WO2023159745A1 - 一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用 - Google Patents

一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用 Download PDF

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WO2023159745A1
WO2023159745A1 PCT/CN2022/089426 CN2022089426W WO2023159745A1 WO 2023159745 A1 WO2023159745 A1 WO 2023159745A1 CN 2022089426 W CN2022089426 W CN 2022089426W WO 2023159745 A1 WO2023159745 A1 WO 2023159745A1
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seq
glpk
propanediol
genetically engineered
gene
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French (fr)
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齐向辉
张宇飞
员君华
窦媛
赵梅
翟彼得
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江苏大学
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Priority claimed from CN202210191504.0A external-priority patent/CN114806984B/zh
Priority claimed from CN202210188871.5A external-priority patent/CN114958928B/zh
Priority claimed from CN202210191491.7A external-priority patent/CN114806983B/zh
Priority claimed from CN202210188882.3A external-priority patent/CN114686413B/zh
Application filed by 江苏大学 filed Critical 江苏大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium co-producing 3-hydroxypropionic acid and 1,3-propanediol and its construction method and application.
  • 3-Hydroxypropionic acid and 1,3-propanediol are two important platform compounds in industry, which are widely used as precursors of biodegradable polymers and food additives.
  • the production of 3-hydroxypropionic acid and 1,3-propanediol has two methods: chemical synthesis and biological method. Most of the chemical methods use non-renewable resources as raw materials. The production process consumes a lot of energy, and many by-products are difficult to separate and purify. The production process produces immeasurable environmental pollution.
  • the biosynthesis of 3-hydroxypropionic acid or 1,3-propanediol mostly uses glucose and glycerol as substrates, and the production of 3-hydroxypropionic acid and 1,3-propanediol using glycerol as a substrate has simple steps, sufficient research, and cheap raw materials. And can solve the problem of excess glycerin.
  • the invention provides a genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol, its construction method and application.
  • Escherichia coli Escherichia coli W3110 DE3
  • the technical means of genetic engineering has been used to construct the genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol, and the constructed gene
  • the fermentation process of engineering bacteria is optimized to realize the efficient co-production of 3-hydroxypropionic acid and 1,3-propanediol.
  • E.coli S10G a kind of genetically engineered bacterium co-producing 3-hydroxypropionic acid and 1,3-propanediol is provided, which is denoted as E.coli S10G; Obtained by recombination of expressed and knocked-out genes;
  • the expressed genes are: glycerol dehydratase and its reactivator (DhaB123-GdrAB), propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isozyme (YqhD) and membrane-bound pyridine core nucleotide transhydrogenase (PntAB);
  • the knockout genes are: soluble pyridine nucleotide transhydrogenase (SthA), lactate dehydrogenase (LdhA), alcohol dehydrogenase (AdhE), pyruvate formate lyase (PflB), pyruvate oxidation
  • SthA soluble pyridine nucleotide transhydrogenase
  • LdhA lactate dehydrogenase
  • AdhE alcohol dehydrogenase
  • PflB pyruvate formate lyase
  • oxidation The genes for enzyme (PoxB), phosphoacetyltransferase-acetate kinase PTA-AckA, and inhibitor of glycerol metabolism (GlpR).
  • nucleotide sequence of the DhaB123-GdrAB is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2.
  • the nucleotide sequence of the GabD4 is shown in SEQ ID No: 3, and the amino acid sequence is shown in SEQ ID No: 4.
  • the nucleotide sequence of the YqhD is shown in SEQ ID No:5, and the amino acid sequence is shown in SEQ ID No:6.
  • the nucleotide sequence of the PntAB is shown in SEQ ID No: 7, and the amino acid sequence is shown in SEQ ID No: 8.
  • the original UTR sequence replacing glycerol kinase (GlpK) in the genome is an artificially designed UTR sequence.
  • the artificially designed UTR sequence is any of the following seven sequences:
  • the present invention also provides a method for constructing the above-mentioned genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol, specifically comprising the following steps:
  • Glycerol dehydratase (DhaB123) gene and glycerol dehydratase reactivator (GdrAB) gene were amplified by PCR, and fusion PCR technology was used to form a complete glycerol dehydratase and its reactivator gene fragment DhaB123-GdrAB, and then cloned into pCDFDuet-1 Plasmid, after transforming E.coli DH5 ⁇ , screening positive clones, plasmid extraction, and sequencing confirmation, the glycerol dehydratase and its reactivation factor plasmid was obtained, named pCDF-DhaB123-GdrAB.
  • the glycerol dehydratase gene and the glycerol dehydratase reactivator gene are derived from Klebsiella pneumoniae.
  • Propionaldehyde dehydrogenase (GabD4) gene, 1,3-propanediol oxidoreductase isoenzyme (YqhD) gene and membrane tubercle-type pyridine nucleotide transhydrogenase (PntAB) gene were amplified by PCR, and the seamless cloning technique was used to amplify
  • the above two genes were cloned into the pRSFuet-1 plasmid, and after transforming E.coli DH5 ⁇ , screening positive clones, plasmid extraction, and sequencing confirmation, the co-expressed propionaldehyde dehydrogenase (GabD4) and 1,3-propanediol oxidoreductase isoforms were obtained.
  • YqhD membrane tuberculosis-type pyridine nucleotide transhydrogenase
  • PntAB membrane tuberculosis-type pyridine nucleotide transhydrogenase
  • the source of the propionaldehyde dehydrogenase (GabD4) gene is derived from the pANY-gabD4 plasmid, the 1,3-propanediol oxidoreductase isoenzyme (YqhD) gene and the membrane tuberculosis type pyridine nucleotide transhydrogenase (PntAB) gene are derived from Escherichia coli (Escherichia coli W3110).
  • Knockout Escherichia coli Esscherichia coli W3110(DE3)
  • soluble pyridine nucleotide transhydrogenase (SthA) gene lactate dehydrogenase (LdhA) gene, alcohol dehydrogenase (AdhE ) gene, pyruvate formate lyase (PflB) gene, pyruvate oxidase (PoxB) gene, phosphoacetyltransferase-acetate kinase PTA-AckA gene and glycerol metabolism inhibitor (GlpR) gene, after PCR verification and sequencing verification , and finally the genetically deficient engineered bacteria, that is, Escherichia coli with by-product knockout.
  • SthA lactate dehydrogenase
  • AdhE alcohol dehydrogenase
  • PflB pyruvate formate lyase
  • PoxB pyruvate oxidase
  • the Glycerol Kinase (GlpK) expression level of Escherichia coli knocked out by UTR engineering technology can be used to artificially modify, use UTR design tool, design UTR artificial sequence, use double plasmid CRIPSR CAS9 tool plasmid, replace gene defect engineering
  • the UTR sequence of the original glycerol kinase (GlpK) gene in the bacterial genome was verified by sequencing, and the engineering bacteria with artificially modified expression of glycerol kinase (GlpK) were obtained.
  • the artificially designed UTR sequence is any one of the following seven sequences:
  • the present invention also provides the application of the above-mentioned genetically engineered bacteria in the co-production of 3-hydroxypropionic acid and 1,3-propanediol by fermenting glycerin.
  • the present invention also provides a method for co-producing 3-hydroxypropionic acid and 1,3-propanediol by fermentation of the above-mentioned genetically engineered bacteria, which specifically includes the following steps:
  • the pH is controlled to be 7.0, and the temperature, ventilation, and stirring rate are adjusted for fermentation and cultivation until the OD600 reaches 4, and IPTG and vitamin B12 are added to continue the cultivation;
  • the pH is controlled to be 8.0, and the temperature, ventilation, and dissolved oxygen value are adjusted for fermentation, and then fed-feed fermentation is carried out every 6 hours to co-produce 3-hydroxypropionic acid and 1,3-propanediol;
  • the ingredients of the supplement contained glycerin and corn steep liquor.
  • the composition of the improved M9-CSL medium is MgSO 4 7H 2 O 0.5g/L, NH 4 Cl 2.0g/L, NaCl 2.0g/L, corn steep liquor 2.5mL/L , glycerol 40g/L and 0.1M potassium phosphate buffer, pH 7.0;
  • the inoculum amount of the EC10S10G primary seed solution is 1% v/v, and the culture conditions are 37° C. and 220 rpm for 12 hours.
  • step (3) the inoculation amount of E.coli S10G secondary seed solution is 5% v/v;
  • the temperature is adjusted to 37°C, the initial ventilation is 2vvm, and the stirring rate is 500rpm for fermentation;
  • the temperature and ventilation volume adjust the temperature and ventilation volume to be adjusted to 3vvm, the stirring rate is 200-800rpm, and the dissolved oxygen value is controlled to be 10% for fermentation;
  • composition of described feed contains 800g/L glycerol and 50mL/L corn steep liquor;
  • the feeding process is as follows: feed every 6 hours to control the glycerin concentration to be maintained at 40g/L.
  • this technology constructs a genetic engineering strain that can efficiently co-produce 3-hydroxypropionic acid and 1,3-propanediol, promotes the cycle of essential cofactors through cofactor engineering, and knocks out byproducts pathways, inactivating metabolic inhibitors, and controlling central metabolic pathways to enhance 3-hydroxypropionate and 1,3-propanediol pathway flux.
  • the constructed genetically engineered bacteria can efficiently co-produce 3-hydroxypropionic acid and 1,3-propanediol through two-stage pH-controlled fed-batch fermentation, using biodiesel waste glycerol as a substrate.
  • the engineered bacteria constructed in the present invention can efficiently transform and metabolize the intermediate metabolite 3-hydroxypropanal, and generate the final products 3-hydroxypropionic acid and 1,3-propanediol; through cofactor engineering, the necessary cofactors and cyclic regeneration can be promoted to solve the problem of 3
  • the problem of insufficient supply of essential cofactors in the production of -hydroxypropionic acid and 1,3-propanediol; moreover, the elimination of by-product production pathways and the weakening of central metabolic pathways make the production of 3-hydroxypropionic acid and 1,3-propanediol Production has higher production yield and less accumulation of by-products, and has broad application prospects and practical significance.
  • Fig. 1 is the plasmid map of pCDF-DhaB123-GDRAB in genetically engineered bacteria E.coli S10G.
  • Figure 2 is a map of the pRSF-GabD4-YqhD-PntAB plasmid in the genetically engineered bacterium E.coli S10G.
  • Figure 3 shows the relative expression level and metabolites of GlpK in genetically engineered E.coli UTR-GlpK.
  • Fig. 4 is a graph showing the yield results of the two-stage pH-controlled fermentation of the present invention.
  • Fig. 5 is a diagram of the fermentation of genetically engineered bacteria E.coli S10G co-producing 3-hydroxypropionic acid and 1,3-propanediol through two-stage pH-controlled fed-batch fermentation according to the present invention.
  • Embodiment 1 Construction of glycerol dehydratase and its reactivator (DhaB123-GdrAB) recombinant plasmid
  • DhaB F GTTTAACTTTAATAAGGAGATATACCatgaaagatcaaacgatttgcagtactg (SEQ ID No: 9);
  • GdrA R CGGCCCCCTCGTTAACACttaattcgcctgaccggccag (SEQ ID No: 10);
  • GrdB F CTGGCCGGTCAGGCGAATTAAgtgttaacgagggggccgtc (SEQ ID No: 11);
  • GrdB R TTATGCGGCCGCAAGCTTGTCGACtcagtttctctcacttaacggcaggac (SEQ ID No: 12).
  • the pCDFDuet-1 plasmid (Miaoling Bio) was double digested with NcoI and BlnI, and the reaction condition was 37°C for 30 minutes; after the reaction, the linearized pCDFDuet-1 plasmid backbone was obtained. Then the DhaB123-GdrAB gene and the linearized pCDFDuet-1 plasmid backbone were subjected to Gibson assembly using 2 ⁇ MultiF Seamless Assembly Mix to obtain recombinant plasmids.
  • the reaction conditions are: 50°C for 30 minutes;
  • Figure 1 is the map of the pCDF-DhaB123-GDRAB plasmid in the genetically engineered bacteria E.coli S10G. It can be seen from the figure that the recombinant plasmid contains the origin of replication of ColDF3, and the gene expression is driven by the T7 promoter.
  • Embodiment 2 the construction of co-expressing propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isozyme (YqhD) and membrane tuberculosis type pyridine nucleotide transhydrogenase (PntAB) recombinant plasmid
  • GabD4 F GTTTAACTTTAAGAAGGAGATACCatgtaccaagatctggcact (SEQ ID No: 14);
  • GabD4 R TTATGCGGCCGCAAGCTTGTCGACttacgcttgggtgatgaact (SEQ ID No: 15);
  • Backbone1 F attagttaagtataagaaggagatatacat (SEQ ID No: 16);
  • Backbone1 R gtggcagcagcctaggttaa (SEQ ID No: 17);
  • YqhD F ATTAGTTAAGTATAAGAAGGAGATATACATatgaacaactttaatctgcacac (SEQ ID No: 18);
  • YqhD R GTGGCAGCAGCCTAGGTTAAttagcgggcggcttcgtat (SEQ ID No: 19);
  • Backbone2 F ttacgcttgggtgatgaacttg (SEQ ID No: 20);
  • Backbone2 R gtcgacaagcttgcggc (SEQ ID No: 21);
  • PntAB F ACCAAGTTCATCACCCAAGCGTAAaaccgatggaagggaatatcatgc (SEQ ID No: 22);
  • PntAB R GCGGCCGCAAGCTTGTCGACttacagagctttcaggattgcatccac (SEQ ID No: 23).
  • the GabD4 gene was amplified using the GabD4F and GabD4R primer pair, the YqhD gene was amplified using the YqhD F and YqhDR primer pair, and the PntAB gene was amplified using the PntAB F and PntA R primer pair.
  • PCR reaction parameters pre-denaturation, 98°C 3min; denaturation, 98°C 10s; annealing, 55°C 10s; extension, 72°C 1.5min; final extension, 72°C 5min; GabD4, YqhD and PntAB genes were obtained after 33 cycles.
  • Figure 2 is a map of the pRSF-GabD4-YqhD-PntAB plasmid, the recombinant plasmid contains the RSF replication origin, the expression of the GabD4 gene and the PntAB gene is driven by T7, and the YqhD gene is driven by a separate T7 promoter.
  • Embodiment 3 the construction of genetically deficient engineered bacteria:
  • sgRNAcas9 According to the soluble pyridine nucleotide transhydrogenase (SthA) gene sequence, use sgRNAcas9 to select a suitable target to generate a suitable sgRNA sequence; according to the sequence characteristics of the above gene sequence and ptargetF plasmid, use Oligo7.0 software to design for Primers for SthA gene knockout:
  • sgRNA-sthA F CCGCGCCATGTACTTATCTAgttttagagctagaaatagc (SEQ ID No: 25);
  • sgRNA-sthAR TAGATAAGTACATGGCGCGGactagtattatacctaggac (SEQ ID No: 26);
  • sthA-up F agttcgtctacgtcgcggaaatgc (SEQ ID No: 27);
  • sthA-up R GCCATTTCGATAAAGTTTTTAcatggtagggcttacctgt (SEQ ID No: 28);
  • sthA-down F AACAGGTAAGCCCTACCATGtaaaactttatcgaaatggccatc (SEQ ID No: 29);
  • sthA-down R tcctcgcgctggtgaaaga (SEQ ID No: 30);
  • E.coli W3110 was purchased from Biobiology, and lysogenized it with a lysogenization kit to obtain E.coli W3110 (DE3)), the E.coli W3110(DE3) containing the pCas9 plasmid was obtained, and then induced by 0.1M L-arabinose to make it competent for electroporation.
  • (6) Based on E.colipCas9 ⁇ sthA, refer to steps (1) to (5) to knock out the lactate dehydrogenase (LdhA) gene, alcohol dehydrogenase (AdhE) gene, pyruvate formate lyase ( PflB) gene, pyruvate oxidase (PoxB) gene, phosphoacetyltransferase-acetate kinase (PTA-AckA) gene and glycerol metabolism inhibitor (GlpR) gene.
  • LdhA lactate dehydrogenase
  • AdhE alcohol dehydrogenase
  • PflB pyruvate formate lyase
  • PoxB pyruvate oxidase
  • PTA-AckA phosphoacetyltransferase-acetate kinase
  • GlpR glycerol metabolism inhibitor
  • the primers for knocking out the lactate dehydrogenase (LdhA) gene are as follows:
  • sgRNA-ldhA F CGACAAGAAGTACCTGCAACgttttagagctagaaatagc (SEQ ID No: 31);
  • sgRNA-ldhAR GTTGCAGGTACTTCTTGTCGactagtattatacctaggac (SEQ ID No: 32);
  • ldhA-up F tttaactttttcgccctga (SEQ ID No: 33);
  • ldhA-up R GCAGGGGAGCGGCAAGATTAcataagactttctccagtgatg (SEQ ID No: 34);
  • ldhA-down F TCACTGGAGAAAGTCTTATGtaatcttgccgctccctgc (SEQ ID No: 35);
  • ldhA-down R taaaagcgtcgatgtccagt (SEQ ID No: 36).
  • the primers for knocking out the alcohol dehydrogenase (AdhE) gene are as follows:
  • sgRNA-adhE F TCGAATCCCACTCGCGAAAAgttttagagctagaaatagc (SEQ ID No: 37);
  • sgRNA-adhE R TTTTCGCGAGTGGGATTCGAactagtattatacctaggac (SEQ ID No: 38);
  • adhE-up F taccaaaaagttgtagaatcgtg (SEQ ID No: 39);
  • adhE-up R CCAGACAGCGCTACTGATTAcataatgctctcctgataatgt (SEQ ID No: 40);
  • adhE-down F ATTATCAGGAGAGCATTATGtaatcagtagcgctgtctgg (SEQ ID No: 41);
  • adhE-down R cagcacagtttcgctctg (SEQ ID No: 42).
  • the primers for knocking out the pyruvate formate lyase (PflB) gene are as follows:
  • sgRNA-pflB F ATGAAAAGTTAGCCACAGCCgttttagagctagaaatagc (SEQ ID No: 43);
  • sgRNA-pflB R GGCTGTGGCTAACTTTTCATactagtattatacctaggac (SEQ ID No: 44);
  • pflB-up F atttgcttctctctggggctga (SEQ ID No: 45);
  • pflB-up R ATTTCAGTCAAATCTAATTAcatgtaacacctaccttct (SEQ ID No: 46);
  • pflB-down F AAGAAGGTAGGTGTTACatgtaattagatttgactgaaatcgt (SEQ ID No: 47);
  • pflB-down R cgtagcggatccacaccttc (SEQ ID No: 48).
  • the primers for knocking out the pyruvate oxidase (PoxB) gene are as follows:
  • sgRNA-poxB F TATCGCCAAAACACTCGAATgttttagagctagaaatagc (SEQ ID No: 49);
  • sgRNA-poxB R ATTCGAGTGTTTTGGCGATAactagtattatacctaggac (SEQ ID No: 50);
  • poxB-up F aaccgtccacaggccgatgt (SEQ ID No: 51);
  • poxB-up R GGGAAATGCCACCCTTTTTAcatggttctccatctcctga (SEQ ID No: 52);
  • poxB-down F TCAGGAGATGGAGAACCATgtaaaaagggtggcatttcccgtc (SEQ ID No: 53);
  • poxB-down R ccagcgaatggcacgtt (SEQ ID No: 54);
  • PTA-AckA phosphoacetyltransferase-acetate kinase
  • sgRNA-pta-ackA F AATAAACAGGAAGCGGCTTTgttttagagctagaaatagc (SEQ ID No: 55);
  • sgRNA-pta-ackAR AAAGCCGCTTCCTGTTTATTactagtattatacctaggac (SEQ ID No: 56);
  • pta-ackA-up F aacacctgtccagactcct (SEQ ID No: 57);
  • pta-ackA-up R TGCGGATGATGACGAGATTAcatggaagtacctataattga (SEQ ID No: 58);
  • pta-ackA-down F CAATTATAGGTACTTCCATgtaatctcgtcatcatccgcag (SEQ ID No: 59);
  • pta-ackA-down R ttctcccataccaaataccg (SEQ ID No: 60).
  • Knockout glycerol metabolism inhibitor (GlpR) gene primers are as follows:
  • sgRNA-glpRF CGTAACGCGATGGTCAATATgttttagagctagaaatagc (SEQ ID No: 61);
  • sgRNA-glpR R ATATTGACCATCGCGTTACGactagtattatacctaggac (SEQ ID No: 62);
  • glpR-up F cgtggtggtgtttattgcc (SEQ ID No: 63);
  • glpR-up R AGCACAGCTCCAGTTGAAtcatttataaatccctggaattatt (SEQ ID No: 64);
  • glpR-down F AATAATTCCAGGGATTTATAAATGattcaactggagctgtg (SEQ ID No: 65);
  • glpR-down R tgaagagaaaaccttttaccc (SEQ ID No: 66).
  • the genetically deficient engineering bacteria obtained are E.coli pCas9 ⁇ sthA ⁇ ldhA ⁇ adhE ⁇ pflB ⁇ poxB ⁇ pta ⁇ ackA ⁇ glpR.
  • Embodiment 4 the construction of the engineered bacterium that the expression level of glycerol kinase (GlpK) is artificially modified
  • sgRNAcas9 According to the glycerol kinase (GlpK) gene sequence, use sgRNAcas9 to select a suitable target to generate a suitable sgRNA sequence; according to the above gene sequence and the sequence characteristics of the ptargetF plasmid (Miaoling Biology), use Oligo7.0 software to design to replace the GlpK gene Primers for UTR:
  • sgRNA-glpKUTR F CTTCGCTGTAATATGACTACgttttagagctagaaatagc (SEQ ID No: 77);
  • sgRNA-glpKUTR R GTAGTCATATTACAGCGAAGactagtattatacctaggac (SEQ ID No: 78);
  • glpK-up F cgcagttgagatggtgattaccg (SEQ ID No: 79);
  • glpK-up R ttacagcgaagctttttgttc (SEQ ID No: 80);
  • glpK-down F atgactgaaaaaaaaatatatcgttgcg (SEQ ID No: 81);
  • glpK-down R atccacttcactttggtgcca (SEQ ID No: 82);
  • glpK-up R1 TTCGTGTTGTCCCGTATAATCTttacagcgaagctttttgttc (SEQ ID No: 83);
  • glpK-up R2 AAACCCTTGTCCCGTAGCTAACttacagcgaagctttttgttc (SEQ ID No: 84);
  • glpK-up R3 TTATGGTTGTCCCGTAAAGAATttacagcgaagctttttgttc (SEQ ID No: 85);
  • glpK-up R4 GGAACTTTGTCCCGTTAGACGTttacagcgaagctttttgttc (SEQ ID No: 86);
  • glpK-up R5 CCGCGTTGTCCCGTAAACCGTttacagcgaagctttttgttc (SEQ ID No: 87);
  • glpK-up R6 ACAGCGTTGTCCCGTAGACGGTttacagcgaagctttttgttc (SEQ ID No: 88);
  • glpK-up R7 ACAGCGTTGTCCCGTAGACCGGttacagcgaagctttttgttc (SEQ ID No: 89);
  • glpK-down F1 AGATTATACGGGACAACACGAAatgactgaaaaaaaaatatatcgttgcg (SEQ ID No: 90);
  • glpK-down F2 GTTAGCTACGGGACAAGGGTTTatgactgaaaaaaaaatatatcgttgcg (SEQ ID No: 91);
  • glpK-down F3 ATTCTTTACGGGACAACCATAAatgactgaaaaaaaaatatatcgttgcg (SEQ ID No: 92);
  • glpK-down F4 ACGTCTAACGGGACAAAGTTCCatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 93);
  • glpK-down F5 ACGGTTTACGGGACAACGCGGatgactgaaaaaaaaatatatcgttgcg (SEQ ID No: 94);
  • glpK-down F6 ACCGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 95);
  • glpK-down F7 CCGGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 96).
  • pTargetF plasmid as a template, use the primer pair sgRNA-glpKUTR F and sgRNA-glpKUTR R to amplify the pTargetF backbone targeting the UTR sequence of the GlpK gene; use Gibson assembly to make it self-ligated, and obtain the recombinant plasmid pTargetF-GlpKUTR.
  • Example 5 Construction of genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol:
  • E.coli UTR1-GlpK E.coli UTR2-GlpK
  • E.coli UTR3-GlpK E.coli UTR4-GlpK
  • E.coli UTR5-GlpK E.coli UTR6-GlpK
  • E.coli UTR7-GlpK genetically engineered bacteria
  • E.coli U1, E. coli U2, E. coli U3, E. coli U4, E. coli U5, E. coli U6, and E. coli U7 E.coli U1, E. coli U2, E. coli U3, E. coli U4, E. coli U5, E. coli U6, and E. coli U7.
  • the genetically engineered bacteria containing the co-production of 3-hydroxypropionic acid and 1,3-propanediol obtained in Example 5 were placed in LB containing 50 ⁇ g/mL spectinomycin and 100 ⁇ g/mL kanamycin respectively.
  • the fermentation metabolites were determined by liquid chromatography, and the specific measurement conditions were: chromatographic column Aminex HPX-87H column (Bio-Rad, 300 ⁇ 7.8mm), mobile phase 0.5mM sulfuric acid, flow rate 0.4mL/min, column temperature 65 °C, UV detector wavelength 210nm, differential refractive index detector 45 °C, detection time 40min.
  • E.coli U3 has the best production performance, and it is further named E.coli U3. .coli S10G.
  • the fermentation medium of E. coli S10G was also improved, and the nitrogen source in the fermentation medium was replaced with cheap corn steep liquor.
  • the yeast extract in the fermentation culture M9 was replaced with different concentrations of corn steep liquor, the content of which was 0.05%, 0.1%, 0.25% and 0.5% (v/v), and the remaining fermentation medium components were: MgSO 4 . 7H 2 O 0.5g/L, NH 4 Cl 2.0g/L, NaCl 2.0g/L and 0.1M potassium phosphate buffer, pH 7.0.
  • the test results show that the medium containing 0.25% corn steep liquor is the optimal concentration, under this concentration, not only the cost of fermentation is reduced, but the yield of 3-hydroxypropionic acid and 1,3-propanediol remains unchanged, and the concentration of by-product acetic acid is reduced.
  • the improved medium was named M9-CSL.
  • the fermentation and production conditions of E.coli S10G are also discussed. Specifically, the entire fermentation process is divided into a growth stage and a production stage, and a two-stage pH control fermentation method is designed for it. Specifically, the large intestine is used in the growth stage The optimal pH of bacilli is 7.0 for biomass accumulation; different pHs are used for testing in the production stage to explore the optimal pH for the production stage.
  • the pH was controlled at 7.0 with 10M NaOH, and when the OD600 reached 4, 0.05mM IPTG and 50 ⁇ M vitamin B12 were added, and the temperature was changed to 35°C; when the growth of the engineered bacteria entered a stable period (OD600 was about 45), the pH was adjusted to 8.0 , the ventilation volume was adjusted to 3vvm, the dissolved oxygen value was controlled to 10%, and metabolites were detected and fed every 6h thereafter, so that the glycerin concentration was maintained at about 40g/L.

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Abstract

提供一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用，属于生物工程技术领域；以大肠杆菌Escherichia coli W3110(DE3)作为出发菌株，利用基因工程的技术手段构建了联产3-羟基丙酸和1,3-丙二醇的基因工程菌，并对所构建的基因工程菌的发酵过程进行优化，实现了3-羟基丙酸和1,3-丙二醇的高效联产。

Description

一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用 技术领域
本发明属于生物工程技术领域，具体涉及一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用。
背景技术
3-羟基丙酸和1,3-丙二醇是工业上两种重要的平台化合物，作为生物可降解性聚合物的前体物质和食品添加剂广泛使用。3-羟基丙酸和1,3-丙二醇的生产有化学合成法和生物法两种。化学法多以不可再生资源作为原料，其生产过程能耗大，产物副产物多难以分离纯化，生产过程产生不可估量的环境污染。生物法合成3-羟基丙酸或1,3-丙二醇多以葡萄糖和甘油作为底物，其中以甘油作为底物生产3-羟基丙酸和1,3-丙二醇步骤简单，研究充分，原料廉价，且能解决甘油过剩的问题。
从甘油生产3-羟基丙酸和1,3-丙二醇，首先，经甘油脱水酶将甘油脱水生成中间代谢物3-羟基丙醛；后经NAD +依赖型醛脱氢酶和NAD(P)H依赖型1,3-丙二醇氧化还原酶/同工酶分别生成3-羟基丙酸和1,3-丙二醇。但是，甘油生产3-羟基丙酸和1,3-丙二醇过程中会产生毒性中间代谢物3-羟基丙醛。此外，可持续甘油发酵生产3-羟基丙酸和1,3-丙二醇还存在毒性中间代谢物3-羟基丙醛积累抑制和必要辅因子供应不足等问题。
目前，甘油代谢抑制，限制了甘油的进一步摄入，使得3-羟基丙酸和1,3-丙二醇生产产量低；并且过多的碳流量流向中心代谢途径将降低3-羟基丙酸和1,3-丙二醇的产率；以上两点是现阶段微生物发酵甘油产3-羟基丙酸和1,3-丙二醇的主要瓶颈。
此外，一方面，微生物发酵甘油生产3-羟基丙酸和1,3-丙二醇的培养基多为改进的低盐培养基，其中添加酵母提取物以维持细胞快速生长，酵母粉是一种昂贵的氮源，这无形中增加了微生物发酵法生产3-羟基丙酸和1,3-丙二醇的成本；另一方面，适宜微生物生长和目标产物合成的pH一般不同，这种矛盾限制了3-羟基丙酸和1,3-丙二醇生产的高效生产。
发明内容
针对现有技术中存在不足，本发明提供了一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌及其构建方法和应用。在本发明中，以大肠杆菌Escherichia coli W3110(DE3)作为出发菌株，利用基因工程的技术手段构建了联产3-羟基丙酸和1,3-丙二醇的基因工程菌，并对所构建的基因工程菌的发酵过程进行优化，实现了3-羟基丙酸和1,3-丙二醇的高效联产。
本发明中首先提供了一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌，记为E.coli S10G； 所述基因工程菌在大肠杆菌E.coli W3110(DE3)的基础上表达和敲除基因重组得到；
所述表达的基因为：甘油脱水酶及其再激活因子(DhaB123-GdrAB)、丙醛脱氢酶(GabD4)、1,3-丙二醇氧化还原酶同工酶(YqhD)和膜结合型吡啶核苷酸转氢酶(PntAB)；
所述敲除的基因为：可溶型吡啶核苷酸转氢酶(SthA)、乳酸脱氢酶(LdhA)、乙醇脱氢酶(AdhE)、丙酮酸甲酸裂解酶(PflB)、丙酮酸氧化酶(PoxB)、磷酸乙酰转移酶-醋酸激酶PTA-AckA和甘油代谢抑制因子(GlpR)的基因。
其中，所述DhaB123-GdrAB的核苷酸序列如SEQ ID No:1所示，氨基酸序列如SEQ ID No:2所示。
所述GabD4的核苷酸序列如SEQ ID No:3所示，氨基酸序列如SEQ ID No:4所示。
所述YqhD的核苷酸序列如SEQ ID No:5所示，氨基酸序列如SEQ ID No:6所示。
所述PntAB的核苷酸序列如SEQ ID No:7所示，氨基酸序列如SEQ ID No:8所示。
优选的，替换基因组中甘油激酶(GlpK)的原始UTR序列为人工设计的UTR序列。
所述人工设计的UTR序列为如下7种序列的任一种：
glpK-U1AGATTATACGGGACAACACGAA(SEQ ID No:70)；
glpK-U2GTTAGCTACGGGACAAGGGTTT(SEQ ID No:71)；
glpK-U3ATTCTTTACGGGACAACCATAA(SEQ ID No:72)；
glpK-U4ACGTCTAACGGGACAAAGTTCC(SEQ ID No:73)；
glpK-U5ACGGTTTACGGGACAACGCGG(SEQ ID No:74)；
glpK-U6ACCGTCTACGGGACAACGCTGT(SEQ ID No:75)；
glpK-U7CCGGTCTACGGGACAACGCTGT(SEQ ID No:76)。
本发明中还提供了上述联产3-羟基丙酸和1,3-丙二醇的基因工程菌的构建方法，具体包括如下步骤：
(1)甘油脱水酶及其再激活因子(DhaB123-GdrAB)重组质粒的构建：
PCR扩增甘油脱水酶(DhaB123)基因、甘油脱水酶再激活因子(GdrAB)基因，并用融合PCR技术融合形成完整的甘油脱水酶及其再激活因子基因片段DhaB123-GdrAB，然后克隆至pCDFDuet-1质粒，经过转化E.coli DH5α、筛选阳性克隆、质粒提取、测序确认，获得甘油脱水酶及其再激活因子质粒，命名为pCDF-DhaB123-GdrAB。
其中，甘油脱水酶基因、甘油脱水酶再激活因子基因来源于肺炎克雷伯氏菌(Klebsiella pneumoniae)。
(2)协同表达丙醛脱氢酶(GabD4)、1,3-丙二醇氧化还原酶同工酶(YqhD)和膜结核型吡啶核苷酸转氢酶(PntAB)重组质粒的构建：
PCR扩增丙醛脱氢酶(GabD4)基因、1,3-丙二醇氧化还原酶同工酶(YqhD)基因和膜结核型吡啶核苷酸转氢酶(PntAB)基因，使用无缝克隆技术将上述两个基因克隆至pRSFuet-1质粒，经过转化E.coli DH5α、筛选阳性克隆、质粒提取、测序确认，获得协同表达丙醛脱氢酶(GabD4)、1,3-丙二醇氧化还原酶同工酶(YqhD)和膜结核型吡啶核苷酸转氢酶(PntAB)的重组质粒，命名为pRSF-GabD4-YqhD-PntAB。
其中，丙醛脱氢酶(GabD4)基因来源与pANY-gabD4质粒，1,3-丙二醇氧化还原酶同工酶(YqhD)基因和膜结核型吡啶核苷酸转氢酶(PntAB)基因来源于大肠杆菌(Escherichia coli W3110)。
(3)副产物敲除的大肠杆菌的构建：
使用双质粒CRIPSR CAS9工具质粒，敲除大肠杆菌(Escherichia coli W3110(DE3))可溶型吡啶核苷酸转氢酶(SthA)基因、乳酸脱氢酶(LdhA)基因、乙醇脱氢酶(AdhE)基因、丙酮酸甲酸裂解酶(PflB)基因、丙酮酸氧化酶(PoxB)基因、磷酸乙酰转移酶-醋酸激酶PTA-AckA基因和甘油代谢抑制因子(GlpR)基因，在PCR验证及测序验证后，最终得到基因缺陷工程菌，即副产物敲除的大肠杆菌。
优选的，可以采用UTR工程技术对副产物敲除的大肠杆菌的甘油激酶(GlpK)表达量进行人工修饰，使用UTR设计工具，设计UTR人工序列，使用双质粒CRIPSR CAS9工具质粒，替换基因缺陷工程菌的基因组中原有的甘油激酶(GlpK)基因的UTR序列，经测序验证后，得到甘油激酶(GlpK)表达量人工修饰的工程菌。
其中，所述人工设计的UTR序列为如下7种序列的任一种：
glpK-U1 AGATTATACGGGACAACACGAA(SEQ ID No:70)；
glpK-U2 GTTAGCTACGGGACAAGGGTTT(SEQ ID No:71)；
glpK-U3 ATTCTTTACGGGACAACCATAA(SEQ ID No:72)；
glpK-U4 ACGTCTAACGGGACAAAGTTCC(SEQ ID No:73)；
glpK-U5 ACGGTTTACGGGACAACGCGG(SEQ ID No:74)；
glpK-U6 ACCGTCTACGGGACAACGCTGT(SEQ ID No:75)；
glpK-U7 CCGGTCTACGGGACAACGCTGT(SEQ ID No:76)。
(4)联产3-羟基丙酸和1,3-丙二醇的基因工程菌的构建：
将pCDF-DhaB123-GdrAB和pRSF-GabD4-YqhD-PntAB同时转化入基因缺陷工程菌的感受态细胞，得到3-羟基丙酸和1,3-丙二醇高效联产基因工程。
其中，步骤(1)～(3)不分先后顺序。
本发明中还提供了上述基因工程菌在发酵甘油联产3-羟基丙酸和1,3-丙二醇中的应用。
本发明中还提供了上述基因工程菌发酵联产3-羟基丙酸和1,3-丙二醇的方法，具体包括如下步骤：
(1)将基因工程菌E.coli S10G在LB培养基中过夜活化，得到E.coli S10G的一级种子液；
(2)将E.coli S10G一级种子液接种至改良M9-CSL培养基中培养，得到E.coli S10G的二级种子液；
(3)将E.coli S10G的二级种子液接种于改良M9-CSL培养基中，将发酵过程划分为生长阶段和生产阶段控制pH值进行补料发酵；
所述生长阶段时pH控制为7.0，调整温度、通气量、搅拌速率发酵培养至OD600达到4时，加入IPTG和维生素B12继续培养；
所述生产阶段时pH控制为8.0，调整温度、通气量、溶氧值进行发酵，然后每6h进行补料发酵联产3-羟基丙酸和1,3-丙二醇；
所述补料的成分含有甘油和玉米浆。
其中，步骤(2)中，所述改良M9-CSL培养基的成分为MgSO 4·7H 2O 0.5g/L,NH  4Cl 2.0g/L,NaCl 2.0g/L,玉米浆2.5mL/L,甘油40g/L和0.1M磷酸钾缓冲液,pH 7.0；
所述EC10S10G一级种子液的接种量为1％v/v，培养条件为37℃、220rpm培养12h。
步骤(3)中，E.coli S10G二级种子液的接种量为5％v/v；
所述生长阶段时，调整温度为37℃，初始通气量为2vvm，搅拌速率为500rpm进行发酵培养；
所述生产阶段时调整温通气量调整为3vvm，搅拌速率为200-800rpm，控制溶氧值为10％进行发酵；
所述补料的成分含有800g/L甘油和50mL/L玉米浆；
所述补料过程为：每6h进行补料控制甘油浓度维持在40g/L。
与现有技术相比，本发明的有益效果在于：
本技术通过代谢工程和合成生物学设计，构建了一株可以高效联产3-羟基丙酸和1,3-丙二醇的基因工程，通过辅因子工程促进必需辅因子循环，并通过敲除副产物途径、失活代谢抑制因子和控制中心代谢途径提高3-羟基丙酸和1,3-丙二醇途径通量。所构建的基因工程菌可以通过两阶段pH控制补料发酵，以生物柴油产物废弃物甘油为底物，高效联产3-羟基丙酸和1,3-丙二醇。
本发明中，摒弃了传统3-羟基丙酸或1,3-丙二醇单独生产的方法，将3-羟基丙酸联产与辅因子工程相结合，将UTR工程运用于代谢通量平衡，具有重要的理论和实践意义，所构建 的基因工程E.coli S10G可高效生产3-羟基丙酸和1,3-丙二醇，具有大规模工业生产潜力。
并且，本发明中构建的工程菌高效转化代谢中间代谢物3-羟基丙醛，生成终产物3-羟基丙酸和1,3-丙二醇；通过辅因子工程促进必需辅因子和循环再生，解决3-羟基丙酸和1,3-丙二醇生产过程中必需辅因子供应不足的问题；此外，由于副产物生产途径的消除和中心代谢途径的削弱，使得3-羟基丙酸和1,3-丙二醇的生产具有较高的生产产率和较少的副产物积累，具有广阔的应用前景与实际意义。
附图说明
图1为基因工程菌E.coli S10G中pCDF-DhaB123-GDRAB质粒图谱。
图2为基因工程菌E.coli S10G中pRSF-GabD4-YqhD-PntAB质粒图谱。
图3为基因工程E.coli UTR-GlpK中GlpK相对表达量及代谢物情况。
图4为本发明所述两阶段pH控制发酵的产量结果图。
图5为本发明所述基因工程菌E.coli S10G通过两阶段pH控制补料发酵联产3-羟基丙酸和1,3-丙二醇的发酵情况图。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步的说明，但本发明的保护范围并不限于此。
下列实施例中未注明具体条件者，皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。除特殊注明外，本发明所采用的均为该领域现有技术。
实施例1：甘油脱水酶及其再激活因子(DhaB123-GdrAB)重组质粒的构建
(1)根据肺炎克雷伯氏菌的甘油脱水酶及其再激活因子(DhaB123-GdrAB)基因序列利用Oligo7.0软件设计引物：
DhaB F：GTTTAACTTTAATAAGGAGATATACCatgaaaagatcaaaacgatttgcagtactg(SEQ ID No:9)；
GdrA R：CGGCCCCCTCGTTAACACttaattcgcctgaccggccag(SEQ ID No:10)；
GrdB F：CTGGCCGGTCAGGCGAATTAAgtgttaacgagggggccgtc(SEQ ID No:11)；
GrdB R：TTATGCGGCCGCAAGCTTGTCGACtcagtttctctcacttaacggcaggac(SEQ ID No:12)。
(2)使用DhaB F和GdrA R引物对扩增DhaB123-GdrA基因，使用GdrB F和GdrB R引物对扩增GdrB基因，PCR反应参数：预变性，98℃3min；变性，98℃10s；退火，55℃10s；延伸，72℃1.5min；终延伸，72℃5min；33个循环后，得到DhaB123-GdrA 基因和GdrB基因。
(3)将所得DhaB123-GdrA基因和GdrB基因片段分别作为模板，使用DhaB F和GdrB R引物对进行融合PCR，PCR反应参数：预变性，98℃3min；变性，98℃10s；退火，55℃10s；延伸，72℃1.5min；终延伸，72℃5min；33个循环后得到完整的DhaB123-GdrAB基因。
(4)将pCDFDuet-1质粒(淼灵生物)使用NcoI和BlnI进行双酶切，反应条件为37℃30min；反应结束后获得线性化pCDFDuet-1质粒骨架。然后将DhaB123-GdrAB基因和线性化pCDFDuet-1质粒骨架使用2×MultiF Seamless Assembly Mix进行吉布森组装，获得重组质粒。反应条件为：50℃30min；
通过标准热激法将上述得到的重组质粒转化进入E.coli DH5α感受态(淼灵生物)中，转化的具体步骤为：
冰浴5min后加入重组质粒，冰上30min，42℃45s，冰上2min，加入1mL无抗生素LB培养基，37℃培养1h后涂布还有50μg/mL壮观霉素的LB平板；挑取适量单菌落培养并提取重组质粒，送苏州金唯智测序验证，验证正确的重组质粒命名为pCDF-DhaB123-GdrAB，其核苷酸序列如SEQ ID No:13所示，质粒图谱如图1所示。
图1为基因工程菌E.coli S10G中pCDF-DhaB123-GDRAB质粒图谱，从图中可以看出，该重组质粒含ColDF3复制起点，基因表达由T7启动子驱动。
实施例2:协同表达丙醛脱氢酶(GabD4)、1,3-丙二醇氧化还原酶同工酶(YqhD)和膜结核型吡啶核苷酸转氢酶(PntAB)重组质粒的构建
(1)根据质粒pANY-gabD4上丙醛脱氢酶(GabD4)基因序列、大肠杆菌中1,3-丙二醇氧化还原酶同工酶(YqhD)基因序列和膜结核型吡啶核苷酸转氢酶(PntAB)基因序列以及pRSFDuet-1质粒的序列特征，利用Oligo7.0软件设计引物：
GabD4 F：GTTTAACTTTAAGAAGGAGATATACCatgtaccaagatctggcact(SEQ ID No:14)；
GabD4 R：TTATGCGGCCGCAAGCTTGTCGACttacgcttgggtgatgaact(SEQ ID No:15)；
Backbone1 F：attagttaagtataagaaggagatatacat(SEQ ID No:16)；
Backbone1 R：gtggcagcagcctaggttaa(SEQ ID No:17)；
YqhD F：ATTAGTTAAGTATAAGAAGGAGATATACATatgaacaactttaatctgcacac(SEQ ID No:18)；
YqhD R：GTGGCAGCAGCCTAGGTTAAttagcgggcggcttcgtat(SEQ ID No:19)；
Backbone2 F：ttacgcttgggtgatgaacttg(SEQ ID No:20)；
Backbone2 R：gtcgacaagcttgcggc(SEQ ID No:21)；
PntAB F：ACCAAGTTCATCACCCAAGCGTAAaaccgatggaagggaatatcatgc(SEQ ID No:22)；
PntAB R：GCGGCCGCAAGCTTGTCGACttacagagctttcaggattgcatccac(SEQ ID No:23)。
(2)使用GabD4F和GabD4R引物对扩增GabD4基因，使用YqhD F和YqhD R引物对扩增YqhD基因，使用PntAB F和PntAB R引物对扩增PntAB基因。PCR反应参数：预变性，98℃3min；变性，98℃10s；退火，55℃10s；延伸，72℃1.5min；终延伸，72℃5min；33个循环后分别得到GabD4、YqhD和PntAB基因。
(3)将pRSFDuet-1质粒使用NcoI和BlnI进行双酶切，反应条件为：37℃、30min；反应结束后获得线性化pRSFDuet-1质粒骨架，然后将GabD4基因和线性化pRSFDuet-1质粒骨架进行吉布森组装，获得重组质粒pRSF-GabD4。
以所得的的重组质粒pRSF-GabD4为模板，使用引物对Backbone1F和Backbone1R进行反向PCR，获得重组质粒pRSF-GabD4骨架；将该骨架与YqhD基因进行吉布森组装，获得重组质粒pRSF-GabD4-YqhD。
以所得的的重组质粒pRSF-GabD4-YqhD为模板，使用引物对Backbone2F和Backbone2R进行反向PCR，获得重组质粒pRSF-GabD4-YqhD骨架；将该骨架与PntAB基因进行吉布森组装，获得重组质粒pRSF-GabD4-YqhD-PntAB，即协同表达丙醛脱氢酶(GabD4)、1,3-丙二醇氧化还原酶同工酶(YqhD)和膜结核型吡啶核苷酸转氢酶(PntAB)重组质粒。所述pRSF-GabD4-YqhD-PntAB的核苷酸序列如SEQ ID No:24所示，质粒图谱如图2所示。
图2为pRSF-GabD4-YqhD-PntAB质粒的图谱，该重组质粒含RSF复制起点，由T7驱动GabD4基因和PntAB基因的表达，另外，YqhD基因由单独T7启动子驱动。
实施例3：基因缺陷工程菌的构建：
(1)根据可溶型吡啶核苷酸转氢酶(SthA)基因序列，使用sgRNAcas9选择合适靶点生成合适的sgRNA序列；根据上述基因序列和ptargetF质粒的序列特性使用Oligo7.0软件设计用于SthA基因敲除的引物：
sgRNA-sthA F：CCGCGCCATGTACTTATCTAgttttagagctagaaatagc(SEQ ID No:25)；
sgRNA-sthA R：TAGATAAGTACATGGCGCGGactagtattatacctaggac(SEQ ID No:26)；
sthA-up F：agttcgtctacgtcgcggaaatgc(SEQ ID No:27)；
sthA-up R：GCCATTTCGATAAAGTTTTAcatggtagggcttacctgt(SEQ ID No:28)；
sthA-down F：AACAGGTAAGCCCTACCATGtaaaactttatcgaaatggccatc(SEQ ID No:29)；
sthA-down R：tcctcgcgctggtgaaaga(SEQ ID No:30)；
(2)以pTargetF质粒为模板，使用引物对sgRNA-sthA F和sgRNA-sthA R扩增获得靶向SthA基因的pTargetF骨架；使用吉布森组装使其自连，获得重组质粒pTargetF-SthA。
(3)使用大肠杆菌E.coli W3110基因组(NC_000913.3)为模板，使用引物对sthA-up F和sthA-up R扩增SthA基因上游序列；使用引物对sthA-down F和sthA-down R扩增SthA基因下游序列；并以SthA基因上游序列和SthA基因下游序列为模板，使用引物对sthA-up F和sthA-down R扩增获得SthA基因上下游同源臂。
(4)将pCas9质粒(淼灵生物)转化进入E.coli W3110(DE3)(E.coli W3110购自百欧生物，使用溶源化试剂盒对其进行溶源化处理，得到E.coli W3110(DE3))中，得到含有pCas9质粒的E.coli W3110(DE3)，然后将其经过0.1M的L-***糖诱导后制作成为电转感受态。接着将得到的100ng的pTargetF-SthA质粒和400ng的SthA基因上下游同源臂使用电转仪转化进入含有pCas9质粒的E.coli W3110(DE3)中，30℃培养2h后涂布含有50μg/mL壮观霉素和50μg/mL卡那霉素的LB平板，30℃培养24-48h。所述电转参数为：电压1800V，电阻200Ω，电容25μF，电转杯1mm，电击时间1.5ms。
挑取培养后的单菌落，使用引物对sthA up F和sthA down R进行菌落PCR验证，将疑似基因缺陷的工程菌送苏州金唯智测序验证，验证正确的基因缺陷工程菌保存备用。
(5)对于上述测序验证正确的基因缺陷工程菌，将其在含有50 50μg/mL卡那霉素的LB液体培养基中进行培养，使用0.5mM IPTG进行诱导，除去pTargetF-SthA质粒，对于除去pTargetF-SthA质粒的工程菌，命名为E.coli pCas9△sthA。
(6)以E.coli pCas9△sthA为基础，参考(1)～(5)的步骤来敲除乳酸脱氢酶(LdhA)基因、乙醇脱氢酶(AdhE)基因、丙酮酸甲酸裂解酶(PflB)基因、丙酮酸氧化酶(PoxB)基因、磷酸乙酰转移酶-醋酸激酶(PTA-AckA)基因和甘油代谢抑制因子(GlpR)基因。
其中，敲除乳酸脱氢酶(LdhA)基因的引物如下所示：
sgRNA-ldhA F：CGACAAGAAGTACCTGCAACgttttagagctagaaatagc(SEQ ID No:31)；
sgRNA-ldhA R：GTTGCAGGTACTTCTTGTCGactagtattatacctaggac(SEQ ID No:32)；
ldhA-up F：tttaactttttcgccctga(SEQ ID No:33)；
ldhA-up R：GCAGGGGAGCGGCAAGATTAcataagactttctccagtgatg(SEQ ID No:34)；
ldhA-down F：TCACTGGAGAAAGTCTTATGtaatcttgccgctcccctgc(SEQ ID No:35)；
ldhA-down R：taaaagcgtcgatgtccagt(SEQ ID No:36)。
敲除乙醇脱氢酶(AdhE)基因的引物如下所示：
sgRNA-adhE F：TCGAATCCCACTCGCGAAAAgttttagagctagaaatagc(SEQ ID No:37)；
sgRNA-adhE R：TTTTCGCGAGTGGGATTCGAactagtattatacctaggac(SEQ ID No:38)；
adhE-up F：taccaaaaagttgtagaatcgtg(SEQ ID No:39)；
adhE-up R：CCAGACAGCGCTACTGATTAcataatgctctcctgataatgt(SEQ ID No:40)；
adhE-down F：ATTATCAGGAGAGCATTATGtaatcagtagcgctgtctgg(SEQ ID No:41)；
adhE-down R：cagcacagtttcgctctg(SEQ ID No:42)。
敲除丙酮酸甲酸裂解酶(PflB)基因的引物如下所示：
sgRNA-pflB F：ATGAAAAGTTAGCCACAGCCgttttagagctagaaatagc(SEQ ID No:43)；
sgRNA-pflB R：GGCTGTGGCTAACTTTTCATactagtattatacctaggac(SEQ ID No:44)；
pflB-up F：atttgcttctctctggggctga(SEQ ID No:45)；
pflB-up R：ATTTCAGTCAAATCTAATTAcatgtaacacctaccttct(SEQ ID No:46)；
pflB-down F：AAGAAGGTAGGTGTTACatgtaattagatttgactgaaatcgt(SEQ ID No:47)；
pflB-down R：cgtagcggatccacaccttc(SEQ ID No:48)。
敲除丙酮酸氧化酶(PoxB)基因的引物如下所示：
sgRNA-poxB F：TATCGCCAAAACACTCGAATgttttagagctagaaatagc(SEQ ID No:49)；
sgRNA-poxB R：ATTCGAGTGTTTTGGCGATAactagtattatacctaggac(SEQ ID No:50)；
poxB-up F：aaccgtccacaggccgatgt(SEQ ID No:51)；
poxB-up R：GGGAAATGCCACCCTTTTTAcatggttctccatctcctga(SEQ ID No:52)；
poxB-down F：TCAGGAGATGGAGAACCATgtaaaaagggtggcatttcccgtc(SEQ ID No:53)；
poxB-down R：ccagcgaatggcacgtt(SEQ ID No:54)；
敲除磷酸乙酰转移酶-醋酸激酶(PTA-AckA)基因引物如下所示：
sgRNA-pta-ackA F：AATAAACAGGAAGCGGCTTTgttttagagctagaaatagc(SEQ ID No:55)；
sgRNA-pta-ackA R：AAAGCCGCTTCCTGTTTATTactagtattatacctaggac(SEQ ID No:56)；
pta-ackA-up F：aacacctgtccagactcct(SEQ ID No:57)；
pta-ackA-up R：TGCGGATGATGACGAGATTAcatggaagtacctataattga(SEQ ID No:58)；
pta-ackA-down F：CAATTATAGGTACTTCCATgtaatctcgtcatcatccgcag(SEQ ID No:59)；
pta-ackA-down R：ttctcccataccaaataccg(SEQ ID No:60)。
敲除甘油代谢抑制因子(GlpR)基因引物如下所示：
sgRNA-glpR F：CGTAACGCGATGGTCAATATgttttagagctagaaatagc(SEQ ID No:61)；
sgRNA-glpR R：ATATTGACCATCGCGTTACGactagtattatacctaggac(SEQ ID No:62)；
glpR-up F：cgtggtggtgtttattgcc(SEQ ID No:63)；
glpR-up R：AGCACAGCTCCAGTTGAAtcatttataaatccctggaattatt(SEQ ID No:64)；
glpR-down F：AATAATTCCAGGGATTTATAAATGattcaactggagctgtg(SEQ ID No:65)；
glpR-down R：tgaagagaaaaccttttaccc(SEQ ID No:66)。
(7)待完成上述所有基因缺陷后，所得基因缺陷工程菌为E.coli pCas9△sthA△ldhA△ adhE△pflB△poxB△pta△ackA△glpR。
实施例4：甘油激酶(GlpK)表达量人工修饰的工程菌的构建
(1)根据大肠杆菌E.coli W3110(DE3)基因组序列，可知甘油激酶(GlpK)基因的原始UTR序列为：TATGACTACGGGACAATTAAAC(SEQ ID No:67)，GlpK基因N端35bp序列为：ATGACTGAAAAAAAATATATCGTTGCGCTCGACCA(SEQ ID No:68)，遵循UTR设计原则，人工设计的UTR序列格式应为NNNNNN TACGGGACAANNNNNN，其中TACGGGACAA为E.coli W3110核糖体结合位点，N代表任意碱基。为了使GlpK表达量达到预期范围，设计了7个强度由强到弱的UTR序列，如表1所示。
表1.不同UTR及其预测的表达强度
名称 5’-UTR序列(5’-3’) 预测的表达量(a.u.)
glpK TATGACTACGGGACAATTAAAC(SEQ ID No:69) 162064.41
glpK-U1 AGATTATACGGGACAACACGAA(SEQ ID No:70) 137030.54
glpK-U2 GTTAGCTACGGGACAAGGGTTT(SEQ ID No:71) 100744.62
glpK-U3 ATTCTTTACGGGACAACCATAA(SEQ ID No:72) 70038.41
glpK-U4 ACGTCTAACGGGACAAAGTTCC(SEQ ID No:73) 30268.08
glpK-U5 ACGGTTTACGGGACAACGCGG(SEQ ID No:74) 10170.17
glpK-U6 ACCGTCTACGGGACAACGCTGT(SEQ ID No:75) 5198.13
glpK-U7 CCGGTCTACGGGACAACGCTGT(SEQ ID No:76) 2008.70
(2)根据甘油激酶(GlpK)基因序列，使用sgRNAcas9选择合适靶点生成合适的sgRNA序列；根据上述基因序列和ptargetF质粒(淼灵生物)的序列特性使用Oligo7.0软件设计用于替换GlpK基因UTR的引物：
sgRNA-glpKUTR F：CTTCGCTGTAATATGACTACgttttagagctagaaatagc(SEQ ID No:77)；
sgRNA-glpKUTR R：GTAGTCATATTACAGCGAAGactagtattatacctaggac(SEQ ID No:78)；
glpK-up F：cgcagttgagatggtgattaccg(SEQ ID No:79)；
glpK-up R：ttacagcgaagctttttgttc(SEQ ID No:80)；
glpK-down F：atgactgaaaaaaaatatatcgttgcg(SEQ ID No:81)；
glpK-down R：atccacttcactttggtgcca(SEQ ID No:82)；
glpK-up R1：TTCGTGTTGTCCCGTATAATCTttacagcgaagctttttgttc(SEQ ID No:83)；
glpK-up R2：AAACCCTTGTCCCGTAGCTAACttacagcgaagctttttgttc(SEQ ID No:84)；
glpK-up R3：TTATGGTTGTCCCGTAAAGAATttacagcgaagctttttgttc(SEQ ID No:85)；
glpK-up R4：GGAACTTTGTCCCGTTAGACGTttacagcgaagctttttgttc(SEQ ID No:86)；
glpK-up R5：CCGCGTTGTCCCGTAAACCGTttacagcgaagctttttgttc(SEQ ID No:87)；
glpK-up R6：ACAGCGTTGTCCCGTAGACGGTttacagcgaagctttttgttc(SEQ ID No:88)；
glpK-up R7：ACAGCGTTGTCCCGTAGACCGGttacagcgaagctttttgttc(SEQ ID No:89)；
glpK-down F1：AGATTATACGGGACAACACGAAatgactgaaaaaaaatatatcgttgcg(SEQ ID No:90)；
glpK-down F2：GTTAGCTACGGGACAAGGGTTTatgactgaaaaaaaatatatcgttgcg(SEQ ID No:91)；
glpK-down F3：ATTCTTTACGGGACAACCATAAatgactgaaaaaaaatatatcgttgcg(SEQ ID No:92)；
glpK-down F4：ACGTCTAACGGGACAAAGTTCCatgactgaaaaaaaatatatcgttgcg(SEQ ID No:93)；
glpK-down F5：ACGGTTTACGGGACAACGCGGatgactgaaaaaaaatatatcgttgcg(SEQ ID No:94)；
glpK-down F6：ACCGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg(SEQ ID No:95)；
glpK-down F7：CCGGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg(SEQ ID No:96)。
(4)以pTargetF质粒为模板，使用引物对sgRNA-glpKUTR F和sgRNA-glpKUTR R扩增获得靶向GlpK基因UTR序列的pTargetF骨架；使用吉布森组装使其自连，获得重组质粒pTargetF-GlpKUTR。
(5)使用大肠杆菌E.coli W3110基因组为模板，使用引物对glpK-up F和glpK-up R扩增GlpK基因上游序列；使用引物对glpK-down F和glpK-down R扩增GlpK基因下游序列。
含UTR-U1的GlpK基因上下游同源臂的获得：
以GlpK基因上游序列为模板，使用引物对glpK-up F和glpK-up R1扩增获得含UTR-U1的GlpK基因上游序列；
以GlpK基因下游序列为模板，使用引物对glpK-down F1和glpK-down R扩增获得含UTR-U1的GlpK基因下游序列；
使用含UTR-U1的GlpK基因上游序列和含UTR-U1的GlpK基因下游序列的基因片段为模板，使用glpK-down F和glpK-down R进行融合PCR，获得含UTR-U1的GlpK基因上下游同源臂。
含UTR-U2的GlpK基因上下游同源臂的获得：
以GlpK基因上游序列为模板，使用引物对glpK-up F和glpK-up R2扩增获得含UTR-U2 的GlpK基因上游序列；
以GlpK基因下游序列为模板，使用引物对glpK-down F2和glpK-down R扩增获得含UTR-U2的GlpK基因下游序列；
使用含UTR-U2的GlpK基因上游序列和含UTR-U2的GlpK基因下游序列的基因片段为模板，使用glpK-down F和glpK-down R进行融合PCR，获得含UTR-U2的GlpK基因上下游同源臂。
(6)将100ng pTargetF-GlpKUTR质粒以及400ng的含UTR-U1、UTR-U2、UTR-U3、UTR-U4、UTR-U5、UTR-U6和UTR-U7的GlpK基因上下游同源臂分别组合转化进入E.coli pCas9△sthA△ldhA△adhE△pflB△poxB△pta△ackA△glpR的电转感受态中，进行孵育、涂布和测序，最后对于成功更换UTR序列的工程菌，即甘油激酶(GlpK)表达量人工修饰的工程菌，分别命名为E.coli pCas9 UTR1-GlpK、E.coli pCas9 UTR2-GlpK、E.coli pCas9 UTR3-GlpK、E.coli pCas9 UTR4-GlpK、E.coli pCas9 UTR5-GlpK、E.coli pCas9 UTR6-GlpK和E.coli pCas9 UTR7-GlpK。
(7)对于上述GlpK基因成功修饰的工程菌，将其分别置于无抗生素LB液体培养基在37℃培养12h以消除pCas9质粒，最终获得的E.coli UTR1-GlpK、E.coli UTR2-GlpK、E.coli UTR3-GlpK、E.coli UTR4-GlpK、E.coli UTR5-GlpK、E.coli UTR6-GlpK和E.coli UTR7-GlpK。
实施例5：联产3-羟基丙酸和1,3-丙二醇的基因工程菌的构建：
将pCDF-DhaB123-GdrAB重组质粒和pRSF-GabD4-YqhD-PntAB重组质粒同时分别转化进入E.coli UTR1-GlpK、E.coli UTR2-GlpK、E.coli UTR3-GlpK、E.coli UTR4-GlpK、E.coli UTR5-GlpK、E.coli UTR6-GlpK、E.coli UTR7-GlpK基因工程菌中，获得联产3-羟基丙酸和1,3-丙二醇的基因工程菌，分别命名为E.coli U1、E.coli U2、E.coli U3E.coli U4、E.coli U5、E.coli U6和E.coli U7。
实施例6：3-羟基丙酸和1,3-丙二醇的联产：
将实施例5中得到的含有不同GlpK基因表达量的联产3-羟基丙酸和1,3-丙二醇的基因工程菌分别在含50μg/mL壮观霉素和100μg/mL卡那霉素的LB平板上37℃活化18-24h，然后分别挑取长势良好的单菌落于含50μg/mL壮观霉素和100μg/mL卡那霉素的LB液体培养基中37℃活化12h，将活化的培养物转接至含有10g/L甘油、50μg/mL壮观霉素和100μg/mL卡那霉素的M9液体培养基(MgSO 4·7H 2O 0.5g/L,NH 4Cl 2.0g/L,NaCl 2.0g/L,酵母提取物1.0g,0.1M磷酸钾缓冲液pH 7.0)中37℃培养12h，获得E.coli U1、E.coli U2、E.coli U3 E.coli U4、E.coli U5、E.coli U6和E.coli U的发酵种子液。
然后将上述得到的发酵种子液分别按1％接种量接种于含30g/L甘油的M9培养基中， 37℃，220rpm培养至OD 600达到0.8，加入0.05mM IPTG诱导重组质粒pCDF-DhaB123-GdrAB和pRSF-GabD4-YqhD-PntAB上的基因表达，同时加入50μM维生素B12以激活甘油脱水酶活性，此后培养温度切换至35℃发酵36h，得到发酵代谢物。
将发酵代谢物通过液相色谱法进行测定，具体测定条件为：色谱柱Aminex HPX-87H column(Bio-Rad,300×7.8mm)，流动相0.5mM硫酸，流速0.4mL/min，柱温65℃，紫外检测器波长210nm，示差折光检测器45℃，检测时间40min。
试验结果表明，无论GlpK表达量高低，都能发酵甘油同时生成3-羟基丙酸和1,3-丙二醇，如图3所示，其中E.coli U3生产性能最佳，将其进一步命名为E.coli S10G。
本实施例中还对E.coli S10G的发酵培养基进行了改进，将发酵培养基中的氮源使用廉价玉米浆替换。具体的，将发酵培养M9中的酵母提取物替换成不同浓度的玉米浆，其含量为0.05％、0.1％、0.25％和0.5％(v/v)，其余发酵培养基成分为：MgSO 4·7H 2O 0.5g/L,NH 4Cl 2.0g/L,NaCl 2.0g/L和0.1M磷酸钾缓冲液,pH 7.0。
试验结果表明，含有0.25％玉米浆的培养基为最优浓度，在此浓度下，发酵不但成本降低，3-羟基丙酸和1，3-丙二醇产量维持不变，且副产物醋酸浓度有所下降，所述改进的培养基命名为M9-CSL。
本实施例中还探讨了E.coli S10G的发酵生产条件，具体地，将整个发酵过程分为生长阶段和生产阶段，为其设计了两阶段pH控制发酵方法，具体为，在生长阶段使用大肠杆菌最适pH 7.0进行生物量积累；在生产阶段使用不同pH进行测试，探索出生产阶段的最适pH。
结果表明，在生产阶段pH 8.0为最适pH，在pH 8.0时，3-羟基丙酸和1,3-丙二醇产量显著高于pH 7.0条件。因此后续发酵试验按生长阶段pH 7.0，生产阶段pH 8.0进行。
(7)最后，在5L发酵罐中使用上述两阶段pH控制的方式对E.coli E.coli S10G进行补料发酵，所补成分含有800g/L甘油和50mL/L玉米浆。首先，使用在M9-CSL培养基中活化的新鲜种子液，按5％接种量接种于5L发酵罐中(实际装液量为2L),温度37℃，初始通气量为2vvm，搅拌速率500rpm，pH使用10M NaOH控制在7.0，培养至OD600达到4时，加入0.05mM IPTG和50μM维生素B12，温度更换为35℃；待工程菌生长进入稳定期(OD600约为45左右)，将pH调整为8.0，通气量调整为3vvm，控制溶氧值为10％，并此后每6h检测一次代谢产物以及进行补料，使甘油浓度维持在40g/L左右。
试验结果如图5所示，所述工程菌E.coli E.coli S10G经66h发酵，其生产3-羟基丙酸77.34g/L和1,3-丙二醇63.16g/L，总产量为140.5g/L。
所述实施例为本发明的优选的实施方式，但本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员能够做出的任何显而易见的改进、替换或变型均 属于本发明的保护范围。

Claims (13)

  1. 一种联产3-羟基丙酸和1,3-丙二醇的基因工程菌，其特征在于，所述基因工程菌在大肠杆菌E.coli W3110(DE3)的基础上表达甘油脱水酶及其再激活因子DhaB123-GdrAB、丙醛脱氢酶GabD4、1,3-丙二醇氧化还原酶同工酶YqhD和膜结合型吡啶核苷酸转氢酶PntAB得到。
  2. 权利要求1所述的联产3-羟基丙酸和1,3-丙二醇的基因工程菌，其特征在于，所述基因工程菌中还敲除了下述基因组合中的一组或几组：
    (1)可溶型吡啶核苷酸转氢酶SthA的基因；
    (2)乳酸脱氢酶LdhA、乙醇脱氢酶AdhE、丙酮酸甲酸裂解酶PflB、丙酮酸氧化酶PoxB、磷酸乙酰转移酶-醋酸激酶PTA-AckA的基因；
    (3)甘油代谢抑制因子GlpR的基因。
  3. 权利要求1或2所述的联产3-羟基丙酸和1,3-丙二醇的基因工程菌，其特征在于，所述基因工程菌中还将甘油激酶GlpK的原始UTR序列替换为人工设计的UTR序列，所述人工设计的UTR序列为如下7种序列的任一种：
    glpK-U1 AGATTATACGGGACAACACGAA(SEQ ID No:70)；
    glpK-U2 GTTAGCTACGGGACAAGGGTTT(SEQ ID No:71)；
    glpK-U3 ATTCTTTACGGGACAACCATAA(SEQ ID No:72)；
    glpK-U4 ACGTCTAACGGGACAAAGTTCC(SEQ ID No:73)；
    glpK-U5 ACGGTTTACGGGACAACGCGG(SEQ ID No:74)；
    glpK-U6 ACCGTCTACGGGACAACGCTGT(SEQ ID No:75)；
    glpK-U7 CCGGTCTACGGGACAACGCTGT(SEQ ID No:76)。
  4. 根据权利要求1所述的联产3-羟基丙酸和1,3-丙二醇的基因工程菌，其特征在于，所述DhaB123-GdrAB的氨基酸序列如SEQ ID No:2所示，编码其的核苷酸如SEQ ID No:1所示；
    所述GabD4的氨基酸序列如SEQ ID No:4所示，编码其的核苷酸如SEQ ID No:3所示；
    所述YqhD的氨基酸序列如SEQ ID No:6所示，编码其的核苷酸如SEQ ID No:5所示；
    所述PntAB的氨基酸序列如SEQ ID No:8所示，编码其的核苷酸如SEQ ID No:7所示。
  5. 权利要求1所述的联产3-羟基丙酸和1,3-丙二醇的基因工程菌的制备方法，其特征在于，包括：
    (1)甘油脱水酶及其再激活因子DhaB123-GdrAB重组质粒的构建：
    PCR扩增甘油脱水酶DhaB123基因、甘油脱水酶再激活因子GdrAB基因，并用融合PCR技术融合形成完整的甘油脱水酶及其再激活因子基因片段DhaB123-GdrAB，然后克隆至pCDFDuet-1质粒，经过转化E.coli DH5α、筛选阳性克隆、质粒提取、测序确认，获得甘油 脱水酶及其再激活因子质粒，命名为pCDF-DhaB123-GdrAB；
    (2)协同表达丙醛脱氢酶GabD4、1,3-丙二醇氧化还原酶同工酶YqhD和膜结核型吡啶核苷酸转氢酶PntAB重组质粒的构建：
    PCR扩增丙醛脱氢酶GabD4基因、1,3-丙二醇氧化还原酶同工酶YqhD基因和膜结核型吡啶核苷酸转氢酶PntAB基因，使用无缝克隆技术将上述两个基因克隆至pRSFuet-1质粒，经过转化E.coli DH5α、筛选阳性克隆、质粒提取、测序确认，获得协同表达丙醛脱氢酶GabD4、1,3-丙二醇氧化还原酶同工酶YqhD和膜结核型吡啶核苷酸转氢酶PntAB的重组质粒，命名为pRSF-GabD4-YqhD-PntAB；
    (3)联产3-羟基丙酸和1,3-丙二醇的基因工程菌的构建：
    将pCDF-DhaB123-GdrAB和pRSF-GabD4-YqhD-PntAB同时转化入大肠杆菌E.coli W3110(DE3)的感受态细胞，得到3-羟基丙酸和1,3-丙二醇高效联产基因工程；
    其中，步骤(1)和(2)不分先后顺序。
  6. 权利要求2所述的联产3-羟基丙酸和1,3-丙二醇的基因工程菌的制备方法，其特征在于，所述敲除的方法为：使用双质粒CRIPSR CAS9工具质粒敲除大肠杆菌E.coli W3110(DE3)的下述基因：可溶型吡啶核苷酸转氢酶SthA、乳酸脱氢酶LdhA、乙醇脱氢酶AdhE、丙酮酸甲酸裂解酶PflB、丙酮酸氧化酶PoxB、磷酸乙酰转移酶-醋酸激酶PTA-AckA和甘油代谢抑制因子GlpR。
  7. 权利要求3所述的的联产3-羟基丙酸和1,3-丙二醇的基因工程菌的制备方法，其特征在于，所述将甘油激酶GlpK的原始UTR序列替换为人工设计的UTR序列的步骤为：
    所述步骤为：采用UTR工程技术对副产物敲除的大肠杆菌的甘油激酶GlpK表达量进行人工修饰，使用UTR设计工具，设计UTR人工序列，使用双质粒CRIPSR CAS9工具质粒，替换基因缺陷工程菌的基因组中原有的甘油激酶GlpK基因的UTR序列，经测序验证后，得到甘油激酶GlpK表达量人工修饰的工程菌；
    所述人工设计的UTR序列为如下7种序列的任一种：
    glpK-U1 AGATTATACGGGACAACACGAA(SEQ ID No:70)；
    glpK-U2 GTTAGCTACGGGACAAGGGTTT(SEQ ID No:71)；
    glpK-U3 ATTCTTTACGGGACAACCATAA(SEQ ID No:72)；
    glpK-U4 ACGTCTAACGGGACAAAGTTCC(SEQ ID No:73)；
    glpK-U5 ACGGTTTACGGGACAACGCGG(SEQ ID No:74)；
    glpK-U6 ACCGTCTACGGGACAACGCTGT(SEQ ID No:75)；
    glpK-U7 CCGGTCTACGGGACAACGCTGT(SEQ ID No:76)。
  8. 权利要求1所述的基因工程菌在发酵甘油联产3-羟基丙酸和1,3-丙二醇中的应用。
  9. 权利要求2所述的基因工程菌在发酵甘油联产3-羟基丙酸和1,3-丙二醇中的应用。
  10. 权利要求3所述的基因工程菌在发酵甘油联产3-羟基丙酸和1,3-丙二醇中的应用。
  11. 根据权利要求8、9或10任一项所述的应用，其特征在于，包括：
    (1)将基因工程菌在LB培养基中过夜活化，得到基因工程菌的一级种子液；
    (2)将基因工程菌的一级种子液接种至改良M9-CSL培养基中培养，得到基因工程菌的二级种子液；
    (3)将基因工程菌的二级种子液接种于改良M9-CSL培养基中，将发酵过程划分为生长阶段和生产阶段控制pH值进行补料发酵；
    所述生长阶段时pH控制为7.0，调整温度、通气量、搅拌速率发酵培养至OD600达到4时，加入IPTG和维生素B12继续培养；
    所述生产阶段时pH控制为8.0，调整温度、通气量、溶氧值进行发酵，然后每6h进行补料发酵联产3-羟基丙酸和1,3-丙二醇；
    所述补料的成分含有甘油和玉米浆。
  12. 根据权利要求11所述的应用，其特征在于，步骤(2)中，所述改良M9-CSL培养基的成分为MgSO 4·7H 2O 0.5g/L,NH 4Cl 2.0g/L,NaCl 2.0g/L,玉米浆2.5mL/L,甘油40g/L和0.1M磷酸钾缓冲液,pH 7.0；
    所述基因工程菌的一级种子液的接种量为1％v/v，培养条件为37℃、220rpm培养12h。
  13. 根据权利要求11所述的应用，其特征在于，步骤(3)中，二级种子液的接种量为5％v/v；
    所述生长阶段时，调整温度为37℃，初始通气量为2vvm，搅拌速率为500rpm进行发酵培养；
    所述生产阶段时调整温通气量调整为3vvm，搅拌速率为200-800rpm，控制溶氧值为10％进行发酵；
    所述补料的成分含有800g/L甘油和50mL/L玉米浆；
    所述补料过程为：每6h进行补料控制甘油浓度维持在40g/L。
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