Construction method of Halomonas J9U and its derivatives, PHA preparation method and application

Abstract

The present invention provides a construction method of Halomonas J9U and its derivatives, a PHA preparation method and application, and belongs to the field of genetic engineering technology. The present invention connects the upstream and downstream homology arm sequences of the upp gene with the suicide plasmid pK18mobsacB to obtain a targeting vector pK18Δupp, which is transferred into the aspiring Halomonas J9, and two homologous recombinations occur to obtain J9U. The homologous recombination efficiency of this construction method is extremely high, up to 100%. The maximum yield of PHA produced by the J9UΔxylD‑P8xylA strain using xylose as the sole carbon source is 2.81g / L, and the strain is an advantageous chassis strain in the co-utilization of xylose and glucose. It has superior growth and PHA yield advantages in lignocellulose bioconversion, greatly reducing production costs.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a construction method of Halomonas J9U and its derivatives, and a PHA preparation method and application. Background Art

[0002] Industrial biotechnology refers to the conversion of raw materials into desired chemicals, materials, and energy sources under mild conditions through microbial fermentation or cell-free biocatalysis. It can effectively alleviate environmental pollution and achieve sustainable development. However, existing industrial biotechnology suffers from numerous drawbacks: susceptibility to microbial contamination; stringent requirements for bioreactors, operators, fermentation conditions, and procedures; slow microbial growth; expensive raw materials; low substrate-to-product conversion rates; difficulty in continuous production; difficulty in product isolation and purification; and high freshwater and energy consumption. These shortcomings result in high production costs for industrial biotechnology, making it difficult to compete with the traditional chemical industry, which relies on low-cost petroleum. Next-generation industrial biotechnology is a low-cost bioprocessing technology that leverages extreme microorganisms that thrive in specialized environments. Halomonas spp. can grow rapidly in high-salt and alkaline environments, providing a dual guarantee for limiting the growth of contaminants. Developing next-generation industrial biotechnology based on Halomonas spp. can effectively conserve freshwater and energy, reduce the complexity of fermentation processes, and reduce production costs.

[0003] Polyhydroxyalkanoate (PHA) is a class of granular polymer biomaterials that are widely present in microorganisms. PHAs are polyesters formed by the ester polymerization of different R-type hydroxyalkanoic acid monomers, but all have the same general structural formula and can be divided into two categories based on the side chain composition of the monomers: monomers composed of 3 to 5 carbon atoms are called short-chain PHAs, and monomers composed of 6 to 14 carbon atoms or more are called long-chain PHAs. There are also some mixed types of PHAs composed of medium-long and short chains, such as polyhydroxybutyrate glycolate, and copolymers composed of 3HB and other 3-hydroxy fatty acids with more than 6 carbon atoms. Among them, the homopolymer 3-hydroxybutyrate (PHB) is a common PHA homopolymer in microorganisms.

[0004] Currently, researchers have developed a variety of gene editing tools and regulatory elements for Halomonas to rapidly obtain superior target traits. Halomonas bluephagenesis was isolated by Chen Guoqiang's team at Tsinghua University from Ayding Lake in Xinjiang. This strain can grow in seawater for weeks to months without sterilization and without contamination. H. bluephagenesis TD01 is an ideal platform for the development of next-generation industrial biotechnology for the production of PHA and other high-value compounds using glucose as the primary carbon source. Fu et al. established a method for plasmid conjugation and transformation into halophilic bacteria, and developed a marker-free gene knockout technology using the suicide vector pRE1126I-SceI. This is a recombination system—the I-SceI gene knockout system—that combines the characteristics of homologous recombination and site-specific recombination. I-SceI is a non-fixed intron-encoded endonuclease found in S. cerevisiae. Like Flp / FRT and Cre / loxP, this enzyme recognizes an 18-bp specific recognition site. However, unlike the other two, this endonuclease causes double-strand breaks in the DNA molecule at the recognition site, thereby inducing the strain genome's own SOS emergency repair system and achieving seamless gene knockout. However, this method is currently time-consuming and inefficient, and cannot knock out large genomic fragments, limiting its scope of application. A more efficient gene editing method is urgently needed.

[0005] Finding cheap carbon sources as substrates for PHA fermentation can effectively reduce the production cost of PHAs and improve the market competitiveness of PHAs. At the same time, it can achieve the recycling and reuse of some waste resources to solve a series of environmental pollution problems, such as lignocellulose. At present, lignocellulose is the most abundant and cheapest renewable biomass resource on the earth. It is widely found in forests and agricultural waste such as wheat straw, corn straw and rice straw. The ability to effectively utilize these waste biomass resources to convert and produce high-value-added chemicals PHAs can greatly reduce the substrate production cost of PHAs. Usually, lignocellulose includes 40-50% cellulose, 20-50% hemicellulose and 20-30% lignin. Before using lignocellulose as a substrate, it must first be pretreated with acid or alkali to destroy the structure of lignocellulose and then enzymatically hydrolyzed by cellulase and hemicellulase to obtain lignocellulose hydrolyzate. Finally, microorganisms can use these fermentable sugars to produce PHAs.

[0006] In addition to glucose, xylose is the most abundant pentose in lignocellulose hydrolysates. Cellobiose is also an abundant disaccharide in lignocellulose hydrolysates. The ability to simultaneously and effectively utilize glucose, xylose, or cellobiose to produce PHA is crucial for the fermentation and production of lignocellulose hydrolysates. Currently, Tan et al. used Halomonas bacterium H. bluephagenesis to modify the xylose metabolic pathway. Since it cannot naturally utilize xylose, they first introduced four different xylose-specific transporters. However, xylose can only be transported but not consumed. Further expression of the xylose isomerase xylA from Escherichia coli enabled the cells to utilize xylose to accumulate 0.39 g / L of poly-3-hydroxybutyrate (PHB). Additional overexpression of xylulose kinase to direct carbon flux into the pentose phosphate pathway reduced PHB production. Subsequently, a ribulose-1-phosphate pathway was constructed by introducing D-tagatose-3-epimerase, fucokinase, and fucose phosphate aldolase. The recombinant strain produced 0.87 g / L of PHB and a cell dry weight of 2.09 g / L. Finally, another carbon-efficient phosphoketolase pathway was introduced by overexpressing an exogenous phosphoketolase. This pathway increased the cell dry weight and PHB titer to 1.88 g / L and 1.2 g / L, respectively. This is the first report on xylose utilization by H. bluephagenesis and will provide a useful engineering strategy for the production of industrial polyhydroxyalkanoates from xylose. However, the cell dry weight after this modification was still low, and the presence of glucose inhibited xylose utilization in a mixed sugar fermentation medium of 10 g / L glucose and 10 g / L xylose, thus significantly limiting its application in the fermentation of lignocellulose hydrolysates. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a construction method of Halomonas J9U and its derivatives, a PHA preparation method and application, wherein the Halomonas J9U derivatives can utilize xylose to produce polyhydroxyalkanoates, and the obtained polyhydroxyalkanoates have a high yield.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for constructing Halomonas J9U, comprising the following steps:

[0010] The suicide plasmid pK18mobsacB was digested to obtain the linearized plasmid pK18mobsacB;

[0011] The upstream and downstream homology arm sequences of the upp gene were ligated with the linearized plasmid pK18mobsacB to obtain the targeting vector pK18Δupp;

[0012] The host cells containing the targeting vector pK18Δupp were transformed into Halomonas aspirans J9 to obtain recombinant bacteria. The recombinant bacteria underwent two homologous recombination steps to obtain Halomonas J9U.

[0013] Preferably, the host cell is Escherichia coli S17-1λpir;

[0014] The method of the two homologous recombination comprises screening the single-exchange strain, performing the first homologous recombination on the recombinant bacteria to obtain a double-band strain, and then performing the second homologous recombination on the double-band strain; the method of screening the single-exchange strain comprises culturing the recombinant bacteria in a high-salt medium containing kanamycin sulfate resistance; the method of screening the double-exchange strain comprises culturing the double-band strain in a high-salt medium containing 5-fluorouracil;

[0015] The high-salt culture medium includes a 60LB culture medium, and the preparation method of the 60LB culture medium is as follows: 25.0g / L LB Mix and 60.0g / L NaCl are mixed; the concentration of the kanamycin sulfate is greater than 100μg / mL; and the concentration of the 5-fluorouracil is greater than 100μg / mL.

[0016] The present invention also provides a Halomonas J9U derivative bacterium, wherein the xylD gene in the genome of the Halomonas J9U derivative bacterium is inactivated, and the Halomonas J9U derivative bacterium includes the xylA gene; the starting strain of the Halomonas J9U derivative bacterium is the Halomonas J9U obtained by the above-mentioned construction method.

[0017] Preferably, the nucleotide sequence of the xylD gene is shown as SEQ ID No.31, and the nucleotide sequence of the xylA gene is shown as SEQ ID No.33; a promoter for upregulating the xylA gene is inserted upstream of the xylA gene, and the promoter is the P8 promoter, and the nucleotide sequence of the P8 promoter is shown as SEQ ID No.32.

[0018] The present invention also provides a method for constructing the above-mentioned Halomonas J9U derivative, characterized in that it comprises the following steps:

[0019] The upp gene was ligated with the linearized plasmid pK18mobsacB to obtain the recombinant plasmid pKJU;

[0020] The recombinant plasmid pKJU was digested with enzymes to obtain the linearized plasmid pKJU;

[0021] The upstream and downstream homology arm sequences of the xylD gene were ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-ΔxylD;

[0022] The P8xylA gene sequence containing upstream and downstream homology arms was ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-P8xylA;

[0023] The targeting vector pKJU-ΔxylD was transferred into the Halomonas J9U obtained by the above construction method to obtain the recombinant J9U. After two homologous recombination steps, the recombinant J9UΔxylD was obtained.

[0024] The targeting vector pKJU-P8xylA was transferred into the recombinant bacterium J9UΔxylD to obtain the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA. After two homologous recombination steps, the derivative bacterium of Halomonas J9U was obtained.

[0025] Preferably, the two homologous recombination methods include using a single-exchange strain to screen the recombinant bacteria J9U or the recombinant bacteria J9UΔxylD containing the targeting vector pKJU-P8xylA for a first homologous recombination to obtain a strain with a double band, and then performing a second homologous recombination on the strain with the double band; the single-exchange strain screening method includes culturing the recombinant bacteria J9U or the recombinant bacteria J9UΔxylD containing the targeting vector pKJU-P8xylA in a high-salt medium containing kanamycin sulfate resistance; the double-exchange strain screening method includes culturing the strain with the double band in a high-salt medium containing 5-fluorouracil;

[0026] The high-salt culture medium includes a 60LB culture medium, and the 60LB culture medium is prepared by mixing 25.0 g / L LB Mix and 60.0 g / L NaCl; the concentration of kanamycin sulfate is greater than 100 μg / mL; the concentration of 5-fluorouracil is greater than 100 μg / mL;

[0027] Preferably, the upp gene is a promoter-containing upp gene, and the nucleotide sequence of the promoter-containing upp gene is shown as SEQ ID No. 35; the nucleotide sequence of the P8xylA is shown as SEQ ID No. 36.

[0028] The present invention also provides a use of the above-mentioned Halomonas J9U derivative in the preparation of polyhydroxyalkanoates.

[0029] The present invention also provides a method for preparing polyhydroxyalkanoate, which comprises culturing the above-mentioned Halomonas J9U derivative in a fermentation medium containing xylose to obtain polyhydroxyalkanoate.

[0030] Preferably, the fermentation medium further comprises glucose; preferably, the fermentation medium further comprises NH4Cl; preferably, the fermentation medium further comprises glucose and NH4Cl;

[0031] Preferably, the concentration of xylose is 10 to 30 g / L;

[0032] Preferably, the concentration of glucose is 8 to 12 g / L;

[0033] Preferably, the concentration of NH4Cl is 10-20 g / L;

[0034] Preferably, the culture is open fermentation culture.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a method for constructing Halomonas J9U and its derivatives, a method for preparing PHA and its application. The present invention utilizes fusion PCR technology to construct upstream and downstream homology arm sequences of the upp gene, and connects them with the suicide plasmid pK18mobsacB to construct a targeting vector pK18Δupp, which is inserted into the eager Halomonas J9. Through two homologous recombinations, Halomonas J9U is obtained. The homologous recombination efficiency of this construction method is extremely high, up to 100%. The maximum yield of PHA produced by the Halomonas J9U derivative of the present invention using xylose as the sole carbon source is 2.81g / L. Moreover, the Halomonas J9U derivative also exhibits more superior characteristics in the co-utilization of xylose and glucose. The continuous consumption of xylose promotes the co-utilization of glucose. The xylose consumption increases from 9.5g / L to 17g / L, and the glucose consumption also increases by 4g / L. OD 600 After 72 hours, it reached approximately 20.0, approximately 2.5 times that of the wild type. The Halomonas J9U derivative is a superior chassis strain for the co-utilization of xylose and glucose, and exhibits superior growth and PHA production in lignocellulose bioconversion, significantly reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the upp gene knockout process;

[0038] Figure 2 This is the verification result of wild type H. cupida J9 resistance to 5-FU;

[0039] Figure 3 This is the electrophoresis diagram of PCR verification of the construction of upp knockout vector pK18Δupp. Lane M is Marker III, and lanes 1 to 6 are positive clones of Escherichia coli DH5α containing plasmid pK18Δupp.

[0040] Figure 4Electrophoresis diagram of single-crossover strain PCR verification for J9U construction, lane M is Marker III; lanes 1 to 24 are the selected transformants; lane 25 is wild-type J9;

[0041] Figure 5 This is the electrophoresis diagram of double-crossover PCR verification of the chassis strain J9U. Lane M is Marker III; lanes 1 to 15 are picked clones; lane 16 is wild-type J9.

[0042] Figure 6 The results show the resistance of wild-type H. cupida J9 and H. cupida J9U to 5-FU.

[0043] Figure 7 This is a construction map of the targeting vector pKJU;

[0044] Figure 8 Schematic diagram of gene knockout in H. cupida J9U using the reverse selection marker upp combined with a suicide plasmid (pKJU);

[0045] Figure 9 Schematic diagram of gene insertion in H. cupida J9U using the reverse selection marker upp combined with a suicide plasmid (pKJU);

[0046] Figure 10 Electrophoresis diagram of PCR verification of the J9UΔxylD-P8xylA engineered strain. Lanes: M, Marker; 1 and 2 represent the engineered strain J9UΔxylD-P8xylA and wild strain J9U, respectively.

[0047] Figure 11 RT-PCR detection of the transcription of the exogenous gene xylA in the engineered strain J9UΔxylD-P8xylA. Lanes: M, Marker; 1, genomic DNA as PCR template; 2, cDNA as PCR template; 3, mRNA as PCR template; 4, dH2O as PCR template;

[0048] Figure 12 The biomass accumulation of the engineered strain J9UΔxylD-P8xylA in fermentation medium with different xylose concentrations;

[0049] Figure 13 Figure 2 is the growth curve and xylose consumption curve of different engineered strains. A is the growth curve of different xylose engineered strains in a xylose fermentation medium containing 30 g / L xylose; B is the xylose consumption curve of different engineered strains in a xylose fermentation medium containing 30 g / L xylose.

[0050] Figure 14The shake flask fermentation results of wild-type J9U, engineered strain J9U-P8xylA, and J9UΔxylD-P8xylA are shown;

[0051] Figure 15 Figure GC-MS analysis of the monomer composition of PHA produced by the engineered strain J9UΔxylD-P8xylA using xylose fermentation. A is the total ion chromatogram (TIC) of all PHA monomers in the xylose fermentation product of J9UΔxylD-P8xylA; B is the extracted ion chromatogram (EI) of methyl 3-hydroxybutyrate; C is the extracted ion chromatogram (EI) of methyl 3-hydroxylaurate;

[0052] Figure 16 Figure 2 shows the growth curves and shake flask fermentation results of xylose engineering strains when excess nitrogen source was added. Figure 2 shows the growth curves of J9U-P8xylA and J9UΔxylD-P8xylA in nitrogen-excess xylose fermentation medium. Figure 2 shows the shake flask fermentation results of J9UΔxylD-P8xylA in nitrogen-excess xylose fermentation medium.

[0053] Figure 17 Figure 3 shows the growth curve and xylose consumption curve of the xylose engineering strain J9UΔxylD-P8xylA in xylose fermentation medium with different nitrogen source concentrations. Figure 3 shows the growth curve and xylose consumption curve of the xylose engineering strain J9UΔxylD-P8xylA in xylose fermentation medium with different nitrogen source concentrations. Figure 3 shows the growth curve of J9UΔxylD-P8xylA in xylose fermentation medium with NH4Cl concentrations of 2, 5, 10, 15, 20, and 30 g / L (30 g / L xylose). Figure 3 shows the growth curve and xylose consumption curve of J9UΔxylD-P8xylA in xylose fermentation medium with NH4Cl concentrations of 2, 5, 10, 15, 20, and 30 g / L (30 g / L xylose).

[0054] Figure 18 Figure 2 shows the growth curves and monosaccharide consumption curves of the wild type and engineered strains in mixed sugar fermentation medium. Figure 2 shows the growth curves and monosaccharide consumption curves of J9U in a mixed sugar fermentation medium containing 20 g / L xylose and 10 g / L glucose. Figure 2 shows the growth curves and monosaccharide consumption curves of J9UΔxylD-P8xylA in a mixed sugar fermentation medium containing 20 g / L xylose and 10 g / L glucose.

[0055] Figure 19 The fermentation results of xylose engineering strain J9UΔxylD-P8xylA in mixed sugar fermentation medium and mixed sugar nitrogen fermentation medium;

[0056] Figure 20 These are the fermentation results of the xylose-engineered strain J9UΔxylD-P8xylA in corn straw hydrolysate. DETAILED DESCRIPTION

[0057] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] The present invention provides a method for constructing Halomonas J9U, comprising the following steps:

[0059] The suicide plasmid pK18mobsacB was digested to obtain the linearized plasmid pK18mobsacB;

[0060] The upstream and downstream homology arm sequences of the upp gene were ligated with the linearized plasmid pK18mobsacB to obtain the targeting vector pK18Δupp;

[0061] The host cells containing the targeting vector pK18Δupp were transformed into Halomonas aspirans J9 to obtain recombinant bacteria. The recombinant bacteria underwent two homologous recombination steps to obtain Halomonas J9U.

[0062] In the present invention, the suicide plasmid pK18mobsacB is digested with EcoRI to obtain a linearized plasmid pK18mobsacB. After obtaining the linearized plasmid pK18mobsacB, the upstream and downstream homology arm sequences of the upp gene are ligated with the linearized plasmid pK18mobsacB to obtain a targeting vector pK18Δupp. The ligation is carried out by homologous recombination. The upstream homology arm sequence of the upp gene is obtained by PCR amplification using Δupp-UF / UR with genomic DNA of Halomonas aspirans J9 as a template; the downstream homology arm sequence of the upp gene is obtained by PCR amplification using Δupp-DF / DR with genomic DNA of Halomonas aspirans J9 as a template. The upstream and downstream homology arm sequences of the upp gene are obtained by PCR amplification of the upstream and downstream homology arm sequences using fusion PCR technology. After obtaining the targeting vector pK18Δupp, the host cell containing the targeting vector pK18Δupp is transferred into Halomonas aspirans J9 to obtain a recombinant bacterium. The recombinant bacterium undergoes two homologous recombination steps to obtain Halomonas J9U. The host cell is preferably Escherichia coli S17-1λpir. The transfer is carried out by conjugation transfer. The two homologous recombination steps include screening the recombinant bacterium with a single exchange strain for a first homologous recombination to obtain a double-band strain, and then performing a second homologous recombination on the double-band strain. The single exchange strain screening step includes culturing the recombinant bacterium in a high-salt culture medium containing kanamycin sulfate resistance. The double exchange strain screening step includes culturing the double-band strain in a high-salt culture medium containing 5-fluorouracil.

[0063] The high-salt culture medium includes a 60LB culture medium, and the preparation method of the 60LB culture medium is as follows: 25.0g / L LB Mix and 60.0g / L NaCl are mixed; the concentration of the kanamycin sulfate is greater than 100μg / mL; and the concentration of the 5-fluorouracil is greater than 100μg / mL.

[0064] The present invention also provides a Halomonas J9U obtained according to the above construction method.

[0065] The present invention also provides a Halomonas J9U derivative bacterium, wherein the xylD gene in the genome of the Halomonas J9U derivative bacterium is inactivated, and the Halomonas J9U derivative bacterium includes the xylA gene; the starting strain of the Halomonas J9U derivative bacterium is the Halomonas J9U obtained by the above-mentioned construction method.

[0066] In the present invention, the nucleotide sequence of the xylD gene is shown in SEQ ID No.31, and the nucleotide sequence of the xylA gene is shown in SEQ ID No.33; a promoter for upregulating the xylA gene is inserted upstream of the xylA gene, and the promoter is the P8 promoter, and the nucleotide sequence of the P8 promoter is shown in SEQ ID No.32.

[0067] The present invention also provides a method for constructing the above-mentioned Halomonas J9U derivative, characterized in that it comprises the following steps:

[0068] The upp gene was ligated with the linearized plasmid pK18mobsacB to obtain the recombinant plasmid pKJU;

[0069] The recombinant plasmid pKJU was digested with enzymes to obtain the linearized plasmid pKJU;

[0070] The upstream and downstream homology arm sequences of the xylD gene were ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-ΔxylD;

[0071] The P8xylA gene sequence containing upstream and downstream homology arms was ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-P8xylA;

[0072] The targeting vector pKJU-ΔxylD was transferred into the Halomonas J9U obtained by the above construction method to obtain the recombinant J9U. After two homologous recombination steps, the recombinant J9UΔxylD was obtained.

[0073] The targeting vector pKJU-P8xylA was transferred into the recombinant bacterium J9UΔxylD to obtain the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA. After two homologous recombination steps, the derivative bacterium of Halomonas J9U was obtained.

[0074] In the present invention, the upp gene is ligated with the linearized plasmid pK18mobsacB to obtain the recombinant plasmid pKJU. The upp gene is preferably a promoter-containing upp gene, the nucleotide sequence of which is shown in SEQ ID No. 35. The ligation site is an EcoRI site. The recombinant plasmid pKJU is digested with the restriction endonuclease EcoRI to obtain the linearized plasmid pKJU.

[0075] In the present invention, the upstream and downstream homology arm sequences of the xylD gene are connected to the linearized plasmid pKJU to obtain a targeting vector pKJU-ΔxylD, and the connection is connected by homologous recombination. The upstream homology arm sequence of the xylD gene is obtained by PCR amplification using the genomic DNA of the above-mentioned Halomonas J9U as a template and using ΔxylD-UF / UR (SEQ ID No. 13-14); the downstream homology arm sequence of the ΔxylD gene is obtained by PCR amplification using the genomic DNA of the Halomonas J9U as a template and using ΔxylD-DF / DR (SEQ ID No. 15-16). The upstream and downstream homology arm sequences of the upp gene are obtained by PCR amplification of the upstream homology arm sequence and the downstream homology arm sequence using a fusion PCR technology.

[0076] In the present invention, the P8xylA gene sequence containing upstream and downstream homology arms is connected to the linearized plasmid pKJU to obtain the targeting vector pKJU-P8xylA, and the connection is connected by homologous recombination. The upstream homology arm sequence of the P8xylA gene is obtained by PCR amplification using the genomic DNA of the above-mentioned Halomonas J9U as a template and using P8xylA-UF / UR (SEQ ID No. 19-20); the downstream homology arm sequence of the P8xylA gene is obtained by PCR amplification using the genomic DNA of Halomonas J9U as a template and using P8xylA-DF / DR (SEQ ID No. 25-26); the nucleotide sequence of the P8 gene is shown in SEQ ID No. 32. The nucleotide sequence of the P8xylA is shown in SEQ ID No. 36. The nucleotide sequence of the xylA gene is shown in SEQ ID No. 33. The P8xylA gene sequence containing upstream and downstream homology arms is obtained by PCR amplification of the upstream homology arm sequence, the nucleotide sequence of the P8 gene, the nucleotide sequence of the xylA gene and the downstream homology arm sequence using a fusion PCR technique.

[0077] In the present invention, the targeting vector pKJU-ΔxylD is transferred into the Halomonas J9U obtained by the above-mentioned construction method to obtain the recombinant bacterium J9U, and after two homologous recombination, the recombinant bacterium J9UΔxylD is obtained. The two homologous recombination methods include using a single-exchange strain to screen the recombinant bacterium J9U for a first homologous recombination to obtain a strain with a double-layer band, and then screening the double-layer band strain for a second homologous recombination; the single-exchange strain screening method includes culturing the recombinant bacterium J9U in a high-salt medium containing kanamycin sulfate resistance; the double-exchange strain screening method includes culturing the double-layer band strain in a high-salt medium containing 5-fluorouracil;

[0078] The high-salt culture medium includes a 60LB culture medium, and the preparation method of the 60LB culture medium is as follows: 25.0g / L LB Mix and 60.0g / L NaCl are mixed; the concentration of the kanamycin sulfate is greater than 100μg / mL; and the concentration of the 5-fluorouracil is greater than 100μg / mL.

[0079] In the present invention, after obtaining the recombinant bacterium J9UΔxylD, the targeting vector pKJU-P8xylA is transferred into the recombinant bacterium J9UΔxylD to obtain the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA. After two homologous recombination processes, a derivative bacterium of Halomonas J9U is obtained. The two homologous recombination processes include screening the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA with a single exchange strain for a first homologous recombination to obtain a strain with a double-layer band, and then screening the double-layer band with a double-layer band for a second homologous recombination. The single exchange strain screening process includes culturing the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA in a high-salt culture medium containing kanamycin sulfate resistance. The double exchange strain screening process includes culturing the strain with a double-layer band in a high-salt culture medium containing 5-fluorouracil.

[0080] The high-salt culture medium includes a 60LB culture medium, and the preparation method of the 60LB culture medium is as follows: 25.0g / L LB Mix and 60.0g / L NaCl are mixed; the concentration of the kanamycin sulfate is greater than 100μg / mL; and the concentration of the 5-fluorouracil is greater than 100μg / mL.

[0081] The present invention also provides a Halomonas J9U derivative obtained according to the above construction method.

[0082] The present invention also provides a use of the above-mentioned Halomonas J9U derivative in the preparation of polyhydroxyalkanoates.

[0083] The present invention also provides a method for preparing polyhydroxyalkanoate, which comprises culturing the above-mentioned Halomonas J9U derivative in a fermentation medium containing xylose to obtain polyhydroxyalkanoate.

[0084] In the present invention, the xylose concentration is preferably 10-30 g / L. The fermentation medium also includes glucose, and the glucose concentration is preferably 8-12 g / L. Preferably, the fermentation medium also includes NH4Cl, and the NH4Cl concentration is preferably 10-20 g / L. Preferably, the fermentation medium further includes glucose and NH4Cl. The culture is preferably an open fermentation culture. The culture temperature is preferably 35-40°C.

[0085] In the present invention, the Halomonas J9U derivative can produce PHA by fermenting xylose, and can also produce PHA by simultaneously utilizing xylose and glucose. The yield of the obtained PHA is high, which greatly reduces the production cost.

[0086] In the following examples, Escherichia coli DH5α was purchased from Nanjing Novozyme Biotechnology Co., Ltd., Catalog No. C502. Escherichia coli S17-1λpir was purchased from Belgrade Technology Tianjin Co., Ltd., Catalog No. DL2010S. pK18mobsacB was purchased from Shanghai Baifeng Biological Co., Ltd. LB Mix was purchased from BD Biosciences, USA. Xylose and cellobiose were purchased from Beijing Dingguo Bioengineering Co., Ltd., and the xylose standard solution was purchased from the Institute of Biology, Shandong Academy of Sciences.

[0087] Example 1 A method for constructing Halomonas J9U

[0088] 1.1.1 Primer sequence information

[0089] The primer sequence information used in this example is shown in Table 1.

[0090] Table 1 Primers and sequence information

[0091]

[0092] 1.1.2 Culture media and solutions

[0093] 1.1.2.1 Culture medium

[0094] Luria-Bertani (LB) medium: Weigh 25.0 g of LB Mix and dissolve in distilled water to 1 L. If a solid medium is needed, add 1.8% to 2% (w / v) agar powder. Autoclave at 121°C for 20 minutes. Store at room temperature.

[0095] 60LB medium containing salt: Weigh 25.0g LB Mix, add 60.0g NaCl, and dilute to 1L with distilled water. Adjust to pH 9.0 with NaOH. If a solid medium is required, add 1.8%–2% (w / v) agar powder. Autoclave at 121°C for 20 minutes. Store at room temperature.

[0096] 1.1.2.2 Solution

[0097] 50× TAE running buffer: Weigh 242.0 g Tris, 37.2 g Na₂EDTA·2H₂O, add 5.71% (v / v) glacial acetic acid, and finally add deionized water to 1 L, pH 8.2. Store at room temperature until ready to use. For agarose gel electrophoresis, dilute 50-fold to 1× TAE running buffer.

[0098] Antibiotic stock solution and usage concentration:

[0099] (1) Kanamycin sulfate (Kan): The stock solution concentration is 50 mg / mL. Dissolve 0.5 g of Kanamycin sulfate powder in 10 mL of deionized water. After fully dissolving, filter sterilize using a 0.22 μm water filter and store at -20°C in the dark until used. The working concentration for Escherichia coli screening is 50 μg / mL; the working concentration for Halomonas cupida J9 screening is 50 μg / mL.

[0100] (2) 5-Fluorouracil (5-FU): The stock solution concentration is 40 mg / mL. Weigh 0.4 g of 5-FU powder into 10 mL of dimethyl sulfoxide. After it is fully dissolved, store at -20°C in the dark until ready to use. The working concentration is 100 μg / mL.

[0101] 1.2 Experimental methods

[0102] 1.2.1 5-FU sensitivity test of wild-type Halomonas cupida J9

[0103] Halomonas cupida J9 stored in a -80°C glycerol tube was streaked onto 60LB solid medium and cultured overnight at 37°C. Single colonies were picked from the solid plate and inoculated into 5 mL of 60LB liquid medium containing 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL 5-fluorouracil, respectively. The culture was shaken at 37°C and 180 rpm, and the OD was measured after overnight culture. 600 .

[0104] Whole-genome sequencing revealed that J9 contains a single copy of the upp gene (encoding uracil phosphoribosyltransferase), which catalyzes the reaction of 5-fluorouracil (5-FU), a toxic antimetabolite commonly used as an anti-tumor drug, with uridine to produce 5-fluoro-UMP (5-fluoro-UMP). 5-fluoro-UMP can then be converted into 5-fluoro-dUMP through the microorganism's own metabolism. 5-fluoro-dUMP is an inhibitor of thymidylate synthase, hindering the production of thymidylate. Thymidylate is an adenine deoxyribonucleoside essential for microbial DNA synthesis. A lack of this deoxyribonucleoside impairs DNA synthesis, ultimately leading to cell death.

[0105] The growth of wild type Halomonas cupida J9 strain in 60LB tubes containing different concentrations of 5-FU after overnight culture is shown in Figure 2. Figure 2 The results showed that wild-type Halomonas cupida J9 cells were insensitive to low concentrations of 5-FU and exhibited some resistance. However, as the 5-FU concentration increased, the survival rate of J9 cells gradually decreased, particularly at concentrations above 100 μg / mL, where J9 cells were virtually incapable of growth. This experiment provides a reference value for the 5-FU concentration used in subsequent double-exchange screening: adding 100 μg / mL of 5-FU was sufficient for screening.

[0106] 1.2.2 Extraction of the wild-type Halomonas cupida J9 (H. cupida J9 or Halomonas aspirans J9) genome

[0107] 1. Extract the genome of H. cupida J9 according to the kit instructions of Vazyme. Use Vazyme's bacterial genomic DNA rapid isolation kit to extract the genomic DNA of H. cupida J9.

[0108] 1.2.3 Amplification and purification of DNA fragments

[0109] Vazyme high-fidelity polymerase was used to PCR amplify the upstream and downstream homology arms of the upp gene using Δupp-UF / UR and Δupp-DF / DR (see Table 1 for details), respectively. Each arm was about 600 bp, and fragments 1 and 2 were obtained, respectively.

[0110] The PCR amplification system is shown in Table 2:

[0111] Table 2 PCR amplification system for upstream and downstream homology arms of upp gene

[0112]

[0113] The fusion PCR reaction system is shown in Table 3:

[0114] Table 3 Fusion PCR reaction system

[0115]

[0116]

[0117] PCR reaction conditions are shown in Table 4:

[0118] Table 4 PCR reaction conditions

[0119]

[0120] The fragments amplified by PCR were subjected to agarose gel electrophoresis (agarose content 0.8% to 1.0%), and Marker III was used as a tool to measure the molecular weight of DNA. DNA fragments were recovered using Vazyme's product purification kit. Specific extraction information can be found in the instructions to obtain upstream and downstream homology arms.

[0121] 1.2.4 Construction of pK18mobsacB-Δupp knockout plasmid (pK18Δupp)

[0122] The upstream and downstream homology arms obtained by fusion PCR were integrated into the EcoRI site of the suicide plasmid pK18mobsacB, as shown in Figure 1 First, the plasmid pK18mobsacB was digested with restriction endonuclease EcoRI to obtain a linearized plasmid.

[0123] Enzyme digestion system Table 5:

[0124] Table 5 Enzyme digestion system

[0125]

[0126] The above enzyme digestion system was mixed by pipetting and placed in a 37°C water bath for 45 minutes.

[0127] The target DNA fragment was connected to the linearized plasmid by homologous recombination ligation, and the vector was constructed using the seamless cloning kit of Vazyme.

[0128] The recombination connection system is shown in Table 6:

[0129] Table 6 Recombination connection system

[0130]

[0131] The above-mentioned recombination ligation system was pipetted and mixed, and the reaction was allowed to proceed at 37°C for 30 min. The ligation product (pK18mobsacB plasmid containing upstream and downstream homology arms, i.e., pK18Δupp) was transformed into E. coli DH5α competent cells, and positive clones were verified by colony PCR.

[0132] 2.2.4 Chemical transformation of plasmid DNA and identification of positive clones

[0133] 1. Thaw the competent E. coli DH5α and S17-1λpir stored at -80°C on ice;

[0134] 2. Under sterile conditions, gently add 10 μL of the ligation product (pK18Δupp) to the freshly thawed E. coli competent cells and incubate on ice for 30 minutes;

[0135] 3. Heat shock the system in a 42°C metal bath for 30 seconds, then immediately place it in an ice bath for 2 minutes.

[0136] 4. Aseptically add 1 mL of LB liquid medium to the upper system and incubate at 37°C, 180 rpm for 1 hour.

[0137] 5. Centrifuge the revived product at 4°C, 5000 rpm for 5 min. Discard the supernatant under sterile conditions and retain the bacteria. Resuspend the bacteria in about 100 μL of supernatant, spread on LB plates containing 50 μg / mL kan, and culture in a 37°C incubator overnight.

[0138] 6. Pick a single colony from the plate and place it in LB liquid medium containing the corresponding resistance. After recovery at 37°C and 180 rpm for 3 hours, use the detection primers pK-JF / JR of the plasmid pK18mobsacB (see Table 1) to perform bacterial liquid PCR to identify positive clones and transformants.

[0139] The colony PCR system is shown in Table 7:

[0140] Table 7 Colony PCR system

[0141]

[0142] Colony PCR reaction conditions are shown in Table 8:

[0143] Table 8 Colony PCR reaction conditions

[0144]

[0145] The PCR band size was detected by 0.8% agarose gel electrophoresis, and the positive clones with the correct band size were transferred to 5 mL LB liquid culture medium containing the corresponding resistance and cultured at 37°C and 180 rpm overnight.

[0146] 1.2.5 Plasmid DNA extraction

[0147] The plasmid was extracted using the FastPure Plasmid Mini Kit from Vazyme. After sequencing and alignment, the extracted plasmid was transformed into E. coli S17-1λpir using the chemical transformation method described in 1.2.4.

[0148] Colony PCR was performed using the detection primers pK-JF / JR of plasmid pK18mobsacB to verify the electrophoresis patterns of E. coli DH5α and S17-1λpir containing plasmid pK18Δupp. Figure 3 As shown, the amplified band was approximately 1400 bp, proving that the targeting vector pK18Δupp was successfully constructed.

[0149] 1.2.6 Joint transfer

[0150] Conjugative transfer is an effective method for transferring plasmids into H. cupida J9. The specific transformation method is as follows:

[0151] 1. Streak the H. cupida J9 strain and the E. coli S17-1λpir strain containing different vectors stored at -80°C onto LB plates;

[0152] 2. Pick E. coli S17-1λpir and H. cupida J9 and their derivatives containing the target plasmid into 5 mL LB and 60 LB liquid culture medium respectively, and culture at 37°C, 180 rpm for 12 h;

[0153] 3. Take the bacterial solution from step 2 that has been cultured overnight and transfer it to 50mL LB and 60mL LB liquid culture medium containing relevant antibiotics at a ratio of 2%, and culture at 37℃ and 180r / min for 6-8h to make OD 600 Reach 1.5-2.0;

[0154] 4. Take 1 mL of the cultured bacterial solution from step 3 and place it in a sterile 1.5 mL EP tube. Centrifuge at 5000 rpm for 5 minutes and discard the supernatant.

[0155] 5. Add LB and 60LB solution of equal volume to the bacterial suspension to the EP tube, gently resuspend the bacteria to elute the antibiotics, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0156] 6. Repeat step 5;

[0157] 7. Add 60 μL of LB and 60 LB liquid respectively, gently pipette to resuspend the bacteria, and gently mix the bacterial suspensions of E. coli S17-1λpir and H. cupida J9 and its derivatives in a ratio of 1:1, 2:1, and 3:1, and dropwise onto the 0.22 μm filter attached to the 60 LB solid plate and culture overnight;

[0158] 8. Pick up the filter and dissolve it in 1 ml of 60LB liquid culture medium. Shake thoroughly for 2 minutes. Pipette 100 μL and spread it on a 60LB plate containing 100 μg / mL Kan. Incubate at 37°C for 36 hours. Perform colony PCR on the grown colonies to screen for the correct transformants.

[0159] 1.2.7 Screening of upp gene deletion mutant strain J9U

[0160] 1.2.7.1 Screening of single-crossover strains

[0161] The suicide plasmid pK18mobsacB used in this example is a shuttle-type suicide plasmid with kanamycin resistance. This plasmid can replicate freely in commercial Escherichia coli, but when the host bacteria is Halomonas, it does not have the ability to replicate independently and can only be site-specifically integrated and inserted into the chromosome of the host bacteria. Otherwise, it will be cut and degraded by the host strain as an exogenous fragment. When the upp gene in the Halomonas cupida J9 genome is successfully knocked out, the mutant strain has 5-FU resistance. Therefore, the above-mentioned reverse screening marker can be used to screen the conjugates to obtain Halomonas cupida J9 with a deletion of the upp gene. The principle is as follows Figure 1 The operation steps are as follows:

[0162] 1. The upp gene targeting vector pK18Δupp was transformed into H. cupida J9 strain by conjugation and plated on 60LB plates containing 100 μg / mL Kan resistance medium at 37°C for 36 h.

[0163] 2. Under sterile conditions, single colonies were picked from the plates and cultured overnight in 60 LB liquid medium containing 100 μg / mL Kan resistance. Single crossover was verified by bacterial liquid PCR using primers Δupp-UF / DR to obtain a strain with double bands.

[0164] However, H. cupida J9 is a moderately halophilic bacterium. To withstand high-salt environments, the sticky surface area of ​​its cell membrane lipid bilayer is unusually thick and uniform, making it difficult to lyse the cells. Before colony PCR, the bacterial solution must be pretreated using Vazyme's Rapid Room-Temperature Lysis Kit. The specific steps are as follows:

[0165] (1) Take 2 μL of bacterial solution and add it to 10 μL Lysis Buffer. Centrifuge briefly to mix, and incubate at room temperature for 3 min to allow the cells to fully lyse.

[0166] (2) Add Stablizing Buffer of equal volume to Lysis Buffer to the above mixture and centrifuge briefly to mix thoroughly to eliminate the inhibitory effect of inhibitory factors in the lysed sample on the downstream PCR reaction, so that the lysed DNA solution can be stored stably for a long time;

[0167] (3) Using the cell lysate obtained in the previous step as a template and corresponding primers, perform colony PCR according to the PCR system and reaction conditions described in 2.2.4.

[0168] The constructed pK18Δupp knockout vector can be transduced into the recipient strain wild-type Halomonas cupida J9 through the pili of the donor bacterium Escherichia coli S17-1λpir by conjugation transfer. At the same time, because the pK18Δupp knockout vector contains upstream and downstream homology arm sequences on both sides of the upp gene, it can stimulate the host strain's own RecA homologous recombination system, causing it to be site-specifically inserted into the chromosome of Halomonas cupida J9 and exist as a linearized DNA fragment, that is, the first homologous recombination occurs (see for details). Figure 1 This also makes Halomonas cupida J9, which is originally sensitive to high concentrations of kanamycin, resistant to kanamycin. Furthermore, J9 itself can tolerate high salt levels, so the above principle can be used to screen for single-crossover strains that are both salt-tolerant and kanamycin-resistant.

[0169] Because the targeting vector pK18Δupp was inserted into Halomonas cupida J9 in a linearized form, when the primers Δupp-UF / DR (see Table 1) were used for bacterial liquid PCR amplification, the strain that underwent single crossover would obtain two amplified target bands: one with a size of approximately 1.9 kb and the other with a length of approximately 1.2 kb. 24 transformants were picked on a 60LB plate containing Kan resistance, and more than 70% of the strains had undergone the first homologous recombination (see Figure 4 ).

[0170] 1.2.7.2 Screening of double-crossover strains

[0171] 1. Transfer the strain with double-layer bands to a 60LB tube without resistance and culture at 37°C and 180 rpm for 24 hours to promote double exchange and lose the suicide plasmid without resistance screening pressure, thus obtaining the cultured bacterial liquid.

[0172] 2. Take 100 μL of the cultured bacterial suspension, dilute it 100-fold with 60 LB liquid medium, and then spread it on 60 LB solid medium containing 100 μg / mL 5-Fu resistance for cultivation. Verify the double crossover by PCR using the genomic detection primers Δupp-JF / JR, J9-F, and J9-R (see Table 1) using the bacterial suspension PCR method described in 1.2.4. Sequence the PCR product of the double crossover strain.

[0173] 3. The knockout strain that was sequenced correctly was stored in a glycerol tube at -80°C to obtain Halomonas J9U.

[0174] After the first homologous recombination, the suicide plasmid is integrated into the chromosome of the host strain, resulting in two pairs of homologous DNA fragments in the genome of the single-crossover strain. Therefore, the single-crossover strain will spontaneously undergo a second crossover, thereby achieving the knockout of the upp gene (see Figure 1 ). Halomonas cupida J9, which was originally sensitive to 5-FU, obtained the upp gene deletion mutant strain J9U due to the deletion of the upp gene, which is resistant to 5-FU. J9U can grow in a high-salt culture medium containing 5-FU, so the target strain Halomonas cupida J9Δupp (J9U) is screened using a 60LB plate containing 5-FU. When the detection primers Δupp-JF / JR on the genome are used for bacterial liquid PCR amplification, the strain that has undergone two recombinations and successfully knocked out the upp gene can amplify a target band of about 1.4kb, while in the strain without upp gene deletion, the band size is about 2.2kb. The J9U double-exchange bacterial liquid PCR verification results are as follows Figure 5 As shown, a total of 16 5-FU-resistant transformants were selected, and all were verified to be upp knockout strains, indicating that the homologous recombination efficiency of J9 is extremely high, reaching 100%. PCR amplification using specific detection primers J9-F and J9-R in the J9 genome (see Table 1) confirmed that all upp knockout strains that successfully underwent double crossover were Halomonas cupida J9U.

[0175] 1.2.8 5-FU sensitivity test of upp gene deletion mutant strain J9Δupp (J9U)

[0176] The activated J9U and the control strain J9 were streaked onto 60LB plates containing 100 μg / mL 5-fluorouracil, and the 5-FU sensitivity of the constructed upp gene-deficient strain was verified.

[0177] The results are as follows Figure 6 As shown, the wild-type J9 did not grow at all, while the J9U with the upp gene knocked out grew well on a plate containing 100 μg / mL 5-FU, had very stable resistance to 5-FU, and could be used as a starting strain for subsequent transformation.

[0178] Example 2

[0179] 2.1.1 Primer sequence information

[0180] The primer sequence information used in this example is shown in Table 9.

[0181] Table 9 Primer sequence information

[0182]

[0183] 2.1.2 Culture media and solutions

[0184] 2.1.2.1 Culture medium

[0185] Fermentation medium: KH2PO4 1.5 g / L, Na2HPO4·12H2O 9.65 g / L, yeast extract 1.0 g / L, NaCl 80.0 g / L, MgSO4 0.2 g / L, NH4Cl 2 g / L, trace element mother solution I 10 mL / L, trace element mother solution II 1 mL / L, balance distilled water, pH adjusted to 9.0 with NaOH;

[0186] Xylose fermentation medium: xylose is added to the above fermentation medium, and the final concentration of the xylose is 10-30 g / L.

[0187] Mixed sugar fermentation medium: Glucose was added to the above fermentation medium at a final concentration of 10 g / L and xylose was added to a final concentration of 20 g / L.

[0188] Mixed sugar pernitrogen fermentation medium: Add glucose to the above fermentation medium with a final concentration of 10 g / L and xylose to a final concentration of 20 g / L, and replace the NH4Cl 2 g / L in the fermentation medium with 20 g / L.

[0189] Pernitrogenated xylose fermentation medium: The difference from the above xylose fermentation medium is that the pernitrogenated xylose fermentation medium contains 5 to 30 g of NH4Cl, and the other components and their contents are the same as those of the above xylose fermentation medium.

[0190] 2.1.2.2 Solution

[0191] Xylose mother liquor: For a mother liquor concentration of 500 g / L, weigh 50.0 g of xylose and add distilled water to 100 mL. Autoclave at 115°C for 20 min and store at 4°C until ready to use. The working concentration is 10-30 g / L.

[0192] Glucose mother solution: For a 500 g / L stock solution, weigh 50.0 g of glucose and dilute to 100 mL with distilled water. Autoclave at 115°C for 30 minutes. Store at 4°C. The working concentration is 10-30 g / L.

[0193] Trace Element Solution I: Weigh 5.0 g ferric ammonium citrate and 2.0 g CaCl2, dissolve in 1 M HCl, and dilute to 1 L. Sterilize by 0.22 μm filtration and store at 4°C. The working concentration is 10 μL / mL.

[0194] Trace element solution II: Dissolve 100 mg ZnSO4·7H2O, 30 mg MnCl2·4H2O, 300 mg H3BO4, 200 mg CoCl2·6H2O, 10 mg CuSO4·5H2O, 20 mg NiCl2·6H2O, and 30 mg NaMoO4·2H2O in 1 M HCl and adjust the volume to 1 L. Sterilize by 0.22 μm filter and store at 4°C. The working concentration is 1 μL / mL.

[0195] MgSO4 stock solution: For a stock solution concentration of 20 g / L, weigh 2.0 g of MgSO4 and dilute to 100 mL with distilled water. Autoclave at 121°C for 20 minutes. Store at 4°C. The working concentration is 0.2 g / L.

[0196] NH4Cl stock solution: For a stock solution concentration of 200 g / L, weigh 20.0 g of NH4Cl and dilute to 100 mL with distilled water. Autoclave at 121°C for 20 minutes. Store at 4°C. The working concentration is 2 g / L.

[0197] Esterification solution: Weigh 0.1 g of benzoic acid, add 97 mL of chromatographic grade methanol and 3 mL of sulfuric acid to make up to 100 mL, and store at 4°C until ready to use.

[0198] The other culture media and solutions used in this example are the same as those in 1.1.2 of Example 1.

[0199] 2.2.1 Construction of Halomonas J9U derivatives

[0200] To achieve PHA production from xylose fermentation by Halomonas J9U, we first knocked out the xylose dehydrogenase xylD (xylD nucleotide sequence is shown in SEQ ID No. 31) in the xylose metabolism branch, and compensated for the lack of xylA (from E. coli MG1655) in J9U, adjusting the xylose metabolic shunt in the xylose metabolic network to direct the carbon source toward PHA production.

[0201] Compared to plasmid-based expression, chromosomal expression of xylose metabolism genes is stable but expression levels are lower. The exogenous promoter P8 can enhance the expression of endogenous genes in J9U, so P8 was used to further enhance their expression levels. The nucleotide sequence of the exogenous promoter P8 is shown in SEQ ID No. 32. The xylA sequence was amplified from the E. coli MG1655 genome by PCR, resulting in the xylA gene sequence shown in SEQ ID No. 33. An RBS sequence (TAAGGAGGTTTTCTA, SEQ ID No. 34) was added upstream of xylA. In this example, the insertion vector pKJU-P8xylA for the xylose metabolism genes and the knockout vector pKJU-ΔxylD for the xylose metabolism branch were successfully constructed. The insertion and knockout vectors were then transformed into H. cupida J9U via conjugation.

[0202] 2.2.1.1 Construction of gene knockout / insertion vectors

[0203] Construction of the targeting vector pKJU: Using the Halomonas cupida J9 genome as a template, primers Pupp-F / Pupp-R (see Table 1, SEQ ID Nos. 7-8) were designed based on the genome sequence to amplify the J9 upp gene and its own promoter to obtain the upp gene containing the promoter (SEQ ID No. 35). PCR amplification and purification were performed according to the conditions and procedures of 1.2.3. The resulting fragment was ligated with the plasmid pK18mobsacB digested with the restriction endonuclease EcoRI (see Table 1). Figure 7 The recombinant plasmid was verified by electrophoresis using the detection primers pK-JF / JR of the plasmid pK18mobsacB. The amplified band was approximately 800 bp, indicating that the targeting vector pKJU was successfully amplified.

[0204] When constructing a gene fragment knockout vector, first extract the Halomonas J9U genome according to the method of 1.2.2 of Example 1, then amplify and purify the upstream and downstream homologous arms UP and DN of the gene to be knocked out in the J9 genome (the length of the homologous arms is determined by the length of the gene) using primers ΔxylD-UF / UR and ΔxylD-DF / DR according to the method of 1.2.3 of Example 1, and then integrate the fused upstream and downstream homologous arms into the EcoRI site of the targeting vector pKJU by the homologous recombination method in 1.2.3 of Example 1 to construct the gene knockout vector pKJU-ΔxylD. Wherein, the knocked-out gene X is xylD, and the schematic diagram of the gene fragment knockout vector construction is shown in FIG. Figure 8 .

[0205] When constructing the gene fragment insertion vector, primers P8xylA-UF / UR, P8xylA-DF / DR, P8xylA-P8F / P8R and P8xylA-AF / AR (see Table 2) were used, respectively, with genomic DNA extracted from J9U, genomic DNA extracted from J9U, genomic DNA extracted from Pseudomonas putida KT2440 and genomic DNA extracted from E. coli MG1655 as templates to amplify the upstream and downstream homologous arms UP and DN of the genomic insertion site, as well as the insert fragment promoter P8 and gene xylA. After fusion PCR, the four amplified fragments were used as templates by the homologous recombination method in 1.2.3 of Example 1. PCR amplification was performed by P8xylA-UF and P8xylA-DR, and the upstream and downstream homologous arms P8xylA were integrated into the EcoR I site of the targeting vector pKJU to construct the gene fragment insertion vector pKJU-P8xylA. The inserted fragment is P8xylA. The schematic diagram of the gene fragment insertion vector construction is shown in Figure 9 The nucleotide sequence of P8xylA is shown in SEQ ID No.36.

[0206] After the plasmid was connected, it was first transformed into E. coli DH5α for replication and amplification according to the method of 1.2.4 of Example 1. Colony PCR verification and sequencing confirmed that the recombinant plasmid was successfully constructed. The recombinant plasmid was then extracted and transformed into E. coli S17-1λpir.

[0207] 2.2.1.2 Construction of Halomonas J9U derivatives

[0208] S1. Screening of single-crossover strains

[0209] 1. The plasmid containing pKJU-ΔxylD was transformed into the Halomonas J9U prepared in Example 1 by conjugation and plated on a 60LB plate containing 100 μg / mL Kan resistance medium and cultured at 37°C for 36 h.

[0210] 2. Under sterile conditions, single colonies were picked from the plates and cultured overnight in 60 LB liquid medium containing 100 μg / mL Kan resistance. Single crossover was verified by PCR using primers to obtain a single crossover strain with double bands, namely the recombinant strain J9UΔxylD.

[0211] S2. Screening of double-crossover strains

[0212] 1. Cultivate the single-crossover strain verified as a double-layer band at 37°C and 180 rpm for 24 hours to promote the occurrence of double crossover, thereby losing the suicide plasmid without resistance screening pressure, and obtain the cultured bacterial liquid;

[0213] 2. Take 100 μL of the cultured bacterial solution, dilute it 100-fold with 60 LB liquid medium, and then spread it on 60 LB solid medium containing 100 μg / mL 5-Fu resistance for cultivation. Use the detection primers on the genome to verify the double crossover and sequence the PCR product of the double crossover strain;

[0214] 3. The correctly sequenced strain was stored in a glycerol tube at -80°C to obtain the Halomonas J9U derivative (also known as the engineered strain J9UΔxylD-P8xylA).

[0215] The results are as follows Figure 10 As shown, the control strain is J9U, and the PCR test results show that the recombinant strain J9UΔxylD-P8xylA strain was successfully constructed. The sequencing results all matched 100%.

[0216] In order to verify the transcription of the exogenous gene xylA introduced into the Halomonas J9U derivative (J9UΔxylD-P8xylA) under the promotion of the exogenous promoter P8, the transcription of the xylA gene was detected by RT-qPCR.

[0217] Figure 11 Results showed that the specific PCR bands obtained using J9UΔxylD-P8xylA genomic DNA and cDNA as templates matched the size of the introduced exogenous gene. However, no target PCR bands were produced using negative control mRNA and ddH2O as templates. This indicates that the xylA exogenous gene integrated into the J9U genome was successfully transcribed.

[0218] In order to detect the growth curve and xylose consumption curve of the derivative of Halomonas J9U in xylose fermentation medium, the seed liquid of the constructed engineered bacteria J9UΔxylD-P8xylA was transferred to xylose fermentation medium containing 10 g / L, 20 g / L and 30 g / L xylose, respectively, with J9U as the control. The culture was carried out at 37°C and 180 rpm for 72 h, and its biomass accumulation was measured.

[0219] Depend on Figure 12 The results showed that, in contrast to J9U, the cell dry weight of the engineered bacterium J9UΔxylD-P8xylA increased significantly with increasing xylose concentration. This phenomenon suggests that these two steps of genetic modification may have successfully adjusted the xylose metabolic flux of J9.

[0220] In order to explore the reason why the xylose-engineered strain J9UΔxylD-P8xylA produces PHA, the intermediate single knockout strain J9UΔxylD and the single insertion strain J9U-P8xylA were inoculated into the fermentation medium containing 30 g / L xylose, and cultured at 37°C and 180 rpm for 60 h. 1 mL of samples were taken every 12 h to measure the OD value. 600 and residual sugar, and the wild type J9U was used as the control to observe its growth.

[0221] The xylose consumption curve was determined as follows:

[0222] The xylose-engineered strain produces PHA using xylose as a substrate, and substrate consumption is monitored using a biosensor analyzer. The SBA-40D biosensor analyzer uses an enzymatic reaction for quantitative analysis. After manually injecting the sample by replacing the xylose enzyme membrane, the entire measurement process is automatically controlled by a microcomputer, with a measurement cycle of approximately one minute per sample.

[0223] Determination of xylose in the culture medium: During and after the culture, take 1 mL of the culture medium, centrifuge at 12,000 rpm for 10 min, take the supernatant to a new EP tube, dilute 50 times, and then test.

[0224] Detection of xylose by biosensor analyzer: Turn on the biosensor analyzer, wait for the instrument to stabilize, flush the xylose enzyme membrane and pipeline with buffer, and then calibrate with 100 mg / dL xylose standard solution. After the calibration is passed, the prepared xylose sample can be measured.

[0225] The results are as follows Figure 13As shown, knocking out xylD alone affects the growth of J9U, indicating that this branch producing xylonic acid will eventually go to TCA, but it can still use xylose as the only carbon source for weak growth (it can grow without yeast powder), indicating that there are other xylose metabolic pathways to be explored; single insertion of P8xylA has no effect on the growth of J9, proving that the combined effect of ΔxylD and P8xylA will enable J9U to synthesize PHA, probably because more xylose carbon sources flow to the PHB synthesis pathway rather than xylonic acid metabolism.

[0226] To evaluate the fermentation performance of the engineered xylose-fermenting strains, wild-type J9U was used as a control, and the engineered strains J9U-P8xylA and J9UΔxylD-P8xylA were cultured in a xylose fermentation medium containing 30 g / L xylose and 2 g / L NH₄Cl at 37°C with shaking at 180 rpm for 72 h. CDW represents cell dry weight; wt% is defined as the proportion of PHA to cell dry weight. Values ​​are mean ± SD of three independent replicates. ** and **** indicate P < 0.01 and P < 0.0001, respectively, using a Student's t-test. Cell samples after esterification and fermentation were analyzed for PHA content and monomer composition using GC-MS.

[0227] The specific steps of shake flask fermentation are as follows:

[0228] 1. Take the H. cupida J9 strain stored at -80℃ and activate it on 8% LB plates;

[0229] 2. Pick a single colony and inoculate it into 5 mL of 8% LB liquid and culture overnight with shaking;

[0230] 3. Transfer 1 mL of the bacterial solution from the previous step to 100 mL of the corresponding 8% LB liquid medium and culture overnight with shaking to obtain the seed solution;

[0231] 4. Inoculate the seed liquid into 100 mL of the corresponding liquid fermentation medium at a 5% (v / v) inoculation rate and culture with shaking for 48 to 60 hours.

[0232] 5. After fermentation is complete, collect all the bacterial liquid, centrifuge at 7000 rpm for 20 min at 4°C, discard the culture medium, transfer all the bacteria to a clean glass culture dish, mark the sample, and seal it with tin foil with small holes. After freezing the sample at -80°C for 2 hours, place the plate in a freeze dryer and dry overnight.

[0233] Collect the freeze-dried cells, weigh them, record the cell dry weight, and store them in a sealed container at room temperature for future use.

[0234] Gas chromatography-mass spectrometry (GC-MS) was used to determine the monomer composition and content of PHA. The specific steps for sample preparation and quantification are as follows:

[0235] 1. Collect the cells from the fermentation broth by centrifugation and freeze-dry them in a freeze dryer for 24 hours. Weigh 0.1g of freeze-dried cell sample and PHA monomer standard and place them in a sealed high-temperature esterification tube that has been soaked in chloroform overnight.

[0236] 2. Add 4 mL of esterification solution, then add an equal volume of chloroform and shake to mix;

[0237] 3. Place the esterification tube in a boiling water bath at 100°C and esterify for 4 hours;

[0238] 4. After the esterification tube has cooled to room temperature, add 4 mL of ddH2O and shake thoroughly for 2 minutes. Let it stand for 2 hours until the aqueous phase and the organic phase separate. Take 1 mL of the lower organic phase, filter and sterilize it, and then place it into a sealed brown vial;

[0239] 5. The sample obtained in the previous step was subjected to GC-MS detection under the following detection conditions: carrier gas is helium (flow rate 1.0555mL / min), injection port temperature is 250℃, transfer tube temperature is 230℃, automatic injection volume is 1μL, and split mode (split ratio 30:1) is used. The column temperature starts at 80℃, stays for 1 minute, then increases to 250℃ at a rate of 10℃ / min, stays for 3 minutes, and scans in the range of 50-280m / z. The column pressure starts at 10psi, stays for 1.5min, then increases to 20psi at 2.5psi / min, and stays for 1.5min;

[0240] 6. The PHA monomer composition was determined by analyzing the NIST database and comparing the retention times with the standards. The internal standard method (methyl benzoate) was used to quantify the PHA. The formula is as follows:

[0241]

[0242] PHA production (g / L) = PHA content (wt%) × cell dry weight (g / L)

[0243] Fermentation results such as Figure 14 As shown in Figure 2, compared with the wild-type J9U, the cell dry weight of the engineered strain J9U-P8xylA did not increase, but the accumulation of PHA achieved a breakthrough from 0 to 1, and was able to accumulate 16.21wt% of PHA. The OD of the final engineered strain J9UΔxylD-P8xylA was 600The cell dry weight increased from 2.1 g / L to 4.2 g / L, the xylose consumption increased from 10 g / L to 13.5 g / L, and 33.8 wt% PHA could be produced from xylose. The PHA yield could reach 1.42 g / L, and the xylose conversion rate increased from 3.7% to 10.5%. In addition, the PHA produced by the bacteria was a short-chain-medium-chain copolymer composed of 31.49 wt% 3-hydroxybutyric acid (3HB) and 2.31 wt% 3-hydroxylauric acid (3HDD) (see Figure 15 ), therefore, more exploratory experiments to improve PHA production were conducted using J9UΔxylD-P8xylA.

[0244] The effect of excess nitrogen source on the production of PHA using xylose by the engineered bacterium J9UΔxylD-P8xylA was further studied:

[0245] Although an imbalance in the carbon-nitrogen ratio, i.e., insufficient nitrogen source, is an important factor in promoting PHA production by limiting cell growth, this example attempted to add 20 g / L NH 4 Cl as an excess nitrogen source for shake flask fermentation.

[0246] (1) Using xylose fermentation medium (xylose concentration of 30 g / L) as a control, xylose engineering strains J9U-P8xylA and J9UΔxylD-P8xylA were cultured in xylose fermentation medium with nitrogen permeation and NH4Cl concentration of 20 g / L at 37°C and 180 rpm for 60 h. 1 mL of sample was taken every 12 h to measure the OD 600 , determine the growth curve.

[0247] (2) Using xylose fermentation medium (xylose concentration of 30 g / L) as a control, the xylose engineered strain J9UΔxylD-P8xylA was cultured and fermented in a nitrogen-permeated xylose fermentation medium with an NH4Cl concentration of 20 g / L at 37°C and 180 r / min with shaking for 72 h; CDW is cell dry weight; wt% is defined as the ratio of PHA to cell dry weight; the values ​​are the mean ± SD of three independent repeated experiments (* and ** indicate P < 0.05 and P < 0.01, respectively), and the shake flask fermentation results were determined using the Student's t-test.

[0248] Growth curves and fermentation results are shown in Figure 2. Figure 16As shown, the results showed that, with J9U-P8xylA as the control, the addition of 20g / L NH4Cl as an excess nitrogen source had no effect on its growth. However, for the strain J9UΔxylD-P8xylA, which can already use xylose to produce PHA, the addition of excess nitrogen source significantly increased its biomass accumulation. At the same time, xylose consumption increased to 19g / L, and the cell dry weight also increased from 4.2g / L to 6.3g / L. GC-MS determination showed that the PHA content increased from 33.8wt% to 44.63wt%, and the substrate conversion rate increased to 14.8%. This is the highest yield of PHA produced by Halomonas using xylose as the sole carbon source, i.e. 2.81g / L, to date.

[0249] In this example, the effects of different concentrations of NH4Cl on the production of PHA by xylose fermentation by the engineered bacterium J9UΔxylD-P8xylA were tested. Shake flask fermentations were performed in a xylose fermentation medium containing 30 g / L xylose (control, expressed as 2N) and in a xylose fermentation medium containing 30 g / L xylose with final NH4Cl concentrations of 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 30 g / L as nitrogen-permeated xylose fermentation medium (expressed as 5N, 10N, 15N, 20N, and 30N, respectively). The cultures were shaken at 37°C and 180 rpm for 84 h, and 1 mL of samples were taken every 12 h to measure the OD values. 600 and residual sugar, and the growth curve and xylose consumption curve were determined, respectively.

[0250] Figure 17 The results showed that the addition of 10-20 g / L NH4Cl to the fermentation medium containing 30 g / L xylose was beneficial to the accumulation of biomass and the consumption of xylose.

[0251] In terms of the co-utilization of xylose and glucose, glucose may bind to the xylose transporter, resulting in the inhibition of xylose transport; in addition, due to the glucose effect CCR, the decomposition metabolites of glucose may also inhibit the transcription of genes related to xylose metabolism, thereby inhibiting the utilization of xylose. Therefore, for most microorganisms, the presence of glucose will inhibit the transport and utilization of xylose. In order to explore whether the presence of glucose will inhibit the transport and utilization of xylose, and to better reflect the effect of xylose pathway modification, this example uses J9U as a control strain, and the constructed engineered bacteria J9UΔxylD-P8xylA is transferred into a mixed sugar fermentation medium containing 20g / L xylose and 10g / L glucose for shake flask fermentation. The culture is shaken at 37°C and 180r / min for 72h, and 1mL samples are taken every 12h to measure the OD 600 and residual sugar to explore whether the presence of glucose would inhibit the transport and utilization of xylose, and the growth curve and monosaccharide consumption curve were measured respectively.

[0252] Figure 18The results showed that in the early stage of fermentation, for both wild and engineered bacteria, xylose could still be transported and utilized in the presence of glucose, indicating that the presence of glucose did not inhibit the transport and utilization of xylose, and that glucose and xylose could be utilized simultaneously. For engineered bacteria, in the late stage of fermentation, the continuous consumption of xylose also promoted the co-utilization of glucose, with xylose consumption increasing from 9.5 g / L to 17 g / L and glucose consumption also increasing by 4 g / L. 600 The expression of xylose and glucose in the engineered strain J9UΔxylD-P8xylA was about 20.0 in 72 hours, which was about 2.5 times that of the wild type.

[0253] To investigate the fermentation performance of the engineered strain J9UΔxylD-P8xylA in mixed sugar fermentation medium and mixed sugar pernitrogen fermentation medium, xylose fermentation medium (xylose concentration of 30 g / L) was used as a control. J9UΔxylD-P8xylA was cultured and fermented at 37°C and 180 rpm with shaking for 72 h in a mixed sugar fermentation medium containing 20 g / L xylose, 10 g / L glucose, and 2 g / L NH4Cl (20 g / L NH4Cl during pernitrogenation). CDW represents cell dry weight; wt% is defined as the proportion of PHA to cell dry weight. Values ​​are the mean ± SD of three independent replicates (* and ** indicate P < 0.05 and P < 0.01, respectively), and Student's t-test was used for analysis.

[0254] Figure 19 The results showed that compared to pure xylose fermentation medium (represented as 30X), the engineered bacteria exhibited a significant growth advantage in mixed sugar fermentation medium (represented as 20X + 10G), achieving a cell dry weight of 6.6 g / L and a PHA content of 42.63 wt%. In mixed sugar fermentation medium (represented as 20X + 10G (20N)), the cell dry weight reached 7.3 g / L, and the PHA content further increased to 49.58 wt%. These results further demonstrate that strain J9UΔxylD-P8xylA is a superior strain for PHA production from lignocellulose hydrolysate.

[0255] Lignocellulose hydrolysate is commonly used for microbial PHA production. Since corn straw hydrolysate is rich in reducing sugars and amino compounds, heat sterilization will cause the Maillard reaction, which seriously affects the growth of the strain. At the same time, halophilic bacteria are suitable as chassis cells for bioconversion of lignocellulosic biomass because they inhibit contamination by miscellaneous bacteria and allow open, unsterilized fermentation. After acid treatment and enzymatic hydrolysis, the corn straw hydrolysate was measured to contain 28.5g / L xylose and 10g / L glucose. This treatment method is low in cost and causes the xylose content to be approximately 3 times that of the glucose content. This example further studies the use of corn straw hydrolysate as a biomass raw material and the xylose engineering strain J9UΔxylD-P8xylA for PHA fermentation.

[0256] The method for preparing the corn straw hydrolyzate is as follows:

[0257] 1. Dry corn stalks (purchased and ground to 40-50 mesh) in an oven to constant weight;

[0258] 2. Use 2% H2SO4 for acid hydrolysis with a liquid-to-solid ratio of 10:1 in a water bath at 100°C for 2 hours;

[0259] 3. After cooling to room temperature, use NaOH to adjust the pH to 4.8-5.2;

[0260] 4. Cellulase Cellic CTec3 HS (Novozymes, Denmark) was added at a ratio of 30 FPU / g-CS and enzymatic hydrolysis was carried out at 50°C and 150 rpm for 36 h.

[0261] 5. Adjust the pH to 7.0 with NaOH and centrifuge at 12,000 rpm for 20 minutes.

[0262] 6. Subsequently, the supernatant was detoxified and decolorized by adding 0.5% (w / v) activated carbon (100 mesh, Aladdin Reagent Co., Ltd, Shanghai, China) at 50°C and 150 rpm for 60 min.

[0263] 7. Centrifuge the hydrolyzate from the previous step at 20,000 rpm for 20 minutes, then filter to completely remove insoluble solids;

[0264] 8. The pretreated hydrolyzate was not sterilized and was directly used as the base liquid for preparing the open fermentation medium. After the glucose and xylose contents were determined, the hydrolyzate was stored at 4°C to obtain the corn straw hydrolyzate.

[0265] Using wild-type J9U as a control, the xylose-engineered strain J9UΔxylD-P8xylA was fermented in a fermentation medium containing corn straw hydrolysate at 37°C and 180 rpm for 72 h. CDW is cell dry weight; wt% is defined as the ratio of PHA to cell dry weight. Values ​​are mean ± SD of three independent replicates (* and ** indicate P < 0.05 and P < 0.01, respectively) and were analyzed using Student's t-test.

[0266] Figure 20 The results showed that after the fermentation, the OD 600 The OD value of the engineered strain J9UΔxylD-P8xylA was 12.71, and the contents of residual xylose and glucose were 8.5 g / L and 4 g / L, respectively. 600 The PH value of the wild-type strain J9U was 18.01, with residual xylose and glucose contents of 6 g / L and 2.5 g / L, respectively. The wild-type strain J9U produced 5.4 g / L CDW and 1.28 g / L PHA. In contrast, the engineered strain J9UΔxylD-P8xylA produced 7.0 g / L CDW and 2.45 g / L PHA, approximately double that of the wild-type strain. Furthermore, the engineered strain exhibited higher xylose and glucose consumption and conversion rates.

[0267] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A derivative of Halomonas J9U, characterized in that: The xylD gene in the genome of the Halomonas J9U derivative is inactivated, and the Halomonas J9U derivative includes the xylA gene; the starting strain of the Halomonas J9U derivative is the Halomonas J9U obtained by the construction method of the Halomonas J9U; The nucleotide sequence of the xylD gene is shown in SEQ ID No. 31; The construction method of the Halomonas sp. J9U comprises the following steps: The suicide plasmid pK18mobsacB was digested to obtain the linearized plasmid pK18mobsacB; The upstream and downstream homology arm sequences of the upp gene were ligated with the linearized plasmid pK18mobsacB to obtain the targeting vector pK18Δupp; The host cells containing the targeting vector pK18Δupp were transformed into Halomonas aspirans J9 to obtain recombinant bacteria. The recombinant bacteria underwent two homologous recombination steps to obtain Halomonas J9U.

2. The Halomonas J9U derivative according to claim 1, characterized in that The nucleotide sequence of the xylA gene is shown in SEQ ID No. 33; a promoter for upregulating the xylA gene is inserted upstream of the xylA gene, the promoter is the P8 promoter, and the nucleotide sequence of the P8 promoter is shown in SEQ ID No.

32.

3. A method for constructing a derivative of Halomonas J9U according to claim 1 or 2, characterized in that: The following steps are involved: Connecting the upp gene with the linearized plasmid pK18mobsacB described in claim 1 to obtain the recombinant plasmid pKJU; The recombinant plasmid pKJU was digested with enzymes to obtain the linearized plasmid pKJU; The upstream and downstream homology arm sequences of the xylD gene were ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-ΔxylD; The P8xylA gene sequence containing upstream and downstream homology arms was ligated with the linearized plasmid pKJU to obtain the targeting vector pKJU-P8xylA; The targeting vector pKJU-ΔxylD is introduced into the Halomonas J9U obtained by the construction method of claim 1 to obtain the recombinant J9U, and after two homologous recombination steps, the recombinant J9UΔxylD is obtained; The targeting vector pKJU-P8xylA was transferred into the recombinant bacterium J9UΔxylD to obtain the recombinant bacterium J9UΔxylD containing the targeting vector pKJU-P8xylA. After two homologous recombination, the derivative bacterium of Halomonas J9U was obtained.

4. The construction method according to claim 3, characterized in that The upp gene is a promoter-containing upp gene, and the nucleotide sequence of the promoter-containing upp gene is shown in SEQ ID No.35; the nucleotide sequence of the P8xylA is shown in SEQ ID No.

36.

5. Use of the Halomonas J9U derivative constructed according to the construction method of claim 3 or 4 in the preparation of polyhydroxyalkanoates.

6. A method for preparing polyhydroxyalkanoate, characterized in that: The Halomonas J9U derivative constructed by the construction method according to claim 3 or 4 is cultured in a fermentation medium containing xylose to obtain polyhydroxyalkanoate.

7. The preparation method according to claim 6, characterized in that The fermentation medium also includes glucose.

8. The preparation method according to claim 6, characterized in that The fermentation medium also includes NH4Cl.

9. The preparation method according to claim 6, characterized in that The fermentation medium also includes glucose and NH4Cl.

10. The preparation method according to claim 6, characterized in that The concentration of xylose is 10-30 g / L.

11. The preparation method according to claim 9, characterized in that The concentration of the glucose is 8-12 g / L.

12. The preparation method according to claim 8 or 9, characterized in that: The concentration of the NH4Cl is 10-20 g / L.

13. The preparation method according to claim 6, characterized in that The culture is open fermentation culture.

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