Strain for producing high-performance polyhydroxyalkanoate as well as construction method and application of strain

Abstract

The invention provides a strain for producing polyhydroxyalkanoate as well as a construction method and application of the strain. The strain expresses an aldD gene, a yahK gene, an orfZ gene and a phaC gene or a mutant thereof. The poly3-hydroxybutyric acid-4-hydroxybutyric acid, the poly3-hydroxybutyric acid-5-hydroxyvaleric acid, the poly3-hydroxybutyric acid-6-hydroxyhexanoic acid, the poly3-hydroxybutyric acid-4-hydroxybutyric acid-5-hydroxyvaleric acid, the poly3-hydroxybutyric acid-6-hydroxyhexanoic acid, the poly3-hydroxybutyric acid-4- Poly3-hydroxybutyrate-4-hydroxybutyrate-6-hydroxyhexanoic acid and poly3-hydroxybutyrate-5-hydroxyvalerate-6-hydroxyhexanoic acid are obviously superior to P3HB in mechanical property, good tensile strength is kept, meanwhile, the flexibility and ductility of the material are remarkably improved, the defects that traditional P3HB is high in brittleness and low in elongation at break are overcome, and the service life of the material is prolonged. Therefore, the method has wider processing adaptability and application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain for producing high-performance polyhydroxy fatty acid esters, its construction method, and its applications. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of natural polymers synthesized by microorganisms. They possess excellent biodegradability and biocompatibility, and are considered important candidate materials to replace petroleum-based plastics. PHAs exhibit a rich variety of monomer structures, with over 160 monomers reported to date. Different monomer compositions significantly affect the chemical structure, mechanical properties, and processing performance of the materials. Therefore, developing novel PHA materials with specific properties by controlling the composition of PHA monomers has become a research hotspot in materials science and microbial synthesis. Among them, various PHA materials, such as poly(3-hydroxybutyrate-6-hydroxyhexanoate) copolyester (P3HB-6HHX), poly(3-hydroxybutyrate-5-hydroxyvalerate) copolyester (P3HB-5HV), and poly(3-hydroxybutyrate-4-hydroxybutyrate) copolyester (P3HB-4HB), have shown good application potential and can be further extended to ternary copolymer systems, such as P3HB-4HB-5HV and P3HB-4HB-6HHX. However, there is still a lack of research on P3HB-6HHX and its copolymerization with 5HV to form P3HB-5HV-6HHX, and there is currently a lack of mature, economical and efficient biosynthetic strategies.

[0003] In reported technical solutions, increasing the proportion of non-3HB monomers such as 4HB, 5HV, or 6HHX through engineered strains can improve the performance of PHA materials. However, when the proportion of non-3HB monomers increases, the total PHA content usually decreases significantly, often falling below 40%, resulting in insufficient material yield and limiting industrial applications. Halophilic bacteria, due to their advantages such as not requiring strict aseptic operation, high substrate utilization, and ability to ferment at high densities, are considered an important class of chassis microorganisms for the economical and sustainable production of novel PHAs, possessing enormous development potential.

[0004] Therefore, how to coordinate and increase the proportion of high-value-added monomers such as 4HB, 5HV, and 6HHX in PHA, while maintaining a high overall PHA synthesis efficiency and enriching the types of PHA to meet different application needs, is a key technical problem that urgently needs to be solved in the current field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a strain for producing high-performance polyhydroxy fatty acid esters, its construction method, and its applications.

[0006] This invention provides a strain for producing polyhydroxyalkanoates, wherein the strain expresses a nucleotide sequence as shown in SEQ ID NO. 8. aldDThe gene and nucleotide sequence are as shown in SEQ ID NO. 9. yahK The gene and nucleotide sequence are as shown in SEQ ID NO. 10. orfZ Genes and phaC Gene; The phaC The gene differs from the amino acid sequence shown in SEQ ID NO.15 in any of the following ways: (a) The isoleucine at position 241 is mutated to threonine; (b) The isoleucine at position 241 is mutated to proline; (c) The histidine at position 493 is mutated to threonine; (d) The histidine at position 493 is mutated to tyrosine; (e) The isoleucine at position 494 is mutated to serine; (f) The isoleucine at position 494 is mutated to serine; (g) The threonine at position 500 is mutated to arginine; (h) Threonine at position 500 is mutated to aspartic acid.

[0007] In some embodiments, the strain is *Haloxylon ammodendron*.

[0008] In some embodiments, the halomonas is Or its derivatives.

[0009] In a specific embodiment of the present invention, the *Haloxymonas* is preferably... LY01, LY02, LY03, LY04.

[0010] The present invention also provides a method for constructing the strain, comprising the following steps: (1) phaC Gene, aldD Gene, yahK Genes and orfZ Genes were constructed in an expression vector using the same expression module to obtain recombinant plasmids; (2) Introduce the recombinant plasmid described in step (1) into the strain.

[0011] In some implementations, the recombinant plasmid in step (1) is a high-copy plasmid or a low-copy plasmid.

[0012] In some implementations, the high-copy plasmids include pSEVA341, ColE1, pMB1, pUC (such as pUC18, pUC19, etc.), pBluescript (such as pBluescript II KS+, pBluescript II SK+, etc.), pTZ57R, pGEM, etc.

[0013] In some implementations, the low-copy plasmids include the pRE series (such as pRE112), pSEVA321, pSC101, pACYC184, pWE15, and the pET series (such as pET-15b and pET-28a).

[0014] In some embodiments, the expression vector in step (1) includes a promoter, which is any one of J23105, J23116, J23106 or J23110.

[0015] The present invention also provides a method for producing polyhydroxyalkanoates, the method comprising the following steps: fermenting the strain; In some embodiments, the diol substrate is any one or more of 1,3-propanediol, 1,2-propanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 1,3-hexanediol, or 1,6-hexanediol.

[0016] In some embodiments, the polyhydroxy fatty acid ester comprises any one or more hydroxy fatty acid monomers; The hydroxy acid monomer is a C2-C6 hydroxy fatty acid or a derivative thereof.

[0017] Preferably, the polyhydroxy fatty acid ester comprises any one or more monomers selected from 3-hydroxypropionic acid, 2-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 5-hydroxyvalerate, 2-hydroxyvalerate, 3-hydroxyhexanoic acid, and 6-hydroxyhexanoic acid.

[0018] The production of polyhydroxyalkanoates can be carried out by batch fermentation, continuous fermentation, or fed-batch fermentation. Fermentation can be optionally carried out for the desired duration, for example, from 12 to 120 hours, such as 24 hours, 48 ​​hours, 60 hours, 72 hours, etc., but is not limited to these. Those skilled in the art can appropriately select the fermentation time according to specific circumstances. These fermentation methods are well known to those skilled in the art, and they can appropriately select any fermentation method to carry out the method of the present invention according to the desired purpose and experimental conditions.

[0019] In the above-described method for producing PHA, the fermentation medium used for fermentation can be sterilized before being added to the fermentation system, or it can be added directly to the fermentation system without any treatment. Furthermore, the fermentation system can be maintained under open, sterile conditions (i.e., it can be directly exposed to the environment without the need for closed fermentation conditions to avoid contamination) or under closed conditions to avoid contamination.

[0020] The fermentation medium can be selected based on the characteristics of the bacteria and the microbial culture techniques known in the art. It can typically include nutrient sources (such as carbon and / or nitrogen sources), energy sources, and essential minerals. For example, conventional mineral media, LB medium, MM medium, MM-G medium, or beef extract peptone, etc., can be used, or media modified based on these media according to the desired purpose can be used.

[0021] The fermentation medium can be liquid, solid, or semi-solid.

[0022] The fermentation medium may contain carbon and nitrogen sources, and / or carbon and / or nitrogen sources may be added to the fermentation system during the fermentation process. Carbon sources include, but are not limited to, monosaccharides, disaccharides, organic acids, fatty acids, polyols, alcohols, hydrocarbons and their derivatives. Specific carbon sources include glucose, fructose, sucrose, lactose, xylose, arabinose, glycerol, ethanol, methanol, lactic acid, acetic acid, propionic acid, butyric acid, decanoic acid, oleic acid, palmitic acid, stearic acid, or mixtures thereof. Nitrogen sources are inorganic nitrogen sources, organic nitrogen sources, or combinations thereof, including but not limited to urea, ammonium sulfate, nitrates, ammonium chloride, peptone, yeast extract, peptone, corn steep liquor powder, amino acids, casein, and soybean meal hydrolysate.

[0023] In this invention, the accumulation of PHA is achieved by regulating the nitrogen source concentration, including induced synthesis under nitrogen-limited conditions, or PHA synthesis can still be achieved under specific nitrogen-rich conditions.

[0024] The present invention also provides the application of the strain in the production of polyhydroxy fatty acid esters.

[0025] In some embodiments, the polyhydroxyalkanoate is composed of 3 Copolymers of hydroxybutyric acid and one or more hydroxy fatty acid monomers; including but not limited to poly(3-hydroxybutyric acid-4-hydroxybutyric acid copolymer (P34HB), poly(3-hydroxybutyric acid-5-hydroxyvalerate copolymer (P3HB5HV), poly(3-hydroxybutyric acid-6-hydroxyhexanoic acid copolymer (P3HB6HHX), poly(3-hydroxybutyric acid-4-hydroxybutyric acid-5-hydroxyvalerate copolymer (P3HB4HB5HV), poly(3-hydroxybutyric acid-4-hydroxybutyric acid-6-hydroxyhexanoic acid copolymer (P3HB4HB6HHX) and poly(3-hydroxybutyric acid-5-hydroxyvalerate copolymer (P3HB5HV6HHX)).

[0026] The recombinant strain constructed in this invention can precisely control the proportion of non-3HB monomers such as 4HB, 5HV, and 6HHX while maintaining a high total PHA content, thus enabling the preparation of poly(3HB) polymers from the obtained PHA monomers. Hydroxybutyric acid 4-hydroxybutyric acid, poly(3-hydroxybutyric acid) Hydroxybutyric acid 5-hydroxyvalerate, poly(3-hydroxyvalerate) Hydroxybutyric acid 6-hydroxyhexanoic acid and its multi-component copolymers exhibit excellent tensile strength and ductility, while also possessing good flexibility and toughness.

[0027] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention modifies the polyhydroxy fatty acid polymerase (PhaC) to maintain a high total PHA content while maintaining a high proportion of non-3HB monomers, significantly improving the production efficiency and yield of PHA. Compared with the prior art, the copolymer prepared by this invention not only obtains a rich combination of monomers (such as 4HB, 5HV, 6HHX), improving the flexibility, toughness and elasticity of the material, but also maintains a high total PHA content, thereby overcoming the problem of decreased total PHA under a high proportion of non-3HB monomers, and improving the feasibility and economic benefits of industrial production.

[0028] (2) The poly(3-hydroxybutyric acid-4-hydroxybutyric acid), poly(3-hydroxybutyric acid-5-hydroxyvalerate acid), poly(3-hydroxybutyric acid-6-hydroxyhexanoic acid), poly(3-hydroxybutyric acid-4-hydroxybutyric acid-5-hydroxyvalerate acid), poly(3-hydroxybutyric acid-4-hydroxybutyric acid-6-hydroxyhexanoic acid) and poly(3-hydroxybutyric acid-5-hydroxyvalerate acid-6-hydroxyhexanoic acid) produced by the strain of the present invention have significantly better mechanical properties than the monomer P3HB. They not only maintain good tensile strength, but also significantly improve the flexibility and ductility of the material, overcoming the shortcomings of traditional P3HB such as high brittleness and low elongation at break, thus having a wider range of processing adaptability and application potential.

[0029] (3) By regulating the expression intensity of polyhydroxy fatty acid polymerase mutant, this invention further increases the ratio of total PHA content to 4HB, 5HV, and 6HHX monomers, thereby improving PHA production efficiency and ensuring the industrial controllability of the required monomer ratio, providing a feasible path for the large-scale production of high-performance biodegradable plastics. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a diagram of the overall metabolic pathway.

[0032] Figure 2 The results of plasmid colony PCR electrophoresis in Example 1 of this invention are shown.

[0033] Figure 3 Example 1pSEVA321-J23105 of the present invention phaC-aldD-yahK-orfZ Plasmid map.

[0034] Figure 4 This is the result of PCR electrophoresis of the integrated plasmid colonies in Example 4 of the present invention.

[0035] Figure 5 Example 4 of the present invention: pRE112-J23105- phaC (M5)- aldD-yahK-orfZ Integral plasmid map.

[0036] Figure 6 The results of PCR electrophoresis of the integrated strain in Example 4 of this invention are shown. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0038] The present invention uses LY01 is disclosed in patent CN116396886A, with accession number GDMCCNO.62635. LY02 is disclosed in patent CN116925981A, with accession number GDMCC NO.63381. LY03 is disclosed in patent CN116970538A, with accession number GDMCC NO. 63382. LY04 is disclosed in patent CN117551585A, with accession number GDMCC NO.63383.

[0039] Example 1: Enhanced Expression phaC-aldD-yahK-orfZ The module promotes the production of PHA by recombinant halomonas. Since Halomonas endogenously possesses enzymes related to diol metabolism and PHA synthesis, but lacks acetyltransferase, we constructed an enhanced expression module for the corresponding endogenous gene and an exogenous acyl-CoA transferase module to promote efficient PHA synthesis through diol utilization metabolism.

[0040] 1. phaC-aldD-yahK-orfZ Enhanced expression plasmid construction by Using the LY03 genome as a template, PCR was performed using SEQ ID NO. 1~6 sequences as primers to obtain fragments. phaC (The nucleotide sequence is shown in SEQ ID NO.7) aldD (The nucleotide sequence is shown in SEQ ID NO.8) yahK (The nucleotide sequence is shown in SEQ ID NO.9), chemically synthesized by BGI Genomics. orfZ Using the sequence (nucleotide sequence as shown in SEQ ID NO. 10) as a template, PCR was performed using the sequences in SEQ ID NO. 11-12 as primers to obtain fragments. orfZ Simultaneously, using pSEVA321-J23105 plasmid as a template and SEQ ID NO.13~14 sequences as primers, PCR amplification was performed to obtain the pSEVA321-J23105 backbone; then, the pSEVA321 backbone and gene fragment were ligated using Gibson assembly technology, and the ligation product was electroporated into Escherichia coli S17-1, and single clones were verified by colony PCR.

[0041] Colony PCR results as follows Figure 2 As shown, the size is as expected. The target band was sent to a biotechnology company for sequencing, and the successfully constructed plasmid was labeled pSEVA321-J23105- phaC-aldD-yahK-orfZ Plasmid information such as Figure 3 As shown. PCR, Gibson assembly, and electroporation were performed according to standard procedures.

[0042] 2. Construction of recombinant strains The plasmid pSEVA321-J23105- phaC-aldD-yahK-orfZ Recombinant strain LY03-PAY1 was obtained by conjugation of introduced strain LY03, and the conjugation was performed using conventional methods.

[0043] 3. Recombinant strain shake-flask fermentation test The recombinant strain with enhanced expression module obtained in step 2 was streaked onto a plate and activated at 37°C for 24 hours. Primary seed culture: Random single clones from the activated plate were inoculated into 5 mL of 60 LB seed medium and cultured at 37°C with shaking at 220 rpm for 12 hours. Secondary seed culture: The primary seed culture was inoculated at 1% into 20 mL of fresh seed medium and cultured at 37°C with shaking at 220 rpm for 12 hours. Fermentation culture: The secondary seed culture was inoculated at 5% into 50 mL of fermentation medium and fermented at 37°C with shaking at 220 rpm for 48 hours. Cell collection after fermentation: 20 mL of fermentation broth was centrifuged at 9000 rpm for 5 minutes to collect the cells. The cells were resuspended in 20 mL of ultrapure water and centrifuged again at 9000 rpm for 5 minutes to collect the cells. Finally, the collected cells were freeze-dried for 24 hours until constant weight was achieved and weighed.

[0044] The above-mentioned 60LB seed culture medium consists of: yeast extract 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L; and water.

[0045] The fermentation medium consists of: glucose 35 g / L; sodium chloride 50 g / L; yeast extract 1.0 g / L; urea 1.0 g / L; anhydrous magnesium sulfate 0.2 g / L; potassium dihydrogen phosphate 1.5 g / L; ferric ammonium citrate 0.05 g / L; anhydrous calcium chloride 0.0015 g / L; zinc sulfate heptahydrate 0.1 mg / L; manganese sulfate tetrahydrate 0.03 mg / L; boric acid 0.3 mg / L; copper sulfate pentahydrate 0.01 mg / L; and sodium molybdate 0.03 mg / L. For P34HB production, an additional 5-10 g / L of 1,4-butanediol (BDO) is added to the fermentation medium; for P3HB5HV production, an additional 5-10 g / L of 1,5-pentanediol (PDO) is added; and for P3HB6HHX production, an additional 5-10 g / L of 1,6-hexanediol (HDO) is added.

[0046] PHA content determination: Approximately 30 mg of lyophilized bacterial cells were added to 2 mL of esterification solution (methanol containing 3% sulfuric acid and 0.1% benzoic acid) and 2 mL of chloroform, and reacted at 99°C for 4 h. After the reaction, the mixture was cooled to room temperature, 1 mL of distilled water was added, and the mixture was shaken thoroughly (1600 rpm, 3 min) and allowed to stand for 3 h to separate into layers. The lower organic phase was collected and analyzed using a gas chromatograph (GC-2010 Pro, Shimadzu, Japan). The column was an SH-5 column (30 m × 0.25 mm inner diameter, 0.1 μm film thickness). The chromatographic program was as follows: initial temperature 70°C, held for 4 min; temperature increased at 30°C / min to 140°C and held for 2 min; then increased at 40°C / min to 240°C and held for 2 min. The internal standard method was used, with PHB (Sigma-Aldrich), γ-butyrolactone (Sigma-Aldrich), δ-valerolactone (Sigma-Aldrich), and ε-caprolactone (Sigma-Aldrich) as standards, to quantitatively analyze the contents of PHB, P34HB, P3HB5HV, and P3HB6HHX, respectively.

[0047] The fermentation results are shown in Table 1: Table 1. Fermentation results of recombinant strains producing multiple types of PHA

[0048] * Indicates the significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample size n=3.

[0049] PHA production was measured in the recombinant plasmid expressing the enhancement module. The results showed that the recombinant strain LY03-PAY1 produced significantly higher PHA under all conditions than the control strain LY03, and also increased the proportion of the corresponding hydroxy acids under different diol addition conditions. These results indicate that PHA production through expression... phaC-aldD-yahK-orfZ The enhancement module can effectively improve the host's ability to synthesize PHA and the ability to synthesize PHA with high proportions of 4HB, 5HV, and 6HHX.

[0050] Example 2: Construction and testing of recombinant bacteria carrying polyhydroxy fatty acid polymerase variants 1. Design and construction of polyhydroxy fatty acid polymerase mutants The nucleotide sequence is as shown in SEQ ID NO.7. phaCThe sequence was input into the InterPro website (https: / / www.ebi.ac.uk / interpro / ) for domain and active site prediction. Based on the prediction results, four active sites were identified: Ile241, His493, Ile494, and Thr500. Site-directed mutagenesis was performed using the amino acid sequence shown in SEQ ID NO.15, and the following mutations were designed: The isoleucine at position 241 is mutated to threonine (I241T, M1). The isoleucine at position 241 is mutated to proline (I241P, M2). The histidine at position 493 is mutated to threonine (H493T, M3). The histidine at position 493 is mutated to tyrosine (H493Y, M4). The isoleucine at position 494 is mutated to serine (I494S, M5). The isoleucine at position 494 is mutated to serine (I494S, M6). The threonine at position 500 is mutated to arginine (T500R, M7). The threonine at position 500 is mutated to aspartic acid (T500D, M8).

[0051] The above mutation was introduced using primers to obtain the plasmid pSEVA321-J23105-. phaC (M1~M8) -aldD-yahK- orfZ .

[0052] 2. Construction of recombinant strains and testing of PHA dimer fermentation performance The plasmid expressing the mutant was introduced into the host bacterium LY03 via conjugation to obtain recombinant strains LY03-PAY2~9. The corresponding recombinant strains were then subjected to fermentation tests, following the same procedure as described in Example 1. Post-fermentation processing, freeze-drying, and PHA detection were all performed according to the procedures described in Example 1. The fermentation results for different mutants are shown in Table 2.

[0053] Table 2. Strains containing different phaC Fermentation results of mutants

[0054] * Indicates the significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample size n=3.

[0055] The test results showed that the proportion of non-3HB monomers in the mutant recombinant strains LY03-PAY2, LY03-PAY5, LY03-PAY6, and LY03-PAY8 was significantly increased. Among them, LY03-PAY6 performed the best, increasing the proportion of 4HB in P34HB to 29.3%, the proportion of 5HV in P3HB5HV to 32.3%, and the proportion of 6HHX in P3HB6HHX to 39.7%. Moreover, its dry weight and PHA ratio decreased less than those of other mutant recombinant strains.

[0056] 3. Performance test of recombinant strain LY03-PAY6 in producing trimeric PHA The recombinant strain LY03-PAY6 was subjected to fermentation tests for the production of PHA trimer, and the fermentation method was the same as that described in Example 1. Post-fermentation processing, freeze-drying, and PHA detection were all performed according to the procedures described in Example 1. The fermentation results are shown in Table 3. The results indicate that the recombinant strain LY03-PAY6 also has the ability to increase the proportion of non-3HB monomers in the synthesis of PHA trimer, demonstrating its stability.

[0057] Table 3 Results of trimeric compound production by fermentation of LY03-PAY6

[0058] The significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample number n=3.

[0059] Example 3: Expressing different intensities M1~M8 aldD-yahK-orfZ Controlling the synthesis of different proportions of PHA polymers by recombinant strains 1. Construction of recombinant strains By adjusting M1~M8- aldD-yahK-orfZ The expression intensity plasmid was further used to control the proportion of non-3HB monomers in dimer and trimer PHA. Therefore, pSEVA321-J23105- phaC (M5) -aldD-yahK-orfZ The J23105 promoter was replaced with J23116, J23106, and J23110 promoters, respectively. The transcriptional strength of J23116 was lower than that of J23105, J23106 was twice as strong as J23105, and J23110 was four times as strong as J23105. Primers were used to introduce these promoters to obtain the plasmid pSEVA321-J23116-. phaC (M5) -aldD-yahK-orfZ pSEVA321-J23116- phaC (M5) -aldD- yahK-orfZ pSEVA321-J23106- phaC (M5) -aldD-yahK-orfZ pSEVA321-J23110- phaC (M5) -aldD-yahK-orfZ The plasmid was conjugated into the LY03 strain to construct recombinant strains LY03-PAY10, LY03-PAY11, and LY03-PAY12.

[0060] 2. PHA test for dimerization and trimerization produced by recombinant strain fermentation The corresponding recombinant strains were subjected to fermentation tests, and the fermentation method was the same as that described in Example 1. Post-fermentation processing, freeze-drying, and PHA detection were all performed according to the procedures described in Example 1.

[0061] The results of fermentation of dimeric PHA by recombinant strains with plasmids of different strengths are shown in Table 4.

[0062] The results showed that at low expression intensity, the proportion of non-3HB monomers was lower than that of the control LY03-PAY6, decreasing by about 10%, but the dry weight and PHA yield were increased. At high expression intensity, the proportion of 4HB monomers reached a maximum of 41.7%, 5HV monomers reached a maximum of 46.9%, and 6HHX reached 50.1%, with the total PHA content of all experimental strains exceeding 45%. The recombinant strains showed consistency in both trimeric and dimeric PHA synthesis, with a lower proportion of non-3HB monomers and increased dry weight and PHA yield under low expression intensity conditions compared to the control LY03-PAY6, demonstrating their stability.

[0063] Table 4 Results of PHA dimerization and trimerization produced by strains with different strengths during fermentation

[0064] The significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample number n=3.

[0065] Example 4: PHA synthesis by genome-integrating strains 1. phaC - aldD-yahK-orfZ Integral plasmid construction pSEVA321-J23116- phaC (M5)- aldD-yahK-orfZ、 pSEVA321-J23105- phaC (M5)- aldD-yahK-orfZ、 pSEVA321-J23110- phaC (M5)- aldD-yahK-orfZUsing SEQ ID NO. 16~17 as primers, PCR was performed to obtain fragment J23116-. phaC (M5)- aldD-yahK-orfZ、 J23105- phaC (M5)- aldD-yahK-orfZ、 J23106- phaC (M5)- aldD-yahK-orfZ、 J23110- phaC (M5)- aldD- yahK-orfZ Using pRE112-L15-R15 plasmid as a template and SEQ ID NO.18~19 sequences as primers, PCR was performed to obtain the suicide plasmid backbone fragment. The fragment J23116- was then assembled using Gibson assembly technology. phaC (M5)- aldD-yahK-orfZ、 J23105- phaC (M5)- aldD-yahK-orfZ、 J23106- phaC (M5)- aldD-yahK-orfZ、 J23110- phaC (M5)- aldD-yahK-orfZ Ligated with the pRE112-L15-R15 backbone fragment and introduced into E. coli via electroporation. E.coli S17-1, and colony PCR verification of single clones was performed using primers SEQ ID NO. 20~21. The electrophoresis results are as follows. Figure 4 As shown.

[0066] The target band was then sent to a biotechnology company for sequencing. The plasmid with correct sequencing was labeled pRE112-J23116- phaC (M5)- aldD-yahK-orfZ、 pRE112-J23105- phaC (M5)- aldD-yahK-orfZ、 pRE112-J23106- phaC (M5)- aldD-yahK-orfZ、 pRE112-J23110- phaC (M5)- aldD-yahK-orfZ Plasmid information such as Figure 5 As shown.

[0067] 2. Construction of integrated recombinant strains Genome integration was performed using homologous recombination, with plasmid pRE112-J23116- phaC (M5)- aldD- yahK-orfZ、 pRE112-J23105- phaC (M5- aldD-yahK-orfZ、 pRE112-J23106- phaC (M5)- aldD-yahK-orfZ、 pRE112-J23110- phaC (M5)- aldD-yahK-orfZ The LY03 strain was introduced via conjugation. Colony PCR was performed on the resulting single colonies using primers SEQ ID NO.16 and SEQ ID NO.17. The banded strains were passaged every 24 hours under antibiotic-free conditions. The culture medium from each generation was diluted and plated. Clones detached from chloramphenicol resistance were screened from the resulting single colonies. Colony PCR verification was performed using primers SEQ ID NO.22-23. The verification results are as follows: Figure 6 As shown.

[0068] The 6.2 kJ band was sequenced for verification; the correctly sequenced strain was identified as having integrated J23116- phaC (M5)- aldD-yahK-orfZ J23105- phaC (M5)- aldD-yahK-orfZ J23106- phaC (M5)- aldD-yahK-orfZ J23110- phaC (M5)- aldD-yahK-orfZ The recombinant bacteria of the module were named LY03-PAY13, LY03-PAY14, LY03-PAY15, and LY03-PAY16, respectively.

[0069] 3. Integrated strain fermentation test The corresponding recombinant strains were subjected to fermentation tests, and the fermentation method was the same as that described in Example 1. The post-fermentation processing, freeze-drying process, and PHA detection process were all performed according to the procedures described in Example 1. The results are shown in Table 5. The dry weight and total PHA percentage of all integrated strains showed a slight increase compared to the plasmid strains, with dry weight increasing by 0.2-1 g / L and PHA increasing by 1-5%. Although the proportion of non-3HB components in each polymer decreased, the proportion remained above 15%.

[0070] Table 5. Results of PHA production by fermentation of strains with different integration strengths

[0071] The significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample number n=3.

[0072] Example 5: Application in the production of PHA by other Halomonas bacteria The plasmid pSEVA321-J23105- phaC (M5) -aldD-yahK-orfZ Connect and import separately Halomonas yellowing MDF-9 LY01 LY02 LY04 was used to construct recombinant strains MDF9-M5, LY01-M5, LY02-M5, and LY04-M5.

[0073] The corresponding recombinant strains were subjected to fermentation tests, and the fermentation method was the same as that described in Example 1. Post-fermentation processing, freeze-drying, and PHA testing were all performed according to the procedures described in Example 1.

[0074] Table 6 Results of PHA production by fermentation of different Halomonas strains

[0075] The significance level of the difference in the proportion of non-3HB in PHA among strains (t-test): * indicates P<0.05; sample number n=3.

[0076] The fermentation test results are shown in Table 6. All recombinant strains were able to synthesize the corresponding dimer and trimer PHAs, and the proportion of non-3HB was not less than 20%, indicating the universality of this technology in producing high proportion of non-3HB PHAs in different Halomonas bacteria.

[0077] Example 6: Tensile property test of PHA P3HB, polymerized from 3HB, is typically a brittle material with high Young's modulus and tensile strength, but its elongation at break is low, only about 5%, indicating poor ductility, which significantly limits its applications. Therefore, the dimer and trimer PHA obtained in the examples were subjected to material mechanics tests to characterize their tensile strength and elongation at break. The following P3HB samples obtained from fermentation in Example 3 were extracted and purified: 20.1% 4HB in P3HB4HB, 21.1% 5HV in P3HB5HV, 35.5% 5HV in P3HB5HV, 25.8% 6HHX in P3HB6HHX, 37.7% 6HHX in P3HB6HHX, 21.8% 5HV and 29.4% 6HV in P3HB5HV6HHX, and 31.9% 5HV and 35.5% 6HV in P3HB5HV6HHX. The P3HB sample was obtained by fermentation of strain LY03 without the addition of diol. The extraction and purification method was as follows: intracellular PHA was extracted from frozen stem cells using a Soxtec 2050 Soxhlet extractor, the sample was then redissolved in chloroform, precipitated with cold anhydrous ethanol, and finally dried and evaporated in a fume hood. The obtained PHA was used to prepare a thin film: 1 gram of sample was dissolved in 20 mL of chloroform and placed on a glass petri dish at room temperature, then placed in a fume hood for 48 hours. The film was cut into dumbbell shapes with a width of 5 mm and a thickness of 0.5 mm and then subjected to mechanical analysis. Stress-strain measurements were performed using an Instron 3365 (Instron, USA) tensile strength measuring device at a tensile speed of 10 mm / min.

[0078] Table 7. Mechanical test results of PHA materials

[0079] The results are shown in Table 7. In the dimerized PHA, when the proportions of 4HB, 5HV, and 6HHX are approximately 20%, the elongation at break of the material can reach over 1000%. When the proportions are approximately 40%, the tensile properties of the material decrease. This indicates that P3HB4HB, P3HB5HV, and P3HB6HHX produced by the strain have high tensile strength and ductility, and thus high application value.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain for producing polyhydroxyalkanoates, characterized in that, The strain expresses the nucleotide sequence shown in SEQ ID NO.

8. aldD The gene and nucleotide sequence are as shown in SEQ ID NO.

9. yahK The gene and nucleotide sequence are as shown in SEQ ID NO.

10. orfZ Genes and phaC Gene; The phaC The gene differs from the amino acid sequence shown in SEQ ID NO.15 in any of the following ways: (a) The isoleucine at position 241 is mutated to threonine; (b) The isoleucine at position 241 is mutated to proline; (c) The histidine at position 493 is mutated to threonine; (d) The histidine at position 493 is mutated to tyrosine; (e) The isoleucine at position 494 is mutated to serine; (f) The isoleucine at position 494 is mutated to serine; (g) The threonine at position 500 is mutated to arginine; (h) Threonine at position 500 is mutated to aspartic acid.

2. The strain according to claim 1, characterized in that, The strain is *Haloxylon ammodendron*.

3. The strain according to claim 2, characterized in that, The halometa is Or its derivatives.

4. The method for constructing the strain according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) phaC Gene, aldD Gene, yahK Genes and orfZ Genes were constructed in an expression vector using the same expression module to obtain recombinant plasmids; (2) Introduce the recombinant plasmid described in step (1) into the strain.

5. The construction method according to claim 4, characterized in that, The expression vector in step (1) contains a promoter, which is any one of J23105, J23116, J23106 or J23110.

6. A method for producing polyhydroxyalkanoates, characterized in that, The method includes the following steps: fermenting the strain described in claim 1; The culture medium used for the fermentation culture contains a diol substrate.

7. The method according to claim 6, characterized in that, The diol substrate is any one of 1,3-propanediol, 1,2-propanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 1,3-hexanediol, or 1,6-hexanediol.

8. The method according to claim 6, characterized in that, The polyhydroxy fatty acid ester contains any one or more hydroxy fatty acid monomers; The hydroxy fatty acid monomer is a C2-C6 hydroxy fatty acid or its derivative.

9. The use of the strain according to any one of claims 1 to 3 in the production of polyhydroxyalkanoates.

10. The application according to claim 9, characterized in that, The polyhydroxy fatty acid ester is composed of 3 A copolymer of hydroxybutyric acid and one or more hydroxy fatty acid monomers.

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