A fusion gene, a genetically engineered bacterium, and a method for producing PHB

Abstract

The present invention discloses a genetically engineered bacterium and a method for producing PHB. The present invention also discloses a fusion gene, which comprises a nucleotide encoding the Ncgl1289 signal peptide and the phaC gene, and the sequences of the nucleotide encoding the Ncgl1289 signal peptide and the phaC gene are respectively shown as SEQ ID NO:6 and SEQ ID NO:4. The genetically engineered bacterium is a Corynebacterium glutamicum engineered bacterium, which comprises the fusion gene and secretes and expresses PHA synthase, can efficiently produce PHB, and can improve the yield of poly-3-hydroxybutyrate produced using substrates derived from lignocellulose.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and relates to a genetically engineered Corynebacterium glutamicum strain secreting and expressing PHA synthase, a method for producing poly-3-hydroxybutyrate using the same, a method for constructing the genetically engineered strain, and its applications. Background Art

[0002] Polyhydroxyalkanoates (PHAs) are a class of microbial fully synthesized high molecular polyesters. This polymeric material has thermochemical properties and physical strength comparable to those of petroleum-based polypropylene materials, and has good biocompatibility and biodegradability. Therefore, it has the potential to replace traditional petroleum-based plastics and alleviate the "white pollution" problem brought by traditional petroleum-based plastics to the environment. Depending on the composition monomer type and degree of polymerization, PHAs have different material properties. The simplest PHA is poly-3-hydroxybutyrate, abbreviated as PHB, which is polymerized from a single monomer of 3-hydroxybutyryl coenzyme A.

[0003] Currently, for the technology of microbial fermentation to produce PHB, food starch is used as the raw material source of fermentable monosaccharides, and its cost accounts for about half of the total cost of PHB. Producing PHB from food starch not only greatly increases the production cost but also has the problem of "competing with people for food", making it impossible to achieve the replacement of petroleum-based polymers by PHB. Among the non-food carbohydrate raw materials of non-food fermentable monosaccharides, lignocellulosic biomass such as corn stover, wheat straw, rice straw, bagasse, and forestry waste has the characteristics of low price and wide source. Obtaining fermentable monosaccharides such as glucose and xylose through advanced biorefining and then using them as sugar raw materials to ferment and produce PHB not only has the advantages of sufficient raw material sources and low costs but also has great potential in various aspects such as reducing environmental pollution, carbon neutralization technology, and promoting the upgrading of the agricultural industry.

[0004] The synthesis process of PHB is mainly driven by three different genes: PhaA, PhaB, and PhaC. Among them, the PHA synthase encoded by the phaC gene is responsible for the last polymerization reaction in PHB synthesis and is considered to play a key role in PHB synthesis. As an intracellular product, in the industrial production process, PHB needs to be extracted from the cells through large-scale cell disruption and separation operations. Such complex operations account for approximately 30% of the total production cost of PHB. If the secretory production of PHB can be achieved, the production cost of PHB can be greatly reduced. Summary of the Invention

[0005] To overcome the above-mentioned disadvantages / defects, the object of the present invention is to provide a genetically engineered Corynebacterium glutamicum (C. glutamicum) strain that secretes and expresses PHA synthase to increase the production of poly-3-hydroxybutyrate. The secretion and expression of PHA synthase is achieved through the Ncgl1289 signal peptide (abbreviated as Ncgl) that mediates the Sec pathway. Ultimately, the engineered strain constructed in the present invention can efficiently produce PHB, for example, efficiently produce PHB using lignocellulose hydrolysate.

[0006] To solve the above technical problems, in the first aspect of the present invention, there is provided a fusion gene, which comprises a nucleotide encoding the Ncgl1289 signal peptide and the phaC gene, and the sequences of the nucleotide encoding the Ncgl1289 signal peptide and the phaC gene are shown in SEQ ID NO:6 and SEQ ID NO:4 respectively.

[0007] To solve the above technical problems, in the second aspect of the present invention, there is provided an expression cassette, which includes the fusion gene as described in the first aspect of the present invention, and the promoters of the expression cassette include Peftu, Psod, and PH3. Preferably, the promoter is Peftu, and its nucleotide sequence is shown in SEQ ID NO:5.

[0008] To solve the above technical problems, in the third aspect of the present invention, there is provided a recombinant expression vector, which includes a backbone plasmid and the expression cassette as described in the second aspect of the present invention. Preferably, the backbone plasmid is selected from pTRCmob or pEC-XK99E.

[0009] To solve the above technical problems, in the fourth aspect of the present invention, there is provided a genetically engineered bacterium, which contains the recombinant expression vector as described in the third aspect of the present invention.

[0010] Preferably, the genetically engineered bacterium overexpresses the PhaA gene and the PhaB gene. More preferably, the genetically engineered bacterium also overexpresses the xylAB gene.

[0011] To solve the above technical problems, in the fifth aspect of the present invention, there is provided a method for constructing the genetically engineered bacterium as described in the fourth aspect of the present invention, and the method includes:

[0012] (1) Inserting the fusion gene as described in the first aspect of the present invention into a backbone plasmid to obtain a recombinant expression vector. Preferably, the backbone plasmid is selected from pTRCmob or pEC-XK99E.

[0013] (2) Transferring the recombinant expression vector into a starting strain, and obtaining the genetically engineered bacterium after screening. Preferably, the starting strain is Corynebacterium glutamicum. More preferably, the starting strain is C. glutamicum JH02.

[0014] To solve the above technical problems, a sixth aspect of the present invention provides a method for producing PHB, the method comprising: fermenting the genetically engineered bacterium as described in the fourth aspect of the present invention using pentose and / or hexose as substrates in a culture medium.

[0015] Preferably, the culture medium comprises ammonium sulfate and magnesium sulfate; and / or, the hexose is glucose and the pentose is xylose.

[0016] In a specific embodiment, the conditions for the fermentation satisfy one or more of the following: the fermentation is carried out under the following conditions: temperature 28-32 °C, rotation speed 500-700 rpm, pH 6.8-7.2; and / or,

[0017] the content of glucose is 90-110 g / L, the content of xylose is 0-40 g / L; and / or,

[0018] the content of ammonium sulfate is 18-22 g / L, the content of magnesium sulfate is 0.2-0.3 g / L.

[0019] Preferably, the temperature is 30 °C, the rotation speed is 600 rpm, and the pH is 7.0; and / or,

[0020] the content of glucose is 92.7 g / L, the content of xylose is 39.8 g / L.

[0021] More preferably, the culture medium comprises 30.0 g / L glucose, 1.0 g / L potassium dihydrogen phosphate, 3.0 g / L urea, 0.6 g / L magnesium sulfate, 5.0 g / L yeast extract and 10.0 g / L peptone.

[0022] In a specific embodiment, the substrate is derived from a hydrolysate of natural raw materials, and the natural raw materials are agricultural waste or forestry waste. Preferably, the agricultural waste includes wheat straw, corn straw, rice straw, and bagasse.

[0023] In a specific embodiment, the method for preparing the hydrolysate of the natural raw materials comprises the following steps:

[0024] Subject the natural raw materials to pretreatment, detoxification, enzymatic hydrolysis, solid-liquid separation to remove solids, and collect the clear liquid to obtain the hydrolysate of the natural raw materials. The toxic inhibitors that have an adverse effect on the fermentation of PHB by the strain are removed through the detoxification treatment. The pretreatment natural raw materials or the enzymatic hydrolysate can be detoxified by a strain capable of metabolizing the toxic inhibitors, thereby removing the toxic inhibitors. That is, the detoxification method can be biological detoxification.

[0025] Conventional pretreatment methods include acid pretreatment, alkali pretreatment, steam explosion pretreatment, hot water pretreatment, and ionic liquid pretreatment, but the present invention is not limited to the above-mentioned pretreatment method ranges because of this.

[0026] Preferably, the solid-liquid separation methods include centrifugation and filtration; the rotation speed of the centrifugation is preferably 10,000 - 15,000 rpm, and the duration of the centrifugation is preferably 8 - 15 minutes.

[0027] More preferably, the clear liquid is sterilized and filtered.

[0028] To solve the above technical problems, the seventh aspect of the present invention provides an application of the fusion gene as described in the first aspect of the present invention, the expression cassette as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, or the genetically engineered bacterium as described in the fourth aspect of the present invention in the production of PHB.

[0029] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0030] The reagents and raw materials used in the present invention are all commercially available.

[0031] The positive and progressive effects of the present invention are as follows:

[0032] The engineered strain constructed by the present invention can efficiently produce PHB and can efficiently produce PHB using lignocellulose hydrolysate. Detailed implementation manners

[0033] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples described herein. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0034] The strains used in the present invention include Escherichia coli DH5α for the construction of expression plasmids; C. glutamicum S9114 for providing the Ncgl1289 signal peptide (abbreviated as Ncgl, and the nucleotide sequence encoding the Ncgl1289 signal peptide is shown in SEQ ID NO: 6); and the starting strain is C. glutamicum JH02.

[0035] The construction process of C. glutamicum JH02:

[0036] (1) Integrate the phaC gene (as shown in SEQ ID NO: 4), phaA gene (as shown in SEQ ID NO: 12), and phaB gene (as shown in SEQ ID NO: 13) into the ldhA2, pdh, and MscCG sites of the genome of C. glutamicum XyltoGA01 (CN202011331840.8) respectively: Construct the integration plasmids for phaC, phaA gene, and phaB gene respectively. Then, transfer the three constructed integration plasmids pK18-Δpdh::phaC, pK18-Δpdh::phaA, and pK18-Δpdh::phaB into C. glutamicum XyltoGA01 by electroporation successively. Furthermore, screen out the strains with correct homologous recombination through primary recombination verification and secondary recombination verification by colony PCR, and name them C. glutamicum JH01;

[0037] (2) Integrate the gene encoding the heterologous transporter araE (as shown in SEQ ID NO: 3) into the genome: First, construct the integration plasmid containing araE. Then, transfer the integration plasmid pK18-Δack::araE containing araE into C. glutamicum JH01 by electroporation. Furthermore, screen out the strains with correct homologous recombination through primary recombination verification and secondary recombination verification by colony PCR to obtain the recombinant Corynebacterium glutamicum, named C. glutamicum JH02.

[0038] Reagents include:

[0039] Restriction endonucleases are used to cut plasmids or gene fragments to generate sticky ends and are purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase is used to amplify gene fragments, and DNA ligase is used to ligate the digested gene fragments and plasmid vectors. Both of these enzymes are purchased from Takara (Otsu, Japan). The plasmid extraction kit, PCR product purification and recovery kit, and gel recovery kit are purchased from Shanghai Jierui Biotechnology (Shanghai, China). Other chemical reagents are purchased from local suppliers. The PHB standard product is used to prepare the liquid phase calibration curve and is purchased from Sigma-Aldrich (Germany).

[0040] Media include:

[0041] LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract;

[0042] LK medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 50 μg / L kanamycin.

[0043] LBG competent medium: 6 g / L peptone, 6 g / L sodium chloride, 3 g / L yeast powder, 30 g / L glycine, 1 g / L Tween-80, 20 g / L glucose.

[0044] Source of backbone plasmid:

[0045] pTRCmob (purchase link: http: / / www.app17.com / c156980 / products / d10333788.html); pEC-XK99E (purchase link: http: / / www.app17.com / c156980 / products / d10333789.html).

[0046] Example 1 Overexpression of phaC gene fused with Ncgl secretion expression signal peptide

[0047] 1.1 Preparation of competent cells of Corynebacterium glutamicum C.glutamicum JH02

[0048] ① Take out the cryotube containing Corynebacterium glutamicum C.glutamicum JH02 from the -80 °C refrigerator, dip a little bacterial liquid with an inoculation loop and streak it on an LB plate. The streaked plate is placed in an incubator at 30 °C for 2 days.

[0049] ② Pick a single colony of Corynebacterium glutamicum from the well-grown plate and inoculate it into a test tube containing 5 mL of LB medium. Place the test tube in a shaker at 30 °C overnight. Add the cultured bacteria in the test tube to a 250 mL flask containing 30 mL of LBG competent medium at an addition amount of 1%, and place it in a shaker at 30 °C for 6 hours.

[0050] ③ Transfer the cultured bacteria into a 50 mL centrifuge tube and pre-cool it in ice water for 20 minutes. After pre-cooling, centrifuge the centrifuge tube containing the bacteria at 4 °C and 5000 rpm for 10 minutes, discard the supernatant, add 30 mL of pre-cooled 10% glycerol to the centrifuge tube, and pipette the bacteria to make them evenly suspended in the glycerol.

[0051] ④ Repeat the third step twice. After discarding the supernatant for the last time, add 1 mL of pre-cooled 10% glycerol, and mix the bacteria thoroughly. Transfer the well-mixed bacteria into a centrifuge tube and place it in ice water for standby.

[0052] 1.2 Overexpression of phaC gene fused with Ncgl secretion expression signal peptide

[0053] Construct the expression plasmid of (Ncgl)phaC. Using the genome of C. glutamicum S9114 as a template, the Ncgl secretion signal peptide was amplified by PCR using primers Ncgl-F (shown in SEQ ID NO:7) and Ncgl-R (shown in SEQ ID NO:8). Using the pPeftu-phaCAB expression plasmid as a template, the phaC gene was amplified by PCR using primers phaC-F (shown in SEQ ID NO:9) and phaC-R (shown in SEQ ID NO:10). Then, the secretion signal peptide and the phaC gene were ligated by fusion PCR to obtain the Ncgl_phaC fusion fragment. Subsequently, the fusion fragment was inserted into the pPeftumob expression vector by restriction enzyme digestion and ligation to obtain pPeftu-(Ncgl)phaC. Then, the expression plasmid pPeftu-(Ncgl)phaC was transferred into C. glutamicum JH02 by electroporation and cultured on an LK medium plate at 30 °C for 2 days. Using the universal primers plasmid-F (shown in SEQ ID NO:1) and plasmid-R (shown in SEQ ID NO:2) on the expression plasmid as verification primers, the strain with the correct band was verified by colony PCR and considered as the recombinant Corynebacterium glutamicum containing the target expression plasmid, named C. glutamicum JH02:Peftu-(Ncgl)phaC. Other promoters in the art, such as Psod or PH36, can also be used in the present invention.

[0054] Example 2 Fermentation production of PHB in wheat straw hydrolysate using the engineered strain obtained by transformation

[0055] The wheat straw was purchased from Nanyang, Henan in the spring of 2021. After two-step acid hydrolysis, it was determined to contain 30.5% cellulose, 24.9% hemicellulose, 21.6% lignin and 9.7% ash. The hydrolysate obtained by dry pretreatment, detoxification and enzymatic hydrolysis of the collected wheat straw was centrifuged at 12000 rpm for 10 min using a high-speed centrifuge, and the supernatant was collected. After autoclaving and filtration, the wheat straw hydrolysate containing glucose and xylose was obtained.

[0056] The engineered strain C. glutamicum JH02:Peftu-(Ncgl)phaC and the control strain C. glutamicum JH02 were fermented using wheat straw hydrolysate in a 3 L fermenter. The concentrations of glucose and xylose in the wheat straw hydrolysate were 92.7 g / L and 39.8 g / L respectively, the fermentation temperature was 30 °C, the rotation speed was 600 rpm, 25% ammonia water was used to control the pH at 7.0, the aeration rate was 1.4 vvm, and the fermentation time was 72 h. The results are shown in Table 1.

[0057] Table 1

[0058]

[0059] The results are shown in Table 1. It can be seen from the fermentation results that the control strain C. glutamicum JH02 can produce 6.9 g / L of PHB, and the PHB content is 24.8 wt%; the PHB content of the secreted expression strain JH02:Peftu-(Ncgl)phaC is 39.0 wt%, and the highest PHB yield is 16.2 g / L. Compared with the control, the PHB yield and intracellular content are increased by 134.8% and 57.3% respectively. These fermentation results indicate that the engineered strain obtained in the present invention has significantly improved the PHB yield by secreting and expressing PHA synthase.

[0060] Example 3 Production of PHB by the engineered strain obtained by modification in synthetic medium

[0061] Components of synthetic medium: 30.0 g / L glucose, 1.0 g / L potassium dihydrogen phosphate, 3.0 g / L urea, 0.6 g / L magnesium sulfate, 5.0 g / L yeast extract and 10.0 g / L peptone; the rest is water. Fermentation was carried out in a 250 mL shake flask at a fermentation temperature of 30 °C, controlling the pH at 7.0 with sodium hydroxide solution and a rotation speed of 200 rpm for 48 hours. The results are shown in Table 2.

[0062] Comparative Example 1

[0063] A fusion protein was constructed using the CGR_RS04950 signal peptide (SEQ ID NO: 11) and the phaC gene, and the other conditions were the same as in Example 1. The effect of the strain obtained in Comparative Example 1 on fermentative production of PHB was examined under the same medium and fermentation conditions as in Example 3 to verify the influence of the signal peptide on PHB production. The results are shown in Table 2.

[0064] Table 2

[0065]

[0066] The comparative experiments in Table 2 were batch fermentations carried out in shake flasks, while the hydrolysate fermentation experiment was fed-batch fermentation carried out on a fermenter. Therefore, there is a large gap in the final yield, but the production differences between different strains are still obvious.

[0067] As shown in Table 2, the yield of PHB produced by the genetically engineered bacterium of the present invention is higher than that of the genetically engineered bacterium using the CGR_RS04950 signal peptide. Specifically, it can be seen from the table that the PHB yield of the genetically engineered bacterium of the present invention is 1.7 g / L, which is 2.125 times that of the genetically engineered bacterium using the CGR signal peptide and 5.67 times that of the starting strain; the intracellular PHB content is 14.6%, which is 1.8 times that of the genetically engineered bacterium using the CGR signal peptide and 2.39 times that of the starting strain; the cell dry weight is 11.7 g / L, which is 1.125 times that of the genetically engineered bacterium using the CGR signal peptide and 2.34 times that of the starting strain.

[0068] The above specifically describes the operation examples of the technical solution of the present invention and is not regarded as a limitation on the application of the present invention. Any equivalent replacement of operating conditions is within the protection scope of the present invention. SEQUENCE LISTING <110> Shanghai Kaisai Biotechnology Co., Ltd. CIBT USA Inc. <120> A Fusion Gene, Genetically Engineered Bacterium and Method for Producing PHB <130> P21016726C <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> plasmid-F <400> 1 ttgacagctt atcatcaaaa gctgggtacc tctatctggt 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> plasmid-R <400> 2 tttgcgatca aataatgaca tggatcccat gctactccta 40 <210> 3 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> araE <400> 3 atggttacta tcaatacgga atctgcttta acgccacgtt ctttgcggga tacgcggcgt 60 atgaatatgt ttgtttcggt agctgctgcg gtcgcaggat tgttatttgg tcttgatatc 120 ggcgtaatcg ccggagcgtt gccgttcatt accgatcact ttgtgctgac cagtcgtttg 180 caggaatggg tggttagtag catgatgctc ggtgcagcaa ttggtgcgct gtttaatggt 240 tggctgtcgt tccgcctggg gcgtaaatac agcctgatgg cgggggccat cctgtttgta 300 ctcggttcta tagggtccgc ttttgcgacc agcgtagaga tgttaatcgc cgctcgtgtg 360 gtgctgggca ttgctgtcgg gatcgcgtct tacaccgctc ctctgtatct ttctgaaatg 420 gcaagtgaaa acgttcgcgg taagatgatc agtatgtacc agttgatggt cacactcggc 480 atcgtgctgg cgtttttatc cgatacagcg ttcagttata gcggtaactg gcgcgcaatg 540 ttgggggttc ttgctttacc agcagttctg ctgattattc tggtagtctt cctgccaaat 600 agcccgcgct ggctggcgga aaaggggcgt catattgagg cggaagaagt attgcgtatg 660 ctgcgcgata cgtcggaaaa agcgcgagaa gaactcaacg aaattcgtga aagcctgaag 720 ttaaaacagg gcggttgggc actgtttaag atcaaccgta acgtccgtcg tgctgtgttt 780 ctcggtatgt tgttgcaggc gatgcagcag tttaccggta tgaacatcat catgtactac 840 gcgccgcgta tcttcaaaat ggcgggcttt acgaccacag aacaacagat gattgcgact 900 ctggtcgtag ggctgacctt tatgttcgcc acctttattg cggtgtttac ggtagataaa 960 gcagggcgta aaccggctct gaaaattggt ttcagcgtga tggcgttagg cactctggtg 1020 ctgggctatt gcctgatgca gtttgataac ggtacggctt ccagtggctt gtcctggctc 1080 tctgttggca tgacgatgat gtgtattgcc ggttatgcga tgagcgccgc gccagtggtg 1140 tggatcctgt gctctgaaat tcagccgctg aaatgccgcg atttcggtat tacctgttcg 1200 accaccacga actgggtgtc gaatatgatt atcggcgcga ccttcctgac actgcttgat 1260 agcattggcg ctgccggtac gttctggctc tacactgcgc tgaacattgc gtttgtgggc 1320 attactttct ggctcattcc ggaaaccaaa aatgtcacgc tggaacatat cgaacgcaaa 1380 ctgatggcag gcgagaagtt gagaaatatc ggcgtctga 1419 <210> 4 <211> 1780 <212> DNA <213> Artificial Sequence <220> <223> phaC <400> 4 aggacataca atggcaaccg gcaagggcgc agctgcttct acccaggaag gtaagtctca 60 gccattcaag gttaccccag gcccattcga tccagcaacc tggcttgaat ggtcccgtca 120 gtggcagggc accgaaggta acggtcacgc agctgcatcc ggtatcccag gccttgatgc 180 tcttgctggt gttaagatcg ctccagcaca gctcggcgat atacagcagc gttacatgaa 240 ggatttctct gcactttggc aggcaatggc agaaggtaag gctgaagcta ccggcccact 300 tcacgatcgc cgtttcgcag gcgatgcatg gcgtaccaac ctgccatacc gtttcgctgc 360 tgcattctac ctcctcaacg cacgcgctct gaccgaactt gctgatgcag ttgaagcaga 420 tgcaaagacc cgtcagcgta tccgtttcgc aatctctcag tgggttgatg ctatgtcccc 480 agcaaacttc ctcgctacca acccagaagc acagcgcctt cttatcgaat ccggcggtga 540 atccctgcgc gctggtgtgc gtaacatgat ggaggacctg acccgcggca aaatatctca 600 gaccgatgaa tccgcattcg aagtcggccg taacgtggct gtgaccgaag gcgcagttgt 660 tttcgaaaac gaatacttcc agcttcttca gtacaagcca ctgaccgata aggttcacgc 720 acgcccactc cttatggttc caccatgcat caacaagtac tacatccttg atcttcagcc 780 agaatcttcc cttgtgcgtc acgtcgtgga acagggtcac accgtgttcc tcgtgtcttg 840 gcgtaaccca gatgcatcta tggctggttc cacctgggat gattacatcg aacacgctgc 900 tatccgcgca atcgaagttg cacgcgacat atccggtcag gataagatca acgtcctggg 960 tttctgcgtc ggcggaacta tcgtgtctac cgcactcgct gtcctcgctg ctcgtggtga 1020 acacccagca gcatctgtga cccttctgac caccctcctt gatttcgctg atactggaat 1080 cctggatgtg ttcgtggatg aaggccacgt tcagcttcgt gaagcaaccc tgggcggcgg 1140 cgctggtgct ccatgcgcac tcctccgcgg tctggaactc gcaaacacct tctctttcct 1200 ccgcccaaac gatcttgttt ggaactacgt tgtcgataac taccttaagg gtaacacccc 1260 agtgccattc gatctccttt tctggaacgg tgatgcaacc aacctgccag gtccttggta 1320 ctgctggtat ctgcgtcaca cctaccttca gaacgaactt aaggttccag gtaaattgac 1380 cgtttgcggt gtgccagttg atcttgcttc tatcgatgtg ccaacctaca tctacggttc 1440 ccgcgaagat cacatcgtgc cttggaccgc tgcatacgca tccaccgcac tcctcgctaa 1500 caagctccgt ttcgtcctcg gcgcttccgg tcacatcgca ggtgttatca acccaccagc 1560 taagaacaag cgttctcact ggaccaacga tgcactgcca gaatccccac agcagtggct 1620 tgctggtgct atcgaacacc acggctcttg gtggccagat tggaccgcat ggcttgcagg 1680 tcaggcaggt gcaaagcgtg ctgctccagc taactacggt aacgctcgtt accgcgctat 1740 cgaaccagct ccaggccgtt acgtcaaggc taaggcttaa 1780 <210> 5 <211> 371 <212> DNA <213> Artificial Sequence <220> <223> pPeftu <400> 5 atccaccatg gaattccgaa aagcaatttg cttttcgacg ccccaccccg cgcgttttag 60 atccaccatg gaattccgaa aagcaatttg cttttcgacg ccccaccccg cgcgttttag 60 cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct tgaaatcggc 120 cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct tgaaatcggc 120 tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat gtccacaggg 180 tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat gtccacaggg 180 tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg gcacattttg 240 tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg gcacattttg 240 taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga aagcaaaacc 300 taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga aagcaaaacc 300 aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac agagctcggt 360 aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac agagctcggt 360 acccggggat c 371 acccggggat c 371 <210> 6<210> 6 <211> 105<211> 105 <212> DNA<212> DNA <213> Artificial Sequence<213> Artificial Sequence <220><220> <223> Ncgl<223> Ncgl <400> 6 <400> 6 atgaaatatg aatttaataa tagattccga acgaaatcgg tgttagcgtc tgtgctgaaa 60 atgaaatatg aatttaataa tagattccga acgaaatcgg tgttagcgtc tgtgctgaaa 60 caatctaatc gcgtttcagg acacctacat gatcaggagc tcttt 105 caatctaatc gcgtttcagg acacctacat gatcaggagc tcttt 105 <210> 7 <210> 7 <211> 51 <211> 51 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence <220> <220> <223> Ncgl-F <223> Ncgl-F <400> 7 <400> 7 acgcgtcgac atgaaatatg aatttaataa tagattccga acgaaatcgg t 51 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Ncgl-R <400> 8 ccggttgcca taaagagctc ctgatcatgt aggtgtc 37 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaC-F <400> 9 aggagctctt tatggcaacc ggcaaggg 28 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> phaC-R <400> 10 acgcgtcgac acgaaaggat tttttaccca tgtcattatt tgatcgca 48 <210> 11 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> CGR signal peptide <400> 11 atgcaaataa accgccgagg cttcttaaaa gccaccgcag gacttgccac tatcggcgct 60 gccagcatgt ttatgccaaa ggccaacgcc cagggagca 99 <210> 12 <211> 1194 <212> DNA <213> Artificial Sequence <220> <223> phaA <400> 12 ggaggacata caatgaccga tgttgtcatc gtttctgctg cacgtaccgc tgttggtaag 60 ttcggcggct ctcttgctaa gatcccagct ccagaacttg gcgctgttgt catcaaggca 120 gcactcgaac gcgctggcgt gaagccagaa caggtttccg aagtgatcat gggtcaggtt 180 ctcaccgctg gctccggtca gaacccagca cgtcaggctg caatcaaggc aggtcttcca 240 gcaatggtgc cagctatgac catcaacaag gtttgcggtt ctggcctcaa ggcagttatg 300 cttgcagcta acgctatcat ggcaggtgat gctgaaatcg ttgtggctgg tggtcaggaa 360 aacatgtctg ctgctccaca cgtccttcca ggttcccgcg atggtttccg tatgggtgat 420 gcaaagctcg tggataccat gatcgtcgat ggcctttggg atgtgtacaa ccagtaccac 480 atgggtatca ccgctgaaaa cgtcgcaaag gaatacggta tcacccgcga agcacaggat 540 gagtttgcag tcggctctca gaacaaggca gaagcagctc agaaggctgg taagttcgat 600 gaagaaatcg tgccagtcct tatcccacag cgtaagggcg atccagtcgc attcaagacc 660 gatgagttcg tgcgtcaggg cgctaccctg gattctatgt ccggccttaa gccagcattc 720 gataaggctg gcactgtgac cgctgcaaac gcatccggcc ttaacgatgg cgcagctgcg 780 gtcgttgtta tgtctgctgc aaaggcaaag gaactcggcc tgaccccact cgcaaccatc 840 aagtcctacg ctaacgctgg tgttgatcca aaggttatgg gcatgggccc agtgccagca 900 tctaagcgtg ctctgtcccg cgctgaatgg accccacagg atcttgatct catggaaatc 960 aacgaggcat tcgcagcaca ggcactcgca gttcaccagc agatgggttg ggatacctct 1020 aaggttaacg ttaacggcgg tgcaatcgct atcggtcacc caatcggtgc ttccggttgc 1080 cgtatcctcg ttaccctcct tcacgaaatg aagcgccgtg atgctaagaa gggtcttgct 1140 tccctgtgca tcggcggcgg tatgggtgtg gcacttgcag tggaacgtaa gtaa 1194 <210> 13 <211> 752 <212> DNA <213> Artificial Sequence <220> <223> phaB <400> 13 gaggacatac aatgacccag cgtatcgcat acgtgacggg gggtatgggc ggcatcggta 60 cggcaatctg ccagcgtctt gcaaaggatg gtttccgcgt cgttgcaggt tgcggtccaa 120 actccccacg ccgcgaaaag tggctcgaac agcagaaggc actgggcttc gatttcatcg 180 cttccgaagg taacgttgct gattgggatt ctaccaagac cgcattcgat aaggtcaagt 240 ccgaagtcgg cgaagtggat gtccttatca acaacgctgg tatcacccgc gatgttgtgt 300 tccgtaagat gacccgtgct gattgggatg cagttatcga taccaacctc acctctctgt 360 tcaacgttac caagcaggtg atcgatggta tggcagatcg tggctggggt cgtatcgtta 420 acatctcctc tgtgaacggt cagaagggtc agttcggcca gaccaactac tctaccgcta 480 aggcaggcct gcacggcttc acaatggcac ttgctcagga agtcgcaacc aagggtgtga 540 ccgtgaacac cgtgtctcca ggttacatcg ctaccgatat ggttaaggca atccgtcagg 600 atgtgctcga taagatcgtc gcaaccatcc cagtcaagcg ccttggcctg ccagaagaaa 660 tcgcttctat ctgcgcttgg ctgtcctctg aagaatccgg tttctccacg ggcgctgatt 720 tctcccttaa cggcggcctg cacatgggct aa 752

Claims

1. A genetically engineered bacterium, characterized in that, Comprising a recombinant expression vector; the recombinant expression vector includes a backbone plasmid and an expression cassette, the expression cassette includes a fusion gene containing a nucleotide encoding the Ncgl1289 signal peptide and the phaC gene, and the promoter of the expression cassette is Peftu; the sequences of the nucleotide encoding the Ncgl1289 signal peptide and the phaC gene are shown in SEQ ID NO:6 and SEQ ID NO:4 respectively; The genetically engineered bacterium overexpresses the phaA gene and the phaB gene, and the genetically engineered bacterium also overexpresses the xylAB gene, and a gene encoding a heterologous transporter araE is integrated into its genome; The starting strain of the genetically engineered bacterium is C.glutamicum JH02; the C.glutamicum JH02 is constructed by the following steps: 1) Integrate the phaC gene, the phaA gene with the sequence shown in SEQ ID NO:12, and the phaB gene with the sequence shown in SEQ ID NO:13 into the ldhA2, pdh, and MscCG sites of the C.glutamicum XyltoGA01 genome recorded in Chinese Patent CN202011331840.8 respectively: Construct integration plasmids for the phaC gene, the phaA gene, and the phaB gene respectively, and transfer the three constructed integration plasmids into C.glutamicum XyltoGA01 successively, and then obtain C.glutamicum JH01; 2) Integrate the gene encoding the heterologous transporter araE with the sequence shown in SEQ ID NO:3 into the genome: First construct an integration plasmid containing araE, and then transfer the integration plasmid containing araE into C.glutamicum JH01, and then obtain the recombinant Corynebacterium glutamicum C.glutamicum JH02.

2. The genetically engineered bacterium according to claim 1, wherein The nucleotide sequence of the Peftu is shown in SEQ IDNO:

5.

3. The genetically engineered bacterium according to claim 1, wherein The backbone plasmid is pTRCmob or pEC-XK99E.

4. A method for constructing a genetically engineered bacterium as described in any one of claims 1-3, characterized in that, The method includes: (1) Insert the fusion gene into the backbone plasmid to obtain a recombinant expression vector; (2) Transfer the recombinant expression vector into the starting strain, and obtain the genetically engineered bacterium after screening.

5. The method according to claim 4, characterized in that, The starting strain is C.glutamicum JH02; the C.glutamicum JH02 is constructed by the following steps: 1) Integrate the phaC gene, the phaA gene with the sequence shown in SEQ ID NO:12, and the phaB gene with the sequence shown in SEQ ID NO:13 into the ldhA2, pdh, and MscCG sites of the C. glutamicum XyltoGA01 genome described in Chinese Patent CN202011331840.8 respectively: Construct the integration plasmids for the phaC gene, the phaA gene, and the phaB gene respectively. Transfer the three constructed integration plasmids into C. glutamicum XyltoGA01 successively, and then obtain C. glutamicum JH01; 2) Integrate the gene encoding the heterologous transporter araE with the sequence shown in SEQ ID NO:3 on the genome: First, construct the integration plasmid containing araE, and then transfer the integration plasmid containing araE into C. glutamicum JH01 to obtain the recombinant Corynebacterium glutamicum C. glutamicum JH02.

6. A method for producing PHB, characterized in that, The method includes: fermenting the genetically engineered bacterium according to any one of claims 1 - 3 using pentose and / or hexose as substrates in a medium.

7. The method according to claim 6, characterized in that, The medium includes ammonium sulfate and magnesium sulfate; and / or, the hexose is glucose, and the pentose is xylose.

8. The method according to claim 7, characterized in that, The fermentation is carried out under the following conditions: temperature 28 - 32°C, rotation speed 500 - 700 rpm, pH 6.8 - 7.2; and / or, The content of glucose is 90 - 110 g / L, and the content of xylose is 0 - 40 g / L; and / or, The content of ammonium sulfate is 18 - 22 g / L, and the content of magnesium sulfate is 0.2 - 0.3 g / L.

9. The method according to claim 8, wherein The temperature is 30°C, the rotation speed is 600 rpm, and the pH is 7.0; and / or, The content of glucose is 92.7 g / L, and the content of xylose is 39.8 g / L.

10. The method according to any one of claims 6-9, characterized in that, The substrate is derived from the hydrolysate of natural raw materials, and the natural raw materials are agricultural waste or forestry waste.

11. The method according to claim 10, characterized in that The agricultural waste includes wheat straw, corn straw, rice straw, and bagasse.

12. The method according to claim 10, characterized in that, The preparation method of the hydrolysate of the natural raw materials includes the following steps: Subject the natural raw materials to pretreatment, detoxification, enzymatic hydrolysis, separate the solid and liquid to remove the solid, and collect the clear liquid to obtain the hydrolysate of the natural raw materials.

13. The method according to claim 12, wherein The methods of solid - liquid separation include: centrifugation and filtration.

14. The method according to claim 13, wherein The rotation speed of the centrifugation is 10000 - 15000 rpm.

15. The method according to claim 13, wherein The duration of the centrifugation is 8 - 15 minutes.

16. The method according to claim 12, wherein The clear liquid is sterilized and filtered.

17. Use of the genetically engineered bacterium according to any one of claims 1 - 3 in the production of PHB.

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