WO2019071668A1 - 一种l-高丝氨酸的生产菌株及其构建方法和应用 - Google Patents

一种l-高丝氨酸的生产菌株及其构建方法和应用 Download PDF

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WO2019071668A1
WO2019071668A1 PCT/CN2017/109009 CN2017109009W WO2019071668A1 WO 2019071668 A1 WO2019071668 A1 WO 2019071668A1 CN 2017109009 W CN2017109009 W CN 2017109009W WO 2019071668 A1 WO2019071668 A1 WO 2019071668A1
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thra
homoserine
strain
seq
psoe
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French (fr)
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谢新开
黄晓飞
杜好勉
季鹏
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苏州引航生物科技有限公司
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Definitions

  • the invention belongs to the field of genetic engineering, and particularly relates to a production strain of L-homoserine and a construction method and application thereof.
  • L-homoserine is a naturally occurring non-protein amino acid that is present in small amounts in many species as a common intermediate for the biosynthesis of threonine, methionine and lysine. Since L-homoserine has the basic skeleton of L-type- ⁇ amino acid and its ⁇ -hydroxy group has various chemical activities, L-homoserine and its derivatives are important as pharmaceutical intermediates in pharmacology and physiology. Application prospects.
  • a chemical chiral separation method uses a mixed serine homoserine to react with a chiral reagent to form a difference in the nature of the diastereomer, thereby isolating L-homoserine.
  • the method has low yield, high reagent cost and requires a large amount of organic solvent, which has a great pollution threat to the environment.
  • a biological enzyme catalytic method utilizes pyruvic acid and formaldehyde to form an amino acid homoserine under the action of an acetalase and an L-amino acid dehydrogenase.
  • the main problem of this method is high cost, the use of toxic raw materials such as formaldehyde and formic acid, and the use of expensive coenzymes.
  • the method has many advantages such as low cost, mild condition, less environmental pollution, and the like, and has become the preferred process for producing various amino acids in recent years.
  • the current L-homoserine fermentation relies on a plasmid-based gene overexpression system, which has the disadvantage of poor cell passage stability.
  • the first technical problem to be solved by the present invention is to provide a stable strain producing L-homoserine.
  • a second technical problem to be solved by the present invention is to provide a genetic engineering method for constructing an L-homoserine producing strain.
  • the present invention provides a production strain of L-homoserine characterized in that it has a single stable chromosome, does not contain a plasmid form or other free DNA vector, and one or more of its chromosomal DNA and L-threonine
  • the synthetically related gene is knocked out or weakened, and/or mutated to enhance, and/or its chromosomal DNA integrates one or more genes associated with the L-homoserine metabolic pathway.
  • the knocked or weakened gene is thrB and/or thrL; the gene mutated to enhance is thrA and/or rhtA, and the integrated copy of the gene is thrA*, ppc, pntA, pntB, One or more of asd, aspA, and aspC.
  • the L-homoserine producing strain belongs to the genus Escherichia coli.
  • the strain is Escherichia coli K-12 wild type MG1655.
  • the present invention also provides a method for constructing the L-homoserine producing strain, which comprises One or more steps in the next step:
  • One or more genes associated with the L-homoserine metabolic pathway are knocked out or weakened in the constructed strain
  • One or more genes associated with the L-homoserine metabolic pathway in the constructed strain are overexpressed or enhanced by point mutation;
  • One or more genes associated with the L-homoserine metabolic pathway in the constructed strain are integrated into the chromosomal DNA for overexpression in multiple copies.
  • the knocked or weakened gene described in the step A is thrB and/or thrL;
  • the mutated gene described in the step B is thrA and/or rhtA;
  • the thrA and/or rhtA genes are mutated to the thrA* and/or rthA23 genes, respectively.
  • the mutant thrA* has the property of inhibiting feedback inhibition of the product, and the mutant rhtA23 has an effect of increasing the expression amount;
  • the gene overexpressed by increasing the chromosome copy number described in the step C is one or more of thrA*, ppc, pntA, pntB, apsA, aspC and asd.
  • the constructed strain described in the above step belongs to the genus Escherichia coli.
  • the strain is Escherichia coli K-12 wild type MG1655.
  • the present invention also provides the use of the L-homoserine producing strain for producing L-homoserine.
  • the present invention achieves the goal of stable high yield by integrating and expressing multiple copies of a specific gene related to the L-homoserine metabolic pathway.
  • the strain of the present invention has good passage stability, stable fermentation results, and higher yield than before, as reported by the previously reported overexpression of the plasmid vector.
  • the L-homoserine producing strain constructed by the present invention has less bacterial by-products in the form of a plasmid and a high conversion rate of sugar acid during the fermentation process.
  • the L-homoserine producing strain of the present invention does not require the use of antibiotics throughout the fermentation process, and avoids the environmental pollution of the antibiotics and the possibility of potential resistance genes flowing into the environment.
  • E. coli MG1655 was used to construct an L-homoserine producing strain, and knocking and editing of the genes in the E. coli genome were mainly based on Lambda-Red recombination, FLP-FRT recombination and CRISPR/Cas9 technology.
  • the temperature sensitive plasmid used in the examples has the DNA sequence structure shown by the thermosensitive plasmid in the literature Cell Fact. 2016 Dec1; 15(1):205.
  • the original strain Escherichia coli MG1655 was purchased from ATCC; The above materials are all commercially available, For the purpose of the present invention, products of different manufacturers and different specifications do not affect the implementation of the present invention.
  • the arabinose-inducing condition is specifically: induction culture was carried out at 30 ° C in an LB medium containing 20 mM arabinose.
  • the thrB gene was knocked out, the rhtA gene was overexpressed, the thrL gene was knocked out, and the mutant thrA gene-enhanced strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA* (G433R)) was constructed.
  • the L-homoserine producing strain constructed in the present example is a construct of a strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA* (G433R)) which knocks out the thrB gene, overexpresses the rhtA gene, knocks out the thrL gene, and mutates the thrA gene.
  • the construction method can be seen in the Chinese patent application CN201710106474.8.
  • the L-homoserine producing strain constructed in this example is a genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA which simultaneously integrates thrA*, ppc, aspA, pntA and pntB on chromosomal DNA.
  • *-ppc-aspA-pntAB the construction method can be referred to the literature: Microb Cell Fact.2016 Dec 1; 15(1): 205, specifically including the following steps:
  • the primer pSOE-thrA-f has the sequence shown as SEQ ID No. 1, the primer pSOE-ppc-thrA-r SEQ ID No. 2, the primer pSOE-thrA-ppc -f has the sequence shown as SEQ ID No. 3, the primer pSOE-aspA-ppc-r has the sequence shown as SEQ ID No. 4, and the primer pSOE-ppc-aspA-f has the SEQ ID In the sequence shown in No. 5, the primer pSOE-pntA-aspA-r has the sequence shown in SEQ ID No. 6, and the primer pSOE-aspA-pntA-f has the sequence shown in SEQ ID No. 7. a sequence, the primer pntB-r having the sequence set forth in SEQ ID No. 8,
  • SEQ ID No. 1 5'-GCTCATATTGGCACTGGAAG-3';
  • SEQ ID No. 2 5'-CTTATGGAAATGTTAAAAAATCGCCAAGCTTTACGCGAACGAG-3';
  • SEQ ID No. 3 5'-CTCGTTCGCGTAAAGCTTGGCGATTTTTTAACATTTCCATAAG-3';
  • SEQ ID No. 4 5'-CATTTTCCTTTATGTCTATATTGAGATTTCCCTTAAGGATATCTGAAGGTATATTCAG-3';
  • SEQ ID No. 5 5'-CTGAATATACCTTCAGATATCCTTAAGGGAAATCTCAATATAGACATAAAGGAAAATG-3';
  • SEQ ID No. 6 5'-GTACTGAAAATTATGCCTGTGATCTGTGCCTTTTTTATTTGTACTACCCT-3';
  • SEQ ID No. 7 5'-AGGGTAGTACAAATAAAAAAGGCACAGATCACAGGCATAATTTTCAGTAC-3';
  • SEQ ID No. 8 5'-TCTCAATAAAGAGTGACGGC-3'.
  • thermosensitive plasmid containing three Lambda phage recombinases, Cas9 endonuclease by induction of expression of Gam, Bet and Exo;
  • the primer pSOE-cadA-H1-f has the sequence shown as SEQ ID No. 9, and the primer pSOE-cadA-H1-r has the sequence shown as SEQ ID No. 10, the primer pSOE-cadA-H2-f has the sequence shown as SEQ ID No. 11, the primer pSOE-cadA-H2-r has the sequence shown as SEQ ID No. 12, and the primer pcadA-N20-f has The primer pcadA-N20-r has the sequence shown in SEQ ID No. 14, as shown in SEQ ID No. 13.
  • SEQ ID No. 9 5'-GTGCAGCGCCAGAGCCAC-3';
  • SEQ ID No. 10 5'-GCCGTCACTCTTTATTGAGAGCTTTAGTCAGCGGAGGC-3';
  • SEQ ID No. 11 5'-CTTCCAGTGCCAATATGAGCACTGTTTAAATATGTTCGTGAAGG-3';
  • SEQ ID No. 12 5'-GAGCTGGATGGATTTCACATCCAGTG-3';
  • SEQ ID No. 13 5'-AGCGGCCTCCGCTGACTAAAGCAT-3';
  • SEQ ID No. 14 5'-AAACATGCTTTAGTCAGCGGAGGC-3';
  • the integrated inserted DNA fragment has the sequence shown in SEQ ID No. 15, as follows:
  • the mutant colonies confirmed by the screening in the step (3) were cultured at 42 ° C to eliminate the CRISPR/Cas9 plasmid, and at the same time, the kanamycin resistance caused by the plasmid was eliminated, that is, the strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA* (G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB).
  • the L-homoserine producing strain constructed in this example is a genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA which simultaneously expresses thrA*, ppc, aspA, pntA and pntB on chromosomal DNA. ::thrA*-ppc-aspA Construction of -pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB), construction of the strain was carried out by using the CRISPR/Cas9 gene editing technique, and the fragment of SEQ ID No.
  • Example 15 described in Example 2 was inserted downstream of the yidJ gene.
  • the construction method is basically similar to the method of Example 2, except that the product strain of the original bacteria Example 2 is operated as an operation target, and the integration position is downstream of the yidJ gene.
  • the primer used in the step (2) described in Example 2 is composed of the pSOE-cadA-H1-f, pSOE-cadA-H1-r, pSOE-cadA-H2-f, and pSOE-cadA-H2.
  • the primer pSOE-yidJ-H1-f has the sequence shown as SEQ ID No. 16
  • the primer pSOE-yidJ-H1-r has the sequence shown as SEQ ID No. 17
  • the primer pSOE -yidJ-H2-f has the sequence shown as SEQ ID No. 18
  • the primer pSOE-yidJ-H2-r has the sequence shown as SEQ ID No. 19
  • the primer pyidJ-N20-f has The sequence shown in SEQ ID No. 20
  • the primer pyidJ-N20-r has the sequence shown as SEQ ID No. 21, specifically as follows:
  • SEQ ID No. 16 5'-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3';
  • SEQ ID No. 17 5'-CTTCCAGTGCCAATATGAGCCGGCCTACCGACACAAG-3';
  • SEQ ID No. 18 5'-GGCCGTCACTCTTTATTGAGAGTGTCATAGTCGCGTACAAC-3';
  • SEQ ID No. 19 5'-GAGCCGGCGATGTATGACGACATC-3';
  • SEQ ID No. 20 5'-AGCGGTTGTACGCGACTATGACAC-3';
  • SEQ ID No. 21 5'-AAACGTGTCATAGTCGCGTACAAC-3'.
  • chromosomal DNA Three copies express thrA*, ppc, aspA, pntA and pntB Genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB, ⁇ atpC::thrA*-ppc- The construction of aspA-pntAB).
  • the L-homoserine producing strain constructed in this example is a genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA which simultaneously expresses thrA*, ppc, aspA, pntA and pntB three times on chromosomal DNA: :thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB, ⁇ atpC::thrA*-ppc-aspA-pntAB), using CRISPR/Cas9 gene editing technology to construct the strain,
  • the primers used in the step (2) described in Example 2 are composed of the pSOE-cadA-H1-f, pSOE-cadA-H1-r, pSOE-cadA-H2-f, and pSOE-cadA-H2.
  • the primer pSOE-atpC-H1-f has the sequence shown as SEQ ID No. 22, and the primer pSOE-atpC-H1-r has the sequence shown as SEQ ID No. 23, the primer pSOE -atpC-H2-f has the sequence shown as SEQ ID No. 24, the primer pSOE-atpC-H2-r has the sequence shown as SEQ ID No. 25, and the primer paptC-N20-f has The sequence shown in SEQ ID No. 26, the primer paptC-N20-r has the sequence shown in SEQ ID No. 27, specifically as follows:
  • SEQ ID No. 22 5'-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3';
  • SEQ ID No. 23 5'-CTTCCAGTGCCAATATGAGCGAAAGCGCCATTTTCGACC-3';
  • SEQ ID No. 24 5'-GCCGTCACTCTTTATTGAGAGTCAGCGGGGATAATCCGT-3';
  • SEQ ID No. 25 5'-GAGCCATAACCGGTCGGATCATCC-3';
  • SEQ ID No. 26 5'-AGCGGTTGTACGCGACTATGACAC-3';
  • SEQ ID No. 27 5'-AAACGTGTCATAGTCGCGTACAAC-3'.
  • the genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB, ⁇ atpC, which continues to integrate thrA* in chromosomal DNA Construction of ::thrA*-ppc-aspA-pntAB, ⁇ dacA::thrA*).
  • the L-continued integration of thrA* genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA -pntAB, ⁇ atpC::thrA*-ppc-aspA-pntAB, ⁇ dacA::thrA*), using CRISPR/Cas9 gene editing technology to construct the strain, inserting the thrA* expression cassette into the dacA gene, and its construction method and examples
  • the method of 2 is basically similar, except that the original strain of the product strain of Example 4 is manipulated, and the integration position is reverse insertion into the inside of the dacA gene.
  • the insert was obtained by PCR amplification from the MG1655 genome using primers pSOE-thrA-f and pSOE-thrA-r, and the primer used in the step (2) described in Example 2 was used from the pSOE-cadA.
  • the primer pSOE-thrA-f has the sequence shown as SEQ ID No. 1.
  • the primer pSOE-thrA-r has the sequence shown as SEQ ID No. 28, and the primer pSOE-dacA-H1-f has the sequence shown as SEQ ID No. 29, the primer pSOE-dacA-H1 -r has the sequence shown as SEQ ID No. 30, the primer pSOE-dacA-H2-f has the sequence shown as SEQ ID No. 31, and the primer pSOE-dacA-H2-r has the SEQ ID The sequence shown in No. 32, wherein the primer pdacA-N20-f has the sequence shown in SEQ ID No. 33, and the primer pdacA-N20-r has the sequence shown in SEQ ID No. 34, specifically as follows :
  • SEQ ID No. 28 5'-AAGCTTTACGCGAACGAGC-3';
  • SEQ ID No. 29 5'-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3';
  • SEQ ID No. 30 5'-GCTCGTTCGCGTAAAGCTTGTACGTCAGGGATCAATGC-3';
  • SEQ ID No. 31 5'-CTTCCAGTGCCAATATGAGCCGAATGAATACTCGATCTATAAAGAAAAAG-3';
  • SEQ ID No. 32 5'-GAGCAACCAAACCAGTGATGGAAC-3';
  • SEQ ID No. 33 5'-AGCGAGTATTCATTCGGTACGTCA-3';
  • SEQ ID No. 34 5'-AAACTGACGTACCGAATGAATACT-3'.
  • the genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB, ⁇ atpC, which continues to integrate thrA* in chromosomal DNA Construction of ::thrA*-ppc-aspA-pntAB, ⁇ dacA::thrA*, ⁇ bcsB::thrA*).
  • the CRISPR/Cas9 gene editing technology was used to construct the strain, and the thrA* expression cassette was inserted into the bcsB gene, and the construction method thereof was basically similar to the method of Example 5, except that the original strain Example 5 product strain was The manipulation target is integrated into the interior of the bcsB gene.
  • the primer used in the step (2) described in Example 5 consists of the pSOE-dacA-H1-f, pSOE-dacA-H1-r, pSOE-dacA-H2-f, pSOE-dacA-H2-r, pdacA -N20-f, pdacA-N20-r is replaced by pSOE-bcsB-H1-f, pSOE-bcsB-H1-r, pSOE-bcsB-H2-f, pSOE-bcsB-H2-r, pbcsB-N20-f, pbcsB-N20-r;
  • primer pSOE-bcsB-H1-f has the sequence shown as SEQ ID No. 35
  • the primer pSOE-bcsB-H1-r has the sequence shown as SEQ ID No. 36
  • the primer pSOE -bcsB-H2-f has the sequence shown as SEQ ID No. 37
  • the primer pSOE-bcsB-H2-r has the sequence shown as SEQ ID No. 38
  • the primer pbcsB-N20-f has The sequence shown in SEQ ID No. 39
  • the primer pbcsB-N20-r has the sequence shown in SEQ ID No. 40, specifically as follows:
  • SEQ ID No. 35 5'-GTGCTCGTTACTGCCTGTCCAGTC-3';
  • SEQ ID No. 36 5'-GCTCGTTCGCGTAAAGCTTCAGTTTGACGTAAGGATTCTGC-3';
  • SEQ ID No. 37 5'-CTTCCAGTGCCAATATGAGCCTGGTGGTGTTTGGTCGTGACG-3';
  • SEQ ID No. 38 5'-GAGCGCTGGAAGGTAATACAGTTATCCAC-3';
  • SEQ ID No. 39 5'-AGCGGAATCCTTACGTCAAACTGC-3';
  • SEQ ID No. 40 5'-AAACGCAGTTTGACGTAAGGATTC-3';
  • This example constructs a genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB in chromosomal DNA integration aspC , ⁇ atpC::thrA*-ppc-aspA-pntAB, ⁇ dacA::thrA*, ⁇ menH::aspC).
  • the aspC expression cassette was inserted into the menH gene using the CRISPR/Cas9 gene editing technology.
  • the construction method was basically similar to the method of Example 5, except that the strain produced in the original bacteria Example 6 was used as a manipulation target, and the aspC gene under the natural promoter was reversely inserted into the inside of the menH gene.
  • the insert was obtained by PCR amplification from the MG1655 genome by primers pSOE-aspC-f and pSOE-aspC-r, and the primer used in the step (2) of the example was obtained from the pSOE-dacA-H1-f, pSOE-dacA-H1-r, pSOE-dacA-H2-f, pSOE-dacA-H2-r, pdacA-N20-f, pdacA-N20-r replaced with pSOE-menH-H1-f, pSOE-menH-H1 -r, pSOE-menH-H2-f, pSOE-menH-H2-r, pmenH
  • the primer pSOE-aspC-f has the sequence shown in SEQ ID No. 41
  • the primer pSOE-aspC-r has the sequence shown as SEQ ID No. 42
  • the primer pSOE-menH-H1 -f has the sequence shown as SEQ ID No. 43
  • the primer pSOE-menH-H1-r has the sequence shown as SEQ ID No. 44
  • the primer pSOE-menH-H2-f has the SEQ ID
  • the primer pSOE-menH-H2-r has the sequence shown in SEQ ID No. 46
  • the primer pmenH-N20-f has the sequence shown in SEQ ID No. 47
  • the primer pmenH-N20-r has the sequence shown in SEQ ID No. 48, as follows:
  • SEQ ID No. 41 5'-CTGACCGTACCAACCTGCA-3';
  • SEQ ID No. 42 5'-CCTGATAAGCGTAGCGCAT-3';
  • SEQ ID No. 43 5'-GTGCGTTTGCCGACTACTCACG-3';
  • SEQ ID No. 44 5'-TGCAGGTTGGTACGGTCAGGCGCTAAGGTT AGCACGTAAAT-3’;
  • SEQ ID No. 45 5'-ATGCGCTACGCTTATCAGGCCGCACATTTGCGTTTTATTAT-3';
  • SEQ ID No. 46 5'-GAGCACATTCAGGTGATGTACGCC-3';
  • SEQ ID No. 47 5'-AGCGACGCAAATGTGCGGGCGCTA-3';
  • SEQ ID No. 48 5'-AAACTAGCGCCCGCACATTTGCGT-3'.
  • This example constructs a genetically engineered strain MG1655 ( ⁇ thrB, rhtA23, ⁇ thrL, thrA*(G433R), ⁇ cadA::thrA*-ppc-aspA-pntAB, ⁇ yidJ::thrA*-ppc-aspA-pntAB in chromosomal DNA integration asd , ⁇ atpC::thrA*-ppc-aspA-pntAB, ⁇ dacA::thrA*, ⁇ bcsB::thrA*, ⁇ menH::aspC, ⁇ yddB::asd).
  • Insertion of the asd expression cassette into the yddB gene was carried out using the CRISPR/Cas9 gene editing technology.
  • the method of construction was basically similar to the method of Example 5, except that the strain produced in the original bacteria Example 7 was used as a manipulation target, and the asd gene under the natural promoter was inserted into the yddB gene.
  • the insert was obtained by PCR amplification from the MG1655 genome by primers pSOE-asd-f and pSOE-asd-r, and the primer used in the step (2) of the example was obtained from the pSOE-menH-H1-f, pSOE-menH-H1-r, pSOE-menH-H2-f, pSOE-menH-H2-r, pmenH-N20-f, pmenH-N20-r replaced with pSOE-yddB-H1-f, pSOE-yddB-H1 -r, pSOE-yddB-H2-f, pSOE-yddB-H2-r, pyddB-N20-f, pyddB-N20-r;
  • the primer pSOE-asd-f has the sequence shown as SEQ ID No. 49
  • the primer pSOE-asd-r has the sequence shown as SEQ ID No. 50
  • the primer pSOE-yddB-H1 -f has the sequence shown as SEQ ID No. 51
  • the primer pSOE-yddB-H1-r has the sequence shown as SEQ ID No. 52
  • the primer pSOE-yddB-H2-f has the SEQ ID
  • the primer pSOE-yddB-H2-r has the sequence shown in SEQ ID No. 53
  • the primer pSOE-yddB-H2-r has the sequence shown in SEQ ID No. 54
  • the primer pyddB-N20-f has the sequence shown in SEQ ID No. 55.
  • the primer pyddB-N20-r has the sequence shown in SEQ ID No. 56, as follows:
  • SEQ ID No. 41 5'-GGATCCATAATCAGGATCAATAAAACT-3';
  • SEQ ID No. 42 5'-AGGATCCGCAAAATGGCC-3';
  • SEQ ID No. 43 5'-GTGCGGCTCTTCAGGAAGTACTTATTAC-3';
  • SEQ ID No. 44 5'-GTTTTATTGATCCTGATTATGGATCCCAGTCCACGCATCGGAATAG-3';
  • SEQ ID No. 45 5'-GGCCATTTTGCGGATCCTAACGCCATAACCAGTCTGAAT-3';
  • SEQ ID No. 46 5'-GAGCCTGAACGAATGCGTTTTGG-3';
  • SEQ ID No. 47 5'-AGCGATGGCGTTCAGTCCACGCAT-3';
  • SEQ ID No. 48 5'-AAACATGCGTGGACTGAACGCCAT-3'.
  • the strain prepared in the first embodiment is used for the fermentative production of L-homoserine, and the production method thereof can be specifically referred to the Chinese patent application CN201710106474.8. Fermentation yielded a high serine 5.3 g/L.
  • the strain finally prepared in the above Example 1 can also be replaced with the strain finally obtained in Examples 2-8.
  • Example 1 and Example 4 and Example 8 were preferably used for the fermentation production of L-homoserine, compared with MG1655.
  • the production method is completely identical to the production method described in Example 10.
  • the experiment numbering of the strain prepared by using the strain prepared in Example 4 was Hom4, and the experiment using the strain prepared in Example 8 for fermentation was numbered Hom8, and the yield of L-homoserine was detected and calculated, respectively. as follows:
  • the yield of L-homoserine fermented by the strain of the present invention is up to 100 g/L, which indicates that the present invention has obvious effects on knockout, weakening, enhancement, overexpression and mutation of each gene.

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Abstract

提供了一种L-高丝氨酸的生产菌株，其特征在于：其具有单一稳定的染色体，不含质粒形式或其他游离DNA载体，其染色体DNA上一个或多个与L-苏氨酸合成相关的基因被敲除或弱化，和/或被突变以增强，和/或其染色体DNA上整合拷贝一个或多个与L-高丝氨酸代谢途径相关的基因。

Description

一种L-高丝氨酸的生产菌株及其构建方法和应用 技术领域
本发明属于基因工程领域，具体涉及一种L-高丝氨酸的生产菌株及其构建方法和应用。
背景技术
L-高丝氨酸是一种天然存在的非蛋白氨基酸，作为生物合成苏氨酸、蛋氨酸和赖氨酸共有的中间体而少量存在于很多物种中。由于L-高丝氨酸具有L-型-α氨基酸的基本骨架，并且其γ-羟基具有多样的化学活性，因此，L-高丝氨酸及其衍生物作为医药中间体在药物学、生理学等方面有重要应用前景。
目前，国内外生产L-高丝氨酸主要有以下几种方法：
(1)化学法；该方法采用相对昂贵的L-蛋氨酸为原料，并使用具有严重生物毒性的碘甲烷或溴甲烷作为甲基化试剂，经亲核进攻以保护氨基，再在弱碱性条件下水解得到产物。该方法不仅成本高，并且需要使用碘化物、生成硫化物，对环境不友好；
(2)化学手性拆分方法；该方法是利用混旋的高丝氨酸与手性试剂反应后，会形成非对应异构体的性质差异，由此分离出L-高丝氨酸。该方法产率低，试剂成本高且需使用大量有机溶剂，对环境有较大污染威胁。
(3)生物酶催化法；该方法利用丙酮酸和甲醛在缩醛酶和L-氨基酸脱氢酶的共同作用下生成氨基酸高丝氨酸。该方法主要问题是成本高，需要使用有毒原料甲醛和甲酸，并需使用价格昂贵的辅酶等。
(4)微生物发酵法；该方法具有成本低、条件温和、环境污染少等诸多优点，近年来已经成为生产各类氨基酸的首选工艺。但是，目前的L-高丝氨酸发酵依赖于以质粒为载体的基因过表达体系，其劣势是菌种传代稳定性差。
发明内容
本发明要解决的第一个技术问题是提供一种稳定的L-高丝氨酸的生产菌株。
本发明要解决的第二个技术问题是提供一种构建L-高丝氨酸生产菌株的基因工程方法。
因此，本发明提供一种L-高丝氨酸的生产菌株，其特征在于：其具有单一稳定的染色体，不含质粒形式或其他游离DNA载体，其染色体DNA上一个或多个与L-苏氨酸合成相关的基因被敲除或弱化，和/或被突变以增强，和/或其染色体DNA上整合拷贝一个或多个与L-高丝氨酸代谢途径相关的基因。
优选地，所述被敲除或弱化的基因为thrB和/或thrL；所述被突变以增强的基因为thrA和/或rhtA，所述整合拷贝的基因为thrA*、ppc、pntA、pntB、asd、aspA和aspC中的一个或多个。
优选地，所述L-高丝氨酸生产菌株属于大肠杆菌种属。
进一步优选地，所述菌株为大肠杆菌K-12野生型MG1655。
本发明还提供所述的L-高丝氨酸生产菌株的构建方法，包括以 下步骤中一步或多步：
A、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因被敲除或弱化；
B、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因通过点突变过表达或增强功能；
C、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因以多拷贝的形式整合到染色体DNA上过表达。
优选地，所述步骤A中所述的被敲除或弱化的基因为thrB和/或thrL；
所述步骤B中所述的被突变的基因为thrA和/或rhtA；
所述步骤B中，thrA和/或rhtA基因分别被突变为thrA*和/或rthA23基因。且所述突变thrA*具有抑制产物的反馈抑制的特性，突变rhtA23具有增加表达量的效果；
所述步骤C中所述的通过提高染色体拷贝数来过表达的基因为thrA*、ppc、pntA、pntB、apsA，aspC和asd中的一个或多个。
优选地，上述步骤中所述的构建菌株属于大肠杆菌种属。
进一步优选地，所述菌株为大肠杆菌K-12野生型MG1655。
本发明还提供所述的L-高丝氨酸生产菌株在生产L-高丝氨酸中的应用。
本发明的上述技术方案，相比现有技术具有以下优点：
(1)本发明通过对L-高丝氨酸代谢途径相关的特定基因的多拷贝整合表达，可实现稳定高产的目的。相对之前报道的通过质粒载体过表达的方式，本发明的菌株传代稳定性好，发酵结果稳定，并且产量较之前高。
(2)本发明所构建的L-高丝氨酸生产菌株在发酵过程中，相对带质粒形式的菌种副产物少，糖酸转化率高。
(3)本发明的L-高丝氨酸的生产菌株，发酵全程无需使用抗生素，避免了抗生素对环境的污染以及潜在的抗性基因流入环境的可能。
具体实施方式
实施例中均采用大肠杆菌MG1655构建L-高丝氨酸生产菌株，对所述大肠杆菌基因组中的基因进行敲除和编辑主要是基于Lambda-Red重组、FLP-FRT重组和CRISPR/Cas9技术。可参照文献“Lambda Red Recombination One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.”Proc Natl Acad Sci USA.2000Jun 6；97(12):6640-5.和“Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9.”Microb Cell Fact.2016 Dec 1；15(1):205.。
实施例中采用的所述温敏质粒具有文献Cell Fact.2016 Dec1；15(1):205.中的温敏质粒所示的DNA序列结构；所述原始菌株大肠杆菌MG1655购买自ATCC；需要说明的是，上述各材料均为市售，对 于实现本发明的目的来说，不同厂家、不同规格的产品并不影响本发明的实施。实施例中，所述阿拉伯糖诱导条件具体为：在含20mM阿拉伯糖的LB培养基中，30℃诱导培养。
实施例1
敲除thrB基因、过表达rhtA基因、敲除thrL基因、突变thrA基因增强菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R))的构建。
本实施例构建的L-高丝氨酸生产菌株为敲除thrB基因、过表达rhtA基因、敲除thrL基因、突变thrA基因的菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R))的构建，其构建方法可见中国专利申请CN201710106474.8。
实施例2
在染色体DNA上单拷贝表达thrA*、ppc、aspA、pntA和pntB的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB)的构建。
本实施例构建的L-高丝氨酸生产菌株为染色体DNA上同时整合表达thrA*、ppc、aspA、pntA和pntB的基因工程菌MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB)，其构建方法可参考文献:Microb Cell Fact.2016 Dec 1；15(1):205，具体包括如下步骤：
(1)以实施例1中所得菌种染色体DNA为模板，以pSOE-thrA-f、pSOE-ppc-thrA-r；pSOE-thrA-ppc-f、pSOE-aspA-ppc-r；pSOE-ppc-aspA-f、pSOE-pntA-aspA-r；pSOE-aspA-pntA-f、pntB-r为引物，通过重叠延伸PCR法(即SOE-PCR法)进行扩增，将模板菌中位于对应天然启动子下的thrA*、ppc、AspA、pntAB等基因连接起来，得到整合插入的基因DNA片段；
其中，所述引物pSOE-thrA-f具有如SEQ ID No.1所示的序列，所述引物pSOE-ppc-thrA-r SEQ ID No.2所示的序列，所述引物pSOE-thrA-ppc-f具有如SEQ ID No.3所示的序列，所述引物pSOE-aspA-ppc-r具有如SEQ ID No.4所示的序列，所述引物pSOE-ppc-aspA-f具有如SEQ ID No.5所示的序列，所述引物pSOE-pntA-aspA-r具有如SEQ ID No.6所示的序列，所述引物pSOE-aspA-pntA-f具有如SEQ ID No.7所示的序列，所述引物pntB-r具有如SEQ ID No.8所示的序列，
具体如下：
SEQ ID No.1:5’-GCTCATATTGGCACTGGAAG-3’；
SEQ ID No.2:5’-CTTATGGAAATGTTAAAAAATCGCCAAGCTTTACGCGAACGAG-3’；
SEQ ID No.3:5’-CTCGTTCGCGTAAAGCTTGGCGATTTTTTAACATTTCCATAAG-3’；
SEQ ID No.4:5’-CATTTTCCTTTATGTCTATATTGAGATTTCCCTTAAGGATATCTGAAGGTATATTCAG-3’；
SEQ ID No.5:5’-CTGAATATACCTTCAGATATCCTTAAGGGAAATCTCAATATAGACATAAAGGAAAATG-3’；
SEQ ID No.6:5’-GTACTGAAAATTATGCCTGTGATCTGTGCCTTTTTTATTTGTACTACCCT-3’；
SEQ ID No.7:5’-AGGGTAGTACAAATAAAAAAGGCACAGATCACAGGCATAATTTTCAGTAC-3’；
SEQ ID No.8:5’-TCTCAATAAAGAGTGACGGC-3’。
(2)以MG1655基因组DNA为模板，使用pSOE-cadA-H1-f、pSOE-cadA-H1-r；pSOE-cadA-H2-f、pSOE-cadA-H2-r为引物，分别获得cadA的上下游整合同源臂，再通过重叠延伸PCR法(即SOE-PCR法)与步骤(1)中得到的所述片段DNA进行扩增，构成整合供体DNA。结合pcadA-N20-f、pcadA-N20-r克隆到温敏质粒中，所述温敏质粒含有通过诱导表达Gam、Bet以及Exo 3种Lambda噬菌体重组酶、Cas9DNA内切酶；
所述引物pSOE-cadA-H1-f具有如SEQ ID No.9所示的序列，所述引物pSOE-cadA-H1-r具有如SEQ ID No.10所示的序列，所述引物 pSOE-cadA-H2-f具有如SEQ ID No.11所示的序列，所述引物pSOE-cadA-H2-r具有如SEQ ID No.12所示的序列，所述引物pcadA-N20-f具有如SEQ ID No.13所示的序列，所述引物pcadA-N20-r具有如SEQ ID No.14所示的序列，
具体如下：
SEQ ID No.9:5’-GTGCAGCGCCAGAGCCAC-3’；
SEQ ID No.10:5’-GCCGTCACTCTTTATTGAGAGCTTTAGTCAGCGGAGGC-3’；
SEQ ID No.11:5’-CTTCCAGTGCCAATATGAGCACTGTTTAAATATGTTCGTGAAGG-3’；
SEQ ID No.12:5’-GAGCTGGATGGATTTCACATCCAGTG-3’；
SEQ ID No.13:5’-AGCGGCCTCCGCTGACTAAAGCAT-3’；
SEQ ID No.14:5’-AAACATGCTTTAGTCAGCGGAGGC-3’；
所述整合插入的基因DNA片段具有如SEQ ID No.15所示的序列，具体如下：
SEQ ID No.15：5’-GCTCATATTGGCACTGGAAGCCGGGGCATAAACTTTAACCATGTCAGACTCCTAACTTCCATGAGAGGGTACGTAGCAGATCAGCAAAGACACCGGCAGCTGTAACGTCATTGCCCGCACCATATCCGCGCAGTACCAACGGCAGCGGCTGATAATAGTGGCTATAGAAGGCCAGGGCGTTTTCGCCATTTTTCACTTTGAACAGCGGATCATTA CCATCCACTTCGGCAATCTTCACGCGGCAGACGCCATCTTCATCAATATTGCCAACATAGCGCAAAACTTTTCCTTCATCACGGGCCTTCGCCACGCGCGCGGCAAAGAGATCGTCGAGTTGTGACAGATTCGCCATAAAAGCGGCAACATCACCCTCGGCGTTAAACTCTGCGGGCAGCACAGGTTCAATTTCAATATCCGCCAGCTCCAGTTCACGTCCCGTTTCACGAGCGAGAATCAATAGTTTACGCGCCACATCCATACCAGAAAGATCATCTCGCGGGTCCGGTTCGGTATAACCCATTTCCCGCGCCAGCGTGGTCGCCTCGGAGAAACTCATGCCTTCGTCTAACTTGCCGAAGATATAAGAAAGCGAACCAGAAAGAATGCCGGAGAACTTCATCAATTCATCACCTGCATTGAGCAGATTTTGCAGGTTCTCAATAACCGGTAATCCAGCCCCAACGTTGGTGTCATAGAGGAATTTACGCCGCGATTTTTCCGCCGCATAACGCAACTGATGGTAGTAATCCATCGACGAGGTGTTGGCCTTTTTGTTCGGCGTGACAACGTGGAAACCTTCGCGCAGGAAGTCGGCATATTGATCCGCCACTGCCTGGCTGGAAGTGCAGTCAACAATGACCGGGTTCAGCAGATGATATTCTTTCACGAGGCGAATTAAGCGCCCGAGATTAAACGGCTCTTTGGCTTGCGCCAGTTCTTCCTGCCAGTTTTCCAGATTAAGGCCATGTACATTGGTGAGCAGAGCCTTCGAGTTGGCAACACCGCAGACACGTAAGTCGATATGTTTATTCTTCAGCCAGCTTTGCTGACGCTTCAGTTGCTCCAGCAGCGCACCGCCAACGCCACCGACGCCAATCACAAACACTTCGATAACCTGATCGGTATTGAACAGCATCTGATGAGTAACGCGCACGCCAGTGGTCGCATCATCGTTATTTACCACGACAGAGATTGAGCGTTCAGAAGATCTCTGAGCAATGGCGACAATGTTGATATTGGCGCGGGCCAGTGCGGCAAAGAATTTCGCCGAGATCCCACGCAAGGTGCGCATACCATCACCTACCACCGAGATAATGGCCAGCCGTTCCGTCACTGCCAGCGGCTCCAGTAAGCCTTCTTTCAGTTCCAGGTAGAACTCTTCCTGCATTGCCCGTTCAGCTCGCACACAGTCGCTTTGTGGAACGCAGAAACTGATGCTGTATTCGGAAGATGATTGCGTAATCAGCACCACGGAAATACGGGCGCGTGACATCGCTGCAAAGACGCGCGCCGC CATGCCGACCATCCCTTTCATCCCCGGACCAGAAACGCTGAACATTGCCATGTTATTCAGATTGGAAATGCCCTTGACCGGTAATTCGTCTTCATCACGGCTGGCACCAATGAGCGTACCTGGTGCTTGAGGATTTCCGGTATTTTTAATCAGGCAAGGGATCTGGAACTGGGCGATGGGGGTAATGGTGCGGGGGTGAAGAACTTTAGCGCCGAAGTAGGAAAGCTCCATCGCTTCCTGGTAGGACATCGACTTCAACAACCTCGCATCGGGCACCTGACGCGGGTCGCAGGTATAGACCCCGTCAACGTCCGTCCAAATCTCGCAACAATCGGCGCGTAAACAGGCAGCCAGCACCGCAGCAGAGTAGTCGGAACCGTTGCGTCCAAGCACCACCAGTTCGCCTTTTTCATTACCGGCGGTGAAACCTGCCATCAGCACCATGTGATCAGCCGGAATGCGGCTTGCCGCAATACGGCGGGTGGACTCAGCAATATCGACGGTAGATTCGAGGTAATGCCCCACTGCCAGCAGTTTTTCGACCGGATCGATAACAGTAACGTTGTGACCGCGCGCTTCTAATACGCCGGCCATAATGGCGATCGACATTTTCTCGCCACGGCAAATCAGCGCAGCGTTGATGCTATCCGGGCACTGCCCCAACAAACTAATGCCATGCAGGACATGTTTTATTTGGGCAAATTCCTGATCGACGAAAGTTTTCAATTGCGCCAGCGGGAACCCCGGCTGGGCGGCGGCGAGTCCCGTCAAAAGTTCGGCAAAAATACGTTCGGCATCGCTGATATTGGGTAAAGCATCCTGGCCGCTAATGGTTTTTTCAATCATCGCCACCAGGTGGTTGGTGATTTTGGCGGGGGCAGAGAGGACGGTGGCCACCTGCCCCTGCCTGGCATTGCTTTCCAGAATATCGGCAACACGCAGAAAACGTTCTGCATTTGCCACTGATGTACCGCCGAACTTCAACACTCGCATGGTTGTTACCTCGTTACCTTTGGTCGGACTCTAGAGTCTTTATCTGTCTGTGCGCTATGCCTATATTGGTTAAAGTATTTAGTGACCTAAGTCAATAAAATTTTAATTTACTCACGGCAGGTAACCAGTTCAGAAGCTGCTATCAGACACTCTTTTTTTAATCCACACAGAGACATATTGCCCGTTGCAGTCAGAATGAAAAGCTGAAAAATACTTACTAAGGCGTTTTTTATTTGGTGATATTTTTTTCAATATCATGCAGCAAACGGTGCAACATTGCCGTGTCTCGTTGCTCTAAAAGCCCC AGGCGTTGTTGTAACCAGTCGACCAGTTTTATGTCATCTGCCACTGCCAGAGTCGTCAGCAATGTCATGGCTCGTTCGCGTAAAGCTTGGCGATTTTTTAACATTTCCATAAGTTACGCTTATTTAAAGCGTCGTGAATTTAATGACGTAAATTCCTGCTATTTATTCGTTTGCTGAAGCGATTTCGCAGCATTTGACGTCACCGCTTTTACGTGGCTTTATAAAAGACGACGAAAAGCAAAGCCCGAGCATATTCGCGCCAATGCGACGTGAAGGATACAGGGCTATCAAACGATAAGATGGGGTGTCTGGGGTAATATGAACGAACAATATTCCGCATTGCGTAGTAATGTCAGTATGCTCGGCAAAGTGCTGGGAGAAACCATCAAGGATGCGTTGGGAGAACACATTCTTGAACGCGTAGAAACTATCCGTAAGTTGTCGAAATCTTCACGCGCTGGCAATGATGCTAACCGCCAGGAGTTGCTCACCACCTTACAAAATTTGTCGAACGACGAGCTGCTGCCCGTTGCGCGTGCGTTTAGTCAGTTCCTGAACCTGGCCAACACCGCCGAGCAATACCACAGCATTTCGCCGAAAGGCGAAGCTGCCAGCAACCCGGAAGTGATCGCCCGCACCCTGCGTAAACTGAAAAACCAGCCGGAACTGAGCGAAGACACCATCAAAAAAGCAGTGGAATCGCTGTCGCTGGAACTGGTCCTCACGGCTCACCCAACCGAAATTACCCGTCGTACACTGATCCACAAAATGGTGGAAGTGAACGCCTGTTTAAAACAGCTCGATAACAAAGATATCGCTGACTACGAACACAACCAGCTGATGCGTCGCCTGCGCCAGTTGATCGCCCAGTCATGGCATACCGATGAAATCCGTAAGCTGCGTCCAAGCCCGGTAGATGAAGCCAAATGGGGCTTTGCCGTAGTGGAAAACAGCCTGTGGCAAGGCGTACCAAATTACCTGCGCGAACTGAACGAACAACTGGAAGAGAACCTCGGCTACAAACTGCCCGTCGAATTTGTTCCGGTCCGTTTTACTTCGTGGATGGGCGGCGACCGCGACGGCAACCCGAACGTCACTGCCGATATCACCCGCCACGTCCTGCTACTCAGCCGCTGGAAAGCCACCGATTTGTTCCTGAAAGATATTCAGGTGCTGGTTTCTGAACTGTCGATGGTTGAAGCGACCCCTGAACTGCTGGCGCTGGTTGGCGAAGAAGGTGCCGCAGAACCGTATCGCTATCTGATGAAAAACCTGCGTTCTCGCCTGATGGCGACACA GGCATGGCTGGAAGCGCGCCTGAAAGGCGAAGAACTGCCAAAACCAGAAGGCCTGCTGACACAAAACGAAGAACTGTGGGAACCGCTCTACGCTTGCTACCAGTCACTTCAGGCGTGTGGCATGGGTATTATCGCCAACGGCGATCTGCTCGACACCCTGCGCCGCGTGAAATGTTTCGGCGTACCGCTGGTCCGTATTGATATCCGTCAGGAGAGCACGCGTCATACCGAAGCGCTGGGCGAGCTGACCCGCTACCTCGGTATCGGCGACTACGAAAGCTGGTCAGAGGCCGACAAACAGGCGTTCCTGATCCGCGAACTGAACTCCAAACGTCCGCTTCTGCCGCGCAACTGGCAACCAAGCGCCGAAACGCGCGAAGTGCTCGATACCTGCCAGGTGATTGCCGAAGCACCGCAAGGCTCCATTGCCGCCTACGTGATCTCGATGGCGAAAACGCCGTCCGACGTACTGGCTGTCCACCTGCTGCTGAAAGAAGCGGGTATCGGGTTTGCGATGCCGGTTGCTCCGCTGTTTGAAACCCTCGATGATCTGAACAACGCCAACGATGTCATGACCCAGCTGCTCAATATTGACTGGTATCGTGGCCTGATTCAGGGCAAACAGATGGTGATGATTGGCTATTCCGACTCAGCAAAAGATGCGGGAGTGATGGCAGCTTCCTGGGCGCAATATCAGGCACAGGATGCATTAATCAAAACCTGCGAAAAAGCGGGTATTGAGCTGACGTTGTTCCACGGTCGCGGCGGTTCCATTGGTCGCGGCGGCGCACCTGCTCATGCGGCGCTGCTGTCACAACCGCCAGGAAGCCTGAAAGGCGGCCTGCGCGTAACCGAACAGGGCGAGATGATCCGCTTTAAATATGGTCTGCCAGAAATCACCGTCAGCAGCCTGTCGCTTTATACCGGGGCGATTCTGGAAGCCAACCTGCTGCCACCGCCGGAGCCGAAAGAGAGCTGGCGTCGCATTATGGATGAACTGTCAGTCATCTCCTGCGATGTCTACCGCGGCTACGTACGTGAAAACAAAGATTTTGTGCCTTACTTCCGCTCCGCTACGCCGGAACAAGAACTGGGCAAACTGCCGTTGGGTTCACGTCCGGCGAAACGTCGCCCAACCGGCGGCGTCGAGTCACTACGCGCCATTCCGTGGATCTTCGCCTGGACGCAAAACCGTCTGATGCTCCCCGCCTGGCTGGGTGCAGGTACGGCGCTGCAAAAAGTGGTCGAAGACGGCAAACAGAGCGAGCTGGAGGCTATGTGCCGCGATTGGCCATTCT TCTCGACGCGTCTCGGCATGCTGGAGATGGTCTTCGCCAAAGCAGACCTGTGGCTGGCGGAATACTATGACCAACGCCTGGTAGACAAAGCACTGTGGCCGTTAGGTAAAGAGTTACGCAACCTGCAAGAAGAAGACATCAAAGTGGTGCTGGCGATTGCCAACGATTCCCATCTGATGGCCGATCTGCCGTGGATTGCAGAGTCTATTCAGCTACGGAATATTTACACCGACCCGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCAGAAAAAGAAGGCCAGGAACCGGATCCTCGCGTCGAACAAGCGTTAATGGTCACTATTGCCGGGATTGCGGCAGGTATGCGTAATACCGGCTAATCTTCCTCTTCTGCAAACCCTCGTGCTTTTGCGCGAGGGTTTTCTGAAATACTTCTGTTCTAACACCCTCGTTTTCAATATATTTCTGTCTGCATTTTATTCAAATTCTGAATATACCTTCAGATATCCTTAAGGGAAATCTCAATATAGACATAAAGGAAAATGGCAATAAAAGGTAACCAGCGCAAAGGTTTCTCCTGTAATAGCAGCCGGTTAACCCCGGCTACCTGAATGGGTTGCGAATCGCGTTTAGCTTATATTGTGGTCATTAGCAAAATTTCAAGATGTTTGCGCAACTATTTTTGGTAGTAATCCCAAAGCGGTGATCTATTTCACAAATTAATAATTAAGGGGTAAAAACCGACACTTAAAGTGATCCAGATTACGGTAGAAATCCTCAAGCAGCATATGATCTCGGGTATTCGGTCGATGCAGGGGATAATCGTCGGTCGAAAAACATTCGAAACCACATATATTCTGTGTGTTTAAAGCAAATCATTGGCAGCTTGAAAAAGAAGGTTCACATGTCAAACAACATTCGTATCGAAGAAGATCTGTTGGGTACCAGGGAAGTTCCAGCTGATGCCTACTATGGTGTTCACACTCTGAGAGCGATTGAAAACTTCTATATCAGCAACAACAAAATCAGTGATATTCCTGAATTTGTTCGCGGTATGGTAATGGTTAAAAAAGCCGCAGCTATGGCAAACAAAGAGCTGCAAACCATTCCTAAAAGTGTAGCGAATGCCATCATTGCCGCATGTGATGAAGTCCTGAACAACGGAAAATGCATGGATCAGTTCCCGGTAGACGTCTACCAGGGCGGCGCAGGTACTTCCGTAAACATGAACACCAACGAAGTGCTGGCCAATATCGGTCTGGAACTGATGGGTCACCAAAAAGGTGAATATCAGTACCTGAACC CGAACGACCATGTTAACAAATGTCAGTCCACTAACGACGCCTACCCGACCGGTTTCCGTATCGCAGTTTACTCTTCCCTGATTAAGCTGGTAGATGCGATTAACCAACTGCGTGAAGGCTTTGAACGTAAAGCTGTCGAATTCCAGGACATCCTGAAAATGGGTCGTACCCAGCTGCAGGACGCAGTACCGATGACCCTCGGTCAGGAATTCCGCGCTTTCAGCATCCTGCTGAAAGAAGAAGTGAAAAACATCCAACGTACCGCTGAACTGCTGCTGGAAGTTAACCTTGGTGCAACGGCAATCGGTACTGGTCTGAACACGCCGAAAGAGTACTCTCCGCTGGCAGTGAAAAAACTGGCTGAAGTTACTGGCTTCCCATGCGTACCGGCTGAAGACCTGATCGAAGCGACCTCTGACTGCGGCGCTTATGTTATGGTTCACGGCGCGCTGAAACGCCTGGCTGTGAAGATGTCCAAAATCTGTAACGACCTGCGCTTGCTCTCTTCAGGCCCACGTGCCGGCCTGAACGAGATCAACCTGCCGGAACTGCAGGCGGGCTCTTCCATCATGCCAGCTAAAGTAAACCCGGTTGTTCCGGAAGTGGTTAACCAGGTATGCTTCAAAGTCATCGGTAACGACACCACTGTTACCATGGCAGCAGAAGCAGGTCAGCTGCAGTTGAACGTTATGGAGCCGGTCATTGGCCAGGCCATGTTCGAATCCGTTCACATTCTGACCAACGCTTGCTACAACCTGCTGGAAAAATGCATTAACGGCATCACTGCTAACAAAGAAGTGTGCGAAGGTTACGTTTACAACTCTATCGGTATCGTTACTTACCTGAACCCGTTCATCGGTCACCACAACGGTGACATCGTGGGTAAAATCTGTGCCGAAACCGGTAAGAGTGTACGTGAAGTCGTTCTGGAACGCGGTCTGTTGACTGAAGCGGAACTTGACGATATTTTCTCCGTACAGAATCTGATGCACCCGGCTTACAAAGCAAAACGCTATACTGATGAAAGCGAACAGTAATCGTACAGGGTAGTACAAATAAAAAAGGCACAGATCACAGGCATAATTTTCAGTACGTTATAGGGCGTTTGTTACTAATTTATTTTAACGGAGTAACATTTAGCTCGTACATGAGCAGCTTGTGTGGCTCCTGACACAGGCAAACCATCATCAATAAAACCGATGGAAGGGAATATCATGCGAATTGGCATACCAAGAGAACGGTTAACCAATGAAACCCGTGTTGCAGCAACGCCAAAAACAGTGGAACAGCTGCTGAAA CTGGGTTTTACCGTCGCGGTAGAGAGCGGCGCGGGTCAACTGGCAAGTTTTGACGATAAAGCGTTTGTGCAAGCGGGCGCTGAAATTGTAGAAGGGAATAGCGTCTGGCAGTCAGAGATCATTCTGAAGGTCAATGCGCCGTTAGATGATGAAATTGCGTTACTGAATCCTGGGACAACGCTGGTGAGTTTTATCTGGCCTGCGCAGAATCCGGAATTAATGCAAAAACTTGCGGAACGTAACGTGACCGTGATGGCGATGGACTCTGTGCCGCGTATCTCACGCGCACAATCGCTGGACGCACTAAGCTCGATGGCGAACATCGCCGGTTATCGCGCCATTGTTGAAGCGGCACATGAATTTGGGCGCTTCTTTACCGGGCAAATTACTGCGGCCGGGAAAGTGCCACCGGCAAAAGTGATGGTGATTGGTGCGGGTGTTGCAGGTCTGGCCGCCATTGGCGCAGCAAACAGTCTCGGCGCGATTGTGCGTGCATTCGACACCCGCCCGGAAGTGAAAGAACAAGTTCAAAGTATGGGCGCGGAATTCCTCGAGCTGGATTTTAAAGAGGAAGCTGGCAGCGGCGATGGCTATGCCAAAGTGATGTCGGACGCGTTCATCAAAGCGGAAATGGAACTCTTTGCCGCCCAGGCAAAAGAGGTCGATATCATTGTCACCACCGCGCTTATTCCAGGCAAACCAGCGCCGAAGCTAATTACCCGTGAAATGGTTGACTCCATGAAGGCGGGCAGTGTGATTGTCGACCTGGCAGCCCAAAACGGCGGCAACTGTGAATACACCGTGCCGGGTGAAATCTTCACTACGGAAAATGGTGTCAAAGTGATTGGTTATACCGATCTTCCGGGCCGTCTGCCGACGCAATCCTCACAGCTTTACGGCACAAACCTCGTTAATCTGCTGAAACTGTTGTGCAAAGAGAAAGACGGCAATATCACTGTTGATTTTGATGATGTGGTGATTCGCGGCGTGACCGTGATCCGTGCGGGCGAAATTACCTGGCCGGCACCGCCGATTCAGGTATCAGCTCAGCCGCAGGCGGCACAAAAAGCGGCACCGGAAGTGAAAACTGAGGAAAAATGTACCTGCTCACCGTGGCGTAAATACGCGTTGATGGCGCTGGCAATCATTCTTTTTGGCTGGATGGCAAGCGTTGCGCCGAAAGAATTCCTTGGGCACTTCACCGTTTTCGCGCTGGCCTGCGTTGTCGGTTATTACGTGGTGTGGAATGTATCGCACGCGCTGCATACACCGTTGATGTCGGTCACCAACGCGATTTC AGGGATTATTGTTGTCGGAGCACTGTTGCAGATTGGCCAGGGCGGCTGGGTTAGCTTCCTTAGTTTTATCGCGGTGCTTATAGCCAGCATTAATATTTTCGGTGGCTTCACCGTGACTCAGCGCATGCTGAAAATGTTCCGCAAAAATTAAGGGGTAACATATGTCTGGAGGATTAGTTACAGCTGCATACATTGTTGCCGCGATCCTGTTTATCTTCAGTCTGGCCGGTCTTTCGAAACATGAAACGTCTCGCCAGGGTAACAACTTCGGTATCGCCGGGATGGCGATTGCGTTAATCGCAACCATTTTTGGACCGGATACGGGTAATGTTGGCTGGATCTTGCTGGCGATGGTCATTGGTGGGGCAATTGGTATCCGTCTGGCGAAGAAAGTTGAAATGACCGAAATGCCAGAACTGGTGGCGATCCTGCATAGCTTCGTGGGTCTGGCGGCAGTGCTGGTTGGCTTTAACAGCTATCTGCATCATGACGCGGGAATGGCACCGATTCTGGTCAATATTCACCTGACGGAAGTGTTCCTCGGTATCTTCATCGGGGCGGTAACGTTCACGGGTTCGGTGGTGGCGTTCGGCAAACTGTGTGGCAAGATTTCGTCTAAACCATTGATGCTGCCAAACCGTCACAAAATGAACCTGGCGGCTCTGGTCGTTTCCTTCCTGCTGCTGATTGTATTTGTTCGCACGGACAGCGTCGGCCTGCAAGTGCTGGCATTGCTGATAATGACCGCAATTGCGCTGGTATTCGGCTGGCATTTAGTCGCCTCCATCGGTGGTGCAGATATGCCAGTGGTGGTGTCGATGCTGAACTCGTACTCCGGCTGGGCGGCTGCGGCTGCGGGCTTTATGCTCAGCAACGACCTGCTGATTGTGACCGGTGCGCTGGTCGGTTCTTCGGGGGCTATCCTTTCTTACATTATGTGTAAGGCGATGAACCGTTCCTTTATCAGCGTTATTGCGGGTGGTTTCGGCACCGACGGCTCTTCTACTGGCGATGATCAGGAAGTGGGTGAGCACCGCGAAATCACCGCAGAAGAGACAGCGGAACTGCTGAAAAACTCCCATTCAGTGATCATTACTCCGGGGTACGGCATGGCAGTCGCGCAGGCGCAATATCCTGTCGCTGAAATTACTGAGAAATTGCGCGCTCGTGGTATTAATGTGCGTTTCGGTATCCACCCGGTCGCGGGGCGTTTGCCTGGACATATGAACGTATTGCTGGCTGAAGCAAAAGTACCGTATGACATCGTGCTGGAAATGGACGAGATCAATGATGACTTT GCTGATACCGATACCGTACTGGTGATTGGTGCTAACGATACGGTTAACCCGGCGGCGCAGGATGATCCGAAGAGTCCGATTGCTGGTATGCCTGTGCTGGAAGTGTGGAAAGCGCAGAACGTGATTGTCTTTAAACGTTCGATGAACACTGGCTATGCTGGTGTGCAAAACCCGCTGTTCTTCAAGGAAAACACCCACATGCTGTTTGGTGACGCCAAAGCCAGCGTGGATGCAATCCTGAAAGCTCTGTAACCCTGACGGCCTCTGCTGAGGCCGTCACTCTTTATTGAGA-3’；
(3)筛选步骤(2)中得到的所述温敏质粒，并将正确的质粒转入实施例1中得到的所述菌株的感受态细胞中，然后，在30℃下进行培养，将成功转入质粒的菌株置于阿拉伯糖诱导条件下继续培养，使其表达Lambda噬菌体重组酶、gRNA和Cas9DNA内切酶，再筛选目标突变体菌落；
(4)将步骤(3)中筛选确认的所述突变体菌落在42℃下培养消除CRISPR/Cas9质粒，同时消除质粒所导致的卡纳霉素抗性，即得到所述菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB)。
实施例3
在染色体DNA上二拷贝整合表达thrA*、ppc、aspA、pntA和pntB的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB)的构建。
本实施例构建的L-高丝氨酸生产菌株为在染色体DNA上同时二次整合表达thrA*、ppc、aspA、pntA和pntB的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA -pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB)的构建，采用CRISPR/Cas9基因编辑技术实现菌株的构建，对yidJ基因的下游插入实施例2中所述SEQ ID No.15片段，其构建方法与实施例2的方法基本类似，区别仅在于：以原始菌实施例2产物菌株为操作对象，整合位置为yidJ基因下游。具体为：将实施例2中所述步骤(2)中采用的引物由所述pSOE-cadA-H1-f、pSOE-cadA-H1-r、pSOE-cadA-H2-f、pSOE-cadA-H2-r、pcadA-N20-f、pcadA-N20-r,替换为pSOE-yidJ-H1-f、pSOE-yidJ-H1-r、pSOE-yidJ-H2-f、pSOE-yidJ-H2-r、pyidJ-N20-f、pyidJ-N20-r；
其中，所述引物pSOE-yidJ-H1-f具有如SEQ ID No.16所示的序列，所述引物pSOE-yidJ-H1-r具有如SEQ ID No.17所示的序列，所述引物pSOE-yidJ-H2-f具有如SEQ ID No.18所示的序列，所述引物pSOE-yidJ-H2-r具有如SEQ ID No.19所示的序列，所述引物pyidJ-N20-f具有如SEQ ID No.20所示的序列，所述引物pyidJ-N20-r具有如SEQ ID No.21所示的序列，具体如下：
SEQ ID No.16:5’-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3’；
SEQ ID No.17：5’-CTTCCAGTGCCAATATGAGCCGGCCTACCGACACAAG-3’；
SEQ ID No.18：5’-GGCCGTCACTCTTTATTGAGAGTGTCATAGTCGCGTACAAC-3’；
SEQ ID No.19：5’-GAGCCGGCGATGTATGACGACATC-3’；
SEQ ID No.20：5’-AGCGGTTGTACGCGACTATGACAC-3’；
SEQ ID No.21：5’-AAACGTGTCATAGTCGCGTACAAC-3’。
实施例4
在染色体DNA上三拷贝表达thrA*、ppc、aspA、pntA和pntB 的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB)的构建。
本实施例构建的L-高丝氨酸生产菌株为在染色体DNA上同时三次整合表达thrA*、ppc、aspA、pntA和pntB的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB)的构建，采用CRISPR/Cas9基因编辑技术实现菌株的构建，对atpC基因的下游插入实施例2中所述SEQ ID No.15片段，其构建方法与实施例2的方法基本类似，区别仅在于：以原始菌实施例3产物菌株为操作对象，整合位置为atpC基因下游。具体的：将实施例2中所述步骤(2)中采用的引物由所述pSOE-cadA-H1-f、pSOE-cadA-H1-r、pSOE-cadA-H2-f、pSOE-cadA-H2-r、pcadA-N20-f、pcadA-N20-r,替换为pSOE-atpC-H1-f、pSOE-atpC-H1-r、pSOE-atpC-H2-f、pSOE-atpC-H2-r、patpC-N20-f、patpC-N20-r；
其中，所述引物pSOE-atpC-H1-f具有如SEQ ID No.22所示的序列，所述引物pSOE-atpC-H1-r具有如SEQ ID No.23所示的序列，所述引物pSOE-atpC-H2-f具有如SEQ ID No.24所示的序列，所述引物pSOE-atpC-H2-r具有如SEQ ID No.25所示的序列，所述引物patpC-N20-f具有如SEQ ID No.26所示的序列，所述引物patpC-N20-r具有如SEQ ID No.27所示的序列，具体如下：
SEQ ID No.22:5’-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3’；
SEQ ID No.23：5’-CTTCCAGTGCCAATATGAGCGAAAGCGCCATTTTCGACC-3’；
SEQ ID No.24：5’-GCCGTCACTCTTTATTGAGAGTCAGCGGGGATAATCCGT-3’；
SEQ ID No.25：5’-GAGCCATAACCGGTCGGATCATCC-3’；
SEQ ID No.26：5’-AGCGGTTGTACGCGACTATGACAC-3’；
SEQ ID No.27：5’-AAACGTGTCATAGTCGCGTACAAC-3’。
实施例5
在染色体DNA继续整合thrA*的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA：：thrA*)的构建。
本实施例构建的L-继续整合thrA*的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA：：thrA*)，采用CRISPR/Cas9基因编辑技术实现菌株的构建，在dacA基因内部插入thrA*表达盒，其构建方法与实施例2的方法基本类似，区别仅在于：以原始菌实施例4产物菌株为操作对象，整合位置为反向插入dacA基因内部。具体的：插入片段是使用引物pSOE-thrA-f和pSOE-thrA-r从MG1655基因组通过PCR扩增获得，且将实施例2中所述步骤(2)中采用的引物由所述pSOE-cadA-H1-f、pSOE-cadA-H1-r、pSOE-cadA-H2-f、pSOE-cadA-H2-r、pcadA-N20-f、pcadA-N20-r,替换为pSOE-dacA-H1-f、pSOE-dacA-H1-r、pSOE-dacA-H2-f、pSOE-dacA-H2-r、pdacA-N20-f、pdacA-N20-r；
其中，所述引物pSOE-thrA-f具有如SEQ ID No.1所示的序列， 所述引物pSOE-thrA-r具有如SEQ ID No.28所示的序列，所述引物pSOE-dacA-H1-f具有如SEQ ID No.29所示的序列，所述引物pSOE-dacA-H1-r具有如SEQ ID No.30所示的序列，所述引物pSOE-dacA-H2-f具有如SEQ ID No.31所示的序列，所述引物pSOE-dacA-H2-r具有如SEQ ID No.32所示的序列，所述引物pdacA-N20-f具有如SEQ ID No.33所示的序列，所述引物pdacA-N20-r具有如SEQ ID No.34所示的序列，具体如下：
SEQ ID No.28:5’-AAGCTTTACGCGAACGAGC-3’；
SEQ ID No.29:5’-GTGCGAAATTTTCATTGTTGCTCCTTTGC-3’；
SEQ ID No.30：5’-GCTCGTTCGCGTAAAGCTTGTACGTCACGGATCAATGC-3’；
SEQ ID No.31：5’-CTTCCAGTGCCAATATGAGCCGAATGAATACTCGATCTATAAAGAAAAAG-3’；
SEQ ID No.32：5’-GAGCAACCAAACCAGTGATGGAAC-3’；
SEQ ID No.33：5’-AGCGAGTATTCATTCGGTACGTCA-3’；
SEQ ID No.34：5’-AAACTGACGTACCGAATGAATACT-3’。
实施例6
在染色体DNA继续整合thrA*的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA::thrA*，ΔbcsB::thrA*)的构建。
本实施例采用CRISPR/Cas9基因编辑技术实现菌株的构建，在bcsB基因内部插入thrA*表达盒，其构建方法与实施例5的方法基本类似，区别仅在于：以原始菌实施例5产物菌株为操作对象，整合位置为反向插入bcsB基因内部。具体的：插入片段同例5，且将 实施例5中所述步骤(2)中采用的引物由所述pSOE-dacA-H1-f、pSOE-dacA-H1-r、pSOE-dacA-H2-f、pSOE-dacA-H2-r、pdacA-N20-f、pdacA-N20-r替换为pSOE-bcsB-H1-f、pSOE-bcsB-H1-r、pSOE-bcsB-H2-f、pSOE-bcsB-H2-r、pbcsB-N20-f、pbcsB-N20-r；
其中，所述引物pSOE-bcsB-H1-f具有如SEQ ID No.35所示的序列，所述引物pSOE-bcsB-H1-r具有如SEQ ID No.36所示的序列，所述引物pSOE-bcsB-H2-f具有如SEQ ID No.37所示的序列，所述引物pSOE-bcsB-H2-r具有如SEQ ID No.38所示的序列，所述引物pbcsB-N20-f具有如SEQ ID No.39所示的序列，所述引物pbcsB-N20-r具有如SEQ ID No.40所示的序列，具体如下：
SEQ ID No.35:5’-GTGCTCGTTACTGCCTGTCCAGTC-3’；
SEQ ID No.36:5’-GCTCGTTCGCGTAAAGCTTCAGTTTGACGTAAGGATTCTGC-3’；
SEQ ID No.37：5’-CTTCCAGTGCCAATATGAGCCTGGTGGTGTTTGGTCGTGACG-3’；
SEQ ID No.38：5’-GAGCGCTGGAAGGTAATACAGTTATCCAC-3’；
SEQ ID No.39：5’-AGCGGAATCCTTACGTCAAACTGC-3’；
SEQ ID No.40：5’-AAACGCAGTTTGACGTAAGGATTC-3’；
实施例7
在染色体DNA整合aspC的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA::thrA*，ΔbcsB::thrA*，ΔmenH::aspC)的构建。
本实施例构建在染色体DNA整合aspC的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA：：thrA*，ΔmenH::aspC)。采用CRISPR/Cas9基因编辑技术实现在menH基因内部插入aspC表达盒。其构建方法与实施例5的方法基本类似，区别仅在于：以原始菌实施例6产生的菌株为操作对象，将天然启动子下的aspC基因反向插入到menH基因内部。具体的：插入片段由引物pSOE-aspC-f和pSOE-aspC-r从MG1655基因组通过PCR扩增获得，且将实施例步骤(2)中采用的引物由所述pSOE-dacA-H1-f、pSOE-dacA-H1-r、pSOE-dacA-H2-f、pSOE-dacA-H2-r、pdacA-N20-f、pdacA-N20-r替换为pSOE-menH-H1-f、pSOE-menH-H1-r、pSOE-menH-H2-f、pSOE-menH-H2-r、pmenH-N20-f、pmenH-N20-r；
其中，所述引物pSOE-aspC-f具有如SEQ ID No.41所示的序列，所述引物pSOE-aspC-r具有如SEQ ID No.42所示的序列，所述引物pSOE-menH-H1-f具有如SEQ ID No.43所示的序列，所述引物pSOE-menH-H1-r具有如SEQ ID No.44所示的序列，所述引物pSOE-menH-H2-f具有如SEQ ID No.45所示的序列，所述引物pSOE-menH-H2-r具有如SEQ ID No.46所示的序列，所述引物pmenH-N20-f具有如SEQ ID No.47所示的序列，所述引物pmenH-N20-r具有如SEQ ID No.48所示的序列，具体如下：
SEQ ID No.41:5’-CTGACCGTACCAACCTGCA-3’；
SEQ ID No.42:5’-CCTGATAAGCGTAGCGCAT-3’；
SEQ ID No.43:5’-GTGCGTTTGCCGACTACTCACG-3’；
SEQ ID No.44：5’-TGCAGGTTGGTACGGTCAGGCGCTAAGGTT AGCACGTAAAT-3’；
SEQ ID No.45：5’-ATGCGCTACGCTTATCAGGCCGCACATTTGCGTTTTATTAT-3’；
SEQ ID No.46：5’-GAGCACATTCAGGTGATGTACGCC-3’；
SEQ ID No.47：5’-AGCGACGCAAATGTGCGGGCGCTA-3’；
SEQ ID No.48：5’-AAACTAGCGCCCGCACATTTGCGT-3’。
实施例8
在染色体DNA整合aspC的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA::thrA*，ΔbcsB::thrA*，ΔmenH::aspC，ΔyddB::asd)的构建。
本实施例构建在染色体DNA整合asd的基因工程菌株MG1655(ΔthrB,rhtA23,ΔthrL,thrA*(G433R)，ΔcadA::thrA*-ppc-aspA-pntAB，ΔyidJ::thrA*-ppc-aspA-pntAB，ΔatpC::thrA*-ppc-aspA-pntAB，ΔdacA::thrA*，ΔbcsB::thrA*，ΔmenH::aspC，ΔyddB::asd)。采用CRISPR/Cas9基因编辑技术实现在yddB基因内部插入asd表达盒。其构建方法与实施例5的方法基本类似，区别仅在于：以原始菌实施例7产生的菌株为操作对象，将天然启动子下的asd基因插入到yddB基因内部。具体的：插入片段由引物pSOE-asd-f和pSOE-asd-r从MG1655基因组通过PCR扩增获得，且将实施例步骤(2)中采用的引物由所述pSOE-menH-H1-f、pSOE-menH-H1-r、pSOE-menH-H2-f、pSOE-menH-H2-r、pmenH-N20-f、pmenH-N20-r替换为pSOE-yddB-H1-f、pSOE-yddB-H1-r、 pSOE-yddB-H2-f、pSOE-yddB-H2-r、pyddB-N20-f、pyddB-N20-r；
其中，所述引物pSOE-asd-f具有如SEQ ID No.49所示的序列，所述引物pSOE-asd-r具有如SEQ ID No.50所示的序列，所述引物pSOE-yddB-H1-f具有如SEQ ID No.51所示的序列，所述引物pSOE-yddB-H1-r具有如SEQ ID No.52所示的序列，所述引物pSOE-yddB-H2-f具有如SEQ ID No.53所示的序列，所述引物pSOE-yddB-H2-r具有如SEQ ID No.54所示的序列，所述引物pyddB-N20-f具有如SEQ ID No.55所示的序列，所述引物pyddB-N20-r具有如SEQ ID No.56所示的序列，具体如下：
SEQ ID No.41:5’-GGATCCATAATCAGGATCAATAAAACT-3’；
SEQ ID No.42:5’-AGGATCCGCAAAATGGCC-3’；
SEQ ID No.43:5’-GTGCGGCTCTTCAGGAAGTACTTATTAC-3’；
SEQ ID No.44：5’-GTTTTATTGATCCTGATTATGGATCCCAGTCCACGCATCGGAATAG-3’；
SEQ ID No.45：5’-GGCCATTTTGCGGATCCTAACGCCATAACCAGTCTGAAT-3’；
SEQ ID No.46：5’-GAGCCTGAACGAATGCGTTTTGG-3’；
SEQ ID No.47：5’-AGCGATGGCGTTCAGTCCACGCAT-3’；
SEQ ID No.48：5’-AAACATGCGTGGACTGAACGCCAT-3’。
实施例9
L-高丝氨酸生产菌株发酵生产L-高丝氨酸
本实施例采用实施例1中制备得到的菌株进行L-高丝氨酸的发酵生产，其生产方法具体可参见中国专利申请CN201710106474.8， 发酵获得高丝氨酸5.3g/L。
作为本实施例可替换的实施方式，所述实施例1最终制备得到的菌株还可替换为实施例2-8中最终制备得到的菌株。
实施例10 L-高丝氨酸生产菌株发酵的结果统计
本实施例优选采用实施例1和实施例4、实施例8构建菌株进行L-高丝氨酸的发酵生产，与MG1655菌对比。其生产方法与实施例10中所述的生产方法完全一致。
按照采用实施例4中制备得到的菌株进行发酵的实验编号为Hom4、采用实施例8中制备得到的菌株进行发酵的实验编号为Hom8，分别对L-高丝氨酸的产量进行检测和计算，其结果如下：
菌种 L-高丝氨酸产量(g/L)
MG1655 0
Hom1 4.8±1.2
Hom4 53.8±5.9
Hom8 88.1±11.4
由上述结果可知，采用本发明的菌株发酵生产L-高丝氨酸的产量最高达近100g/L，表明本发明对各基因的敲除、弱化、增强、过表达、突变起到了明显的效果。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种L-高丝氨酸的生产菌株，其特征在于，其具有单一稳定的染色体，不含质粒形式或其他游离DNA载体，其染色体DNA上一个或多个与L-苏氨酸合成相关的基因被敲除或弱化，和/或被突变以增强，和/或其染色体DNA上整合拷贝一个或多个与L-高丝氨酸代谢途径相关的基因。
  2. 如权利要求1所述的L-高丝氨酸的生产菌株，其特征在于，所述被敲除或弱化的基因为thrB和/或thrL；所述被突变以增强的基因为thrA和/或rhtA，所述整合拷贝的基因为thrA*、ppc、pntA、pntB、asd、aspA和aspC中的一个或多个。
  3. 如权利要求1所述的L-高丝氨酸的生产菌株，其特征在于，所述L-高丝氨酸生产菌株由大肠杆菌种属构建。
  4. 如权利要求3所述的L-高丝氨酸的生产菌株，其特征在于，所述菌株为大肠杆菌K-12野生型MG1655。
  5. 一种L-高丝氨酸生产菌株的构建方法，包括以下步骤中一步或多步：
    A、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因被敲除或弱化；
    B、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因通过点突变过表达或增强功能；
    C、构建菌株中一个或多个与L-高丝氨酸代谢途径相关的基因以 多拷贝的形式整合到染色体DNA上过表达。
  6. 如权利要求5所述的构建方法，其特征在于，所述步骤A中所述的被敲除或弱化的基因为thrB和/或thrL；所述步骤B中所述的被突变的基因为thrA和/或rhtA；所述步骤C中所述的通过提高染色体拷贝数来过表达的基因为thrA*、ppc、pntA、pntB、asd、aspA和aspC中的一个或多个。
  7. 如权利要求6所述的构建方法，其特征在于，步骤B中，thrA和/或rhtA基因分别被突变为thrA*和/或rthA23基因。
  8. 如权利要求5所述的构建方法，其特征在于，所述的构建菌株属于大肠杆菌种属。
  9. 如权利要求8所述的构建方法，其特征在于，所述菌株为大肠杆菌K-12野生型MG1655。
  10. 如权利要求1-4所述的L-高丝氨酸生产菌株在生产L-高丝氨酸中的应用。
PCT/CN2017/109009 2017-10-13 2017-11-01 一种l-高丝氨酸的生产菌株及其构建方法和应用 WO2019071668A1 (zh)

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