WO2022174597A1 - 一种用于l-肌氨酸生产的基因工程菌及构建方法与应用 - Google Patents

一种用于l-肌氨酸生产的基因工程菌及构建方法与应用 Download PDF

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WO2022174597A1
WO2022174597A1 PCT/CN2021/123534 CN2021123534W WO2022174597A1 WO 2022174597 A1 WO2022174597 A1 WO 2022174597A1 CN 2021123534 W CN2021123534 W CN 2021123534W WO 2022174597 A1 WO2022174597 A1 WO 2022174597A1
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gene
escherichia coli
seq
sarcosine
site
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French (fr)
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范晓光
周宇航
曹华杰
谢沛
杨军
田俊宇
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天津科技大学
新泰市佳禾生物科技有限公司
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Definitions

  • the invention belongs to the technical field of genetic engineering, in particular to a genetically engineered bacterium for L-sarcosine production and a construction method and application.
  • L-sarcosine (N-methyl-L-glycine) is an N-methylated amino acid, and its sodium salt, sarcosinate, is often used as a precursor for the synthesis of some important products.
  • L-sarcosine is an important amino acid product. Sarcosine has a considerable effect on the damage and repair of the brain and muscles. At the same time, L-sarcosine can provide the body with quick-acting energy, thereby relieving the body's fatigue. .
  • Sodium sarcosinate and its downstream products are basically low in toxicity, and are easily degraded in nature and are environmentally friendly. Therefore, L-sarcosine has broad application prospects in the fields of aquaculture, food, health care and medicine.
  • the existing L-sarcosine production is mainly produced by acidification of sodium sarcosinate, and the preparation route of sodium sarcosinate is mainly chemical synthesis method (CN201310676025.9, CN200610155348.3), and there are harsh reaction conditions and product separation difficulties. Shortcomings.
  • Imine reductase DpkA is an enzyme that can actively reduce imine bonds in cyclic and aliphatic compounds, and can reduce piperidine-2-carboxylic acid to L-piperidine acid. Requires NADPH to provide reducing power. Studies have shown that imine reductase DpkA can also catalyze the synthesis of L-sarcosine from glyoxylate and methylamine. Melanie Mindt et al.
  • the recombinant bacterium can synthesize 8.7g/L L-sarcosine with xylose and acetic acid as carbon sources through 126h stream-adding methylamine fermentation.
  • Escherichia coli Compared with Corynebacterium glutamicum, Escherichia coli has the advantages of simple culture conditions, short production cycle and low fermentation cost, and has better industrial application value.
  • this patent uses a novel and efficient imine reductase, which is derived from Brevibacterium linens ATCC9172 and has a higher activity for catalyzing L-sarcosine synthesis.
  • a new metabolic engineering strategy was used to construct a genetically engineered Escherichia coli strain with glucose as the carbon source and high production of L-sarcosine.
  • the object of the present invention is to overcome the deficiencies of the prior art, and to provide a genetically engineered bacterium for L-sarcosine production and a construction method and application.
  • a novel and efficient imine reductase is derived from Brevibacterium linens ATCC9172, and the nucleotide sequence of its encoding gene dpkA is SEQ ID NO.1.
  • a plasmid-free genetically engineered bacterium that efficiently synthesizes L-sarcosine with a cheap carbon source as a substrate the genetically engineered bacterium is Escherichia coli Escherichia coli SAR, and the Escherichia coli SAR is Escherichia coli Escherichia coli ATCC27325
  • the host was obtained by the following transformations: integrating a single copy of the imine reductase gene dpkA on its genome, which is controlled by the T7 promoter; a single copy of the citrate synthase gene gltA, which is controlled by the trc promoter; knockout B Aldehyde cycle inhibitor gene iclR; knockout malate synthase gene aceB; single-copy isocitrate lyase gene aceA, which is controlled by the trc promoter; single-copy membrane-bound transhydrogenase gene pntAB, which is controlled by
  • the imine reductase gene dpkA is derived from Brevibacterium linens ATCC 9172, and its nucleotide sequence is SEQ ID NO.1.
  • citrate synthase gene gltA is derived from Escherichia coli ATCC 27325, and its nucleotide sequence is SEQ ID NO.2.
  • the isocitrate lyase gene aceA is derived from Escherichia coli Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.3.
  • the membrane-bound transhydrogenase gene pntAB is derived from Escherichia coli Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.4.
  • the phosphoenolpyruvate carboxylase gene ppc is derived from Escherichia coli Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.5.
  • the above-mentioned construction method of genetically engineered bacteria without plasmids and using cheap carbon sources as substrates to efficiently synthesize L-sarcosine includes the following steps:
  • gene knockout was performed at the aceB site on the genome of Escherichia coli ATCC27325;
  • the construction order of steps to is in no particular order, and can be adjusted according to requirements.
  • Fermentation culture insert the seed liquid of genetically engineered bacteria into fresh fermentation medium according to 15-20% inoculation amount.
  • the fermentation medium is composed of: glucose 15-25g/L, tryptone 1-5g/L, sodium citrate 3-5g/L, KH 2 PO 4 1-5g/L, MgSO 4 7H 2 O 0.1-1g/ L, the remainder is water, pH 7.0-7.2.
  • the present invention uses a novel high-efficiency imine reductase, which is derived from Brevibacterium linens ATCC9172 and has high activity for catalyzing L-sarcosine synthesis.
  • Escherichia coli has no L-sarcosine anabolic pathway.
  • the present invention integrates the imine reductase gene derived from Brevibacterium linens ATCC9172 into the genome of wild-type Escherichia coli, so that carbon metabolism flows to the synthesis of L-sarcosine.
  • the present invention uses the T7 promoter to regulate the expression of the imine reductase gene to achieve the effect of enhancing gene expression.
  • the present invention knocks out the glyoxylate cycle inhibitory gene, the malate synthase gene and the 2-keto acid reductase gene, and introduces The endogenous citrate synthase and isocitrate lyase genes in Escherichia coli greatly increased the metabolic flux of pyruvate to the precursor glyoxylate.
  • the present invention introduces the endogenous membrane-bound transhydrogenase gene of Escherichia coli, so that more intracellular NADH can be converted into NADPH, providing more conditions for L-sarcosine synthesis. required restoring force.
  • the present invention realizes the construction of Escherichia coli genetic engineering bacteria for the first time at home and abroad to synthesize L-sarcosine with glucose and methylamine as the main raw materials, and the yield of L-sarcosine after fermentation in a 5L fermentor for 30 hours It can reach 10g/L, which is the highest value reported so far, and has good industrial application prospects.
  • the present invention provides a genetically engineered bacterium that is free of plasmids and uses cheap carbon sources such as glucose as substrates to efficiently synthesize L-sarcosine.
  • Escherichia coli is used as a host, and a single copy of imine reductase is integrated into its genome.
  • Fig. 1 is the electrophoresis diagram of mbhA::PT7- dpkA integration in the present invention; wherein, M—1kb Maker; 1—upstream homology arm; 2—target gene; 3—downstream homology arm; 4—overlapping fragment; 5— Original bacteria PCR fragment; 6—target bacteria PCR fragment;
  • Fig. 2 is the electrophoretogram of ylbE::P trc -gltA integration in the present invention; wherein, M—1kb Maker; 1—upstream homology arm; 2—target gene; 3—downstream homology arm; 4—overlapping fragment; 5— Original bacteria PCR fragment; 6—target bacteria PCR fragment;
  • Fig. 3 is the electrophoresis diagram of iclR knockout in the present invention.
  • M 1kb Maker
  • 1 upstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 target bacteria PCR fragment
  • Fig. 4 is the electrophoresis diagram of aceB knockout in the present invention.
  • M 1kb Maker
  • 1 upstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 target bacteria PCR fragment
  • Fig. 5 is yeeP::P trc -aceA integration electropherogram in the present invention; wherein, M—1kb Maker; 1—upstream homology arm; 2—target gene; 3—downstream homology arm; 4—overlapping fragment; 5— Original bacteria PCR fragment; 6—target bacteria PCR fragment;
  • Fig. 6 is the electrophoretogram of yghE::P trc -pntAB integration in the present invention; wherein, M—1kb Maker; 1—upstream homology arm; 2—target gene; 3—downstream homology arm; 4—overlapping fragment; 5— Original bacteria PCR fragment; 6—target bacteria PCR fragment;
  • Fig. 7 is the electrophoresis diagram of ycdW knockout in the present invention.
  • M 1kb Maker
  • 1 upstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 target bacteria PCR fragment
  • Fig. 8 is the yeeL::P trc -ppc integration electropherogram in the present invention; wherein, M—1kb Maker; 1—upstream homology arm; 2—target gene; 3—downstream homology arm; 4—overlapping fragment; 5— Original bacteria PCR fragment; 6—target bacteria PCR fragment;
  • Fig. 9 is the electrophoresis diagram of pykF knockout in the present invention.
  • M 1kb Maker
  • 1 upstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria PCR fragment
  • 5 target bacteria PCR fragment
  • Fig. 10 is the fermentation process diagram of Example 2 in the present invention.
  • Figure 11 The L-sarcosine anabolic pathway constructed in Escherichia coli in the present invention.
  • the raw materials used in the present invention are conventional commercial products
  • the methods used in the present invention are conventional methods in the field
  • the quality of each substance used in the present invention is conventionally used quality.
  • a novel and efficient imine reductase is derived from Brevibacterium linens ATCC9172, and the nucleotide sequence of its encoding gene dpkA is SEQ ID NO.1.
  • a plasmid-free genetically engineered bacterium that efficiently synthesizes L-sarcosine with a cheap carbon source as a substrate the genetically engineered bacterium is Escherichia coli Escherichia coli SAR, and the Escherichia coli SAR is Escherichia coli Escherichia coli ATCC27325
  • the host was obtained by the following transformations: integrating a single copy of the imine reductase gene dpkA on its genome, which is controlled by the T7 promoter; a single copy of the citrate synthase gene gltA, which is controlled by the trc promoter; knockout B Aldehyde cycle inhibitor gene iclR; knockout malate synthase gene aceB; single-copy isocitrate lyase gene aceA, which is controlled by the trc promoter; single-copy membrane-bound transhydrogenase gene pntAB, which is controlled by
  • the imine reductase gene dpkA is derived from Brevibacterium linens ATCC 9172, and its nucleotide sequence is SEQ ID NO.1.
  • the citrate synthase gene gltA is derived from Escherichia coli Escherichia coli ATCC 27325, and its nucleotide sequence is SEQ ID NO.2.
  • the isocitrate lyase gene aceA is derived from Escherichia coli Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.3.
  • the membrane-bound transhydrogenase gene pntAB is derived from Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.4.
  • the phosphoenolpyruvate carboxylase gene ppc is derived from Escherichia coli Escherichia coli ATCC27325, and its nucleotide sequence is SEQ ID NO.5.
  • the above-mentioned construction method of genetically engineered bacteria without plasmids and using cheap carbon sources as substrates to efficiently synthesize L-sarcosine includes the following steps:
  • gene knockout was performed at the aceB site on the genome of Escherichia coli ATCC27325;
  • Fermentation culture insert the seed liquid of genetically engineered bacteria into fresh fermentation medium according to 15-20% inoculation amount.
  • the fermentation medium is composed of: glucose 15-25g/L, tryptone 1-5g/L, sodium citrate 3-5g/L, KH 2 PO 4 1-5g/L, MgSO 4 7H 2 O 0.1-1g/ L, the remainder is water, pH 7.0-7.2.
  • Example 1 Construction of genetically engineered strain Escherichia coli SAR:
  • the gene editing method mediated by CRISPR/Cas9 is carried out with reference to the literature (Metabolic Engineering, 2015, 31:13-21.), and the two plasmids used in this method are pGRB and pREDCas9 respectively.
  • pREDCas9 carries gRNA plasmid elimination system, ⁇ phage Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg/L), cultured at 32°C; pGRB plasmid, with pUC18 as backbone, including promoter J23100 , gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg/L), cultured at 37°C.
  • the upstream homology arm primers UP-mbhA-S (SEQ ID NO.6), UP-mbhA-A (SEQ ID NO.7) and downstream primers were designed according to the upstream and downstream sequences of its mbhA gene Homologous arm primers DN-mbhA-S (SEQ ID NO.8), DN-mbhA-A (SEQ ID NO.9), and PCR amplification of the upper and lower homology arm fragments; according to dpkA gene design primer dpkA- S (SEQ ID NO. 10), dpkA-A (SEQ ID NO.
  • the promoter PT7 was designed in the downstream primer of the upstream homology arm and the upstream primer of the dpkA gene.
  • the above-mentioned fragment obtains the integrated fragment of dpkA gene by the method of overlapping PCR (mbhA gene upstream homology arm-P T7 -dpkA-mbhA-gene downstream homology arm), constructs the DNA fragment containing the target sequence that pGRB-mbhA uses by primer gRNA - mbhA-S (SEQ ID NO. 12) and gRNA-mbhA-A (SEQ ID NO.
  • the electropherogram of the construction of the integrated fragment and the PCR verification of the positive strain is shown in Figure 1.
  • the length of the upstream homology arm is 682 bp
  • the length of the dpkA gene fragment is 1006 bp
  • the length of the downstream homology arm is 720 bp
  • the length of the overlapping fragment is 2457 bp.
  • the length of the fragment amplified from the original bacteria should be 1837bp.
  • the promoter P trc was designed in the downstream primer of the upstream homology arm of ylbE gene and the upstream primer of gltA gene.
  • the above-mentioned fragment obtains the integrated fragment of the gltA gene (the upstream homology arm of the ylbE gene-Ptrc-gltA-ylbE gene downstream homology arm) by the method of overlapping PCR, and the DNA fragment containing the target sequence used to construct pGRB-ylbE is passed through the primer gRNA- ylbE-S (SEQ ID NO. 20) and gRNA-ylbE-A (SEQ ID NO. 21) were obtained by annealing, and the recombinant pGRB-ylbE was obtained after recombination with the linearized pGRB vector.
  • the integrated fragment and pGRB-ylbE were electroporated into E.coli SAR1 competent cells containing pREDCas9 vector, and the cultured cells were plated on LB plates containing ampicillin and spectinomycin overnight at 32°C. After culturing, the positive recombinants were verified by PCR, and the pGRB-ylbE and pREDCas9 used for gene editing were eliminated, and finally the strain E.coli SAR2 was obtained.
  • the electropherogram of the construction of the integrated fragment of P trc -gltA and the PCR verification of the positive strain is shown in Fig. 2 .
  • the length of the upstream homology arm should be 601bp
  • the length of the gltA gene fragment should be 1407bp
  • the length of the downstream homology arm should be 547bp
  • the total length of the integrated fragment should be 2474bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 2474bp
  • the length of the PCR amplification fragment of the original bacteria should be 2184bp.
  • design upstream homology arm primers UP-iclR-S (SEQ ID NO.22), UP-iclR-A (SEQ ID NO.23) and downstream according to the upstream and downstream sequences of its iclR gene
  • Homologous arm primers DN-iclR-S (SEQ ID NO.24), DN-iclR-A (SEQ ID NO.25), and amplify the upstream and downstream homology arms of the iclR gene; the above-mentioned fragments pass the method of overlapping PCR
  • iclR gene knockout fragment (iclR gene upstream homology arm-iclR gene downstream homology arm), construct the DNA fragment containing the target sequence used by pGRB-iclR by primer gRNA-iclR-S (SEQ ID NO.26) and The annealing of gRNA-iclR-A (SEQ ID NO.
  • the electropherogram of the construction of the iclR gene knockout fragment and the PCR verification of the positive strain is shown in Figure 3.
  • the length of the upstream homology arm should be 595bp
  • the length of the downstream homology arm should be 532bp
  • the total length of the gene knockout fragment should be 1086bp.
  • the length of PCR amplification fragment of positive bacteria should be 1086bp
  • the length of PCR amplification fragment of original bacteria should be 1745bp.
  • the upstream homology arm primers UP-aceB-S (SEQ ID NO.28), UP-aceB-A (SEQ ID NO.29) and downstream primers were designed according to the upstream and downstream sequences of its aceB gene Homologous arm primers DN-aceB-S (SEQ ID NO.30), DN-aceB-A (SEQ ID NO.31), and amplify the upstream and downstream homology arms of the aceB gene; the above-mentioned fragments are by the method of overlapping PCR
  • aceB gene knockout fragment aceB gene upstream homology arm-aceB gene downstream homology arm
  • the integrated fragment and pGRB-aceB were electro-transformed into E.coli SAR3 competent cells containing pREDCas9 vector, and the cells recovered and cultured after electro-transformation were spread on LB plates containing ampicillin and spectinomycin, overnight at 32°C After culturing, the positive recombinants were verified by PCR, and then pGRB-aceB and pREDCas9 for gene editing were eliminated, and finally the strain E.coli SAR4 was obtained.
  • the electropherogram of the construction of the aceB knockout fragment and the PCR verification of the positive strain is shown in Figure 4.
  • the length of the upstream homology arm should be 538bp
  • the length of the downstream homology arm should be 586bp
  • the total length of the gene knockout fragment should be 1082bp.
  • the length of PCR amplification fragment of positive bacteria should be 1082bp
  • the length of PCR amplification fragment of original bacteria should be 2397bp.
  • the promoter P trc was designed in the downstream primer of the upstream homology arm of yeeP gene and the upstream primer of aceA gene.
  • the above-mentioned fragment obtains the integrated fragment of the aceA gene (the upstream homology arm of the yeeP gene-Ptrc- aceA -yeeP gene downstream homology arm) by the method of overlapping PCR, and the DNA fragment containing the target sequence used by the construction pGRB-yeeP is passed through the primer gRNA- yeeP-S (SEQ ID NO. 40) and gRNA-yeeP-A (SEQ ID NO.
  • the electropherogram of the construction of the integrated fragment of P trc -aceA and the PCR verification of the positive strain is shown in Fig. 5 .
  • the length of the upstream homology arm should be 568bp
  • the length of the aceA gene fragment should be 1428bp
  • the length of the downstream homology arm should be 576bp
  • the total length of the integrated fragment should be 2491bp.
  • the length of the PCR amplification fragment of the positive bacteria should be 2491bp
  • the length of the PCR amplification fragment of the original bacteria should be 1396bp.
  • the upstream homology arm primers UP-yghE-S (SEQ ID NO.42), UP-yghE-A (SEQ ID NO.43) and downstream primers were designed according to the upstream and downstream sequences of its yghE gene Homologous arm primers DN-yghE-S (SEQ ID NO.44), DN-yghE-A (SEQ ID NO.45), and amplify the upper and lower homology arms of yghE gene; according to pntAB gene design primer pntAB- S (SEQ ID NO. 46), pntAB-A (SEQ ID NO.
  • the promoter P trc was designed in the downstream primer of the upstream homology arm of yghE gene and the upstream primer of pntAB gene.
  • the above-mentioned fragment obtains the integrated fragment of the pntAB gene (the upstream homology arm of the yghE gene-P trc -pntAB-yghE gene downstream homology arm) by the method of overlapping PCR, and the DNA fragment containing the target sequence used in the construction of pGRB-yghE is passed through the primer gRNA- yghE-S (SEQ ID NO.
  • gRNA-yghE-A SEQ ID NO. 49
  • the integrated fragment and pGRB-yghE were electro-transformed into E.coli SAR5 competent cells containing pREDCas9 vector, and the cells recovered after electro-transformation were spread on LB plates containing ampicillin and spectinomycin, overnight at 32°C After culturing, the positive recombinants were verified by PCR, and then pGRB-yghE and pREDCas9 for gene editing were eliminated, and finally the strain E.coli SAR6 was obtained.
  • the electropherogram of the construction of the integrated fragment of P trc -pntAB and the PCR verification of the positive strain is shown in Fig. 6 .
  • the length of the upstream homology arm should be 559bp
  • the length of the pntAB gene fragment should be 3050bp
  • the length of the downstream homology arm should be 549bp
  • the total length of the integrated fragment should be 4087bp.
  • the length of PCR amplification fragment of positive bacteria should be 4087bp
  • the length of PCR amplification fragment of original bacteria should be 1547bp.
  • the upstream homology arm primers UP-ycdW-S (SEQ ID NO.50), UP-ycdW-A (SEQ ID NO.51) and downstream primers were designed according to the upstream and downstream sequences of its ycdW gene Homologous arm primers DN-ycdW-S (SEQ ID NO.52), DN-ycdW-A (SEQ ID NO.53), and amplify the upstream and downstream homology arms of the ycdW gene; the above-mentioned fragments pass the method of overlapping PCR Obtain the ycdW gene knockout fragment (ycdW gene upstream homology arm-ycdW gene downstream homology arm), and construct the DNA fragment containing the target sequence used in pGRB-ycdW by primer gRNA-ycdW-S (SEQ ID NO.54) and The annealing of gRNA-ycdW-A
  • the recombinant pGRB-ycdW was obtained after recombination with the linearized pGRB vector.
  • the integrated fragment and pGRB-ycdW were electro-transformed into E.coli SAR6 competent cells containing pREDCas9 vector, and the cells recovered and cultured after electro-transformation were spread on LB plates containing ampicillin and spectinomycin, overnight at 32°C After culturing, the positive recombinants were verified by PCR, and then pGRB-ycdW and pREDCas9 for gene editing were eliminated, and finally the strain E.coli SAR7 was obtained.
  • the electropherogram of the construction of the ycdW gene knockout fragment and the PCR verification of the positive strain is shown in Figure 7.
  • the length of the upstream homology arm should be 642bp
  • the length of the downstream homology arm should be 1428bp
  • the total length of the gene knockout fragment should be 2024bp.
  • the length of PCR amplification fragment of positive bacteria should be 2024bp
  • the length of PCR amplification fragment of original bacteria should be 2604bp.
  • the upstream homology arm primers UP-yeeL-S (SEQ ID NO.56), UP-yeeL-A (SEQ ID NO.57) and downstream primers were designed according to the upstream and downstream sequences of its yeeL gene Homologous arm primers DN-yeeL-S (SEQ ID NO.58), DN-yeeL-A (SEQ ID NO.59), and amplify the upstream and downstream homology arms of yeeL gene; design primers ppc- S (SEQ ID NO. 60), ppc-A (SEQ ID NO. 61), and amplified ppc gene fragment (SEQ ID NO. 5).
  • the promoter P trc was designed in the downstream primer of the upstream homology arm of yeeL gene and the upstream primer of ppc gene.
  • the above-mentioned fragment obtains the integrated fragment of the ppc gene (the upstream homology arm of the yeeL gene-P trc -ppc-yeeL gene downstream homology arm) by the method of overlapping PCR, and the DNA fragment containing the target sequence used to construct the pGRB-yeeL is passed through the primer gRNA- yeeL-S (SEQ ID NO. 62) and gRNA-yeeL-A (SEQ ID NO.
  • the electropherogram of the construction of the integrated fragment of P trc -ppc and the PCR verification of the positive strain is shown in Fig. 8 .
  • the length of the upstream homology arm should be 533bp
  • the length of the ppc gene fragment should be 2770bp
  • the length of the downstream homology arm should be 581bp
  • the total length of the integrated fragment should be 3813bp.
  • the length of PCR amplification fragment of positive bacteria should be 3813bp
  • the length of PCR amplification fragment of original bacteria should be 1613bp.
  • the upstream homology arm primers UP-pykF-S (SEQ ID NO.64), UP-pykF-A (SEQ ID NO.65) and downstream primers were designed according to the upstream and downstream sequences of its pykF gene Homologous arm primers DN-pykF-S (SEQ ID NO.66), DN-pykF-A (SEQ ID NO.67), and amplify the upstream and downstream homology arms of the pykF gene; the above-mentioned fragments pass the method of overlapping PCR Obtain the pykF gene knockout fragment (pykF gene upstream homology arm-pykF gene downstream homology arm), and construct the DNA fragment containing the target sequence used in pGRB-pykF by primer gRNA-pykF-S (SEQ ID NO.68) and The annealing of gRNA-pykF-A
  • the recombinant pGRB-pykF was obtained after recombination with the linearized pGRB vector.
  • the integrated fragment and pGRB-pykF were electro-transformed into E.coli SAR8 competent cells containing pREDCas9 vector, and the cells recovered and cultured after electro-transformation were spread on LB plates containing ampicillin and spectinomycin, overnight at 32°C After culturing, the positive recombinants were verified by PCR, and then pGRB-pykF and pREDCas9 for gene editing were eliminated, and finally the strain E.coli SAR was obtained.
  • the electropherogram of the construction of the pykF gene knockout fragment and the PCR verification of the positive strain is shown in Figure 9.
  • the length of the upstream homology arm should be 471bp
  • the length of the downstream homology arm should be 429bp
  • the total length of the gene knockout fragment should be 856bp.
  • the length of PCR amplification fragment of positive bacteria should be 856bp
  • the length of PCR amplification fragment of original bacteria should be 2180bp.
  • Slope activation culture scrape a ring of bacteria from the -80 °C refrigerator storage tube, spread evenly on the activated slant, cultivate at 37 °C for 12 hours, and continue to cultivate in eggplant-shaped flask for 12 hours;
  • Seed culture Take an appropriate amount of sterile water in an eggplant-shaped bottle, insert the bacterial suspension into the seed medium, the pH is stable at about 7.0, the temperature is constant at 37°C, and the dissolved oxygen is between 25-35%, and cultivate for 6 hours;
  • the slant medium is composed of: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl 2.5g/L, agar 25g/L, the rest is water, pH 7.0;
  • the seed medium was composed of: glucose 25/L, yeast extract 5g/L, tryptone 5g/L, KH 2 PO 4 5g/L, MgSO 4 ⁇ 7H 2 O 2g/L, the rest was water, pH 7.0.
  • the fermentation medium is composed of: glucose 20g/L, yeast extract 4g/L, tryptone 5g/L, sodium citrate 5g/L, KH 2 PO 4 2g/L, MgSO 4 7H 2 O 1g/L, the rest as water, pH 7.0.
  • the yield of L-sarcosine can reach 10g/L after 30h fermentation in a 5L fermenter.
  • the fermentation process curve is shown in Figure 10.

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Abstract

提供了一种用于L-肌氨酸生产的基因工程菌及构建方法与应用，该基因工程菌是以大肠杆菌为宿主，通过在其基因组上整合单拷贝亚胺还原酶基因dpkA；单拷贝柠檬酸合酶基因gltA；敲除乙醛酸循环抑制基因iclR；敲除苹果酸合酶基因aceB；单拷贝异柠檬酸裂解酶基因aceA；单拷贝膜结合转氢酶基因pntAB；敲除2-酮酸还原酶基因ycdW；单拷贝磷酸烯醇丙酮酸羧化酶基因ppc；敲除丙酮酸激酶基因pykF获得。通过系统代谢改造后，该基因工程菌能够以葡萄糖和甲胺为主要原料合成L-肌氨酸。在5L发酵罐发酵30h后L-肌氨酸的产量可达10g/L。

Description

一种用于L-肌氨酸生产的基因工程菌及构建方法与应用 技术领域
本发明属于基因工程技术领域，尤其是一种用于L-肌氨酸生产的基因工程菌及构建方法与应用。
背景技术
L-肌氨酸(N-甲基-L-甘氨酸)是一种N-甲基化氨基酸，其钠盐肌氨酸钠经常被用作一些重要的产品合成前体。L-肌氨酸是一种重要的氨基酸产品，肌氨酸对大脑以及肌肉的损伤修复具有比较可观的作用，同时L-肌氨酸可以为机体提供速效能量，从而起到缓解机体疲劳的功效。肌氨酸钠及其下游产品基本上毒性很低，且在自然界中都易降解，对环境较友好。因此L-肌氨酸在养殖、食品、保健以及医学等领域存在广泛的应用前景。
现有的L-肌氨酸生产主要是由肌氨酸钠酸化而来的，肌氨酸钠制备途径主要为化学合成法(CN201310676025.9、CN200610155348.3)，存在反应条件苛刻和产物分离困难的缺点。
亚胺还原酶DpkA(imine reductases，IRED)是一种能够在环状和脂肪族化合物中主动还原亚胺键的酶，可以将哌啶-2-羧酸还原为L-哌啶酸，该反应需要NADPH提供还原力。研究表明亚胺还原酶DpkA也能催化乙醛酸和甲胺合成L-肌氨酸，Melanie Mindt等人在谷氨酸棒杆菌表达了Pseudomonas putida ATCC12633来源的亚胺还原酶编码基因dpkA(Bioresource Technology,2019:281,135-142)，该重组菌能够以木糖和乙酸为碳源，经126h流加甲胺发酵合成8.7g/L的L-肌氨酸。
与谷氨酸棒杆菌相比，大肠杆菌具有培养条件简单、生产周期短以及发酵成本低廉等优势，具有更加良好的工业应用价值。为了获得更加高产的L-肌氨酸发酵生产菌株，本专利使用了一种新型高效的亚胺还原酶，该酶来源于Brevibacterium linens ATCC9172，具有较高的催化L-肌氨酸合成的活力。同时采用新的代谢工程改造策略，构建出一株以葡萄糖为碳源，高产L-肌氨酸的大肠杆菌基因工程菌。
通过检测，尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明的目的在于克服现有技术的不足之处，提供一种用于L-肌氨酸生产的基因工程菌及构建方法与应用。
本发明解决其技术问题所采用的技术方案是：
一种新型高效的亚胺还原酶，所述亚胺还原酶来源于Brevibacterium linens ATCC9172，其编 码基因dpkA的核苷酸序列为SEQ ID NO.1。
一株无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌，所述基因工程菌为大肠杆菌Escherichia coli SAR，该大肠杆菌Escherichia coli SAR是以大肠杆菌Escherichia coli ATCC27325为宿主，通过以下改造获得：在其基因组上整合单拷贝的亚胺还原酶基因dpkA，该基因由T7启动子控制；单拷贝柠檬酸合酶基因gltA，该基因由trc启动子控制；敲除乙醛酸循环抑制基因iclR；敲除苹果酸合酶基因aceB；单拷贝异柠檬酸裂解酶基因aceA，该基因由trc启动子控制；单拷贝膜结合转氢酶基因pntAB，该基因由trc启动子控制；敲除2-酮酸还原酶基因ycdW；单拷贝磷酸烯醇丙酮酸羧化酶基因ppc，该基因由trc启动子控制；敲除丙酮酸激酶基因pykF。
而且，所述亚胺还原酶基因dpkA来源于Brevibacterium linens ATCC 9172，其核苷酸序列为SEQ ID NO.1。
而且，所述柠檬酸合酶基因gltA来源于大肠杆菌Escherichia coli ATCC 27325，其核苷酸序列为SEQ ID NO.2。
而且，所述异柠檬酸裂解酶基因aceA来源于大肠杆菌Escherichia coli ATCC27325，其核苷酸序列为SEQ ID NO.3。
而且，所述膜结合转氢酶基因pntAB来源于大肠杆菌Escherichia coli ATCC27325，其核苷酸序列为SEQ ID NO.4。
而且，所述磷酸烯醇丙酮酸羧化酶基因ppc来源于大肠杆菌Escherichia coli ATCC27325，其核苷酸序列为SEQ ID NO.5。
如上所述的无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌的构建方法，所述方法采用CRISPR/Cas9介导的基因编辑技术对大肠杆菌进行定向改造，具体包括如下步骤：
为了引入L-肌氨酸的合成代谢，在大肠杆菌Escherichia coli ATCC27325基因组上的mbhA位点单拷贝来源于Brevibacterium linens ATCC 9172的亚胺还原酶基因dpkA，其序列为SEQ ID NO.1，该基因经过密码子优化并且由T7启动子控制；
为了增强草酰乙酸到柠檬酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上ylbE位点单拷贝内源的柠檬酸合酶基因gltA，其序列为SEQ ID NO.2，该基因由trc启动子控制；
为了菌株在正常培养条件下开启乙醛酸循环，对大肠杆菌Escherichia coli ATCC27325基因组上iclR位点进行基因敲除；
为了阻断乙醛酸到苹果酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上aceB 位点进行基因敲除；
为了增强异柠檬酸到乙醛酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeP位点单拷贝内源的异柠檬酸裂解酶基因aceA，其序列为SEQ ID NO.3，该基因由trc启动子控制；
为了增强NADH到NADPH的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yghE位点单拷贝内源的膜结合转氢酶基因pntAB，其序列为SEQ ID NO.4，该基因由trc启动子控制；
为了阻断乙醛酸到乙醇酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上ycdW位点进行基因敲除；
为了增强磷酸烯醇丙酮酸到草酰乙酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeL位点单拷贝内源的磷酸烯醇丙酮酸羧化酶基因ppc，其序列为SEQ ID NO.5，该基因由trc启动子控制；
为了减少磷酸烯醇式丙酮酸到丙酮酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上pykF位点进行基因敲除；
其中，步骤至的构建顺序不分先后，根据需求进行调整即可。
如上所述的无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌在L-肌氨酸生产方面中的应用。
利用如上所述的基因工程菌发酵生产L-肌氨酸的方法，具体步骤如下：
发酵培养：将基因工程菌的种子液按照15-20％接种量接入新鲜的发酵培养基，发酵过程中控制pH稳定在6.8-7.2，温度维持在36.5-37.5℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加700-800g/L的葡萄糖溶液继续培养，并维持发酵培养基中的葡萄糖浓度＜3g/L，当OD 600＝40时，开始以20-25mL/h的流速流加1.5-1.6mol/L的甲胺盐酸盐溶液，流加量为75mL/L培养基，发酵周期28-32h，即得L-肌氨酸；
发酵培养基组成为：葡萄糖15-25g/L，胰蛋白胨1-5g/L，柠檬酸钠3-5g/L，KH 2PO 4 1-5g/L，MgSO 4·7H 2O 0.1-1g/L，其余为水，pH 7.0-7.2。
本发明的优点和积极效果如下：
1、本发明使用了一种新型高效的亚胺还原酶，该酶来源于Brevibacterium linens ATCC9172，具有较高的催化L-肌氨酸合成的活力。
2、大肠杆菌自身没有L-肌氨酸合成代谢途径。为了得到能够直接发酵生产L-肌氨酸的菌株，本发明将Brevibacterium linens ATCC9172来源的亚胺还原酶基因整合到野生型大肠杆菌的基因组上，使得碳代谢流向L-肌氨酸的合成。此外本发明使用T7启动子调控亚胺还 原酶基因的表达以达到增强基因表达的效果。
3、为了增加L-肌氨酸合成前体物乙醛酸的积累，本发明将乙醛酸循环抑制基因、苹果酸合酶基因以及2-酮酸还原酶基因进行了敲除，并引入了大肠杆菌内源的柠檬酸合酶基因和异柠檬酸裂解酶基因，大幅度提高了丙酮酸向前体物乙醛酸的代谢流量。
4、为了增加反应所需的NADPH的供给，本发明引入大肠杆菌内源的膜结合转氢酶基因，使得胞内更多的NADH能够转变为NADPH，为L-肌氨酸合成提供更多所需的还原力。
5、通过系统代谢改造策略，本发明在国内外首次实现了构建大肠杆菌基因工程菌以葡萄糖和甲胺为主要原料合成L-肌氨酸，5L发酵罐发酵30h后L-肌氨酸的产量可达10g/L，为目前报道的最高值，具有很好的工业应用前景。
6、本发明提供一株无质粒、以葡萄糖等廉价碳源为底物高效合成L-肌氨酸的基因工程菌，是以大肠杆菌为宿主，通过在其基因组上整合单拷贝亚胺还原酶基因dpkA；单拷贝柠檬酸合酶基因gltA；敲除乙醛酸循环抑制基因iclR；敲除苹果酸合酶基因aceB；单拷贝异柠檬酸裂解酶基因aceA；单拷贝膜结合转氢酶基因pntAB；敲除2-酮酸还原酶基因ycdW；单拷贝磷酸烯醇丙酮酸羧化酶基因ppc；敲除丙酮酸激酶基因pykF获得。通过系统代谢改造后，工程菌能够以葡萄糖和甲胺为主要原料合成L-肌氨酸。在5L发酵罐发酵30h后L-肌氨酸的产量可达10g/L。
附图说明
图1为本发明中mbhA::P T7-dpkA整合电泳图；其中，M—1kb Maker；1-上游同源臂；2—目的基因；3—下游同源臂；4—重叠片段；5—原菌PCR片段；6—目的菌PCR片段；
图2为本发明中ylbE::P trc-gltA整合电泳图；其中，M—1kb Maker；1—上游同源臂；2—目的基因；3—下游同源臂；4—重叠片段；5—原菌PCR片段；6—目的菌PCR片段；
图3为本发明中iclR敲除电泳图；其中，M—1kb Maker；1—上游同源臂；2—下游同源臂；3—重叠片段；4—原菌PCR片段；5—目的菌PCR片段；
图4为本发明中aceB敲除电泳图；其中，M—1kb Maker；1—上游同源臂；2—下游同源臂；3—重叠片段；4—原菌PCR片段；5—目的菌PCR片段；
图5为本发明中yeeP::P trc-aceA整合电泳图；其中，M—1kb Maker；1—上游同源臂；2—目的基因；3—下游同源臂；4—重叠片段；5—原菌PCR片段；6—目的菌PCR片段；
图6为本发明中yghE::P trc-pntAB整合电泳图；其中，M—1kb Maker；1—上游同源臂；2—目的基因；3—下游同源臂；4—重叠片段；5—原菌PCR片段；6—目的菌PCR片段；
图7为本发明中ycdW敲除电泳图；其中，M—1kb Maker；1—上游同源臂；2—下游同源臂；3—重叠片段；4—原菌PCR片段；5—目的菌PCR片段；
图8为本发明中yeeL::P trc-ppc整合电泳图；其中，M—1kb Maker；1—上游同源臂；2—目的基因；3—下游同源臂；4—重叠片段；5—原菌PCR片段；6—目的菌PCR片段；
图9为本发明中pykF敲除电泳图；其中，M—1kb Maker；1—上游同源臂；2—下游同源臂；3—重叠片段；4—原菌PCR片段；5—目的菌PCR片段；
图10为本发明中实施例2发酵过程图；
图11本发明中在大肠杆菌中构建的L-肌氨酸合成代谢途径。
具体实施方式
下面结合实施例，对本发明进一步说明，下属实施例是叙述性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
本发明中所使用的原料，如无特殊说明，均为常规市售产品，本发明中所使用的方法，如无特殊说明，均为本领域常规方法，本发明所用各物质质量均为常规使用质量。
一种新型高效的亚胺还原酶，所述亚胺还原酶来源于Brevibacterium linens ATCC9172，其编码基因dpkA的核苷酸序列为SEQ ID NO.1。
一株无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌，所述基因工程菌为大肠杆菌Escherichia coli SAR，该大肠杆菌Escherichia coli SAR是以大肠杆菌Escherichia coli ATCC27325为宿主，通过以下改造获得：在其基因组上整合单拷贝的亚胺还原酶基因dpkA，该基因由T7启动子控制；单拷贝柠檬酸合酶基因gltA，该基因由trc启动子控制；敲除乙醛酸循环抑制基因iclR；敲除苹果酸合酶基因aceB；单拷贝异柠檬酸裂解酶基因aceA，该基因由trc启动子控制；单拷贝膜结合转氢酶基因pntAB，该基因由trc启动子控制；敲除2-酮酸还原酶基因ycdW；单拷贝磷酸烯醇丙酮酸羧化酶基因ppc，该基因由trc启动子控制；敲除丙酮酸激酶基因pykF。
较优地，所述亚胺还原酶基因dpkA来源于Brevibacterium linens ATCC 9172，其核苷酸序列为SEQ ID NO.1。
较优地，所述柠檬酸合酶基因gltA来源于大肠杆菌Escherichia coli ATCC 27325，其核苷酸序列为SEQ ID NO.2。
较优地，所述异柠檬酸裂解酶基因aceA来源于大肠杆菌Escherichia coli ATCC27325，其核苷酸序列为SEQ ID NO.3。
较优地，所述膜结合转氢酶基因pntAB来源于大肠杆菌Escherichia coli  ATCC27325，其核苷酸序列为SEQ ID NO.4。
较优地，所述磷酸烯醇丙酮酸羧化酶基因ppc来源于大肠杆菌Escherichia coli ATCC27325，其核苷酸序列为SEQ ID NO.5。
如上所述的无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌的构建方法，所述方法采用CRISPR/Cas9介导的基因编辑技术对大肠杆菌进行定向改造，具体包括如下步骤：
为了引入L-肌氨酸的合成代谢，在大肠杆菌Escherichia coli ATCC27325基因组上的mbhA位点单拷贝来源于Brevibacterium linens ATCC 9172的亚胺还原酶基因dpkA，其序列为SEQ ID NO.1，该基因经过密码子优化并且由T7启动子控制；
为了增强草酰乙酸到柠檬酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上ylbE位点单拷贝内源的柠檬酸合酶基因gltA，其序列为SEQ ID NO.2，该基因由trc启动子控制；
为了菌株在正常培养条件下开启乙醛酸循环，对大肠杆菌Escherichia coli ATCC27325基因组上iclR位点进行基因敲除；
为了阻断乙醛酸到苹果酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上aceB位点进行基因敲除；
为了增强异柠檬酸到乙醛酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeP位点单拷贝内源的异柠檬酸裂解酶基因aceA，其序列为SEQ ID NO.3，该基因由trc启动子控制；
为了增强NADH到NADPH的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yghE位点单拷贝内源的膜结合转氢酶基因pntAB，其序列为SEQ ID NO.4，该基因由trc启动子控制；
为了阻断乙醛酸到乙醇酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上ycdW位点进行基因敲除；
为了增强磷酸烯醇丙酮酸到草酰乙酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeL位点单拷贝内源的磷酸烯醇丙酮酸羧化酶基因ppc，其序列为SEQ ID NO.5，该基因由trc启动子控制；
为了减少磷酸烯醇式丙酮酸到丙酮酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上pykF位点进行基因敲除；
其中，步骤至的构建顺序不分先后，根据需求进行调整即可。可以如图11所示。
如上所述的无质粒、以廉价碳源为底物高效合成L-肌氨酸的基因工程菌在L-肌氨酸 生产方面中的应用。
利用如上所述的基因工程菌发酵生产L-肌氨酸的方法，具体步骤如下：
发酵培养：将基因工程菌的种子液按照15-20％接种量接入新鲜的发酵培养基，发酵过程中控制pH稳定在6.8-7.2，温度维持在36.5-37.5℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加700-800g/L的葡萄糖溶液继续培养，并维持发酵培养基中的葡萄糖浓度＜3g/L，当OD 600＝40时，开始以20-25mL/h的流速流加1.5-1.6mol/L的甲胺盐酸盐溶液，流加量为75mL/L培养基，发酵周期28-32h，即得L-肌氨酸；
发酵培养基组成为：葡萄糖15-25g/L，胰蛋白胨1-5g/L，柠檬酸钠3-5g/L，KH 2PO 4 1-5g/L，MgSO 4·7H 2O 0.1-1g/L，其余为水，pH 7.0-7.2。
具体地，相关制备及检测实施例如下：
实施例1：基因工程菌株大肠杆菌Escherichia coli SAR的构建：
1基因编辑的方法
本发明中采用CRISPR/Cas9介导的基因编辑方法参照文献(Metabolic Engineering,2015,31:13-21.)进行，该方法所用的两个质粒分别为pGRB与pREDCas9。其中pREDCas9携带gRNA质粒消除系统，λ噬菌体的Red重组系统及Cas9蛋白表达系统，奇霉素抗性(工作浓度：100mg/L)，32℃培养；pGRB质粒，以pUC18为骨架，包括启动子J23100，gRNA-Cas9结合区域序列和终止子序列，氨苄青霉素抗性(工作浓度：100mg/L)，37℃培养。
2菌株构建的具体过程
2.1将P T7-dpkA(含有dpkA基因和T7启动子的片段)整合在假基因mbhA位点处
以E.coliATCC27325基因组为模板，根据其mbhA基因的上、下游序列设计上游同源臂引物UP-mbhA-S(SEQ ID NO.6)、UP-mbhA-A(SEQ ID NO.7)和下游同源臂引物DN-mbhA-S(SEQ ID NO.8)、DN-mbhA-A(SEQ ID NO.9)，并PCR扩增其上、下游同源臂片段；根据dpkA基因设计引物dpkA-S(SEQ ID NO.10)、dpkA-A(SEQ ID NO.11)，并扩增dpkA基因片段(SEQ ID NO.1)。启动子P T7则设计在上游同源臂的下游引物和dpkA基因的上游引物中。上述片段通过重叠PCR的方法获得dpkA基因的整合片段(mbhA基因上游同源臂-P T7-dpkA-mbhA-基因下游同源臂)，构建pGRB-mbhA使用的含靶序列的DNA片段通过引物gRNA-mbhA-S(SEQ ID NO.12)和gRNA-mbhA-A(SEQ ID NO.13)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-mbhA。将整合片段和pGRB-mbhA电转化至含有pREDCas9的E.coli ATCC27325感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上， 32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-mbhA，最终获得菌株E.coli SAR1。
P T7-dpkA片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图1。其中上游同源臂长度为682bp，dpkA基因片段长度为1006bp，下游同源臂长度为720bp，重叠片段的长度为2457bp，PCR验证重组子时，阳性重组子所扩增的片段长度应为2457bp，原菌扩增出来的片段长度应为1837bp。
2.2将P trc-gltA(含有gltA基因和trc启动子的片段)整合在假基因位点ylbE处以E.coliATCC27325基因组为模板，根据其ylbE基因的上、下游序列设计上游同源臂引物UP-ylbE-S(SEQ ID NO.14)、UP-ylbE-A(SEQ ID NO.15)和下游同源臂引物DN-ylbE-S(SEQ ID NO.16)、DN-ylbE-A(SEQ ID NO.17)，并扩增ylbE基因的上、下游同源臂；根据gltA基因设计引物gltA-S(SEQ ID NO.18)、gltA-A(SEQ ID NO.19)，并扩增gltA基因片段(SEQ ID NO.2)。启动子P trc则设计在ylbE基因上游同源臂的下游引物和gltA基因的上游引物中。上述片段通过重叠PCR的方法获得gltA基因的整合片段(ylbE基因上游同源臂-P trc-gltA-ylbE基因下游同源臂)，构建pGRB-ylbE使用的含靶序列的DNA片段通过引物gRNA-ylbE-S(SEQ ID NO.20)和gRNA-ylbE-A(SEQ ID NO.21)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-ylbE。将整合片段和pGRB-ylbE电转化至含有pREDCas9载体的E.coli SAR1感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-ylbE及pREDCas9，最终获得菌株E.coli SAR2。
P trc-gltA整合片段的构建和阳性菌株的PCR验证的电泳图见图2。其中，上游同源臂的长度应为601bp，gltA基因片段长度应为1407bp，下游同源臂的长度应为547bp，整合片段的总长应为2474bp。PCR验证时，阳性菌PCR扩增片段长度应为2474bp，原菌PCR扩增片段长度应为2184bp。
2.3将iclR基因敲除
以E.coliATCC27325基因组为模板，根据其iclR基因的上、下游序列设计上游同源臂引物UP-iclR-S(SEQ ID NO.22)、UP-iclR-A(SEQ ID NO.23)和下游同源臂引物DN-iclR-S(SEQ ID NO.24)、DN-iclR-A(SEQ ID NO.25)，并扩增iclR基因的上、下游同源臂；上述片段通过重叠PCR的方法获得iclR基因敲除的片段(iclR基因上游同源臂-iclR基因下游同源臂)，构建pGRB-iclR使用的含靶序列的DNA片段通过引物gRNA-iclR-S(SEQ ID NO.26)和gRNA-iclR-A(SEQ ID NO.27)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-iclR。将整合片段和pGRB-iclR电转化至含有pREDCas9载体的E.coli SAR2感受态细胞中，将电转化 后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-iclR及pREDCas9，最终获得菌株E.coli SAR3。
iclR基因敲除片段的构建和阳性菌株的PCR验证的电泳图见图3。其中，上游同源臂的长度应为595bp，下游同源臂的长度应为532bp，基因敲除片段的总长应为1086bp。PCR验证时，阳性菌PCR扩增片段长度应为1086bp，原菌PCR扩增片段长度应为1745bp。
2.4将aceB基因敲除
以E.coliATCC27325基因组为模板，根据其aceB基因的上、下游序列设计上游同源臂引物UP-aceB-S(SEQ ID NO.28)、UP-aceB-A(SEQ ID NO.29)和下游同源臂引物DN-aceB-S(SEQ ID NO.30)、DN-aceB-A(SEQ ID NO.31)，并扩增aceB基因的上、下游同源臂；上述片段通过重叠PCR的方法获得aceB基因敲除的片段(aceB基因上游同源臂-aceB基因下游同源臂)，构建pGRB-aceB使用的含靶序列的DNA片段通过引物gRNA-aceB-S(SEQ ID NO.32)和gRNA-aceB-A(SEQ ID NO.33)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-aceB。将整合片段和pGRB-aceB电转化至含有pREDCas9载体的E.coli SAR3感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-aceB及pREDCas9，最终获得菌株E.coli SAR4。
aceB基因敲除片段的构建和阳性菌株的PCR验证的电泳图见图4。其中，上游同源臂的长度应为538bp，下游同源臂的长度应为586bp，基因敲除片段的总长应为1082bp。PCR验证时，阳性菌PCR扩增片段长度应为1082bp，原菌PCR扩增片段长度应为2397bp。
2.5将P trc-aceA(含有aceA基因和trc启动子的片段)整合在假基因位点yeeP处以E.coliATCC27325基因组为模板，根据其yeeP基因的上、下游序列设计上游同源臂引物UP-yeeP-S(SEQ ID NO.34)、UP-yeeP-A(SEQ ID NO.35)和下游同源臂引物DN-yeeP-S(SEQ ID NO.36)、DN-yeeP-A(SEQ ID NO.37)，并扩增yeeP基因的上、下游同源臂；根据aceA基因设计引物aceA-S(SEQ ID NO.38)、aceA-A(SEQ ID NO.39)，并扩增aceA基因片段(SEQ ID NO.3)。启动子P trc则设计在yeeP基因上游同源臂的下游引物和aceA基因的上游引物中。上述片段通过重叠PCR的方法获得aceA基因的整合片段(yeeP基因上游同源臂-P trc-aceA-yeeP基因下游同源臂)，构建pGRB-yeeP使用的含靶序列的DNA片段通过引物gRNA-yeeP-S(SEQ ID NO.40)和gRNA-yeeP-A(SEQ ID NO.41)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-yeeP。将整合片段和pGRB-yeeP电转化至含有pREDCas9载体的E.coli  SAR4感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-yeeP及pREDCas9，最终获得菌株E.coli SAR5。
P trc-aceA整合片段的构建和阳性菌株的PCR验证的电泳图见图5。其中，上游同源臂的长度应为568bp，aceA基因片段长度应为1428bp，下游同源臂的长度应为576bp，整合片段的总长应为2491bp。PCR验证时，阳性菌PCR扩增片段长度应为2491bp，原菌PCR扩增片段长度应为1396bp。
2.6将P trc-pntAB(含有pntAB基因和trc启动子的片段)整合在假基因位点yghE处
以E.coliATCC27325基因组为模板，根据其yghE基因的上、下游序列设计上游同源臂引物UP-yghE-S(SEQ ID NO.42)、UP-yghE-A(SEQ ID NO.43)和下游同源臂引物DN-yghE-S(SEQ ID NO.44)、DN-yghE-A(SEQ ID NO.45)，并扩增yghE基因的上、下游同源臂；根据pntAB基因设计引物pntAB-S(SEQ ID NO.46)、pntAB-A(SEQ ID NO.47)，并扩增pntAB基因片段(SEQ ID NO.4)。启动子P trc则设计在yghE基因上游同源臂的下游引物和pntAB基因的上游引物中。上述片段通过重叠PCR的方法获得pntAB基因的整合片段(yghE基因上游同源臂-P trc-pntAB-yghE基因下游同源臂)，构建pGRB-yghE使用的含靶序列的DNA片段通过引物gRNA-yghE-S(SEQ ID NO.48)和gRNA-yghE-A(SEQ ID NO.49)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-yghE。将整合片段和pGRB-yghE电转化至含有pREDCas9载体的E.coli SAR5感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-yghE及pREDCas9，最终获得菌株E.coli SAR6。
P trc-pntAB整合片段的构建和阳性菌株的PCR验证的电泳图见图6。其中，上游同源臂的长度应为559bp，pntAB基因片段长度应为3050bp，下游同源臂的长度应为549bp，整合片段的总长应为4087bp。PCR验证时，阳性菌PCR扩增片段长度应为4087bp，原菌PCR扩增片段长度应为1547bp。
2.7将ycdW基因敲除
以E.coliATCC27325基因组为模板，根据其ycdW基因的上、下游序列设计上游同源臂引物UP-ycdW-S(SEQ ID NO.50)、UP-ycdW-A(SEQ ID NO.51)和下游同源臂引物DN-ycdW-S(SEQ ID NO.52)、DN-ycdW-A(SEQ ID NO.53)，并扩增ycdW基因的上、下游同源臂；上述片段通过重叠PCR的方法获得ycdW基因敲除的片段(ycdW基因上游同源臂-ycdW基因下游同源臂)，构建pGRB-ycdW使用的含靶序列的DNA片段通过引物gRNA-ycdW-S(SEQ ID NO.54)和gRNA-ycdW-A(SEQ ID NO.55)的退火制得，与线性化的pGRB载体重组后获得重组的 pGRB-ycdW。将整合片段和pGRB-ycdW电转化至含有pREDCas9载体的E.coli SAR6感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-ycdW及pREDCas9，最终获得菌株E.coli SAR7。
ycdW基因敲除片段的构建和阳性菌株的PCR验证的电泳图见图7。其中，上游同源臂的长度应为642bp，下游同源臂的长度应为1428bp，基因敲除片段的总长应为2024bp。PCR验证时，阳性菌PCR扩增片段长度应为2024bp，原菌PCR扩增片段长度应为2604bp。
2.8将P trc-ppc(含有ppc基因和trc启动子的片段)整合在假基因位点yeeL处
以E.coliATCC27325基因组为模板，根据其yeeL基因的上、下游序列设计上游同源臂引物UP-yeeL-S(SEQ ID NO.56)、UP-yeeL-A(SEQ ID NO.57)和下游同源臂引物DN-yeeL-S(SEQ ID NO.58)、DN-yeeL-A(SEQ ID NO.59)，并扩增yeeL基因的上、下游同源臂；根据ppc基因设计引物ppc-S(SEQ ID NO.60)、ppc-A(SEQ ID NO.61)，并扩增ppc基因片段(SEQ ID NO.5)。启动子P trc则设计在yeeL基因上游同源臂的下游引物和ppc基因的上游引物中。上述片段通过重叠PCR的方法获得ppc基因的整合片段(yeeL基因上游同源臂-P trc-ppc-yeeL基因下游同源臂)，构建pGRB-yeeL使用的含靶序列的DNA片段通过引物gRNA-yeeL-S(SEQ ID NO.62)和gRNA-yeeL-A(SEQ ID NO.63)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-yeeL。将整合片段和pGRB-yeeL电转化至含有pREDCas9载体的E.coli SAR7感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-yeeL及pREDCas9，最终获得菌株E.coli SAR8。
P trc-ppc整合片段的构建和阳性菌株的PCR验证的电泳图见图8。其中，上游同源臂的长度应为533bp，ppc基因片段长度应为2770bp，下游同源臂的长度应为581bp，整合片段的总长应为3813bp。PCR验证时，阳性菌PCR扩增片段长度应为3813bp，原菌PCR扩增片段长度应为1613bp。
2.9将pykF基因敲除
以E.coliATCC27325基因组为模板，根据其pykF基因的上、下游序列设计上游同源臂引物UP-pykF-S(SEQ ID NO.64)、UP-pykF-A(SEQ ID NO.65)和下游同源臂引物DN-pykF-S(SEQ ID NO.66)、DN-pykF-A(SEQ ID NO.67)，并扩增pykF基因的上、下游同源臂；上述片段通过重叠PCR的方法获得pykF基因敲除的片段(pykF基因上游同源臂-pykF基因下游同源臂)，构建pGRB-pykF使用的含靶序列的DNA片段通过引物gRNA-pykF-S(SEQ ID NO.68) 和gRNA-pykF-A(SEQ ID NO.69)的退火制得，与线性化的pGRB载体重组后获得重组的pGRB-pykF。将整合片段和pGRB-pykF电转化至含有pREDCas9载体的E.coli SAR8感受态细胞中，将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养后利用PCR验证阳性重组子，再消除用于基因编辑的pGRB-pykF及pREDCas9，最终获得菌株E.coli SAR。
pykF基因敲除片段的构建和阳性菌株的PCR验证的电泳图见图9。其中，上游同源臂的长度应为471bp，下游同源臂的长度应为429bp，基因敲除片段的总长应为856bp。PCR验证时，阳性菌PCR扩增片段长度应为856bp，原菌PCR扩增片段长度应为2180bp。
3.菌株构建过程中用到的引物
菌株构建过程中所涉及的所有引物见下表：
Figure PCTCN2021123534-appb-000001
Figure PCTCN2021123534-appb-000002
Figure PCTCN2021123534-appb-000003
Figure PCTCN2021123534-appb-000004
实施例2:菌株E.coli SAR摇瓶发酵生产L-肌氨酸
菌株E.coli SAR在5L发酵罐上的发酵实验：
斜面活化培养：从-80℃冰箱保菌管中刮一环菌种，均匀涂布于活化斜面，37℃培养12h，转茄形瓶继续培养12h；
种子培养：取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中，pH稳定在7.0左右， 温度恒定在37℃，溶氧在25-35％之间，培养6h；
发酵培养：按照15％接种量接入新鲜的发酵培养基，装液量为60％(v培养基/v发酵罐)，发酵过程中控制pH稳定在7.0左右，温度维持在36.5-37.5℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加800g/L的葡萄糖溶液继续培养，并维持发酵培养基中的葡萄糖浓度＜3g/L，当OD 600＝40时开始以25mL/h的流速流加1.6mol/L的甲胺盐酸盐溶液流加量为75mL/L培养基，发酵周期30h；
斜面培养基组成为：葡萄糖1g/L，蛋白胨10g/L，牛肉膏10g/L，酵母粉5g/L，NaCl2.5g/L，琼脂25g/L，其余为水，pH 7.0；
种子培养基组成为：葡萄糖25/L，酵母提取物5g/L，胰蛋白胨5g/L，KH 2PO 45g/L，MgSO 4·7H 2O 2g/L，其余为水，pH 7.0。
发酵培养基组成为：葡萄糖20g/L，酵母提取物4g/L，胰蛋白胨5g/L，柠檬酸钠5g/L，KH 2PO 42g/L，MgSO 4·7H 2O 1g/L，其余为水，pH 7.0。
5L发酵罐发酵30h后L-肌氨酸的产量可达10g/L。发酵过程曲线见图10。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。

Claims (4)

  1. 一种合成L-肌氨酸的基因工程菌，其特征在于：所述基因工程菌为大肠杆菌Escherichia coli SAR，该大肠杆菌Escherichia coli SAR是以大肠杆菌Escherichia coli ATCC27325为宿主，通过以下改造获得：在其基因组上的mbhA位点整合单拷贝的亚胺还原酶基因dpkA，该基因由T7启动子控制；ylbE位点单拷贝柠檬酸合酶基因gltA，该基因由trc启动子控制；敲除乙醛酸循环抑制基因iclR；敲除苹果酸合酶基因aceB；yeeP位点单拷贝异柠檬酸裂解酶基因aceA，该基因由trc启动子控制；yghE位点单拷贝膜结合转氢酶基因pntAB，该基因由trc启动子控制；敲除2-酮酸还原酶基因ycdW；yeeL位点单拷贝磷酸烯醇丙酮酸羧化酶基因ppc，该基因由trc启动子控制；敲除丙酮酸激酶基因pykF；
    所述亚胺还原酶基因dpkA的核苷酸序列为SEQ ID NO.1；
    所述柠檬酸合酶基因gltA的核苷酸序列为SEQ ID NO.2；
    所述异柠檬酸裂解酶基因aceA的核苷酸序列为SEQ ID NO.3；
    所述膜结合转氢酶基因pntAB的核苷酸序列为SEQ ID NO.4；
    所述磷酸烯醇丙酮酸羧化酶基因ppc的核苷酸序列为SEQ ID NO.5。
  2. 如权利要求1所述的合成L-肌氨酸的基因工程菌的构建方法，其特征在于：所述方法采用CRISPR/Cas9介导的基因编辑技术对大肠杆菌进行定向改造，具体包括如下步骤：
    ⑴为了引入L-肌氨酸的合成代谢，在大肠杆菌Escherichia coli ATCC27325基因组上的mbhA位点单拷贝来源于Brevibacterium linens ATCC 9172的亚胺还原酶基因dpkA，其序列为SEQ ID NO.1，该基因经过密码子优化并且由T7启动子控制；
    ⑵为了增强草酰乙酸到柠檬酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上ylbE位点单拷贝内源的柠檬酸合酶基因gltA，其序列为SEQ ID NO.2，该基因由trc启动子控制；
    ⑶为了菌株在正常培养条件下开启乙醛酸循环，对大肠杆菌Escherichia coli ATCC27325基因组上iclR位点进行基因敲除；
    ⑷为了阻断乙醛酸到苹果酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上aceB位点进行基因敲除；
    ⑸为了增强异柠檬酸到乙醛酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeP位点单拷贝内源的异柠檬酸裂解酶基因aceA，其序列为SEQ ID NO.3，该基因由trc启动子控制；
    ⑹为了增强NADH到NADPH的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yghE位点单拷贝内源的膜结合转氢酶基因pntAB，其序列为SEQ ID NO.4，该基因由trc启动子控制；
    ⑺为了阻断乙醛酸到乙醇酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上ycdW位点进行基因敲除；
    ⑻为了增强磷酸烯醇丙酮酸到草酰乙酸的代谢，在大肠杆菌Escherichia coli ATCC27325基因组上yeeL位点单拷贝内源的磷酸烯醇丙酮酸羧化酶基因ppc，其序列为SEQ ID NO.5，该基因由trc启动子控制；
    ⑼为了减少磷酸烯醇式丙酮酸到丙酮酸的代谢，对大肠杆菌Escherichia coli ATCC27325基因组上pykF位点进行基因敲除。
  3. 如权利要求1所述的合成L-肌氨酸的基因工程菌在L-肌氨酸生产方面中的应用。
  4. 利用如权利要求1所述的基因工程菌发酵生产L-肌氨酸的方法，其特征在于：具体步骤如下：
    发酵培养：将基因工程菌的种子液按照15-20％接种量接入新鲜的发酵培养基，发酵过程中控制pH稳定在6.8-7.2，温度维持在36.5-37.5℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加700-800g/L的葡萄糖溶液继续培养，并维持发酵培养基中的葡萄糖浓度＜3g/L，当OD 600＝40时，开始以20-25mL/h的流速流加1.5-1.6mol/L的甲胺盐酸盐溶液，流加量为75mL/L培养基，发酵周期28-32h，即得L-肌氨酸；
    发酵培养基组成为：葡萄糖15-25g/L，胰蛋白胨1-5g/L，柠檬酸钠3-5g/L，KH 2PO 41-5g/L，MgSO 4·7H 2O 0.1-1g/L，其余为水，pH 7.0-7.2。
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