Recombinant halomonas, construction method thereof and application of recombinant halomonas in production of polyhydroxyalkanoate

Abstract

The invention provides recombinant halomonas, a construction method of the recombinant halomonas and application of the recombinant halomonas in production of polyhydroxyalkanoate. The recombinant halomonas is used for expressing the 2-keto acid decarboxylase or the 2-keto acid decarboxylase mutant. According to the invention, a 2-ketonic acid decarboxylase mediated 3HV synthesis path is constructed, alpha-ketoglutaric acid in tricarboxylic acid circulation can be directly converted into 3-hydroxyvaleryl coenzyme A, de novo synthesis of a 3HV monomer is realized, a conventional carbon source can be efficiently utilized to synthesize 3HV, and the traditional technical bottleneck of dependence on an exogenous propionic acid or valeric acid precursor is broken through; the 3HV with a higher proportion can be obtained by regulating and controlling a metabolic pathway under the condition of not adding an exogenous precursor, so that the fermentation process is remarkably simplified, and the raw material cost and the production complexity are reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant halomonas bacterium, its construction method, and its application in the production of polyhydroxy fatty acid esters. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of biodegradable polyesters synthesized by microorganisms. They are mainly composed of various hydroxy fatty acid monomers and possess excellent biocompatibility, biodegradability, and processability. They are currently recognized as a green and environmentally friendly material that can replace petroleum-based plastics. PHAs are widely used in packaging materials, medical devices, agricultural films, and many other fields. The performance of PHAs largely depends on their monomer composition and proportions. Common PHAs include homopolymers such as poly(3-hydroxybutyrate) (PHB), and copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV).

[0003] P3HB3HV significantly improves the brittleness, flexibility, and ductility of materials by adjusting the proportion of 3-hydroxyvalerate (3HV) monomers; however, its production typically relies on adding expensive 3HV precursors (such as propionic acid or valerate), resulting in high production costs and limiting its widespread industrial application. P3HB4HB3HV, on the other hand, introduces 4-hydroxybutyric acid (4HB) monomers, giving the material flexibility similar to low-density polyethylene, and further improves processing performance through 3HV, combining excellent ductility and mechanical strength. However, its synthesis process is complex due to the need for precise control of the three monomer proportions, requiring sophisticated production equipment and control strategies. Furthermore, the poor supply stability and high price of 4HB precursors (such as γ-butyrolactone) further limit the feasibility of large-scale production.

[0004] Currently, research on increasing the 3HV content in PHA copolymers mainly employs methods such as microbial metabolic engineering and enzyme engineering. Existing metabolic engineering strategies mainly include: (1) synthesizing 3HV by exogenously adding propionic acid based on knocking out the propionyl-CoA degradation pathway; (2) directly adding valeric acid to increase the 3HV content; and (3) constructing a de novo synthesis pathway for 3HV, such as introducing exogenous... scpB The gene converts succinyl-CoA in the tricarboxylic acid cycle (TCA) to propionyl-CoA, thereby enhancing the endogenous synthesis capacity of 3HV. For the aforementioned engineered strains, carbon source optimization, gene modification, and fermentation process regulation are usually required to further improve the yield and proportion of 3HV.

[0005] In enzyme engineering, mutant libraries with different enzyme activities are often constructed through mutagenesis, and strains with different proportions of products are obtained through high-throughput screening, thus providing a variety of choices for PHA materials with different performance requirements. However, the existing 3HV synthesis strategy still has several technical bottlenecks: (1) The product regulation pathway has limited selectivity, and currently it mainly relies on the propionic acid / valeric acid pathway, lacking other effective synthesis methods; (2) While the exogenous addition of 3HV precursors can improve the synthesis efficiency of the target product, it often has a toxic effect on the strains and inhibits their normal growth; (3) The addition of precursors leads to complex fermentation processes and high production costs. Moreover, in the process of industrial scale-up, the more complex the raw materials, the easier it is to interfere with the stability of the process, which is not conducive to large-scale promotion.

[0006] Therefore, there is an urgent need to develop a novel PHA copolymer synthesis strategy that requires no additional precursors, has a more efficient and stable synthesis route, exhibits strong strain tolerance, and simplifies the production process, in order to further lower the industrialization threshold of PHA and promote its widespread application in the field of environmentally friendly materials. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a recombinant Halomonas bacterium, its construction method, and its application in the production of polyhydroxy fatty acid esters.

[0008] This invention provides a recombinant halomonas bacterium, wherein the recombinant halomonas bacterium expresses any one of the following: (1) 2-Keto acid decarboxylase; (2) 2-Keto acid decarboxylase mutant; The amino acid sequence of the 2-keto acid decarboxylase is shown in SEQ ID No. 24; The 2-keto acid decarboxylase mutant differs from 2-keto acid decarboxylase in any of the following ways: (a) The 99th amino acid is threonine; (b) The 7th amino acid is serine and the 196th amino acid is histidine; (c) The 202nd amino acid is tryptophan; (d) The 512th amino acid is glycine; (e) The 219th amino acid is isoleucine and the 470th amino acid is arginine; (f) The third amino acid is arginine, the third and second amino acids are valine and the fifth and tenth amino acids are glutamic acid.

[0009] In some embodiments, the 2-keto acid decarboxylase is derived from... Lactococcus or its derivatives Collets gigas or its derivatives Atopostipes suicloacalis Or any one of its derivatives; preferably Lactococcus lactis .

[0010] In some embodiments, the recombinant halomonas also expresses xylB Gene, xylC Gene, xylD Gene, xylX Genes and yqhD Gene.

[0011] In some embodiments, the recombinant Halomonas expresses xylB Gene xylC Gene xylD Gene 、 xylX Genes originate from Caulobacter or its derivatives 、Pseudomonas sp. or its derivatives Azotobacter vinelandii or any one of its derivatives; yqhD Genes originate from E. coli or its derivatives Salmonella enterica or its derivatives Klebsiella pneumoniae or its derivatives; preferably, the xylB, xylC, xylD, xylX Genes of microorganisms originate from Caulobacter crescentus, yqhD Genes originate from E. coli MG1655 .

[0012] In some embodiments, the halomonas is or its derivatives Halomonas bluephagenesis or its derivatives Halomonas campaniensis or its derivatives and Halomonas aydingkolgenesis Or any one of its derivatives.

[0013] In a specific embodiment of the present invention, the *Haloxymonas* is preferably... MDF-9, LY01, LY02, LY03, LY04.

[0014] The present invention also provides a method for constructing the recombinant halomonas bacteria, comprising the following steps: (1) Construct an expression vector containing the 2-keto acid decarboxylase gene or the 2-keto acid decarboxylase mutant gene; (2) The expression vector constructed in (1) is introduced into Halomonas to obtain the recombinant Halomonas.

[0015] The nucleotide sequence of the 2-keto acid decarboxylase gene is shown in SEQ ID NO.1.

[0016] In some embodiments, the expression vector is in plasmid form.

[0017] In some embodiments, the plasmid can integrate a foreign gene into the chromosomal genome for expression or can express a foreign gene on the plasmid; when expressed on the plasmid, the plasmid can be a high-copy plasmid or a low-copy plasmid.

[0018] 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.

[0019] 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).

[0020] The present invention also provides a method for producing polyhydroxy fatty acid esters, the method comprising fermenting and culturing the recombinant halomonas bacteria.

[0021] 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.

[0022] In the above-described method for producing PHA, the fermentation medium 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 requiring closed fermentation conditions to avoid contamination) or under closed conditions to avoid contamination.

[0023] The culture 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.

[0024] The fermentation medium can be liquid, solid, or semi-solid.

[0025] The culture medium may contain carbon and nitrogen sources, and / or carbon and / or nitrogen sources may be added to the culture and fermentation system during the fermentation process. Carbon sources may be, for example, sugars, fats, organic acids and organic acid esters, etc., but are not limited to these. Nitrogen sources may be organic nitrogen sources, inorganic nitrogen sources, or mixtures thereof, such as beef extract, yeast extract, corn steep liquor powder, oilseed meal powder, urea, peptone, gelatin, ammonium sulfate, ammonium chloride, potassium nitrate, etc., but are not limited to these.

[0026] In one specific embodiment of the present invention, the fermentation medium used for fermentation is a 50 mm medium.

[0027] In some embodiments, the fermentation temperature is preferably 30-45°C, and the pH is preferably 6.5-12.

[0028] In some embodiments, the polyhydroxy fatty acid ester is a polyhydroxy fatty acid ester containing a monomer of 3-hydroxybutyric acid.

[0029] In some embodiments, the polyhydroxy fatty acid ester (PHA) is a homopolymer or copolymer of monomers.

[0030] The monomers mentioned include, but are not limited to, 3-hydroxybutyric acid (3-HB), 3-hydroxyvalerate (3-HV), 4-hydroxybutyric acid (4-HB), 4-hydroxyvalerate (4-HV), etc.

[0031] In some embodiments, the polyhydroxy fatty acid ester containing the 3-hydroxybutyric acid monomer is preferably poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV).

[0032] The present invention also provides a method for adjusting the ratio of polyhydroxyalkanoate monomers, the method comprising fermenting and culturing the recombinant halomonas bacteria.

[0033] The present invention also provides polyhydroxy fatty acid esters synthesized using the aforementioned recombinant halomonas bacteria.

[0034] The present invention also provides the application of the recombinant halometazoa in the production of polyhydroxy fatty acid esters.

[0035] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention constructs a 3HV synthesis pathway mediated by 2-keto acid decarboxylase (kivD), which can directly convert α-ketoglutarate in the tricarboxylic acid cycle into 3-hydroxyvalerate coenzyme A, realizing the de novo synthesis of 3HV monomers. It can efficiently utilize conventional carbon sources (such as glucose) to synthesize 3HV, breaking through the technical bottleneck of traditional reliance on exogenous propionic acid or valerate precursors. It realizes that a high proportion of 3HV can be obtained through metabolic pathway regulation without the need for exogenous precursors, which significantly simplifies the fermentation process and reduces raw material costs and production complexity.

[0036] (2) The synthesis strategy in this invention does not require disrupting the original metabolic pathway, which simplifies the strain construction process and improves the genetic stability and growth performance of the strain.

[0037] (3) By mutating kivD, the present invention further increases the content of 3HV monomer and achieves effective regulation of the 3HV ratio in PHA. Attached Figure Description

[0038] 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.

[0039] Figure 1 This is a pathway diagram of the P3HB3HV metabolic pathway. Figure 2 This is a pathway diagram of the P3HB4HB3HV metabolic pathway. Figure 3 Example 1 of the present invention, pSEVA341 kivD Plasmid map; Figure 4 Example 1 of the present invention, pRE341 kivD Electrophoresis verification gel image; Figure 5 Example 3 of the present invention, pSEVA321 xylB-xylC-xylD-xylX Plasmid map; Figure 6 Example 3 of the present invention, pSEVA321 xylB-xylC-xylD-xylX Electrophoresis verification gel image; Figure 7 Example 3 of the present invention, pSEVA341 kivD-yqhD Plasmid mapping; Figure 8 Example 3pSEVA341 of the present invention kivD-yqhD Electrophoresis to verify the gel image. Detailed Implementation

[0040] 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.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0042] 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.

[0043] Example 1: Construction of the 3HV Pathway 1. Construction of the 3HV pathway (1) Plasmid construction PCR amplification using overlap extension kivD Gene and pSEVA341 vector, under the action of Gibson ligase, kivD The gene and the pSEVA341 vector were combined to form a new plasmid and labeled as pSEVA341. kivD Plasmid information such as Figure 3 As shown. Using pSEVA341 kivD Get the template kivD Gene sequence (nucleotide sequence as shown in SEQ ID NO.1), a portion of the product was sent to a biotechnology company for sequencing.

[0044] Among them, PCR amplification kivD The primer sequences (5'-3') for the pSEVA341 backbone are as follows: kivD -F: SEQ ID No. 2; kivD -R: SEQ ID No. 3; pSEVA341-F: SEQ ID No. 4; pSEVA341-R: SEQ ID No. 5.

[0045] The amplification system and amplification procedure are shown in Tables 1 and 2: Table 1 Amplification System

[0046] Table 2 Amplification Procedure

[0047] 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.

[0048] (2) Gibson Assembly method connection 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 pSEVA341 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. Table 3 Gibson Assembly Connection System Table

[0049] Table 4 Gibson Assembly Connector

[0050] (3) Transformation of S17-1 Escherichia coli Step 1: Take the pre-prepared S17-1 Escherichia coli competent cells out of -80℃, thaw them on ice, and wait for the bacterial block to thaw after 5 minutes; Step 2: Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not shake to mix). Note: The conversion volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used; Step 3: Ice bath for 30 min, heat shock in 42℃ water bath for 2 min, then immediately place on ice to cool for 2 min. Note: Shaking will reduce conversion efficiency. Step 4: Add 400 μL of LB medium (antibiotic-free) to the centrifuge tube, mix well, and then place in a 37°C shaker at 200 rpm for 60 min to recover. Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of supernatant, keep 100 μL, gently pipette to resuspend the bacterial block and spread it on LB medium containing the corresponding antibiotic; Step 6: Invert the culture medium and incubate it in a 37°C incubator for 12-16 hours.

[0051] (4) Verification of positive monoclonal colonies Colonies were picked from the corresponding antibiotic LB plates and colony PCR was performed for verification. PCR products with the correct band size were sent to a biotechnology company for sequencing.

[0052] (5) Select single colonies with correct sequences for amplification, and after 12-16 h, combine with... MDF-9 was conjugated on 20 LB plates. After 8 h, a small amount of conjugated cells were picked and spread on 60 LB plates with corresponding resistance. After 36-48 h, a single colony verification was performed.

[0053] (6) PCR verification of strains Colony PCR results showed that this example pSEVA341 was successfully conjugated in the MDF-9 strain. kivD The size of the target product was verified by PCR using plasmids, and the electrophoresis image is shown below. Figure 4 As shown, the target fragment is 3080bp, which meets the expected result. (The fragment will contain pSEVA341.) kivD The strain on the substrate of the plasmid was named MDF-9-1.

[0054] Example 2: Fermentation test of P3HB3HV production by recombinant strain MDF-9-1 (1) Culture medium: 60LB agar medium: yeast extract 0.5%, tryptone 1%, sodium chloride 60%, agar powder 1.8g / 100mL, ampicillin 50 μg / mL, kanamycin 30 g / mL, pH 8.0.

[0055] Fermentation medium (50 mm): glucose 30 g / L, sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 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. 0.3g / L, CoCl2·6H2O 0.2g / L, CuSO4·5H2O 0.01g / L, NiCl2·6H2O 0.02g / L, NaMoO4·2H2O 0.03 g / L.

[0056] (2) Seed liquid preparation ① Activation of microbial strains 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.

[0057] ② Primary seed culture: 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.

[0058] ③ Secondary seed culture: Take 200 μL of primary bacterial culture (1% inoculum) and inoculate it into a 150 mL Erlenmeyer flask (20 mL of 60 LB medium). Incubate on a shaker at 37°C and 220 rpm for 12 h.

[0059] (3) Preparation of fermentation medium Fermentation medium (50 mm): glucose 30 g / L, sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 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. 0.3g / L, CoCl2·6H2O 0.2g / L, CuSO4·5H2O 0.01g / L, NiCl2·6H2O 0.02g / L, NaMoO4·2H2O 0.03 g / L.

[0060] (4) Fermentation culture Inoculate the seed culture at 5% (1 mL) into a 150 mL Erlenmeyer flask and incubate on a shaker at 37°C and 220 rpm for 48 h.

[0061] (5) Determination of cell dry weight and PHA content Cell dry weight (CDW): Place 10-35 mL of fermented bacterial culture into a 50 mL centrifuge tube, centrifuge at room temperature for 6 minutes at 8000 rpm, discard the supernatant; add an appropriate amount of deionized water to restore the original volume, resuspend, ensuring that the precipitate completely disappears, centrifuge under the same conditions, 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 cell dry weight (g / L).

[0062] PHA content determination: Weigh 0.05 g of the fermented dried cells, grind them, 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: Table 5 Program Temperature Settings

[0063] The injection volume was 1 μL, and PHA was quantitatively analyzed using the external standard method. The yield of PHA was calculated based on the peak area.

[0064] (6) Fermentation results The fermentation results of the recombinant strain MDF-9-1 constructed using the 3HV pathway are shown in Table 6: Table 6. Fermentation results of PHA production by strain 6

[0065] The results showed that, compared with the control strain, MDF-9-1 was expressed exogenously. kivD Without the addition of precursors, glucose enters the TCA cycle to generate α-ketoglutarate, which is then further processed... kivD Metabolism produces 3HV monomer, which accounts for 6.63% of the total.

[0066] Example 3: Construction of the 4HB3HV pathway Since the xylose pathway involves many genes, this embodiment expresses the genes of this pathway using two plasmids. Among them, kivD has a dual function, which, in addition to performing its function in the xylose metabolism pathway, also participates in the metabolism of the 3HV pathway.

[0067] 1. xyl BCDXplasmid construction PCR amplification of xyl BCDX With the pSEVA321 backbone, xyl ligase was used to ligate xyl BCDX The fragment and the pSEVA321 backbone recombine to form a new plasmid, named pSEVA321. xylBCDX pSEVA321 was obtained through overlap extension PCR. xylBCDX xyl is obtained from the template BCDX (The nucleotide sequence is shown in SEQ ID No. 6). A portion of the product was sent to a biotechnology company for sequencing. Specific construction and transformation steps are described in Example 1. Plasmid information is as follows: Figure 5 As shown. Verification was performed by checking the size of the target fragment, as... Figure 6 As shown, the target fragment is 5402bp, which is in line with the expected result.

[0068] Among them, amplification xylBCDX Primer sequences of the fragment and pSEVA321 plasmid backbone xylB -F: SEQ ID No. 7; xylB -R: SEQ ID No. 8; xylC -F: SEQ ID No. 9; xylC -R: SEQ ID No. 10; xylD -F: SEQ ID No. 11; xylD -R: SEQ ID No. 12; xylX -F: SEQ ID No. 13; xylX -R: SEQ ID No. 14; pSEVA321-F: SEQ ID No. 15; pSEVA321-R: SEQ ID No. 16.

[0069] 2. kivD - yqhD plasmid construction PCR amplification kivD - yqhD The plasmid pSEVA341 backbone, along with Gibson ligase, was used to ligate it. kivD - yqhD The fragment and the pSEVA341 backbone recombine to form a new plasmid, named pSEVA341. kivD-yqhDpSEVA341 was obtained through overlap extension PCR. kivD-yqhD Get the template kivD - yqhD (The nucleotide sequence is shown in SEQ ID No. 17). A portion of the product was sent to a biotechnology company for sequencing. Specific construction and transformation steps are described in Example 1. Plasmid information is as follows: Figure 7 As shown. Verification was performed by checking the size of the target fragment, as... Figure 8 As shown, the target fragment is 4687 bp, which is consistent with the expected result. Then, the plasmid pSEVA321... xylBCDX and pSEVA341 kivD-yqhD Join to MDF-9-BDO strain (in) Based on the MDF-9 strain, the BDO gene cluster was integrated ( aldD - dhaT-orfZ The nucleotide sequence is shown in SEQ ID No. 25 (1-1520bp is aldD, 1535-2720bp is dhaT, 3046-4336 is orfZ), and strain MDF-9--BDO-1 was obtained.

[0070] kivD -1-F: SEQ ID No. 18; kivD -1-R: SEQ ID No. 19; yqhD -F: SEQ ID No. 20; yqhD -R: SEQ ID No. 21; pSEVA341-1-F: SEQ ID No. 22; pSEVA341-1-R: SEQ ID No. 23.

[0071] Example 4: Fermentation test of P3HB4HB3HV produced by recombinant strain MDF-9-BDO-1 (1) Culture medium For specific steps, please refer to Example 2.

[0072] (2) Seed liquid preparation For specific steps, please refer to Example 2.

[0073] (3) Preparation of fermentation medium Fermentation medium (50 mm): glucose 20 g / L, xylose 10 g / L, sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 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. 0.3g / L, CoCl2·6H2O 0.2g / L, CuSO4·5H2O 0.01g / L, NiCl2·6H2O 0.02g / L, NaMoO4·2H2O0.03 g / L.

[0074] (4) Fermentation culture For specific steps, please refer to Example 2.

[0075] (5) Determination of cell dry weight and PHA content For specific steps, please refer to Example 2.

[0076] (6) Fermentation results The fermentation results of the recombinant strain MDF-9-BDO-1, which constructed the 4HB and 3HV pathways, are shown in Table 7: Table 7. Fermentation results of PHA production by the strain

[0077] The results showed that, compared with the control strain, MDF-9-BDO-1 expressed xyl through exogenous expression. BCDX , kivD and yqhD Without the addition of precursors, the simultaneous addition of glucose and xylose allows for dual-pathway metabolism. The 3HV monomer enters the TCA cycle from glucose to generate α-ketoglutarate, which is then metabolized via kivD, reaching a molar proportion of 3.01%. The 4HB monomer is generated from xylose via xyl... BCDX Path generation.

[0078] Example 5: random mutation of kivD To obtain different proportions of 3HV, kivD was randomly mutated (chemical method) in this embodiment, and strains producing different proportions of 3HV monomers were then screened from the mutants. The specific construction method and steps are the same as in Example 1. The primer sequences for amplifying kivD are the same as in Example 1; The amplification system is shown in Table 8: Table 8 Amplification System

[0079] The main components of the Tris-HCl mix described in Table 8 are Tris-HCl 10 mM, KCl 50 mM, and MgCl2 2 mM.

[0080] (3) After screening and sequencing, the kivD mutants were obtained. The mutants were as follows: serine at position 99 was mutated to threonine and named kivD-1 (S99T); tyrosine at position 7 was mutated to serine and asparagine at position 196 was mutated to histidine and named kivD-2 (Y7S, N196H); leucine at position 202 was mutated to tryptophan and named kivD-3 (L202W); alanine at position 512 was mutated to glycine and named kivD-4 (A512G); phenylalanine at position 219 was mutated to isoleucine and glutamine at position 470 was mutated to arginine and named kivD-5 (F219I, Q470R); threonine at position 3 was mutated to arginine, leucine at position 323 was mutated to valine and lysine at position 510 was mutated to glutamic acid and named kivD-6 (T3R, L323V, K510E).

[0081] The strains containing the six kivD mutants were named MDF-9-kivD-1 (S99T), MDF-9-kivD-2 (Y7S, N196H), MDF-9-kivD-3 (L202W), MDF-9-kivD-4 (A512G), MDF-9-kivD-5 (F219I, Q470R), and MDF-9-kivD-6 (T3R, L323V, K510E), respectively. Fermentation tests were conducted, and the specific fermentation steps were as described in Example 1. The fermentation results are shown in Table 9. Table 9 Fermentation results of the kivD mutant

[0082] The results showed that by randomly mutagenizing kivD, six mutant strains producing different proportions of 3HV monomers were screened, namely MDF-9-kivD-1~6, with 3HV monomer molar ratios of 3.71%, 7.82%, 5.56%, 9.75%, 12.77%, and 8.55%, respectively. The MDF-9-kivD-5 mutant strain had the highest dry weight, PHA, and 3HV, increasing to 12.69 g / L, 84.31%, and 12.77%, respectively. Specifically, the mutation of phenylalanine at position 219 to isoleucine and glutamine at position 470 to arginine in kivD increased cell dry weight and PHA content, and promoted 3HV synthesis. In practical applications of PHA, the rigidity and ductility properties of materials can be altered by adding different amounts of 3HV monomers. PHA with low 3HV content is rigid and has low ductility, and is mainly used in disposable rigid packaging, agricultural film, and 3D printing filaments. PHA with high 3HV content is flexible and has high ductility, and is mainly used in flexible packaging, medical materials, and high value-added products. The 3HV molar ratio obtained in this embodiment is between 3% and 12%, which can provide a certain selection according to the rigidity requirements of the material in practical applications.

[0083] Example 6: Fermentation test of other recombinant strains producing P3HB3HV The strains selected in this experiment were *Halomonas*. LY01 LY02 LY03 LY04.

[0084] (1) Construction of the 3HV pathway In this experiment, the kivD-5 mutant was selected to construct the 3HV pathway for different strains. The recombinant strains after successful pathway construction were named LY01-1, LY02-1, LY03-1 and LY04-1, respectively. For specific steps, please refer to Example 1.

[0085] (2) Culture medium For specific steps, please refer to Example 2.

[0086] (3) Seed liquid preparation For specific steps, please refer to Example 2.

[0087] (4) Preparation of fermentation medium For specific steps, please refer to Example 2.

[0088] (5) Fermentation culture For specific steps, please refer to Example 2.

[0089] (6) Determination of cell dry weight and PHA content For specific steps, please refer to Example 2.

[0090] (7) Fermentation results The fermentation results of other recombinant strains constructing the 3HV pathway are shown in Table 10: Table 10 Fermentation results of PHA production by other recombinant strains

[0091] The results show that the method for constructing the 3HV pathway in this invention is also applicable to other Halomonas bacteria. From the fermentation results of this experiment, the recombinant strain LY03-1 had the highest strain dry weight, PHA content, and 3HV monomer molar ratio, which were 12.53 g / L, 81.89%, and 11.51 mol, respectively.

[0092] Example 7: Fermentation test of other recombinant strains producing P3HB4HB3HV The strains selected in this experiment were those that integrated the BDO gene cluster ( dhaT-aldD - orfZ )of MDF-9-BDO, LY01-BDO LY02-BDO LY03-BDO LY04-BDO.

[0093] (1) Construction of the 4HB3HV pathway In this experiment, the kivD-5 mutant was selected to construct the 4HB3HV pathway for different strains. The recombinant strains after successful pathway construction were named MDF-9-BDO-2, LY01-BDO-1, LY02-BDO-1, LY03-BDO-1, and LY04-BDO-1, respectively. For specific steps, please refer to Example 3.

[0094] (2) Culture medium For specific steps, please refer to Example 2.

[0095] (3) Seed liquid preparation For specific steps, please refer to Example 2.

[0096] (4) Preparation of fermentation medium For specific steps, please refer to Example 4.

[0097] (5) Fermentation culture For specific steps, please refer to Example 2.

[0098] (6) Determination of cell dry weight and PHA content For specific steps, please refer to Example 2.

[0099] (7) Fermentation results The fermentation results of the recombinant strains constructed using the 4HB and 3HV pathways are shown in Table 11: Table 11 Fermentation results of PHA production by other recombinant strains

[0100] The results show that the construction method of the 4HB3HV pathway in this invention is also applicable to other Halomonas bacteria. From the fermentation results of this experiment, the recombinant strain MDF-9-BDO-2 had the highest strain dry weight, PHA content, 4HB monomer molar ratio, and 3HV monomer molar ratio, at 9.67 g / L, 69.35%, 7.72%, and 6.22%, respectively. The recombinant strain LY03-BDO-1 had the next highest strain dry weight, PHA content, 4HB monomer molar ratio, and 3HV monomer molar ratio, at 9.57 g / L, 68.19%, 7.07%, and 5.67%, respectively.

[0101] 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 recombinant halomonas bacterium, characterized in that, The recombinant Halomonas strain expresses any one of the following: (1) 2-Keto acid decarboxylase; (2) 2-Keto acid decarboxylase mutant; The amino acid sequence of the 2-keto acid decarboxylase is shown in SEQ ID No. 24; The 2-keto acid decarboxylase mutant differs from 2-keto acid decarboxylase in any of the following ways: (a) The 99th amino acid is threonine; (b) The 7th amino acid is serine and the 196th amino acid is histidine; (c) The 202nd amino acid is tryptophan; (d) The 512th amino acid is glycine; (e) The 219th amino acid is isoleucine and the 470th amino acid is arginine; (f) The third amino acid is arginine, the third and second amino acids are valine and the fifth and tenth amino acids are glutamic acid.

2. The recombinant Halomonas bacillus according to claim 1, characterized in that, The recombinant Halomonas also expresses xylB Gene, xylC Gene, xylD Gene, xylX Genes and yqhD Gene.

3. The recombinant halometazoa according to claim 2, characterized in that, The xylB Gene xylC Gene xylD Gene xylX Genes originate from Caulobacter or its derivatives 、Pseudomonas sp. or its derivatives Azotobacter vinelandii or any one of its derivatives; The yqhD Genes originate from E. coli or its derivatives Salmonella enterica or its derivatives Klebsiella pneumoniae Or its derivatives.

4. The recombinant halomonas according to claim 1, characterized in that, The halometa is or its derivatives Halomonas bluephagenesis or its derivatives Halomonas campaniensis Or its derivatives and any one of Halomonas aydingkolgenesis or its derivatives.

5. A method for constructing the recombinant halomonas strain according to claim 1, characterized in that, Includes the following steps: (1) Construct an expression vector containing the 2-keto acid decarboxylase gene or the 2-keto acid decarboxylase mutant gene; (2) The expression vector constructed in (1) is introduced into Halomonas to obtain the recombinant Halomonas.

6. A method for producing polyhydroxyalkanoates, characterized in that, The method includes fermentation culture of the recombinant halomonas strain according to any one of claims 1 to 4.

7. The method according to claim 6, characterized in that, The polyhydroxy fatty acid ester is a polyhydroxy fatty acid ester containing a monomer of 3-hydroxybutyric acid.

8. A method for adjusting the ratio of polyhydroxyalkanoate monomers, characterized in that, The method includes fermentation culture of the recombinant halomonas strain according to any one of claims 1 to 4.

9. A polyhydroxy fatty acid ester synthesized using the recombinant halomonas strain according to any one of claims 1 to 4.

10. The use of the recombinant Halomonas strain according to any one of claims 1 to 4 in the production of polyhydroxy fatty acid esters.

Images