum OD reached of H16-4 600 The values ​​of H16-4 and H16-PHA were superior to those of H16-PHA strain. After 120 h of culture, the cell dry weight of H16-4 reached 1.56 g / L, while the cell dry weight of H16 strain was only 0.36 g / L. The PHA content in the fermentation broth of H16-4 and H16-PHA was 1.26 g / L and 0.30 g / L, respectively, and the PHA yield increased by 4.2 times.

[0152] Example 6: Production of PHA by fermentation in a 5L fermenter

[0153] The engineered strain H16-4 was inoculated into MM liquid medium with 10 g / L fructose as the carbon source and cultured until OD500. 600 The seed culture was obtained at a pH of approximately 1.5. The seed culture was inoculated at a rate of 10% (v / v) into a 5L fermenter containing 2.5L of fermentation medium (MM liquid medium). The pH was adjusted to 7.0 with a 35% NaOH aqueous solution, and fermentation began. The temperature of the fermentation broth was controlled at 30°C, and the pressure in the fermenter at 0.05 MPa. The fermentation system was placed in a fume hood, and a hydrogen generator and a 5L fermenter were used to minimize the risk of explosion. The H2:CO2:O2 ratio was 7:1:1, with the aim of achieving a low initial OD. 600 The inoculum level was adjusted to achieve rapid cell growth and product production in one step. Specific steps are detailed in Example 5. After fermentation, the PHA content was detected by GC (detection conditions were the same as in Example 5). The results showed that the cell dry weight of the strain after fermentation was 22.14 g / L, and the PHA content in the fermentation broth was 18.5 g / L.

[0154] sequence

[0155] SEQ ID NO:9

[0156] CCTGGCGCACCAGGCGGAAGCCGGGCTCGTCGGCCGGACGATTCACGATGAACGACA TCACCACCGACTCCACCGTATGCGTCGAATCGAAGGCCGTGACGCGGATGTAGTGGTTGGGGAAGGTGCTGCGCGCGTTGTTGATCTCCAGCAGGATGCCGGCGGCATCGCGCAG GTCGAACATCGGCAGGCCGAACATCTCCCAGTAGGTATTGCGCGGATGCGGGTCGTCG GTGTACTCGATGCCGACCGCCCAGCCCTGGTTCAGGCAGTATTCAAGCTGGCTGGTGATCTGCGCGTCGGTGAGGTCGGGCAGGAAAGAGAAAGTGCCTTGAGTAATACGCATGA TGTCCTCTCGTTCTTTGGGTTCGCTCGTCGTGCCTTGGTGGCGTCGCGCCAGGGCAAG GTCGATCCATTAGGTTTACTAACAGTATATTCTAAATTTCCACCTGTGTCAATAACGGTTTTTATATCCGCTATGATTCAAGAAGAACTCTCAAAAACGCTCAATCCGAAGCAGGTCC AGTACATGAGGATGGATCTTCCTGATCCGAGGAACGGCGAGTATCTTCTGGCTGTGTTTCACCTGATCCCCAGCGGCGAGCTCAACATTATGCAAGCCGCAGCCGAGGTCGCAGC TGAGAGCTCAACAGGCACCAATTTTGCCGTGAAGACCGAGACTCCCTTCTCAAGGGTGATGAACGCCCTCGTCTACAAGGTTGACATCGAGAAGAACCTGGTCTGGATCGCCTAT CCCTGGCGCCTGTTCGATAGAAACGGCAACGTCCAGAACATCATGACCTACATCGCCGGGAACGCCCTCGGCATGAAGGAGATCAAGGCTCTCAAGCTCCTGGACATCTGGTTCC CCCCCTCCATGCTGGAGCAGTACGACGGCCCAAGCTATACACTCGACGACATGAGAACCTACCTCAATGTGCACGACAGGCCGATACTCGGGACCATAATCAAACCCAAGATGGGGCTCACCTCTTCGGAGTACGCCGAGGTCTGCTATGACTTCTGGGTGGGCGGAGGCGA CTTCGTCAAGAACGACGAGCCCCAAGCCGACCAGGACTTCTGCCCCTACGACAAAATGGTCAAGTATGTCAAGATGGCAATGGACAAGGCGGTCAAGGAGACGGGGAAGAAGA AGGTCCACTCGTTCAACGTATCCTCGGCCGATTTCGACACCATGATCGAAAGGTGCGAGATGATCAGGGAGGCGGGCTTCGAGCCGGGTTCATACGCCTTCCTCATAGACGGCATC ACCGCCGGGTGGATGGCGGTGCAGACGCTGAGACGGAGGTATCCGGATGTCTTTTTGCACTTCCACAGGGCAGGCCACGGTGGCTTTACCAGGCCGGAGAACCCCATTGGCTTC TCAGTGCTAGTGCTCTCCAAGTTTGCCAGGTTGGCAGGCGCATCAGGTATCCACACCGGGACGGCAGGCGTCGGCAAGATGGCAGGCAGTCCCGAGGAGGACGTAACTGCGGCG AGGAACATCCTGAAAGTCGAGGGAAAGGGTCACTTCTTCACCCAGAACTGGGGAAGGATCCCGCCGCAGGACGACGATGCCATCAAGATGGTCGAGATGGACGATGCTCACCA CGTTGTCCTGGAGGACGACTCCTGGCGCGGCCTCAAGAAATGCTGCCCGATCATCTCT GGAGGACTAAACCCGACGCTCCTCGAGCCTTTCATCGATGTCATGGGAGGAATCGATTTCATTACGACCATGGGCGCGGGTTGCCACGCTCACCCGAGAGGAACACGCGCTGGTG CGATGGCCCTTGTGCAGGCATGCGAAGCCTACAAGAATAAGATCGACATCGCGGACTATGCGAAGGATCACAGAGAACTCGCCGAGGCGATCGAGTTCTTCAGCAAGAAGAAAAAGAAGTGAGCTTGTCTCCTTGCGTGGTTGAGCGTCGTGGCTGCTGCGTTGATGCAAAGATATAAGCCCACTAACATAAGGTAAATTCAGATATTCTTAGACACCGTATAAGCAATC CCTTAAGTGCGACCCAGACTGCACATCACGGAACCATTCAAAAGCCGCCCCCACTGCTCATGTCCTTCCTGCGCGCCCTCACCCTTCGCCAGTTGCTAGCTTCGTGCGCGCCGCC GAGGAACTGCACCTGACCCAGCCAGCGGTCTCGATGCAGGTCAAGCAGCTCGAATCC GTGGTCGGCCTGGCGCTGTTCGAACGGGTCAAGGGACAGTTCACGCTGCGCCCGGCGACCGCCTGCTGCCACCATGCGTCCCGGATCCTCGGCGAGGTCAAGGACGCCGAGGAA TGCCTGCAGGCCGTCAAGGACGTCAGCAGGGGTCGATCACGATCGGGCTGATCAGCACGTCGAAATACTTCGCGCCCAAGCTGCTCGCGCGGTATACGGAACGC

[0157] SEQ ID NO:10

[0158] MIQEELSKTLNPKQVQYMRMDLPDPRNGEYLLAVFHLIPSGELNIMQAAAEVAAESSTGT NFAVKTETPFSRVMNALVYKVDIEKNLVWIAYPWRLFDRNGVNVQNIMTYIAGNALGMKEIKALKLLDIWFPPSMLEQYDGPSYTLDMRTYNLVHDRPILGTIIKPKMGLTSSEYAEVCY DFWVGGGDFVKNDEPQADQDFCPYDKMVKYVKMAMDKAVKETGKKVHSFNVSSAD FDTMIERCEMIREAGFEPGSYAFLIDGITAGWMAVQTLRRRYPDVFLHFHRAGHGGFTRPENPIGFSVLVLSKFARLAGASGIHTGTAGVGKMAGSPEEDVTAARNILKVEGKGHFFTQN WGRIPPQDDDAIKMVEMDDAHHVVLEDDSWRGLKKCCPIISGGLNPTLLEPFIDVMGGIDFITTMGAGCHAHPRGTRAGAMALVQACEAYKNKIDIADYAKDHRELAEAEFFSKKK K

[0159] SEQ ID NO:21

[0160] GATGTTTCTTGCCCCCATCCCGACGACGGACGCAGTACCGCCGGTGATCGAACGTCTG GTCGAACAGCATCAGGCTGCCTGGGTTGACCAGGATTCCATTGACGACTGGCTGGCTGGCGGCGGCGATTGCGTGCTGTTTATCGCCGGCGACCCGGTGCGCTTTCCCGAGTGCG TGGATGTCGCTGTCGTGTTGCCGGAGTTGCAAAGGGTGTTCTTCAATGGTTTTCGCAT TGGCGTGGCGAAACGTGAACGTGAGCACGAAGATGTGCTGGCGAATCGCTTTGGCACGCAGCGTCGACCGTCGCTGGTGTTCTTGCGAGACGGTGCCTATGTGAGCGTAATCGCC GGCATGCGCGACTGGGACGAATACGTGCGCGAGGTCCGGCGGGCGCTCGCGATGCCGACTTCACGGCCGCCATCCATCGGCATTCCAGTGATTTCGGCGGCAGCCGGCGGCAGTT GCCACTGATCCGTCGAACAATGCCTCACAGGACAACGCGATGAACAGCGTACAACTCGCCCACGGTAGCGGCGGTCAGGCTATGCAGCAGTTGATTAACAGTCTGTTTATGGAGGCTTTTGCTAATCCGTGGCTGGCGGAACAGGAAGATCAGGCGCGTCTGGAGCTGGCGC AACTGGTCGCAGAGGGCGATCGTCTGGCGTTTTCAACCGACAGCTACGTTATCGACCCGTTGTTCTTCCCTGGCGGCAATATTGGCAAGCTGGCTATCTGCGGTACGGCTAACGATG TCGCCGTCAGCGGCGCGATCCCGCGCTACCTCTCCTGTGGTTTTATCCTTGAAGAGGG CCTGCCGATGGAGACGCTGAAAAGCGTCGTCACCAGCATGGCGGCAACCGCACGCGAAGCGGGTATCGCCATCGTGACCGGCGATACCAAAGTCGTGCAGCGTGGCGCAGCGGATAAGCTGTTCATCAACACCGCCGGTATGGGCGCGATCCCCGCCAACATTCACTGGGGCGCGCAAACCCTGAGCGTCGGTGATGTTCTGCTGGTCAGCGGTACGCTCGGCGACCA CGGCGCAACCATTCTTAATCTGCGCGAGCAATTGGGTCTTGATGGCGAACTGGTCAGCGACTGTGCGGTGCTGACGCCGCTTATCCAGACGATACGTGATATACCCGGCGTGAAGG CGCTGCGTGACGCCACCCGTGGCGGCGTGAATGCGGTGACGCACGAGTTTGCCGCAGCCTGCGGATTGGGTATTGAGCTGTCTGAAGCGGCTCTGCCCGTGAAGCCCGCGGTGC GTGGCGTTTGCGAGCTGCTGGGCCTGGATGCGCTCAACTTTGCCAACGAAGGCAAACTGGTGATTGCCGTTGAGCGCCAGGCGGCGGAAAGCGTGCTTGCCGCGCTGCGCGCGC ATCCGTTAGGACGCGATGCGGCAATGATTGGCGAAGTGGTCGAACGTAAAGGGGTACGCCTTGCCGGACTGTACGGCGTGAAACGAACCCTCGATTTACCACACGCCGAACCATT ACCTCGTATATGCATGTGCATAGGTGTTCCGGGTCAAATCCGCACCATTGACGGCAACC AGGCCAAAGTCGACGTTTGCGGCATTCAGCGCGACGTCGACCTGACGCTGGTCGGTAGCTGCGATGAAAACGGTGAGCCGCGCCTGGGCCAGTGGGTACTGGTTCACGTCGGCT TTGCCATGAGCATCATTAATGAAGCAGAAGCGCGCGACACGCTCGACGCGCTGCAAAACATGTTTGACGTTGAGCCGGATGTCGGCGCGTTGCTGTACGGCGAGGAAAAATAAA CGACGCAGGTATGGGGCGCTTCGAAGGCAGTTACCTGGGAGATCGGGGGCGCCTGGCGGCCGACGCGCGGCTGGAATGCAAGATCTGCTGGTGGGAGTACGATCCTGAAGTCGGCGATCCAGTTTGGCAGATTGCGCCCGGCACTTCATTTTCGGCGCTCCCGGCGCACTGGCGGTGTCCGAATTGCGACGGTGAAGCCGAGCAATTTATGGTGCTCGGTCCGCAGGCG TGAGAGACTGCAACGATGTTGCTGGCCAACTATGGAGCCTGTGAAGTGGACCGGGTG CGCCATCTGGAAGAGGCTTTCTGCTGCATTGCGGCAACTCGCATGGCCGATATCCCCGTGGTGAACCGCGCGTTGTCGGTGGAAGCATTGGGGTTCGAGCAGTGTGCCGAATCAG CGGGCGGCTCGGATGGCGAAATGGGGATTCTCATTACTCCCTGGTTCATGAACCTGATTTGGCTAGCGCCATACGGCCCATGCCTCGGAGAGCGGGAT

[0161] SEQ ID NO:22

[0162] MNSVQLAHGSGGQAMQQLINSLFMEAFANPWLAEQEDQARLELAQLVAEGDRLAFSTD SYVIDPLFFPGGNIGKLAICGTANDVAVSGAIPRYLSCGFILEEGLPMETLKSVVTSMAATAREAGIAIVTGDTKVVQRGAADKLFINTAGMGAIPANIHWGAQTLSVGDVLLVSGTLGDH GATILNLREQLGLDGELVSDCAVLTPLIQTIRDIPGVKALRDATRGGVNAVTHEFAAACGLGIELSEAALPVKPAVRGVCELLGLDALNFANEGKLVIAVERQAAESVLAALRAHPLGRDA AMIGEVVERKGVRLAGLYGVKRTLDLPHAEPLPRICMCIGVPGQIRTIDGNQAKVDVCGIQRDVDLTLVGSCDENGEPRLGQWVLVHVGFAMSIINEAEARDTLDALQNMFDVEPDVG ALLYGEEK

[0163] SEQ ID NO:31

[0164] GAATTTTCTGCCAGCGCCATTACCGACGGCTTACCTCAGCCTCAACGGCTCCAGCAAT GCCCCGTCATACTCTCCTCGCGAGATCCGTCCAAGCTCCAGCATCCGCTGCAGGATATAAACCTGCCGCTGCCGCGCCCGCCTCGGATTCGCCACCGGATTATTAGCCGACGGCGCC TTGGGGAGCCCGGCAAGCATCGCACACTCAGCCAGCGTCAACTGATCCAGCCGCTTGCCAAAGTAAGTCCGCGCCGCATCGGCAAAGCCGTACGCCCCCTGGCCCAGGTAGATC TTGTTCATGTACAACTCCAGGATCTCGTCCTTGGTCAGCGCCTTTTCAATCCGGTATGAAAGCAGCACCTCGTACAGCTTGCGGGTATAGGTCTTGTCCCGCGACAGGTAGAAATTG CGTGCCACCTGCATGGTGATGGTCGATGCCCCCTGCGACAGCTCGTCCGACAGGTTGGCAACGCCGGCACGGACGACGCCGATGTAGTCGATGCCGTCATGCACGTAGAAGCGCTCATCCTCGATCGCCACCACGGCGCCGGTCAGCTCGCGCGGGATCTTGCCGATGGGCACGTAGTCGGGCGTATGGCGGAAGGCGGTGAGGGCGTCGAGCGACGGCAGCTGCCGGT TGGCGGCCAGGATGGCGATGGCAGCCAGCAGCGCGCCGCCGATCACGGCGAGCACCAGCAGCTTGATGAAGGCGGACCAGAGTCTTTTCATGGCACGTATTGTGCCAGCCGTGTGCGGCAGGGTGCGAGTCCCGCCGCAGGCGGAACCTCTCGAGGGTTCGTACCTCCTCA GACCCAGCCCAGCCAGCGCCAGTAGGTGGCTGCGAACAGCAGCATCATGGCGTAGCCGATGATCGTCACGGGGATGCCGATGCGCGCGAACTGGCGGCCGTTGAAGGTTTCCGTGCCCAGGCACACCATGTTCTGCGGCGCGTTGATCGGCAGGATAAAGCCGAAGCTGACGGTAAAGCCCAGCAGCATGGTCATGCCCACGCGGTTGATGTCGCCGGGCAGTGTCTG CAGCACCGCGATCAGGATCGGCAGCAGTGCCGCGGTCAGCGCGGTGGCGCTGGCAAAGCCCAGGTGGATCAGGATCAGGAAGGCCGACAGGATCGCAAACACCAGCAGCGCG CCGTGCGCCGCCAGTCCGGAATGCGTGACCACGAACTGCCCCAGCCACTGGCCGGCGTTGGTGGACAGCAGCGCCGTGCCCAGGCTGATGCCGACGCCGAACACGATCAGCGTG CCCCACGGCGTGCGTTGCTGCATGGTCTTCCAGTCCATCACGCCGATGCGCGGCATCA TCAGGATCACCAGGCCGACGAAGGTCACGGTGGCGGTATCGAAGCTGTGCAGCTTGCCTTCGGTGGCCCAGAACAGCAGCAGGCCCAGCGATACCGCGGCCAGCCGCTTCTGCG GCGCGCTCATGGGGCCGAGGGCCGACAGCTCGCGCTGCACGGCTTCCTTGCCGCCGG GGATGGCGTCGGTTTCGGCGGGCAGCAGCAAGCGCACCAGGAAATACAGCACCACCGACATTGCG

[0165] SEQ ID NO:38

[0166] CGAATAGTGACGGCAGAGAGACAATCAAATCATGGCGACCGGCAAAGGCGCGGCAG CTTCCACGCAGGAAGGCAAGTCCCAACCATTCAAGGTCACGCCGGGGCCATTCGATCCAGCCACATGGCTGGAATGGTCCCGCCAGTGGCAGGGCACTGAAGGCAACGGCCAC GCGGCCGCGTCCGGCATTCCGGGCCTGGATGCGCTGGCAGGCGTCAAGATCGCGCCG GCGCAGCTGGGTGATATCCAGCAGCGCTACATGAAGGACTTCTCAGCGCTGTGGCAGGCCATGGCCGAGGGCAAGGCCGAGGCCACCGGTCCGCTGCACGACCGGCGCTTCGC CGGCGACGCATGGCGCACCAACCTCCCATATCGCTTCGCTGCCGCGTTCTACCTGCTCAATGCGCGCGCCTTGACCGAGCTGGCCGATGCCGTCGAGGCCGATGCCAAGACCCGC CAGCGCATCCGCTTCGCGATCTCGCAATGGGTCGATGCGATGTCGCCCGCCAACTTCCTTGCCACCAATCCCGAGGCGCAGCGCCTGCTGATCGAGTCGGGCGGCGAATCGCTGC GTGCCGGCGTGCGCAACATGATGGAAGACCTGACACGCGGCAAGATCTCGCAGACCGACGAGAGCGCGTTTGAGGTCGGCCGCAATGTCGCGGTGACCGAAGGCGCCGTGGTCT TCGAGAACGAGTACTTCCAGCTGTTGCAGTACAAGCCGCTGACCGACAAGGTGCACG CGCGCCCGCTGCTGATGGTGCCGCCGTGCATCAACAAGTACTACATCCTGGACCTGCAGCCGGAGAGCTCGCTGGTGCGCCATGTGGTGGAGCAGGGACATACGGTGTTTCTGGT GTCGTGGCGCAATCCGGACGCCAGCATGGCCGGCAGCACCTGGGACGACTACATCGAGCACGCGGCCATCCGCGCCATCGAAGTCGCGCGCGACATCAGCGGCCAGGACAAGATCAACGTGCTCGGCTTCTGCGTGGGCGGCACCATTGTCTCGACCGCGCTGGCGGTGCT GGCCGCGCGCGGCGAGCACCCGGCCGCCAGCGTCACGCTGCTGACCACGCTGCTGGACTTTGCCGACACGGGCATCCTCGACGTCTTTGTCGACGAGGGCCATGTGCAGTTGCG CGAGGCCACGCTGGGCGGCGGCGCCGGCGCGCCGTGCGCGCTGCTGCGCGGCCTTGAGCTGGCCAATACCTTCTCGTTCTTGCGCCCGAACGACCTGGTGTGGAACTACGTGGT CGACAACTACCTGAAGGGCAACACGCCGGTGCCGTTCGACCTGCTGTTCTGGAACGGCGACGCCACCAACCTGCCGGGGCCGTGGTACTGCTGGTACCTGCGCCACACCTACCT GCAGAACGAGCTCAAGGTACCGGGCAAGCTGACCGTGTGCGGCGTGCCGGTGGACC TGGCCAGCATCGACGTGCCGACCTATATCTACGGCTCGCGCGAAGACCATATCGTGCCGTGGACCGCGGCCTATGCCTCGACCGCGCTGCTGGCGAACAAGCTGCGCTTCGTGCT GGGTGCGTCGGGCCATATCGCCGGTGTGATCAACCCGCCGGCCAAGAACAAGCGCAGCCACTGGACTAACGATGCGCTGCCGGAGTCGCCGCAGCAATGGCTGGCCGGCGCCAT CGAGCATCACGGCAGCTGGTGGCCGGACTGGACCGCATGGCTGGCCGGGCAGGCCGGCGCGAAACGCGCCGCGCCCGCCAACTATGGCAATGCGCGCTATCGCGCAATCGAAC CCGCGCCTGGGCGATACGTCAAAGCCAAGGCATGACGCTTGCATGAGTGCCGGCGTGCGTCATGCACGGCGCCGGCAGGCCTGCAGGTTCCCTCCCGTTTCCATTGAAAGGACTACACAATGACTGACGTTGTCATCGTATCCGCCGCCCGCACCGCGGTCGGCAAGTTTGGC GGCTCGCTGGCCAAGATCCCGGCACCGGAACTGGGTGCCGTGGTCATCAAGGCCGCGCTGGAGCGCGCCGGCGTCAAGCCGGAGCAGGTGAGCGAAGTCATCATGGGCCAGGT GCTGACCGCCGGTTCGGGCCAGAACCCCGCACGCCAGGCCGCGATCAAGGCCGGCCTGCCGGCGATGGTGCCGGCCATGACCATCAACAAGGTGTGCGGCTCGGGCCTGAAGGC CGTGATGCTGGCCGCCAACGCGATCATGGCGGGCGACGCCGAGATCGTGGTGGCCGGCGGCCAGGAAAACATGAGCGCCGCCCCGCACGTGCTGCCGGGCTCGCGCGATGGTTT CCGCATGGGCGATGCCAAGCTGGTCGACACCATGATCGTCGACGGCCTGTGGGACGTGTACAACCAGTACCACATGGGCATCACCGCCGAGAACGTGGCCAAGGAATACGGCAT CACACGCGAGGCGCAGGATGAGTTCGCCGTCGGCTCGCAGAACAAGGCCGAAGCCGCGCAGAAGGCCGGCAAGTTTGACGAAGAGATCGTCCCGGTGCTGATCCCGCAGCGCA AGGGCGACCCGGTGGCCTTCAAGACCGACGAGTTCGTGCGCCAGGGCGCCACGCTGGACAGCATGTCCGGCCTCAAGCCCGCCTTCGACAAGGCCGGCACGGTGACCGCGGCC AACGCCTCGGGCCTGAACGACGGCGCCGCCGCGGTGGTGGTGATGTCGGCGGCCAAGGCCAAGGAACTGGGCCTGACCCCGCTGGCCACGATCAAGAGCTATGCCAACGCCGG TGTCGATCCCAAGGTGATGGGCATGGGCCCGGTGCCGGCCTCCAAGCGCGCCCTGTCGCGCGCCGAGTGGACCCCGCAAGACCTGGACCTGATGGAGATCAACGAGGCCTTTGCCGCGCAGGCGCTGGCGGTGCACCAGCAGATGGGCTGGGACACCTCCAAGGTCAATGTGAACGGCGGCGCCATCGCCATCGGCCACCCGATCGGCGCGTCGGGCTGCCGTATCCTG GTGACGCTGCTGCACGAGATGAAGCGCCGTGACGCGAAGAAGGGCCTGGCCTCGCTGTGCATCGGCGGCGGCATGGGCGTGGCGCTGGCAGTCGAGCGCAAATAAGGAAGGG GTTTTCCGGGGCCGCGCGCGGTTGGCGCGGACCCGGCGACGATAACGAAGCCAATCAAGGAGTGGACATGACTCAGCGCATTGCGTATGTGACCGGCGGCATGGGTGGTATCGG AACCGCCATTTGCCAGCGGCTGGCCAAGGATGGCTTTCGTGTGGTGGCCGGTTGCGG CCCCAACTCGCCGCGCCGCGAAAAGTGGCTGGAGCAGCAGAAGGCCCTGGGCTTCGATTTCATTGCCTCGGAAGGCAATGTGGCTGACTGGGACTCGACCAAGACCGCATTCGA CAAGGTCAAGTCCGAGGTCGGCGAGGTTGATGTGCTGATCAACAACGCCGGTATCACCCGCGACGTGGTGTTCCGCAAGATGACCCGCGCCGACTGGGATGCGGTGATCGACAC CAACCTGACCTCGCTGTTCAACGTCACCAAGCAGGTGATCGACGGCATGGCCGACCGTGGCTGGGGCCGCATCGTCAACATCTCGTCGGTGAACGGGCAGAAGGGCCAGTTCGG CCAGACCAACTACTCCACCGCCAAGGCCGGCCTGCATGGCTTCACCATGGCACTGGCGCAGGAAGTGGCGACCAAGGGCGTGACCGTCAACACGGTCTCTCCGGGCTATATCGC CACCGACATGGTCAAGGCGATCCGCCAGGACGTGCTCGACAAGATCGTCGCGACGAT CCCGGTCAAGCGCCTGGGCCTGCCGGAAGAGATCGCCTCGATCTGCGCCTGGTTGTCGTCGGAGGAGTCCGGTTTCTCGACCGGCGCCGACTTCTCGCTCAACGGCGGCCTGCA TATGGGCTGA

[0167] SEQ ID NO:39

[0168] MTDVVIVSAARTAVGKFGGSLAKIPAPELGAVVIKAALERAGVKPEQVSEVIMGQVLTAG SGQNPARQAAIKAGLPAMVPAMTINKVCGSGLKAVMLAANAIMAGDAEIVVAGGQENMSAAPHVLPGSRDGFRMGDAKLVDTMIVDGLWDVYNQYHMGITAENVAKEYGITREAQD EFAVGSQNKAEAAQKAGKFDEEIVPVLIPQRKGDPVAFKTDEFVRQGATLDSMSGLKPAFDKAGTVTAANASGLNDGAAAVVVMSAAKAKELGLTPLATIKSYANAGVDPKVMGMGPVPASKRALSRAEWTPQDLDLMEINEAFAAQALAVHQQMGWDTSKVNVNGGAIAIGHPI GASGCRILVTLLHEMKRRDAKKGLASLCIGGGMGVALAVERK

[0169] SEQ ID NO:40

[0170] MTQRIAYVTGGMGGIGTAICQRLAKDGFRVVAGCGPNSPRREKWLEQQKALGFDFIASE GNVADWDSTKTAFDKVKSEVGEVDVLINNAGITRDVVFRKMTRADWDAVIDTNLTSLF NVTKQVIDGMADRGWGRIVNISSVNGQKGQFGQTNYSTAKAGLHGFTMALAQEVATKGVTVNTVSPGYIATDMVKAIRQDVLDKIVATIPVKRLGLPEEIASICAWLSSEESGFSTGADF SLNGGLHMG

[0171] SEQ ID NO:41

[0172] MATGKGAAASTQEGKSQPFKVTPGPFDPATWLEWSRQWQGTEGNGHAAASGIPGLDAL AGVKIAPAQLGDIQQRYMKDFSALWQAMAEGKAEATGPLHDRRFAGDAWRTNLPYRFAAAFYLLNARALTELADAVEADAKTRQRIRFAISQWVDAMSPANFLATNPEAQRLLIESGG ESLRAGVRNMMEDLTRGKISQTDESAFEVGRNVAVTEGAVVFENEYFQLLQYKPLTDKVHARPLLMVPPCINKYYILDLQPESSLVRHVVEQGHTVFLVSWRNPDASMAGSTWDDYIEHAAIRAIEVARDISGQDKINVLGFCVGGTIVSTALAVLAARGEHPAASVTLLTTLLDFADTGILDVFVDEGHVQLREATLGGGAGAPCALLRGLELANTFSFLRPNDLVWNYVVDNYLK GNTPVPFDLLFWNGDATNLPGPWYCWYLRHTYLQNELKVPGKLTVCGVPVDLASIDVPTYIYGSREDHIVPWTAAYASTALLANKLRFVLGASGHIAGVINPPAKNKRSHWTNDALPE SPQQWLAGAIEHHGSWWPDWTAWLAGQAGAKRAAPANYGNARYRAIEPAPGRYVKAK A

Claims

1. A method for constructing engineered bacteria that synthesize polyhydroxy fatty acid esters using carbon dioxide, wherein the bacteria are hookworm-like copper-loving bacteria H16 (… Cupriavidus necator H16) was the starting strain, and the Pj5 promoter was used to start the *Methanotherium soxeoniflorum* GP6 (…). Methanothrix soehngenii The Rubisco gene from GP6 was integrated into the genome of H16 hookworm worm *Citrobacter pumilus* to replace the original Rubisco gene, resulting in strain H16-1; *Citrobacter yangensis* ATCC 29220 ( Citrobacter youngae The hydrogenase gene from ATCC 29220 was integrated into the genome of strain H16-1 to replace the original hydrogenase gene, resulting in strain H16-2. The LDH gene was knocked out of the H16-2 genome to obtain strain H16-3. Finally, a recombinant plasmid containing genes encoding acetyl-CoA C-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway was transformed into strain H16-3 to overexpress these three enzymes, resulting in strain H16-4. The genes encoding acetyl-CoA C-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway were obtained from P... phaC Startup sub-boot.

2. The method according to claim 1, characterized in that: The sequence of the Rubisco gene derived from Methanophora soxe GP6 is shown from position 476 to position 1903 in SEQ ID NO: 9, and the amino acid sequence of the ribulose diphosphate carboxylase it encodes is shown in SEQ ID NO:

10.

3. The method according to claim 1, characterized in that: The sequence of the hydrogenase gene derived from *Citrobacter yangensis* ATCC 29220 is shown from position 501 to position 1781 in SEQ ID NO: 21, and the amino acid sequence of the hydrogenase it encodes is shown in SEQ ID NO:

22.

4. The method according to any one of claims 1-3, characterized in that: The integration of the Rubisco gene from *Methanophora sowii* GP6, the integration of the hydrogenase gene from *Citrobacter yangii* ATCC 29220, and the knockout of the LDH gene were all achieved through a suicide plasmid-mediated target gene deletion strategy.

5. The method according to any one of claims 1-3, characterized in that: The amino acid sequences of acetyl-CoA-acetyltransferase, acetyl-CoA reductase, and PHA synthase in the PHA synthesis pathway are shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively.

6. The engineered bacteria constructed by the method according to any one of claims 1-5.

7. The application of the engineered bacteria described in claim 6 in the synthesis of polyhydroxy fatty acid esters.

8. A method for synthesizing polyhydroxy fatty acid esters using carbon dioxide, comprising the step of fermenting the engineered bacteria of claim 6 to produce polyhydroxy fatty acid esters under conditions where the volume ratio of H2:CO2:O2 is (5-9):(1-3):

1.

9. The method according to claim 8, characterized in that: The fermentation medium used in the fermentation process consisted of 8-10 g / L Na₂HPO₄·12H₂O, 1-2 g / L KH₂PO₄, 0.5-2 g / L (NH₄)₂SO₄, 70-100 mg / L MgSO₄·7H₂O, 0.5-2 mg / L CaSO₄·2H₂O, 0.4-0.6 mg / L NiSO₄·7H₂O, 0.2-0.6 mg / L ferric citrate, 100-300 mg / L NaHCO₃, and 0.5-1.5 mL / L trace elements.

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