Recombinant halophilic bacteria for producing PHBV by using acetate as single carbon source and application of recombinant halophilic bacteria

Abstract

The invention relates to the technical field of bioengineering, in particular to a recombinant halophilic bacterium for producing PHBV by using acetate as a single carbon source and application of the recombinant halophilic bacterium. The expression and / or activity of 2-methyl citrate synthetase and succinate dehydrogenase assembly factor 2 of the recombinant halophilic bacteria provided by the invention are reduced, 3-hydroxyvaleric acid synthetic genes scpA and scpB are overexpressed, an ADP-dependent acetyl coenzyme A synthetase gene acd is overexpressed, and a heterologous monocarboxylic acid transporter gene mctC is expressed. The strain can generate PHBV without adding precursors such as propionic acid and valeric acid, acetic acid or acetate can be used as a sole carbon source to produce PHBV, the yield is close to that when glucose is used as a carbon source, and the problems that the added precursor substances have certain toxicity, so that the growth of thalli is influenced, and the yield is reduced are solved; environmental pollution caused by addition of precursor substances is avoided, and meanwhile, the fermentation production cost of the PHBV is effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant halophilic bacterium that produces PHBV using acetate as a single carbon source and its application. Background Technology

[0002] High energy consumption, high freshwater resource consumption, and complex aseptic operations lead to persistently high production costs, which continue to constrain the development of modern industrial biotechnology. However, the advantages of halophilic bacteria as the latest industrial production substrate bacteria are gradually becoming apparent. Halophilic bacteria are a type of microorganism that can grow over a wide range of salt concentrations, have low nutritional requirements, and easily adapt to harsh environments.

[0003] Halomonas are microorganisms that can tolerate high-salt and high-pH environments, making them valuable research materials and production resources. Currently, next-generation industrial biotechnology based on Halomonas is relatively mature, possessing strong market competitiveness and unique advantages due to its energy-saving, freshwater-reducing, and cost-reducing characteristics.

[0004] The random copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) is one of the most widely studied polyhydroxyalkanoate (PHA) materials, possessing excellent material properties and broad application prospects. Currently, the industrialization of PHBV is still hampered by its high production costs.

[0005] Currently, industrially, PHBV is generally produced by adding propionic acid or valeric acid, or other carbon sources, to a glucose-containing culture medium and then utilizing microbial fermentation (Masood et al., 2012). Taking sodium valerate as an example, its price is relatively high, and the powder is light and has an unpleasant odor, leading to not only unpleasant smells during preparation but also dust pollution. Furthermore, some PHBV precursors are toxic, affecting cell growth, reducing yield, and polluting the environment. This method of producing PHBV by adding substrates also increases production costs and limits output. Therefore, there is an urgent need to develop strains and methods for producing PHBV that do not rely on the addition of precursors such as propionic acid or valeric acid. Summary of the Invention

[0006] This invention provides a recombinant halophilic bacterium that uses acetate as a single carbon source to produce PHBV and its application.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a recombinant halophilic bacterium, wherein the expression and / or activity of 2-methylcitrate synthase and succinate dehydrogenase assembly factor 2 are reduced, the 3-hydroxyvalerate synthase genes scpA and scpB are overexpressed, the ADP-dependent acetyl-CoA synthase gene acd is overexpressed, and the heterologous monocarboxylic acid transporter gene mctC is expressed.

[0009] Existing strains and production methods typically require the addition of precursors such as propionic acid and valeric acid to generate PHBV. To address this issue, this invention develops a recombinant halophilic bacterium capable of producing PHBV using acetic acid or acetate as the sole carbon source. However, halophilic bacteria such as Halomonas have low efficiency in utilizing acetic acid and exhibit low tolerance to it. To address the aforementioned issues, this invention modifies the halophilic bacteria as follows: (1) knocking out the succinate dehydrogenase assembly factor 2 gene sdhE and the 2-methylcitrate synthase gene prpC, and overexpressing scpA and scpB. This modification allows the strain to synthesize PHBV using glucose without the need for additional carbon sources (such as propionic acid, valeric acid, etc.); (2) overexpressing the ADP-dependent acetyl-CoA synthase gene acd to improve the tolerance of Halomonas to acetic acid. Combined with the modification in (1), this allows the strain to fully utilize acetic acid or acetate (such as sodium acetate) as a carbon source to synthesize PHBV; (3) expressing the heterologous monocarboxylic acid transporter MctC to improve the strain's utilization efficiency of acetic acid or acetate, further increasing the yield of PHBV. This invention, in conjunction with the above modifications, constructs a recombinant halophilic bacterium that synthesizes PHBV entirely from acetic acid. It can produce PHBV using inexpensive carbon sources such as acetic acid or acetate without the addition of precursor substances, further reducing fermentation costs.

[0010] The monocarboxylic acid transporter protein described in this invention is derived from Corynebacterium bacteria.

[0011] Preferably, the monocarboxylic acid transporter is derived from Corynebacterium glutamicum.

[0012] More preferably, the monocarboxylic acid transporter has the amino acid sequence shown in SEQ ID NO.7. The gene encoding the monocarboxylic acid transporter has the nucleotide sequence shown in SEQ ID NO.8.

[0013] The ADP-dependent acetyl-CoA synthase described in this invention is an endogenous ADP-dependent acetyl-CoA synthase from the halophilic bacteria.

[0014] Preferably, the ADP-dependent acetyl-CoA synthase has the amino acid sequence shown in SEQ ID NO. 5. The ADP-dependent acetyl-CoA synthase encoding gene acd has the nucleotide sequence shown in SEQ ID NO. 6.

[0015] The protein encoded by scpA in this invention is an endogenous enzyme of the halophilic bacteria, preferably having the amino acid sequence encoded by the sequence shown in SEQ ID NO.1.

[0016] The protein encoded by scpB in this invention is an endogenous enzyme of the halophilic bacteria, preferably having the amino acid sequence encoded by the sequence shown in SEQ ID NO.2.

[0017] Preferably, the 2-methylcitrate synthase has the amino acid sequence encoded by the sequence shown in SEQ ID NO.3.

[0018] Preferably, the succinate dehydrogenase assembly factor 2 has an amino acid sequence encoded by the sequence shown in SEQ ID NO.4.

[0019] Preferably, the expression and / or activity is reduced to inactivation.

[0020] Preferably, in the recombinant halophilic bacteria, the succinate dehydrogenase assembly factor 2 gene sdhE and the 2-methylcitrate synthase gene prpC are knocked out.

[0021] In this invention, the expression of the heterologous monocarboxylic acid transporter gene mctC can be achieved by introducing an expression plasmid carrying the mctC gene into the strain, or by integrating the mctC gene into the genome.

[0022] Preferably, the expression of the heterologous monocarboxylic acid transporter gene mctC is achieved by integrating the mctC gene into the genome.

[0023] The heterologous monocarboxylic acid transporter gene mctC can be expressed under the control of a constitutive promoter or an inducible promoter; exemplary constitutive promoters include the wild-type porin gene porin promoter or its variants porin1, porin3, porin42, porin51, porin58, porin68, porin140, porin141, porin183, porin192, porin194, porin203, porin221, porin226, porin259, and porin278 promoters.

[0024] Preferably, the mctC gene is expressed under the porin140 promoter.

[0025] In this invention, the overexpression of the gene can be achieved through any one or more of the following methods: (1) Increase the copy number of the gene; (2) Replace the transcriptional and / or translational regulatory elements of the gene with more active elements; (3) Mutate one or more bases of the gene to enhance its expression.

[0026] The increase in copy number can be achieved by introducing an expression plasmid carrying the gene into the strain, or by integrating the gene into the genome.

[0027] The transcriptional and / or translational regulatory elements include promoters, RBS sequences, etc.

[0028] The overexpressed gene can be expressed under the control of a constitutive promoter or an inducible promoter; exemplary constitutive promoters include the wild-type porin gene promoter or its variants porin1, porin3, porin42, porin51, porin58, porin68, porin140, porin141, porin183, porin192, porin194, porin203, porin221, porin226, porin259, and porin278 promoters.

[0029] Preferably, the overexpression of acd is achieved by integrating the acd gene into the genome.

[0030] Preferably, the acd gene integrated into the genome is expressed under the control of the porin140 promoter.

[0031] In this invention, the overexpression of scpA and scpB is preferably achieved by increasing the copy number of the genes. Preferably, the recombinant halophilic bacterium contains an expression plasmid carrying the scpA and scpB genes, or the recombinant halophilic bacterium integrates additional copies of the scpA and scpB genes into its genome. The additional copies refer to copies other than the original scpA and scpB genes in the genome. Preferably, there is at least one additional copy.

[0032] Preferably, the expression plasmid is pHbPBC plasmid.

[0033] Preferably, in the overexpression, the scpA and scpB genes are expressed under the control of the porin58, porin278, or porin203 promoters.

[0034] This invention does not impose any particular limitations on genomic integration sites; exemplary integration sites include genomic sites G7, G4, G5, and G49.

[0035] In this invention, the halophilic bacteria include genus Halomonas, genus Haloferax, and genus Alkalibacterium.

[0036] Preferably, the halophilic bacteria are *Halomonas*. The *Halomonas* include, but are not limited to, *Halomonas*. Halomonas bluephagenesis , Halomonas aydingkolgenesis , Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas elongata , Halomonas smyrnensis Preferably, it is *Haloxylon ammodendron*. Halomonas bluephagenesis Among them, the aforementioned Halomonas bluephagenesis Including but not limited to Halomonas bluephagenesis WZY254 Halomonas bluephagenesis TD1.0 Halomonas campaniensis LS21 Halomonas bluephagenesis WZY278, etc.

[0037] Secondly, the present invention provides the application of the recombinant halophilic bacteria described in the first aspect above in the fermentation production of PHBV or in the construction of PHBV production strains.

[0038] Preferably, the fermentation process uses acetic acid and / or acetate as the carbon source.

[0039] The recombinant halophilic bacteria of the present invention can produce PHBV by fermentation using acetic acid and / or acetate as the sole carbon source, and can achieve a high PHBV yield and a 3HV molar ratio.

[0040] Thirdly, the present invention provides a method for fermenting to produce PHBV, the method comprising: fermenting and culturing the above-mentioned recombinant halophilic bacteria using acetic acid and / or acetate as carbon sources, and separating and recovering PHBV from the culture.

[0041] Preferably, the fermentation culture uses acetic acid and / or acetate as the sole carbon source.

[0042] The beneficial effects of this invention include at least the following: the recombinant halophilic bacteria provided by this invention can generate PHBV without the addition of precursors such as propionic acid and valeric acid, and can use cheaper acetic acid or acetate as the sole carbon source to produce PHBV, with a yield close to that when glucose is used as the carbon source. This solves the problem that propionic acid and valeric acid have certain toxicity, which can affect cell growth and reduce yield, and avoids environmental pollution caused by the addition of precursor substances. At the same time, it effectively reduces the fermentation production cost of PHBV. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram of the metabolic pathways related to major genes involved in this invention.

[0045] Figure 2 This is a schematic diagram of the structure of the pGZ01 plasmid in Example 4 of the present invention.

[0046] Figure 3 This is a schematic diagram of the structure of the pGZ02 plasmid in Example 5 of the present invention.

[0047] Figure 4 This is a schematic diagram of the structure of the pGZ03 plasmid in Example 6 of the present invention.

[0048] Figure 5 This is a schematic diagram of the structure of the pGZ04 plasmid in Example 13 of the present invention. Detailed Implementation

[0049] The gene and its encoded protein involved in this invention ( Figure 1 The information is summarized as follows: scpA and scpB are key genes involved in the synthesis of 3-hydroxyvalerate (3HV), one of the monomers of polyhydroxyvalerate (PHV) and polyhydroxybutyrate-hydroxyvalerate copolymer (PHBV).

[0050] scpA encodes β-ketothiolase, which catalyzes the condensation of propionyl-CoA and acetyl-CoA to generate 3-ketovalerate-CoA.

[0051] scpB encodes acetyl-CoA reductase, which reduces 3-ketovalerate-CoA to 3-hydroxyvalerate-CoA (3HV-CoA), a direct precursor of 3HV.

[0052] SdhE: Encodes succinate dehydrogenase assembly factor 2.

[0053] prpC: 2-methylcitrate synthase gene.

[0054] acd: ADP-dependent acetyl-CoA synthase gene, which converts acetate into acetyl-CoA.

[0055] mctC: a monocarboxylic acid transporter gene that mediates the transmembrane transport of acetate.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0057] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0058] The bacteria used in the following examples are Halomonas bluephagenesis WZY254 is an outer membrane-defective strain that can produce high levels of PHB and P3HB4HB in a 7L fermenter. This strain has been published in "Ziyu Wang et al., 2022. Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors[J]. Microbial Biotechnology".

[0059] The Escherichia coli S17-1 competent cells used to construct the plasmids in the following examples were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0060] The CRISPR / Cas9 genome editing technology method described in the following examples is from the literature CRISPR / Cas9 editinggenome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0061] The genome editing sites described in the following examples are from the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0062] The promoter sequences described in the following examples are from the literature "Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis" (Shen et al, ACS Synthetic Biology, 2018).

[0063] The culture medium formulations in the following examples are as follows: LB60 medium: containing 60 g / L NaCl, 10 g / L peptone (purchased from OXIOD, catalog number LP0042), 5 g / L yeast extract (purchased from OXIOD, catalog number LP0021), and water to make up the volume, autoclaved at 121°C.

[0064] MM50 medium (pH 8.0-8.5): 30 g / L glucose or sodium acetate, 50 g / L NaCl, 1 g / L yeast extract, 1 g / L urea, 0.2 g / L MgSO4, 9.65 g / L Na2HPO4•12H2O, 1.5 g / L KH2PO4, 10 ml / L trace element solution I and 1 ml / L trace element solution II; wherein, the composition of trace element solution I is: 5 g / L ferric ammonium citrate, 2 g / L CaCl2, both prepared in 1M HCl. The composition of trace element solution II was as follows: 100 mg / L ZnSO4•7H2O, 30 mg / L MnCl2•4H2O, 300 mg / L H3BO3, 200 mg / L CoCl2•6H2O, 10 mg / L CuSO4•5H2O, 20 mg / L NiCl2•6H2O, and 30 mg / L NaMoO4•2H2O, all prepared in 1M HCl; the final pH of the culture medium was adjusted to 8.5. All reagents were purchased from Sinopharm Chemical Reagent Company.

[0065] The method for preparing seed solution in the following examples is as follows: 1) Activation of microbial strains Take the glycerol tube containing the bacterial culture stored at -80℃, streak it onto an LB60 medium plate, and incubate at 37℃ for 24 h.

[0066] 2) First-level seeds Pick a single colony from the plate after completing step 1), inoculate it into 20 mL of liquid LB60 medium, and incubate at 37°C and 200 rpm for 24 h with shaking.

[0067] 3) Secondary seeds Take the primary seed culture obtained in step 2) and inoculate it into 20 mL of liquid LB60 medium at an inoculation rate of 1%, and culture at 37℃ and 200 rpm for 8-10 h with shaking.

[0068] The shake-flask fermentation method in the following examples is as follows: Secondary seed culture was prepared using the same method as described above. 2% of the seed culture was inoculated into 50 mL of MM50 medium. γ-Butyrolactone could be added at an appropriate concentration as needed. Three replicates were prepared for each experimental group. The culture was incubated at 37°C and 200 rpm for 48 hours. After shake-flask culture, the bacterial culture was centrifuged, and the bacterial cells were freeze-dried, weighed, and their dry weight calculated. The freeze-dried bacterial cells were analyzed by GC to determine the PHA content and monomer ratio, and by GPC to determine the molecular weight.

[0069] The fermentation method in the fermenter is as follows in the following examples: A secondary seed culture was prepared and inoculated into the fermentation medium at a 10% inoculum rate. The initial volume of the culture in a 7.5L fermenter was 3L. The fermentation system was not sterilized and fermentation proceeded directly. The temperature was controlled at 37℃, and the initial dissolved oxygen was maintained at 30%-50%. Dissolved oxygen was controlled by adjusting the turbine speed and aeration. The initial turbine speed was 200 rpm, the maximum speed was 800 rpm, and the maximum aeration rate was 3 vvm. Once the turbine speed and aeration reached their maximum, dissolved oxygen was no longer controlled. During fermentation, the carbon source concentration was controlled between 10-15 g / L through feeding, and the fermentation pH was controlled using 5M NaOH or ammonia. The fermentation process included cell growth and product synthesis: the feeding solution for cell growth consisted of 400 ml of 750 g / L sugar solution with 15.2 g of urea added; once the feeding solution was depleted, product synthesis began. This process required maintaining nitrogen-deficient conditions to produce PHA, so 75% of the sugar solution was continuously added until fermentation ended.

[0070] During fermentation, it is necessary to monitor the fermentation status in real time and control parameters such as temperature, pH, dissolved oxygen, and carbon source concentration within the normal range. The sampling and analysis frequency is 3-5 mL small sample every 2 hours and 30 mL large sample every 4 hours. The small sample is used to determine the carbon source concentration and cell density to monitor the fermentation process and feeding rate, and the large sample is used for subsequent PHA content analysis and cell dry weight determination.

[0071] The freeze-drying method in the following examples is as follows: After fermentation, take 35 mL of cell culture medium, centrifuge at 8000 g for 15 min, collect the cell precipitate, wash with water, and then freeze-dry (place the centrifuge tube containing the cell precipitate at -80℃ for 1 h, and then place it in a vacuum freeze dryer for 36 h) to obtain the freeze-dried product.

[0072] The cell dry weight calculation method is as follows in the following examples: Measured as cell dry weight per liter of the fermented system. The unit of cell dry weight is g / L. Cell dry weight (CDW) = (weight of the freeze-dried centrifuge tube - weight of the original empty centrifuge tube) ÷ 0.035; the weight of the freeze-dried centrifuge tube and the weight of the original empty centrifuge tube are both in g; 0.035 represents 0.035L.

[0073] The detection methods for bacterial PHA content and the content of each monomer in the following examples are as follows: The freeze-dried product was subjected to esterification, and the monomer content was then determined by gas chromatography (GC). Esterification reaction: Take 60-70 mg of the freeze-dried product into an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution (methanol solution containing 1 g / L benzoic acid and 3% concentrated sulfuric acid), mix well, cover and seal, and esterify in a metal bath at 100℃ for 4 h; after cooling to room temperature, add 1 mL of distilled water, shake well and mix thoroughly, and let stand to separate the layers; after the chloroform phase and water are completely separated, take the chloroform phase for gas chromatography analysis; Take 10-100 mg of poly-3-hydroxybutyrate (P3HB) and PHBV and perform esterification reaction to obtain the standard sample; Gas chromatography (GC) analysis parameters: The analytes were separated using an HP-5 column in a Shimadzu GC-2014 gas chromatograph; the GC analysis temperature program was set as follows: injection port temperature (240℃), detector temperature (250℃), initial temperature and hold time (80℃, 1.5 min), first stage temperature increase (temperature increase rate 30℃ / min), second stage temperature increase (temperature increase rate 40℃ / min, hold at 240℃ for 2 min), and total program time 8 min; The corresponding PHA monomer ratio is calculated by reading the peak area of ​​the internal standard, the peak area of ​​the PHA monomer methyl ester in the standard, the peak area of ​​the internal standard in the sample, and the peak area of ​​the PHA monomer methyl ester in the sample obtained by gas chromatography.

[0074] PHA content (wt%) = (mass of 3HB + mass of 3HV) ÷ mass of freeze-dried product × 100%; The molar ratio of 3HB (mol%) = number of moles of 3HB ÷ (number of moles of 3HB + number of moles of 3HV) × 100%; For the calculation method of the molar ratio of 3HV, please refer to the molar ratio of 3HB.

[0075] Example 1 Halomonas bluephagenesis Knockout of the sdhE gene in strain WZY254 In this embodiment, the sdhE gene of strain WZY254 was knocked out using CRISPR / Cas9 gene editing technology. The pQ08 plasmid used is described in the literature (Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp[J]. Metabolic engineering ,2018, 47: 219-229.). The plasmid ΔshdE and the pQ08 plasmid were transformed into Halomonas via conjugation transformation. Halomonas bluephagenesis WZY254. Verified by PCR and sequencing. sdhE The gene was successfully knocked out, yielding strain GZ01. The edited strain was serially passaged in LB60 medium and diluted 10⁻⁶ times. 6 The bacteria were spread onto LB60 plates and grown for 36 hours. Single colonies were then picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify the GZ01 strain with CRISPR plasmid loss.

[0076] Example 2 Halomonas bluephagenesis Knockout of the prpC gene in strain WZY254 In this embodiment, the prpC gene of strain WZY254 was knocked out using CRISPR / Cas9 gene editing technology. The plasmid ΔprpC and plasmid pQ08 were then transformed into *Halomonas* via conjugation transformation. Halomonas bluephagenesis In strain 254. Verification was performed by PCR and sequencing. prpC The gene was successfully knocked out, yielding strain GZ02. The edited strain was continuously passaged in LB60 medium and diluted 10⁻⁶ times. 6 The bacteria were spread onto LB60 plates and grown for 36 hours. Single colonies were then picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify the GZ02 strain with CRISPR plasmid loss.

[0077] Example 3 Halomonas bluephagenesis Knockout of sdhE and prpC genes in strain WZY254 This embodiment utilizes CRISPR / Cas9 gene editing technology to knock out the sdhE and prpC genes in strain WZY254. The plasmid ΔprpC and plasmid pQ08 were then transformed into *Halomonas* via conjugation transformation. Halomonas bluephagenesis In strain GZ01. Verification was performed by PCR and sequencing. prpC The gene was successfully knocked out, yielding strain GZ03. The edited strain was continuously passaged in LB60 medium and diluted 10⁻⁶ times. 6The bacteria were spread onto LB60 plates and grown for 36 hours. Single colonies were then picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify the GZ03 strain with CRISPR plasmid loss.

[0078] Example 4 Overexpression of scpA and scpB genes in strain GZ03 (1) In this embodiment, plasmid pGZ01 ( Figure 2 ) Transformed into Halomonas via conjugation transformation Halomonas bluephagenesis In GZ03, the plasmid backbone of pGZ01 is pHbPBC, and the scpA+scpB genes are expressed using the porin58 promoter (where the sequences of scpA and scpB are shown in SEQ ID NO. 1 and 2, respectively). The successfully constructed strain has a CDW of 14 g / L during shake-flask fermentation and can utilize glucose as the sole carbon source to generate PHBV, with the 3HV ratio reaching 3.99%. The results are shown in Table 1.

[0079] Example 5 Overexpression of scpA and scpB genes in strain GZ03 (2) In this embodiment, plasmid pGZ02 ( Figure 3 ) Transformed into Halomonas via conjugation transformation Halomonas bluephagenesis In GZ03, the plasmid backbone of pGZ02 is pHbPBC, and the scpA+scpB gene is expressed using the porin278 promoter. The successfully constructed strain achieved a CDW of 14 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 4.21%. The results are shown in Table 1.

[0080] Example 6 Overexpression of scpA and scpB genes in strain GZ03 (3) In this embodiment, plasmid pGZ03 ( Figure 4 ) Transformed into Halomonas via conjugation transformation Halomonas bluephagenesis In GZ03, the plasmid backbone of pGZ03 is pHbPBC, and the scpA+scpB gene is expressed using the porin203 promoter. The successfully constructed strain had a CDW of 14 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with a 3HV ratio reaching 3.96%. Fermentation was carried out in a 7L fermenter, and the 3HV ratio reached 6.5% at the end of fermentation. The results are shown in Table 1.

[0081] Example 7 Overexpression of scpA and scpB genes in strain GZ01 (1) In this embodiment, plasmid pGZ01 was transformed into Halomonas bacteria via conjugation transformation. Halomonasbluephagenesis In GZ01, the successfully constructed strain had a CDW of 15.2 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 0.9%. The results are shown in Table 1.

[0082] Example 8 Overexpression of scpA and scpB genes in strain GZ01 (2) In this embodiment, plasmid pGZ02 was transformed into Halomonas bacteria via conjugation transformation. Halomonas bluephagenesis In GZ01, the successfully constructed strain had a CDW of 12.3 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 1.36%. The results are shown in Table 1.

[0083] Example 9 Overexpression of scpA and scpB genes in strain GZ01 (3) In this embodiment, plasmid pGZ03 was transformed into Halomonas bacteria via conjugation transformation. Halomonas bluephagenesis In GZ01, the successfully constructed strain had a CDW of 15.02 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 1.2%. Validation was performed using a 7L fermenter, where the 3HV proportion reached 1.78% at the end of fermentation. The results are shown in Table 1.

[0084] Example 10 Overexpression of scpA and scpB genes in strain GZ02 (1) In this embodiment, plasmid pGZ01 was transformed into Halomonas bacteria via conjugation transformation. Halomonas bluephagenesis In strain GZ02, the successfully constructed strain had a CDW of 15.26 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 0.6%. The results are shown in Table 1.

[0085] Example 11 Overexpression of scpA and scpB genes in strain GZ02 (2) In this embodiment, plasmid pGZ02 was transformed into Halomonas bacteria via conjugation transformation. Halomonas bluephagenesis In GZ02, the successfully constructed strain had a CDW of 14.82 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 1.3%. The results are shown in Table 1.

[0086] Example 12 Overexpression of scpA and scpB genes in strain GZ02 (3) In this embodiment, plasmid pGZ03 was transformed into Halomonas bacteria via conjugation transformation. Halomonasbluephagenesis In GZ02, the successfully constructed strain had a CDW of 15.27 g / L during shake-flask fermentation and could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 0.8%. The results are shown in Table 1.

[0087] Example 13 Overexpression of scpA and scpB genes in strain GZ03 (4) This embodiment utilizes CRISPR / Cas9 gene editing technology to insert the scpA and scpB genes into the G7 site of the halophilic bacteria genome. The plasmid pGZ04 ( Figure 5 The scpA+scpB gene was successfully expressed into *Haloxylon ammodendron* strain GZ03 via conjugation transformation with the pQ08 plasmid. PCR and sequencing confirmed successful expression of the scpA+scpB gene, yielding strain GZ04. The successfully edited strain was serially passaged in LB60 medium and diluted 10⁻⁶ times. 6 The culture was spread onto LB60 plates and grown for 36 hours. Single colonies were then streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify strains with lost CRISPR plasmids. After successful strain construction, shake-flask fermentation experiments were conducted. With a CDW of 16 g / L, the strain could utilize glucose as the sole carbon source to generate PHBV, with the 3HV proportion reaching 0.5%. The results are shown in Table 1.

[0088] The plasmid backbone of pGZ04 is pSEVA341, and the scpA+scpB gene is expressed using porin278.

[0089] Table 1. Results of PHBV production using glucose as the sole carbon source.

[0090] Example 14: Overexpression of the acd gene in strain GZ03 In this embodiment, the acd gene (SEQ ID NO. 6) was inserted into the genome of a halophilic bacterium using CRISPR / Cas9 gene editing technology. Plasmids pGZ05 and pQ08 were transformed into *Halomonas* strain GZ03 via conjugation transformation. Successful integration of the acd gene into the strain's genome was verified by PCR and sequencing, resulting in strain GZ05. The successfully edited strain was continuously passaged in LB60 medium and diluted 10⁻⁶ times. 6 The bacteria were spread onto LB60 plates and grown for 36 hours. Single colonies were then picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify strains that had lost CRISPR plasmids.

[0091] The plasmid backbone of pGZ05 is pSEVA341, and the acd gene is expressed using the porin140 promoter.

[0092] Example 15 Production of PHBV using acetic acid as the sole carbon source Plasmids pGZ01, pGZ02, and pGZ03 were conjugated and transformed into strain GZ05. The resulting strains were then fermented in MM50 medium in shake flasks for 48 h, using 30 g / L sodium acetate as the sole carbon source. Strain GZ03+pGZ01 / pGZ02 / pGZ03 served as a control. Cell dry weight, PHBV content, and 3HV ratio were measured, and the results are shown in Table 2.

[0093] Compared to strains GZ03+pGZ01 / pGZ02 / pGZ03, strains GZ05+pGZ01 / pGZ02 / pGZ03 showed significantly increased dry weight and PHBV content, while the proportion of 3HV in the PHBV was almost identical. This indicates that overexpression of the acd gene can improve the utilization efficiency and tolerance of Halomonas to acetic acid, enabling the strain to utilize acetic acid as a single carbon source to produce more PHBV.

[0094] Example 16 Expression of heterologous mctC gene To further improve the utilization efficiency of acetic acid by Halomonas, the mctC gene (SEQ ID NO.8) derived from Corynebacterium glutamicum was expressed in GZ05 to enhance the cell's ability to transport acetic acid.

[0095] The mctC gene was inserted into the genome of a halophilic bacterium using CRISPR / Cas9 gene editing technology. Plasmids pGZ06 and pQ08 were transformed into *Halomonas* strain GZ05 via conjugation transformation. Successful integration of the mctC gene into the strain's genome was verified by PCR and sequencing, resulting in strain GZ06. The successfully edited strain was serially passaged in LB60 medium and diluted 10⁻⁶ times. 6 The bacteria were spread onto LB60 plates and grown for 36 hours. Single colonies were then picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify strains that had lost CRISPR plasmids.

[0096] The plasmid backbone of pGZ06 is pSEVA341, and the mctC gene is expressed using the porin140 promoter.

[0097] Example 17 Plasmids pGZ01, pGZ02, and pGZ03 were conjugated and transformed into strain GZ06, and fermented in MM50 medium in shake flasks for 48 h, using 30 g / L sodium acetate as the sole carbon source. Cell dry weight, PHBV content, and 3HV ratio were measured, and the results are shown in Table 2.

[0098] Compared to strains GZ05+pGZ01 / pGZ02 / pGZ03, strains GZ06+pGZ01 / pGZ02 / pGZ03 showed significantly increased dry weight and PHBV content, with the highest dry weight reaching 13.85 g / L and the highest PHBV content reaching 63.12%. The PHBV yield was close to that obtained when glucose was the sole carbon source. This indicates that expressing the heterologous mctC gene can enhance the uptake of acetate by *Halomonas*, further improving the utilization and transformation efficiency of acetate.

[0099] Table 2 Results of PHBV production using acetate as the sole carbon source

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant halophilic bacterium, characterized in that, The recombinant halophilic bacteria has reduced expression and / or activity of 2-methylcitrate synthase and succinate dehydrogenase assembly factor 2, overexpresses 3-hydroxyvalerate synthesis genes scpA and scpB, overexpresses ADP-dependent acetyl-CoA synthase gene acd, and expresses heterologous monocarboxylate transporter gene mctC.

2. The recombinant halophilic bacteria according to claim 1, characterized in that, The monocarboxylate transporter is derived from a Corynebacterium bacterium.

3. The recombinant halophilic bacteria according to claim 2, characterized in that, The monocarboxylate transporter is derived from Corynebacterium glutamicum. Preferably, the monocarboxylate transporter has the amino acid sequence shown in SEQ ID NO.

7.

4. The recombinant halophilic bacterium according to any one of claims 1 to 3, wherein The ADP-dependent acetyl-CoA synthase has the amino acid sequence shown in SEQ ID NO.

5.

5. The recombinant halophilic bacterium according to any one of claims 1 to 4, wherein The protein encoded by scpA has the amino acid sequence encoded by the sequence shown in SEQ ID NO.

1. The protein encoded by scpB has the amino acid sequence encoded by the sequence shown in SEQ ID NO.

2. The 2-methylcitrate synthase has the amino acid sequence encoded by the sequence shown in SEQ ID NO.

3. The succinate dehydrogenase assembly factor 2 has the amino acid sequence encoded by the sequence shown in SEQ ID NO.

4.

6. The recombinant halophilic bacterium according to any one of claims 1 to 5, wherein The reduced expression and / or activity is inactivation.

7. The recombinant halophilic bacterium according to any one of claims 1 to 6, wherein The expression of the heterologous monocarboxylate transporter gene mctC is achieved by integrating the mctC gene into the genome. The overexpression of the acd gene is achieved by integrating the acd gene into the genome. Halomonas bluephagenesis 8. The recombinant halophilic bacterium according to any one of claims 1 to 7, wherein The halophilic bacteria is Halomonas sp.; preferably Halomonas sp.

9. Use of the recombinant halophilic bacteria of any one of claims 1-8 in fermentation production of PHBV or construction of PHBV production strains. . Preferably, the fermentation production is with acetic acid and / or acetate as carbon source. The method comprises: culturing the recombinant halophilic bacteria of any one of claims 1-8 in fermentation with acetic acid and / or acetate as carbon source, and isolating and recovering PHBV from the culture.

10. A method for the fermentative production of PHBV, characterized in that, ​

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