Recombinant halomonas sp. for synthesizing p3hb3hp by using glucose and construction method and application thereof

Abstract

This invention relates to the field of microbial metabolic engineering, and particularly to a recombinant *Halomonas* strain that synthesizes P3HB3HP from glucose, its construction method, and its applications. This invention constructs a synthetic pathway in *Halomonas* containing enzymes such as aspartate decarboxylase and 4-aminobutyrate aminotransferase. The resulting recombinant *Halomonas* strain can synthesize P3HB3HP with a high 3HP ratio from glucose alone via the β-alanine pathway, while maintaining a high P3HB3HP yield. The recombinant *Halomonas* strain provided by this invention can produce P3HB3HP using inexpensive carbon sources, significantly reducing substrate costs and achieving high cell density, high yield, high 3HP ratio, and stable P3HB3HP synthesis.

Description

Technical Field

[0001] This invention relates to the field of microbial metabolic engineering technology, and in particular to recombinant halomonas strains that synthesize P3HB3HP using glucose, their construction methods, and applications. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of fully biodegradable biopolyesters synthesized by microorganisms and are considered an ideal alternative to traditional petroleum-based plastics. Poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP), a novel PHA copolymer, exhibits superior material properties compared to traditional PHBV. P3HB3HP shows significantly higher elongation at break than PHBV with similar 3HV content, demonstrating greater flexibility and a wider processing window. The introduction of the 3HP monomer effectively lowers the polymer's melting point while maintaining good thermal stability, facilitating melt processing. Since its degradation products are all naturally occurring substances in living organisms, P3HB3HP possesses unique advantages in high-end biomedical materials (such as drug delivery carriers and tissue engineering scaffolds). Therefore, developing a microbial synthetic pathway that does not rely on expensive or toxic precursors and can efficiently synthesize P3HB3HP using low-cost, renewable carbon sources (such as glucose) is crucial for promoting the large-scale application of this high-performance biomaterial.

[0003] Currently, the microbial synthesis of P3HB3HP mainly relies on the introduction of exogenous 3HP synthesis modules. Existing technologies are primarily based on the glycerol / diol synthesis pathway, utilizing propylene glycol dehydratases and aldehyde dehydrogenases from certain microorganisms to convert glycerol or 1,3-propanediol into 3-hydroxypropanealdehyde (3HPA), which is then further oxidized to 3HP-CoA, subsequently binding with endogenous PHA synthases into the copolymer. This approach suffers from drawbacks such as high substrate cost, lengthy and low-throughput pathways, and highly toxic metabolic intermediates. There are no reports of achieving high cell density, high yield, high 3HP ratio, and stable production of P3HB3HP in potentially industrial-scale hosts using glucose as a single, inexpensive carbon source. Summary of the Invention

[0004] This invention provides a recombinant halomonas strain that synthesizes P3HB3HP using glucose, its construction method, and its application.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a recombinant Halomonas bacterium, wherein the recombinant Halomonas bacterium expresses aspartate decarboxylase and 4-aminobutyric acid aminotransferase; The recombinant halomonas can synthesize P3HB3HP using glucose.

[0007] Preferably, the recombinant halomonas can synthesize P3HB3HP using glucose as the sole carbon source.

[0008] This invention constructs a synthetic pathway in *Haematomyces* to synthesize 3HP from glucose using the β-alanine pathway. This pathway begins with aspartic acid produced from glucose metabolism, which is converted to β-alanine by aspartate decarboxylase. Following steps involving 4-aminobutyrate aminotransferase and malonyl hemialdehyde dehydrogenase, 3HP-CoA is ultimately generated. This pathway combines with endogenous PHA synthase in *Haematomyces* to finally generate P3HB3HP. By screening and optimizing the selection and combination of enzymes involved in this synthetic pathway, efficient synthesis of P3HB3HP from glucose is achieved. This invention reveals that aspartate decarboxylase and 4-aminobutyrate aminotransferase are key catalytic enzymes in the synthesis of 3HP from glucose using the β-alanine pathway, and their combined action endows *Haematomyces* with the ability to synthesize P3HB3HP using glucose as the sole carbon source.

[0009] The aspartate decarboxylase described above is encoded by the panD gene. It is preferably derived from Corynebacterium bacteria.

[0010] The 4-aminobutyric acid aminotransferase described above is encoded by the GabT gene. It is preferably derived from Pseudomonas.

[0011] The Corynebacterium species is preferably Corynebacterium glutamicum. The Pseudomonas species is preferably Pseudomonas aeruginosa.

[0012] Using aspartate decarboxylase and 4-aminobutyric acid aminotransferase from the above sources can work together better to promote the synthesis of 3HP by Halomonas from glucose, thereby increasing the molar ratio of 3HP in the product and the yield of P3HB3HP.

[0013] Preferably, the aspartate decarboxylase has the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.1.

[0014] Preferably, the 4-aminobutyric acid aminotransferase has the amino acid sequence shown in SEQ ID NO.2 or has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.2.

[0015] The consistency is preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.9%.

[0016] In some embodiments of the present invention, the amino acid sequence of the aspartate decarboxylase is shown in SEQ ID NO.1, and the amino acid sequence of the 4-aminobutyric acid aminotransferase is shown in SEQ ID NO.2.

[0017] To further increase the proportion of 3HP in the product P3HB3HP polymer, the recombinant Halomonas strain described above preferably also expresses aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

[0018] Among them, aspartate transaminase can promote the synthesis of aspartic acid from oxaloacetate, while malonyl hemialdehyde dehydrogenase and propionyl-CoA synthase can convert more β-alanine into 3-hydroxypropionyl-CoA. This invention found that, in conjunction with the aforementioned aspartate decarboxylase and 4-aminobutyrate aminotransferase, further expression of aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase can significantly increase the 3HP ratio in the P3HB3HP polymer synthesized by the strain, thereby obtaining a P3HB3HP product with a high 3HP ratio.

[0019] Preferably, the aspartate transaminase is encoded by the aspB gene. It is preferably derived from Corynebacterium bacteria.

[0020] Preferably, the malonyl hemialdehyde dehydrogenase is encoded by the ydfG gene. It is preferably derived from Escherichia coli bacteria.

[0021] Preferably, the propionyl-CoA synthase is encoded by the prpE gene. It is preferably derived from Escherichia coli bacteria.

[0022] The Corynebacterium species is preferably Corynebacterium glutamicum. The Escherichia coli species is preferably Escherichia coli.

[0023] The aspartate transaminase, malonyl hemialdehyde dehydrogenase and propionyl-CoA synthase from the above sources can better cooperate with each other and with Halomonas, promote the efficiency of Halomonas in synthesizing 3HP from glucose, thereby significantly increasing the molar ratio of 3HP in the product and ensuring a high yield of P3HB3HP.

[0024] Preferably, the aspartate transaminase has the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.3.

[0025] Preferably, the malonyl hemialdehyde dehydrogenase has the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.4.

[0026] Preferably, the propionyl-CoA synthase has the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.5.

[0027] The consistency is preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, and more preferably at least 99.9%.

[0028] In some embodiments of the present invention, the amino acid sequence of the aspartate transaminase is shown in SEQ ID NO.3, the amino acid sequence of the malonyl hemialdehyde dehydrogenase is shown in SEQ ID NO.4, and the amino acid sequence of the propionyl-CoA synthase is shown in SEQ ID NO.5.

[0029] In this invention, the expression is achieved by causing the recombinant halomonas to carry an expression plasmid containing the encoding gene of the enzyme, and / or by integrating the encoding gene of the enzyme into the genome.

[0030] In some embodiments of the present invention, the recombinant Halomonas bacteria carries an expression plasmid containing the encoding genes of the aspartate decarboxylase and 4-aminobutyric acid aminotransferase, or carries an expression plasmid containing the encoding genes of the aspartate decarboxylase, 4-aminobutyric acid aminotransferase, aspartate transaminase, malonyl hemialdehyde dehydrogenase and propionyl-CoA synthase.

[0031] The present invention does not impose any particular limitation on the expression plasmid; any plasmid capable of replication and gene expression in *Halomonas* can be used. Exemplary expression plasmids include pHbPBC plasmid, SEVA series plasmids, pBBR1MCS series plasmids, etc.

[0032] In some embodiments of the present invention, the recombinant Halomonas carries a pHbPBC plasmid containing the encoding genes of the aspartate decarboxylase and 4-aminobutyric acid aminotransferase, or carries a pHbPBC plasmid containing the encoding genes of the aspartate decarboxylase, 4-aminobutyric acid aminotransferase, aspartate transaminase, malonyl hemialdehyde dehydrogenase and propionyl-CoA synthase.

[0033] In some embodiments of the present invention, the recombinant Halomonas strain integrates the coding genes for aspartate decarboxylase and 4-aminobutyric acid aminotransferase into its genome, or integrates the coding genes for aspartate decarboxylase, 4-aminobutyric acid aminotransferase, aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

[0034] The present invention does not impose any particular limitations on the genomic integration sites of the encoding genes of the above-mentioned enzymes, and commonly used integration sites can be used. Exemplary integration sites include, but are not limited to, the G7 site, the G51 site, etc.

[0035] In some embodiments of the present invention, the encoding genes for aspartate decarboxylase and 4-aminobutyric acid aminotransferase are integrated into the G7 site of the genome, and the encoding genes for aspartate transaminase, malonyl semialdehyde dehydrogenase and propionyl-CoA synthase are integrated into the G51 site of the genome.

[0036] For the promoters used to express the above-mentioned enzymes, it is preferable to use medium- or high-strength promoters to drive the expression of the gene encoding the enzyme.

[0037] The present invention does not impose any particular restrictions on the specific selection of medium- or high-strength promoters. Commonly used medium- or high-strength promoters in Halomonas can be used, such as the Halomonas porin promoter series (including the wild-type porin gene porin promoter and its variants).

[0038] In some embodiments of the present invention, the promoter is selected from any one of Pporin140, Pporin68, and Pporin141. Promoter regulation enables *Halomonas* to synthesize P3HB3HP with a high 3HP ratio using glucose as a single carbon source.

[0039] The nucleotide sequence of the gene encoding the enzyme can be determined by those skilled in the art based on the amino acid sequence of the enzyme and the codon rules provided above. Due to the degeneracy of codons, the gene sequence encoding a single amino acid sequence is not unique, and all genes capable of encoding the enzyme are within the scope of protection of this invention.

[0040] In some embodiments of the present invention, the sequence of the gene encoding the aspartate decarboxylase is shown in SEQ ID NO. 6. The sequence of the gene encoding the 4-aminobutyrate aminotransferase is shown in SEQ ID NO. 7. The sequence of the gene encoding the aspartate transaminase is shown in SEQ ID NO. 8. The sequence of the gene encoding the malonyl hemialdehyde dehydrogenase is shown in SEQ ID NO. 9. The sequence of the gene encoding the propionyl-CoA synthase is shown in SEQ ID NO. 10.

[0041] In this invention, the *Haloxymonas* can be selected from... Halomonas bluephagenesis , Halomonas aydingkolgenesis , Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas elongata , Halomonas smyrnensisPreferably, it is *Haloxylon ammodendron* (Haloxylon ammodendron). Halomonas bluephagenesis ).

[0042] Secondly, the present invention provides a method for constructing the recombinant halomonas bacteria described above, the method comprising: modifying the halomonas bacteria to express aspartate decarboxylase and 4-aminobutyric acid aminotransferase.

[0043] Preferably, the method further includes modifying the Halomonas bacteria to also express aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

[0044] In this invention, the modification includes genetic engineering or gene editing. The genetic engineering or gene editing can be performed using gene editing methods commonly used in Halomonas, such as CRISPR / Cas9 gene editing technology.

[0045] Preferably, the modification includes introducing an expression plasmid carrying a gene encoding the enzyme into *Halomonas*, or integrating the gene encoding the enzyme into the genome of *Halomonas*.

[0046] For the starting strain used in the construction of recombinant halomonas, it can be selected from halomonas ( Halomonas bluephagenesis WZY254, Halomonas ( Halomonas bluephagenesis TD1.0, Halomonas ( Halomonas campaniensis LS21, Halomonas ( Halomonas bluephagenesis WZY278, etc.

[0047] Thirdly, the present invention provides any one of the following applications of the recombinant halomonas bacteria described above: (1) Fermentation production of P3HB3HP; (2) The starting strain used to construct the strain for fermentation production of P3HB3HP.

[0048] The recombinant halomonas strain provided by this invention can be directly used for fermentation to produce P3HB3HP, or used as a starting strain to construct a strain for fermentation to produce P3HB3HP.

[0049] Fourthly, the present invention provides a method for fermenting and producing P3HB3HP, the method comprising: fermenting and culturing the above-mentioned recombinant halomonas bacteria, and collecting P3HB3HP from the culture.

[0050] Preferably, the fermentation culture uses glucose as the carbon source. More preferably, glucose is the sole carbon source.

[0051] Preferably, the fermentation medium used in the fermentation culture further includes a nitrogen source and inorganic salts.

[0052] In some embodiments of the present invention, the fermentation medium comprises the following components: 20-40 g / L glucose, 40-60 g / L NaCl, 0.5-1.5 g / L yeast extract, 0.5-1.5 g / L urea, 0.1-0.3 g / L MgSO4, 8-11 g / L Na2HPO4•12H2O, 1-2 g / L KH2PO4, 8-12 mL / L trace element solution I, and 0.8-1.2 mL / L trace element solution II. Trace element solution I comprises the following components: 4-6 g / L ferric ammonium citrate and 1-3 g / L CaCl2. Trace element solution II contains the following components: 80-120 mg / L ZnSO4•7H2O, 20-40 mg / L MnCl2•4H2O, 280-320 mg / L H3BO3, 180-220 mg / L CoCl2•6H2O, 8-12 mg / L CuSO4•5H2O, 18-22 mg / L NiCl2•6H2O, and 28-32 mg / L NaMoO4•2H2O. The pH of the fermentation medium is 8.0-8.5.

[0053] Preferably, the fermentation culture temperature is 35-37℃, the pH is 8.0-8.5, and the dissolved oxygen is 30%-50%.

[0054] The beneficial effects of this invention include at least the following: By constructing a metabolic pathway in *Haematomyces* containing aspartate decarboxylase, 4-aminobutyric acid aminotransferase, etc., the recombinant *Haematomyces* obtained can synthesize P3HB3HP with a high 3HP ratio from a single carbon source, glucose, using the β-alanine pathway. The 3HP ratio in the synthesized P3HB3HP can reach 10%-30%, and a high P3HB3HP yield can be guaranteed. The recombinant *Haematomyces* provided by this invention can produce P3HB3HP using inexpensive carbon source glucose, significantly reducing substrate costs and achieving high cell density, high yield, high 3HP ratio, and stable P3HB3HP synthesis. Attached Figure Description

[0055] 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.

[0056] Figure 1 This is a schematic diagram of a portion of the metabolic pathway by which recombinant Halomonas synthesizes P3HB3HP using glucose in this invention.

[0057] Figure 2This is a map of the pK01 plasmid in Example 1 of the present invention.

[0058] Figure 3 This is a spectrum of the pK02 plasmid in Example 2 of the present invention. Detailed Implementation

[0059] Definitions of abbreviations and key terms: PHA: Polyhydroxyalkanoates; 3HP: 3-hydroxypropionic acid; P3HB3HP: Poly-3-hydroxybutyric acid (3HB)-3-hydroxypropionic acid (3HP) ester; AspB: Aspartate aminotransferase; PanD: Aspartate decarboxylase; GabT: 4-Aminobutyrate aminotransferase; YdfG: Malonate hemialdehyde dehydrogenase; PrpE: Propionyl-CoA synthase.

[0060] In a specific embodiment of the present invention, gene editing technology is used to target *Halomonas* (Haloxylon ammodendron). Halomonas bluephagenesis The WZY254 strain was modified by expressing exogenous β-alanine pathway genes on the genome and / or recombinant plasmids. Figure 1 This allows Halomonas to synthesize P3HB3HP polymers using a single carbon source, glucose; wherein the exogenous β-alanine pathway genes include genes encoding aspartate decarboxylase and 4-aminobutyrate aminotransferase, or genes encoding aspartate decarboxylase, 4-aminobutyrate aminotransferase, aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

[0061] In specific embodiments of the present invention, by regulating the expression of exogenous β-alanine pathway genes through promoters of different strengths, P3HB3HP polymers containing different 3HP ratios can be produced, resulting in materials with different properties and applications.

[0062] 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.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent manufacturers or distributors. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0064] The following examples use Halomonas ( Halomonas bluephagenesis WZY254 is an outer membrane-defective strain that can produce high levels of PHB and P3HB4HB in a 7L fermenter. This strain is published in the literature: 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.

[0065] The E. coli S17-1 competent cells used to construct the plasmids in the following examples were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0066] The CRISPR / Cas9 genome editing techniques used in the following examples are described in the literature: Qin et al., 2018. CRISPR / Cas9 editing genome of extremophile Halomonas spp [J].MetabolicEngineering.

[0067] The genome editing sites used in the following examples are referenced in: Qin et al., 2018. CRISPR / Cas9editing genome of extremophile Halomonas spp [J].Metabolic Engineering.

[0068] The promoter sequences used in the following examples are available in the literature: Shen et al., 2018. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis[J]. ACS Synthetic Biology.

[0069] The methods for endogenous plasmid knockout and construction described in the following examples are referenced in: Ren K, Zhao Y, Chen GQ, Aox, Wu Q. Construction of a stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis. Acs Synth Biol., 2024 jan 19,13(1).61-67. The specific 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.

[0070] MM50 medium (pH 8.0-8.5): 30 g / L glucose, 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. Trace element solution I consists of 5 g / L ferric ammonium citrate and 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.

[0071] In the following examples, unless otherwise specified, the method for preparing the seed solution 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.

[0072] 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.

[0073] 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.

[0074] In the following examples, unless otherwise specified, the shake flask culture method is as follows: Secondary seed culture was prepared using the same method as described above. The secondary seed culture was inoculated into 50 mL of MM50 medium at an inoculum volume of 2%. γ-Butyrolactone could be added at an appropriate concentration as needed. Three parallel samples were prepared for each experimental group. The culture was carried out 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 PHA content and monomer ratio of the freeze-dried bacterial cells were determined by gas chromatography (GC).

[0075] In the following examples, unless otherwise specified, the method for culturing in a 7L fermenter is as follows: Secondary seed culture was prepared using the same method as described above. This secondary seed culture was inoculated into the fermentation medium (MM50 medium) at a 10% inoculation rate. The initial volume of the 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 maintained between 10-15 g / L through feeding, and the fermentation pH was maintained at 8.5 using 5M NaOH. The fermentation process included cell growth and product synthesis. The feeding solution for cell growth consisted of 400 mL of 750 g / L glucose 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% glucose was continuously added until fermentation ended.

[0076] 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.

[0077] In the following embodiments, the freeze-drying method 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.

[0078] In the following examples, the cell dry weight is calculated as follows: 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.

[0079] In the following examples, the methods for detecting the PHA content and the content of each monomer in the bacterial cells 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 3HP monomer and carry out esterification reaction to obtain standard sample (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 holding time (80℃, 1.5 min), first stage temperature increase (temperature increase rate 30℃ / min), second stage temperature increase (temperature increase rate 40℃ / min, held 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.

[0080] PHA content (wt%) = (mass of 3HB + mass of 3HP) ÷ 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 3HP)) × 100%; For the calculation method of the molar ratio of 3HP, please refer to the molar ratio of 3HB.

[0081] Example 1: Construction of recombinant Halomonas HP01 synthesizing P3HB3HP using glucose The key steps in the synthesis of 3HP from glucose via the β-alanine pathway are the conversion of aspartic acid to β-alanine and the conversion of β-alanine to malondialdehyde, requiring aspartic acid decarboxylase and 4-aminobutyric acid aminotransferase, respectively. Since *Halomonas* lacks these required enzymes, this invention achieves the synthesis of the *Halomonas* p3HB3HP by overexpressing a foreign gene. First, the aspartic acid decarboxylase gene from *Halomonas* is overexpressed using the pHbPBC recombinant plasmid (refer to Ren K, Zhao Y, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-ntitoxin System of *Halomonas bluephagenesis*[J]. ACS Synth Biol. 2024: 13(1):61-67.). panD and the 4-aminobutyric acid aminotransferase gene of Pseudomonas aeruginosa gabT The specific methods for constructing and validating recombinant bacteria are described below.

[0082] 1. Knock out the endogenous plasmid of strain WZY254 Since the replicon of the pHbPBC plasmid is identical to that of the endogenous plasmid, the endogenous plasmid in strain WZY254 must be removed before transformation with this plasmid. The endogenous plasmid in *Halomonas* was knocked out using CRISPR / Cas9 technology, referring to *CRISPR / Cas9 editing genome of extremophile *Halomonas* spp. (Qin et al., *Metabolic Engineering*, 2018). The pQ08 plasmid and the pLCP-O2-sgE plasmid were sequentially transformed into *Halomonas* via conjugation transformation. Halomonas bluephagenesis In WZY254, complete knockout of the endogenous plasmid was verified by PCR, resulting in the 254LCP strain. The successfully edited strain was then passaged multiple times in liquid culture medium and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify strains with lost CRISPR plasmids.

[0083] 2. Construction of recombinant Halomonas HP01 Pporin140- panD - gabTGene fragments were integrated into the pHbPBC plasmid to construct the pK01 plasmid. Figure 2 The pK01 plasmid was transformed into *Haloxylon ammodendron* strain 254LCP via conjugation transformation. The transformation was successfully verified by PCR, yielding recombinant *Haloxylon ammodendron* HP01.

[0084] 3. Production of P3HB3HP by shake-flask fermentation of recombinant Halomonas HP01 To investigate whether recombinant Halomonas HP01 can synthesize P3HB3HP using glucose as a single carbon source, strains WZY254 and HP01 were inoculated into 20 mL of LB60 / LB60 + spectinomycin medium, respectively. After culturing for 12-16 h, they were transferred at a volume ratio of 1% to a fresh 20 mL LB60 / LB60 + spectinomycin medium and cultured for another 8-12 h. 2.5 mL of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of MM50 medium for a shake-flask experiment. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of culture, cell dry weight, PHA content, and the 3HP molar ratio were measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 1.

[0085] The results showed that the dry weight and PHA content of strain HP01 were slightly lower than those of strain WZY254, but it was able to synthesize P3HB3HP polymer with a 3HP molar ratio of 10.54%. This indicates that the exogenous gene... panD and gabT The introduction of this technology enabled Halomonas to synthesize P3HB3HP using a single glucose carbon source.

[0086] Table 1 Production of P3HB3HP by shake-flask fermentation using strain HP01

[0087] Example 2: Construction of P3HB3HP using recombinant Halomonas HP02 (a strain that synthesizes P3HB3HP from glucose) To increase the proportion of 3HP in the P3HB3HP polymer synthesized by the strain, three additional exogenous genes were overexpressed on the pK01 plasmid, namely the aspartate transaminase gene from Corynebacterium glutamicum. aspB The malonyl hemialdehyde dehydrogenase gene of Escherichia coli ydfG and propionyl-CoA synthase gene prpE The former gene enables more oxaloacetate to be converted into aspartic acid, while the latter two genes enable more β-alanine to be converted into 3-hydroxypropionyl-CoA. The specific methods for constructing and validating the recombinant bacteria are described below.

[0088] 1. Construction of recombinant Halomonas HP02 Pporin68- aspB -ydfG - prpE Gene fragments were inserted into the pK01 plasmid to construct the pK02 plasmid. Figure 3 The pK02 plasmid was transformed into *Haloxylon ammodendron* strain 254LCP via conjugation transformation. The transformation was successfully verified by PCR, yielding recombinant *Haloxylon ammodendron* HP02.

[0089] 2. Production of low molecular weight PHA by shake-flask fermentation of recombinant Halomonas HP02 The HP02 strain was inoculated into 20 mL of LB60 medium supplemented with spectinomycin and cultured for 12-16 h. Then, it was transferred at a 1% volume ratio to a fresh 20 mL LB60 medium supplemented with spectinomycin and cultured for another 8-12 h. 2.5 mL of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of MM50 medium for a shake-flask experiment. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of culture, the cell dry weight, PHA content, and 3HP molar ratio were measured. Each experiment was performed in triplicate, and the results were averaged. The WZY254 strain was used as a control. The results are shown in Table 2.

[0090] The results showed that the dry weight and PHA content of strain HP02 were lower than those of strain WZY254, but the molar ratio of 3HP in the synthesized P3HB3HP polymer increased to 22.24%.

[0091] Table 2 Production of P3HB3HP by shake-flask fermentation using strain HP02

[0092] Example 3: Genome expression of exogenous genes To construct a strain capable of stably producing P3HB3HP polymer, the aforementioned exogenous gene was integrated into the genome of strain WZY254 for expression. Genome editing was performed using CRISPR / Cas9 technology, sequentially introducing Pporin140- [[ID= - ​ The gene fragment was integrated into the G7 site of the WZY254 strain genome, bringing Pporin68- ​ - ​ - ​ The gene fragment was integrated into the G51 site of the genome to obtain recombinant Halomonas HP03. The HP03 strain was subjected to shake-flask fermentation experiments (using the same method as in Example 2), with strain WZY254 as a control. The results are shown in Table 3.

[0093] The results showed that the dry weight and PHA content of strain HP03 were not significantly lower than those of strain WZY254, and the molar ratio of 3HP in the synthesized P3HB3HP polymer was 18.53%. This indicates that the intensity of exogenous gene expression using recombinant plasmids is higher than that of genomic expression.

[0094] Table 3 Production of P3HB3HP by shake-flask fermentation using strain HP03

[0095] Example 4: Regulation of exogenous gene expression via promoter To enhance the expression level of the exogenous gene on the genome and increase the molar ratio of 3HP in the P3HB3HP polymer generated by the strain, the exogenous gene was expressed using the Pporin141 promoter, which has a higher expression intensity. Genome editing was performed using CRISPR / Cas9 technology, sequentially expressing Pporin141- ​ - ​ The gene fragment was integrated into the G7 site of the WZY254 strain genome, bringing Pporin141- ​ - ​ - ​ The gene fragment was integrated into the G51 site of the genome to obtain recombinant Halomonas HP04. The HP04 strain was subjected to a shake-flask experiment, with the WZY254 strain as a control. The results are shown in Table 4.

[0096] The results showed that the dry weight and PHA content of strain HP04 did not decrease significantly compared with strain WZY254, and the molar ratio of 3HP in the synthesized P3HB3HP polymer increased to 26.98%.

[0097] Table 4. Shake-flask production of P3HB3HP by strain HP04

[0098] Example 5: Production of P3HB3HP by Fermentation of Recombinant Halomonas in a Fermenter To investigate the ability of the four recombinant halometabolites to produce P3HB3HP polymer in a 7L fermenter, recombinant halometabolites HP01-HP04 were subjected to fed-batch fermentation in a 7L fermenter to produce P3HB3HP. The fermentation experiment of strain WZY254 was used as a control, and three replicates were set up for each experiment. After fermentation, cell dry weight, PHA content, and 3HP molar ratio were measured, and the results are shown in Table 5.

[0099] The results showed that the dry weight and PHA content of strains HP01-HP04 were slightly lower than those of strain WZY254, but they were able to synthesize P3HB3HP polymer from glucose, a single carbon source, with a 3HP molar ratio of 10%-30%. By expressing the exogenous β-alanine pathway gene through the genome / recombinant plasmid, high cell density, high yield, high 3HP ratio (>20 mol%) and stable P3HB3HP synthesis could be achieved.

[0100] Table 5. Production of P3HB3HP by Recombinant Halomonas Fermentation in a Fermenter

[0101] 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.

[0102] References: 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.

[0103] Kang Ren et al., 2024. Construction of a Stable Expression SystemBased on the Endogenous hbpB / hbpC Toxin-ntitoxin System of Halomonasbluephagenesis[J]. ACS Synth Biol.

Claims

1. A recombinant halomonas bacterium, characterized in that, The recombinant Halomonas strain expresses aspartate decarboxylase and 4-aminobutyric acid aminotransferase. The recombinant halomonas can synthesize P3HB3HP using glucose.

2. The recombinant Halomonas bacillus according to claim 1, characterized in that, The aspartate decarboxylase is composed of panD Gene encoding, preferably derived from Corynebacterium spp.; And / or, the 4-aminobutyrate aminotransferase is composed of gabT The gene encoding is preferably derived from Pseudomonas; Preferably, the Corynebacterium species is Corynebacterium glutamicum; And / or, the Pseudomonas is Pseudomonas aeruginosa.

3. The recombinant halomonas according to claim 1 or 2, characterized in that, The aspartate decarboxylase has the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.1; And / or, the 4-aminobutyric acid aminotransferase has the amino acid sequence shown in SEQ ID NO.2 or has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.

2.

4. The recombinant halomonas according to any one of claims 1 to 3, characterized in that, The recombinant Halomonas also expresses aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

5. The recombinant halometazoa according to claim 4, characterized in that, The aspartate transaminase is composed of aspB Gene encoding, preferably derived from bacteria of the genus Corynebacterium; And / or, the malonyl hemialdehyde dehydrogenase is derived from... ydfG Gene encoding, preferably derived from Escherichia coli bacteria; And / or, the propionyl-CoA synthase is composed of prpE Gene encoding, preferably derived from Escherichia coli bacteria; Preferably, the Corynebacterium species is Corynebacterium glutamicum; And / or, the Escherichia coli bacteria are Escherichia coli.

6. The recombinant halomonas according to claim 4 or 5, characterized in that, The aspartate transaminase has the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.3; And / or, the malonyl hemialdehyde dehydrogenase has the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.4; And / or, the propionyl-CoA synthase has the amino acid sequence shown in SEQ ID NO.5 or has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.

5.

7. The recombinant halomonas according to any one of claims 1 to 6, characterized in that, The expression is achieved by causing the recombinant halomonas to carry an expression plasmid containing the encoding gene of the enzyme, and / or by integrating the encoding gene of the enzyme into the genome; Preferably, the expression of the gene encoding the enzyme is driven by a medium- or high-strength promoter.

8. The method for constructing recombinant halomonas bacteria according to any one of claims 1 to 7, characterized in that, The method includes: modifying Halomonas bacteria to express aspartate decarboxylase and 4-aminobutyrate aminotransferase; Preferably, the method further includes modifying the Halomonas bacteria to also express aspartate transaminase, malonyl hemialdehyde dehydrogenase, and propionyl-CoA synthase.

9. Any one of the following applications of the recombinant halomonas according to any one of claims 1 to 7: (1) Fermentation production of P3HB3HP; (2) The starting strain used to construct the strain for fermentation production of P3HB3HP.

10. A method for producing P3HB3HP by fermentation, characterized in that, The method includes: fermenting and culturing the recombinant halometa according to any one of claims 1 to 7, and collecting P3HB3HP from the culture; Preferably, the fermentation culture uses glucose as the carbon source.

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