A strain for producing polyhydroxyalkanoate and a method for constructing the same and use thereof

Abstract

This invention provides a strain for producing polyhydroxy fatty acid esters, its construction method, and its applications. The strain expresses any one of the following: (1) Escherichia coli-derived... adhE Genes; (2) adhE The 2267th base of the gene is mutated from A to C; (3) adhE The T-base mutation at position 1058 of the gene changes to C; (4) adhE The 616th base of the gene is mutated from G to A, and the 2054th base is mutated from A to G; (5) adhE The 173rd base of the gene is mutated from G to A; (6) adhE adhE The 285th base of the gene is mutated from T to G. This invention, through metabolic engineering, endows *Halomonas* with the ability to simultaneously synthesize multiple PHA monomers. Compared to traditional methods relying on exogenous multi-enzyme cascade pathways, this invention only requires the addition of structure-related precursor substances to synthesize multiple PHAs within the bacteria, greatly simplifying metabolic pathway construction and reducing the metabolic burden on host cells.

Description

Technical Field

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

[0002] Polyhydroxyalkanoates (PHAs) are a class of novel, fully bio-based, and fully biodegradable polymeric materials synthesized by microorganisms, possessing excellent environmental friendliness and application potential. Among them, poly-3-hydroxybutyrate (PHB) was the earliest PHA to be developed and researched, but its application range is limited due to its high brittleness and poor mechanical properties.

[0003] To improve the performance of PHB, researchers have attempted to introduce other monomers into the polymer, such as 4-hydroxybutyric acid (4HB) and 3-hydroxypropionic acid (3HP), to form copolymers such as poly-3-hydroxybutyrate-4-hydroxybutyrate (P34HB), poly-3-hydroxybutyrate-3-hydroxypropionate (P3HB3HP), and the terpolymer poly-3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxypropionate (P34HB3HP). These novel PHB materials exhibit significantly better toughness, ductility, and overall mechanical properties than PHB, thus showing broad application prospects in packaging, agriculture, textiles, chemicals, and the medical field.

[0004] Currently, a common approach to obtaining the aforementioned copolymers is through metabolic engineering of classic chassis microorganisms such as Escherichia coli. The general method involves adding the precursor 1,4-butanediol (BDO) or (and) 1,3-propanediol (PDO) to the culture medium, followed by the sequential catalysis of exogenously introduced alcohol dehydrogenase, aldehyde dehydrogenase, and coenzyme A transferase to synthesize 4HB and 3HP monomers, thereby achieving the synthesis of the PHA copolymer.

[0005] However, existing technologies still have some shortcomings. First, the cultivation process involves high risks of contamination and complex sterilization procedures. Second, the introduction of 4HB or 3HP monomers often relies on exogenous synthetic pathways and typically requires a cascade reaction of three enzymes to complete. This not only increases the complexity of metabolic pathways and the burden on host cells but also significantly reduces the synthesis efficiency of the target product, limiting the large-scale production and application of this type of PHA material.

[0006] Therefore, there is an urgent need for a new strategy that can efficiently synthesize PHA copolymers without the need for complex exogenous metabolic pathways, in order to reduce the host burden and increase the yield of the target product. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a strain for producing polyhydroxy fatty acid esters, its construction method, and its applications.

[0008] This invention provides a strain for producing polyhydroxy fatty acid esters, wherein the strain expresses any one of the following:

[0009] (1) Escherichia coli source adhE Gene;

[0010] (2) Compared with (1), adhE The 2267th base of the gene is mutated from A to C;

[0011] (3) Compared with (1), adhE The T-base mutation at position 1058 of the gene changes to C;

[0012] (4) Compared with (1), adhE The 616th base of the gene is mutated from G to A, and the 2054th base is mutated from A to G.

[0013] (5) Compared with (1), adhE The 173rd base of the gene is mutated from G to A;

[0014] (6) Compared with (1), adhE The 285th base of the gene is mutated from T to G.

[0015] In some embodiments, the adhE The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0016] In some embodiments, the strain is any one of the genera *Halomonas*, *Escherichia*, *Klebsiella*, *Ralstonia*, *Pseudomonas*, and *Akkermansia*.

[0017] Preferably, the strain is or its derivatives Halomonas aydingkolgenesis Or any one of its derivatives.

[0018] In one specific embodiment of the present invention, the strain is preferably MDF-9. LY01 LY02 LY03 LY04.

[0019] The present invention also provides a method for constructing the strain, comprising the following steps:

[0020] (1) The adhE gene was introduced into a plasmid to obtain a recombinant plasmid;

[0021] (2) Introduce the recombinant plasmid described in step (1) into the strain.

[0022] 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.

[0023] 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).

[0024] The present invention also provides a method for producing polyhydroxy fatty acid esters, the method comprising fermenting the strain.

[0025] In some embodiments, the fermentation medium used for the fermentation contains any one or more of 1,4-butanediol and 1,3-propanediol.

[0026] In some embodiments, the fermentation culture temperature is 30~42℃, preferably 32~40℃, and more preferably 35~38℃.

[0027] In some embodiments, the pH of the fermentation culture is 6-11, preferably 7-10.

[0028] The present invention also provides the application of the strain in the production of polyhydroxy fatty acid esters.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The fermentation medium can be liquid, solid, or semi-solid.

[0033] The fermentation medium may contain a carbon source and a nitrogen source, and / or the carbon source and / or nitrogen source may be added to the culture and fermentation system during the fermentation process.

[0034] In some embodiments, the polyhydroxy fatty acid ester is poly-3-hydroxybutyrate-4-hydroxybutyrate (P34HB), poly-3-hydroxybutyrate-3-hydroxypropionate (P3HB3HP), or poly-3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxypropionate (P34HB3HP).

[0035] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0036] (1) This invention, through metabolic engineering, endows Halomonas with the ability to synthesize multiple PHA monomers simultaneously. Compared with traditional methods that rely on exogenous multi-enzyme cascade pathways, this invention only requires the addition of structure-related precursor substances to synthesize multiple PHAs in the bacteria, which greatly simplifies the construction of metabolic pathways and reduces the metabolic burden on host cells.

[0037] (2) The present invention can obtain copolymer PHA composed of multiple monomers such as 4HB and 3HP, and the mechanical properties, toughness and application adaptability of the material are significantly improved; the cumulative amount of PHA produced by fermentation using the strain of the present invention and the proportion of each monomer in the copolymer are significantly improved, thus improving the structural composition of PHA material.

[0038] (3) The substrate bacteria selected in this invention are Halomonas, which can grow stably under high salt and high alkalinity conditions. This characteristic not only effectively avoids the problem of easy contamination by other bacteria in conventional fermentation processes, but also eliminates the need for strict sterilization operations, thereby greatly reducing the complexity and cost of the production process. Attached Figure Description

[0039] 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.

[0040] Figure 1 This is a pathway diagram of the multi-component polymer synthesis route of the present invention;

[0041] Figure 2 This is a diagram of the pSEVA321-adhE1 plasmid from Example 1 of the present invention;

[0042] Figure 3 This is a diagram of the pSEVA321-adhE2 plasmid from Example 1 of the present invention;

[0043] Figure 4 This is a diagram of the pSEVA321-adhE3 plasmid from Example 1 of the present invention;

[0044] Figure 5 This is a diagram of the pSEVA321-adhE4 plasmid from Example 1 of the present invention;

[0045] Figure 6 This is Embodiment 1 of the present invention. adhE1 Fragment electrophoresis image;

[0046] Figure 7 This is Embodiment 1 of the present invention. adhE2 Fragment electrophoresis image;

[0047] Figure 8 This is Embodiment 1 of the present invention. adhE3 Fragment electrophoresis image;

[0048] Figure 9 This is Embodiment 1 of the present invention. adhE4 Fragment electrophoresis image;

[0049] Figure 10 This is embodiment 4pRE112 of the present invention. adhE1 Plasmid diagram;

[0050] Figure 11 This is Embodiment 4 of the present invention. adhE1 Fragment electrophoresis image. Detailed Implementation

[0051] 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.

[0052] This invention introduces exogenous substances from different strains. adhE Genes were developed to enable the incorporation of 4HB monomers or 3HP monomers into polymers by adding the precursors 1,4-butanediol (BDO) and / or 1,3-propanediol (PDO), ultimately achieving the synthesis of multi-component polymers P34HB, P3HB3HP, and P34HB3HP. The optimal performance was determined through screening. adhE Following gene sequencing, random mutations were performed on the gene, enhancing its ability to synthesize various monomers. Unlike previous synthetic pathways that require multiple enzymes, this approach only requires one enzyme to synthesize the target product. This strategy is of great significance for the synthesis of polyhydroxyalkanoates.

[0053] The present invention uses MDF-9 is disclosed in patent CN113801810A, with accession number GDMCC NO.61850. 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.

[0054] Example 1 Aldol dehydrogenase ( adhE ) Filtering

[0055] 1. From different sources adhE Gene expression plasmid construction

[0056] Using PCR to amplify different sources ( adhE1 Derived from E. coli adhE2 Derived from Klebsiella pneumoniae, adhE3 Derived from Salmonella, adhE4 Derived from Shigella, with nucleotide sequences shown in SEQ ID No. 1~4 respectively. adhE Gene fragments and the pSEVA321 backbone were recombined using Gibson ligase to form new plasmids, named pSEVA321-adhE1, pSEVA321-adhE2, pSEVA321-adhE3, and pSEVA321-adhE4. These plasmids were then synthesized using pSEVA321-adhE1, pSEVA321-adhE2, pSEVA321-adhE3, and pSEVA321-adhE4 as templates via PCR. adhE Gene fragments were taken, and a portion of the product was sent to a biotechnology company for sequencing. Plasmid information is as follows: Figure 2~5 As shown.

[0057] The amplification system and amplification procedure are as follows:

[0058] Table 1 Amplification System

[0059]

[0060] Table 2 Amplification Procedure

[0061]

[0062] After the PCR reaction is complete, prepare agarose gel of the appropriate concentration and perform electrophoresis to observe the size of the DNA bands. Place the gel under a UV lamp and quickly cut off the gel containing the target DNA fragment, removing as much excess gel as possible.

[0063] Gibson Assembly method linking:

[0064] The concentration of the recovered DNA was determined, and the DNA addition ratio was calculated based on the length and concentration of the target fragment and the pSEVA321 backbone. Ligation was then performed using a Gibson ligation enzyme mixture. The Gibson Assembly ligation system and procedure are shown in Tables 3 and 4.

[0065] Table 3 Gibson Assembly Connection System

[0066]

[0067] Table 4 Gibson Assembly Connector

[0068]

[0069] (2) Transformation of S17-1 Escherichia coli:

[0070] Remove the pre-prepared S17-1 Escherichia coli competent cells from -80℃ and thaw them on ice. After 5 min, allow the bacterial block to thaw. Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not shake). Incubate on ice for 30 min, then heat shock in a 42℃ water bath for 2 min, and immediately place on ice to cool for 2 min. Add 400 μL of LB medium to the centrifuge tube, mix well, and place in a 37℃ shaker at 200 rpm for 60 min to recover. Centrifuge at 5000 rpm for 5 min to collect the bacteria. Discard 350 μL of supernatant and keep 100 μL. Gently pipette and resuspend the bacterial block and spread it onto LB medium containing the appropriate antibiotic. Invert the medium and incubate at 37℃ for 12–16 h.

[0071] (3) Verification of positive monoclonal colonies

[0072] Colonies were picked out from the corresponding resistant LB plates and colony PCR was performed for verification. PCR products with the correct band size were sent to a biotechnology company for sequencing.

[0073] (4) Select single colonies with correct sequences for amplification. After 12-16 h, conjugate them with MDF-9 on 20 LB plates. After 8 h, pick a small number of conjugated cells and spread them on 60 LB plates with corresponding resistance. After 36-48 h, verify the single colony again.

[0074] (3) Sequencing identification

[0075] The results showed that the MDF-9 strain in this embodiment was successfully transformed with adhE Genes were collected, and the strains were named MDF-9-1, MDF-9-2, MDF-9-3, and MDF-9-4, respectively. Verification was performed based on the size of the target product, as shown below. Figure 6~9 As shown, the target fragments are 2730 bp, 2788 bp, 2733 bp and 2730 bp, which are consistent with the expected results.

[0076] The primer sequences for amplifying each adhE gene fragment and the pSEVA321 plasmid backbone are as follows:

[0077] adhE1 -F: See SEQ ID No. 5;

[0078] adhE1 -R: See SEQ ID No. 6;

[0079] adhE2 -F: See SEQ ID No. 7;

[0080] adhE2 -R: See SEQ ID No. 8;

[0081] adhE3 -F: See SEQ ID No. 9;

[0082] adhE3 -R: See SEQ ID No. 10;

[0083] adhE4 -F: See SEQ ID No. 11;

[0084] adhE4 -R: See SEQ ID No. 12;

[0085] V1-F: See SEQ ID No. 13;

[0086] V1-R: See SEQ ID No. 14.

[0087] Example 2: Fermentation test of recombinant strain trimer P34HB3HP

[0088] (1) Culture medium:

[0089] 60LB agar plate medium: yeast extract 0.5%; tryptone 1%; sodium chloride 60%; agar powder 1.8g / 100mL; pH 8.5.

[0090] Fermentation medium (50 mm): glucose 30 g / L; sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 0.6 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5 g / L, component III: Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, component IV: ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L.

[0091] (2) Specific operating steps:

[0092] Strain activation:

[0093] The bacterial culture was taken from a laboratory freezer at -80℃. The bacterial culture was picked up with a pipette tip and streaked onto a plate solid medium (5 g / L yeast extract; 10 g / L tryptone; 60 g / L sodium chloride, pH 8.5) and incubated at 37℃ for 24 h.

[0094] Primary seed culture:

[0095] Pick a single colony and inoculate it into a 12 mL shaker tube (5 mL 60 LB medium: 5 g / L yeast extract; 10 g / L tryptone; 60 g / L sodium chloride; pH 8.5). Incubate the culture in a shaker at 37°C and 220 rpm for 12 h.

[0096] Secondary seed culture:

[0097] Take 200 μL of primary bacterial culture (1% inoculum) and inoculate it into a 150 mL Erlenmeyer flask (20 mL 60 LB medium). Incubate on a shaker at 37 °C and 220 rpm for 12 h.

[0098] (3) Preparation of fermentation medium

[0099] Fermentation medium (50 mm): glucose 30 g / L; sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 1 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L.

[0100] (4) Fermentation culture

[0101] Inoculate the seed culture at 5% (2.5 mL) into a 500 mL Erlenmeyer flask and incubate on a shaker at 37°C and 220 rpm for 48 h.

[0102] (5) Determination of cell dry weight, PHA content and molar fraction of each monomer

[0103] Cell dry weight (CDW):

[0104] Place 30-35 mL of fermented bacterial culture into a 50 mL centrifuge tube, centrifuge at room temperature for 6 minutes at 8000 rpm, and discard the supernatant; add an appropriate amount of deionized water to restore the original volume, resuspend, and ensure that the precipitate completely disappears. Centrifuge under the same conditions and discard the supernatant; seal the centrifuge tube with sealing film and freeze at -80℃ for 2 hours; dry the centrifuge tube in a vacuum freeze dryer for 12-16 hours; weigh and calculate the dry weight of the cells (g / L).

[0105] Determination of PHA content and molar fraction of each monomer:

[0106] Weigh 0.05 g of the ground, fermented dried cells and place them in a well-sealed esterification tube. Add 2 mL of chloroform, 1700 μL of methanol, and 300 μL of concentrated sulfuric acid. React in an oil bath at 100℃ for 1 h. After cooling to room temperature, add 1 mL of ddH2O, shake thoroughly to mix, and allow to stand for layering. After the aqueous and organic phases are completely separated, take the chloroform layer (usually the lower layer) and filter it through a 0.22 μm organic filter membrane into a liquid chromatography bottle for GC detection. Use a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, 30 m long, 0.25 mm inner diameter and 0.25 μm stationary phase), and flame ionization detection (FID). High-purity nitrogen was used as the carrier gas. The temperature program was set as follows:

[0107] Table 5 Program Temperature Settings

[0108]

[0109] The injection volume was 1 μL. The external standard method was used to quantitatively analyze the PHA content and the molar fraction of each monomer. The PHA content and the molar fraction of each monomer were calculated based on the peak area. For strains MDF-9-1, MDF-9-2, MDF-9-3, and MDF-9-4, 50 μL of chloramphenicol (25 mg / L), 5 g / L of BDO, and 2.5 g / L of PDO were added.

[0110] The fermentation test results are as follows:

[0111] Table 6. Fermentation test of recombinant strain P34HB3HP

[0112]

[0113] Fermentation data showed that after introducing exogenous 4HB and 3HP expression pathways, the strain was able to synthesize the trimer P34HB3HP normally, and compared with the wild-type strain MDF-9, the strain's dry weight and PHA content were both increased. Among them, strain MDF-9-1 (expressing...) adhE1 The gene synthesis of P34HB3HP showed the best results, with the highest dry weight, PHA content, and proportions of each monomer. Therefore, subsequent studies will focus on... adhE1 Research is conducted based on genes.

[0114] Example 3 Aldol dehydrogenase ( adhE1 Random mutation modification

[0115] 1. adhE1 random mutations

[0116] To improve the expression efficiency of aldosterone dehydrogenase, we screened the enzymes in Example 1... adhE1Chemical random mutagenesis (such as dNTP, Tris-HCl mix) is performed to screen for more efficient aldol dehydrogenases. adhE1 For the mutant, please refer to Example 1 for specific steps.

[0117] The PCR amplification system is shown in Table 7:

[0118] Table 7 Amplification System

[0119]

[0120] The main components of the Tris-HCl mix described in Table 7 are Tris-HCl 10 mM, KCl 50 mM, and MgCl2 2 mM. These were obtained through screening and sequencing. adhE1 The positive mutants are: adhE1-1, adhE1-4, adhE1-8, adhE1-12, and adhE1-16.

[0121] wild type adhE1 Nucleotide sequence for reference:

[0122] adhE1-1: adhE1 The 2267th base of the gene is mutated from A to C;

[0123] adhE1-4: adhE1 The T-base mutation at position 1058 of the gene changes to C;

[0124] adhE1-8: adhE1 The 616th base of the gene is mutated from G to A, and the 2054th base is mutated from A to G.

[0125] adhE1-12: adhE1 The 173rd base of the gene is mutated from G to A;

[0126] adhE1-16: adhE1 The 285th base of the gene is mutated from T to G.

[0127] The strains after being transformed into each mutant were named MDF-9-5, MDF-9-6, MDF-9-7, MDF-9-8, and MDF-9-9, respectively.

[0128] 2. Fermentation test of mutant trimer

[0129] Strains MDF-9-5, MDF-9-6, MDF-9-7, MDF-9-8, and MDF-9-9 require the addition of 50 μL chloramphenicol (25 mg / L), 5 g / L BDO, and 2.5 g / L PDO. Refer to Example 1 for specific procedures.

[0130] The fermentation test results are as follows:

[0131] Table 8. Fermentation test of recombinant strain P34HB3HP

[0132]

[0133] Fermentation data showed that all mutant strains had increased cell dry weight and PHA content compared to the non-mutated strains, with strain MDF-9-6 (mutant adhE1-4) showing the greatest increase, exhibiting the highest cell dry weight, PHA content, and proportions of each monomer. Therefore, mutant strains will be used in subsequent studies. adhE1-4 Further investigation will be conducted.

[0134] Example 4: Optimal mutant genome integration and fermentation test

[0135] 1. adhE1 Genome integration at the G51 site of the gene (optimal mutant adhE1-4)

[0136] Integral plasmid pRE112 adhE1 Build:

[0137] PCR amplification adhE1 Gene and pRE112 scaffold with G51 upstream and downstream homologous arms; adhE1 The gene and pRE112 backbone were combined by Gibson ligase to form a new plasmid and labeled as pRE112. adhE1 Subsequently, pRE112 adhE1 Get the template adhE1 Gene sequence; a portion of the product was sent to a biotechnology company for sequencing; plasmid information is as follows: Figure 10 As shown. Refer to Example 1 for specific steps.

[0138] Screening for genome-integrating strains:

[0139] Select single colonies with the correct sequence for propagation. After 12-16 hours, conjugate them with MDF-9 on 20LB plates. After 8 hours, pick a small amount of conjugated cells and spread them on 60LB plates with the corresponding antibiotic resistance. Perform the first validation after 36-48 hours. If the validation is successful, remove the antibiotic.

[0140] Antibiotic removal involves subculturing in a 60LB deep-well plate at 42℃ for 3-7 generations (one generation every 24 hours; plating verification begins from generation 3). After antibiotic removal, a small amount of bacterial culture is added to 60LB (approximately 10...). 5 -10 6 Diluted medium is spread evenly on 60 LB plates. For subculturing, transfer to fresh 60 LB medium (without antibiotics) at a ratio of 0.02%.

[0141] After single colonies grow on plates, streak them onto 60LB and 60LB (cm) plates respectively. After 12 hours of incubation, colonies that grow on 60LB plates but not on 60LB (cm) plates are considered successfully de-antibioticated. Selected colonies for PCR verification are then used for sequencing.

[0142] The results showed that the MDF-9 strain in this embodiment successfully integrated [the enzyme]. adhE1 The gene was used, and the strain was named MDF-9-10. Verification was performed based on the size of the target product, such as... Figure 11 As shown, the target fragment is 2730 bp, which is in line with the expected result.

[0143] Among them, amplification adhE1 Fragment and primer sequences for the pRE112 plasmid backbone:

[0144] adhE-F: See SEQ ID No. 15;

[0145] adhE-R: See SEQ ID No. 16;

[0146] V2-F: See SEQ ID No. 17;

[0147] V2-R: See SEQ ID No. 18.

[0148] 2. Recombinant strains synthesized dimers P34HB and P3HB3HP

[0149] (1) Adding 1,3-propanediol to synthesize dimer P3HB3HP

[0150] The specific steps for the fermentation test are as described in Example 1, wherein 50 μL of chloramphenicol (25 mg / L) needs to be added to MDF-9-6, and 2.5 g / L of PDO needs to be added to both strains MDF-9-6 and MDF-9-10.

[0151] The fermentation test results are as follows:

[0152] Table 9. P3HB3HP fermentation test of recombinant strains

[0153]

[0154] Fermentation data showed that the genome-integrated strain MDF-9-10 produced the dimer P3HB3HP better than the plasmid-expressed strain MDF-9-6, with improvements in cell dry weight, PHA content, and 3HP ratio.

[0155] (2) Adding 1,4-butanediol to synthesize dimer P34HB

[0156] The specific steps for the fermentation test are as described in Example 1, wherein 50 μL of chloramphenicol (25 mg / L) needs to be added to MDF-9-6, and 5 g / L of BDO needs to be added to both strains MDF-9-6 and MDF-9-10.

[0157] The fermentation test results are as follows:

[0158] Table 10 Fermentation test of recombinant strain P34HB

[0159]

[0160] Fermentation data showed that the genome-integrated strain MDF-9-10 produced the dimer P34HB better than the plasmid-expressed strain MDF-9-6, with improvements in cell dry weight, PHA content, and 4HB ratio.

[0161] (3) The recombinant strain synthesized trimer P34HB3HP

[0162] The specific steps for the fermentation test are as described in Example 1. MDF-9-6 requires the addition of 50 μL of chloramphenicol (25 mg / L), while strains MDF-9-6 and MDF-9-10 require the addition of 5 g / L BDO and 2.5 g / L PDO.

[0163] The fermentation test results are as follows:

[0164] Table 11 Fermentation test of recombinant strain P34HB3HP

[0165]

[0166] Fermentation data showed that, similar to the results of the dimer fermentation test, the genome-integrated strain MDF-9-10 produced trimers better than the plasmid-expressed strain MDF-9-6, with improvements in cell dry weight, PHA content, and the proportion of each monomer.

[0167] Example 5: Construction of a recombinant strain producing P34HB3HP

[0168] 1. Construction of recombinant Halomonas strain producing P34HB3HP

[0169] The plasmid pSEVA321 containing the optimal mutant was used. adhE1 Transfected with Halomonas LY01 LY02 LY03 LY04, respectively named LY01-A, LY02-A, LY03-A, and LY04-A.

[0170] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L), along with 5 g / L of BDO and 2.5 g / L of PDO.

[0171] The fermentation test results are as follows:

[0172] Table 12 Fermentation tests of other Halomonas strains (P34HB3HP)

[0173]

[0174] Fermentation test data showed that all four halophilic bacteria could synthesize trimer P34HB3HP through this modification strategy, with strain LY03-A showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other halophilic bacteria.

[0175] 2. Construct other recombinant strains to produce P34HB3HP

[0176] The plasmid pSEVA321 containing the optimal mutant was used. adhE1 The bacteria were respectively introduced into Escherichia coli (EcN), Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 17699), Pseudomonas putida (ATCC 47054), and Akkermansia myxophilus (ATCCBAA-835).

[0177] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L), along with 5 g / L of BDO and 2.5 g / L of PDO.

[0178] The fermentation test results are as follows:

[0179] Table 13 Fermentation tests of other recombinant strains using P34HB3HP

[0180]

[0181] Fermentation test data showed that all five strains could synthesize trimer P34HB3HP through this modification strategy, with Escherichia coli EcN strain showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other strains of non-halophilic bacteria.

[0182] 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 any one of the following: (1) With Escherichia coli adhE Compared to genes, adhE The 2267th base of the gene is mutated from A to C; (2) With Escherichia coli adhE Compared to genes, adhE The T-base mutation at position 1058 of the gene changes to C; (3) With Escherichia coli adhE Compared to genes, adhE The 616th base of the gene is mutated from G to A, and the 2054th base is mutated from A to G. (4) With Escherichia coli adhE Compared to genes, adhE The 173rd base of the gene is mutated from G to A; (5) With Escherichia coli adhE Compared to genes, adhE The 285th base of the gene is mutated from T to G; The adhE The nucleotide sequence of the gene is shown in SEQ ID No. 1; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate-4-hydroxybutyrate, poly-3-hydroxybutyrate-3-hydroxypropionate, or poly-3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxypropionate.

2. The strain according to claim 1, characterized in that, The strain is any one of the genera *Haloxylon*, *Escherichia coli*, *Klebsiella*, *Rochetomyces*, *Pseudomonas*, and *Ackermania*.

3. The method for constructing the strain according to any one of claims 1 to 2, characterized in that, Includes the following steps: (1) adhE Genes are introduced into plasmids to obtain recombinant plasmids; (2) Introduce the recombinant plasmid described in step (1) into the strain.

4. A method for producing polyhydroxy fatty acid esters, characterized in that, The method includes fermenting the strain according to any one of claims 1 to 2; The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate-4-hydroxybutyrate, poly-3-hydroxybutyrate-3-hydroxypropionate, or poly-3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxypropionate.

5. The method according to claim 4, characterized in that, The fermentation medium used for fermentation contains any one or more of 1,4-butanediol and 1,3-propanediol; The amount of 1,4-butanediol used is 2.5~10 g / L, and the amount of 1,3-propanediol used is 2.5~10 g / L.

6. The method according to claim 4, characterized in that, The fermentation culture temperature is 30~42℃.

7. The method according to claim 4, characterized in that, The pH of the fermentation culture was 6-11.

8. The use of the strain according to any one of claims 1 to 2 in the production of polyhydroxyalkanoates, The polyhydroxy fatty acid ester is poly-3-hydroxybutyrate-4-hydroxybutyrate, poly-3-hydroxybutyrate-3-hydroxypropionate, or poly-3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxypropionate.

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