GENETICALLY ENGINEERED BACTERIUM FOR PRODUCING L-HISTIDINE AND USE THEREOF

Abstract

A genetically engineered bacterium includes a genome of the Eschericia coli and a mutant encoding gene hisG* of a Corynebacterium glutamicum ATP phosphoribosyl transferase HisG on the genome, and the gene hisG* is strongly expressed to enhance activity of a key enzyme HisG for histidine synthesis. The gene hisG* has a nucleotide sequence as shown in SEQ ID NO: 1; a copy number of histidine operon genes hisDBCHAFI of the Eschericia coli is further increased on the genome to enhance a terminal synthetic route of histidine; an encoding gene lysE from an arginine/lysine transportprotein of the Corynebacterium glutamicum is further integrated to the genome and strongly expressed to promote the intracellular histidine secrete to the extracellular space; and an encoding gene rocG of glutamate dehydrogenase of Bacillus subtilis is further integrated to the genome and strongly expressed to promote generation of histidine.

Claims

1. A genetically engineered bacterium for producing L-histidine, wherein, the genetically engineered bacterium comprises a genome of Escherichia coli and a mutant encoding gene hisG* of a Corynebacterium glutamicum ATP phosphoribosyl transferase HisG, and the mutant encoding gene hisG* is integrated to the genome and strongly expressed, wherein the mutant encoding gene hisG* has a nucleotide sequence as shown in SEQ ID NO: 1; an expression of histidine operon genes of the Escherichia coli on the genome is further enhanced, wherein the histidine operon genes comprise hisD, hisB, hisC, hisH, hisA, hisF and hisI; an encoding gene lysE from an arginine/lysine transport protein of the Corynebacterium glutamicum is further integrated to the genome and strongly expressed; and an encoding gene rocG of glutamate dehydrogenase of Bacillus subtilis is further integrated to the genome and strongly expressed.

2. The genetically engineered bacterium for producing L-histidine according to claim 1, wherein, the Escherichia coli is E. coli W3110.

3. The genetically engineered bacterium for producing L-histidine according to claim 1, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to at least two gene loci on the genome and is initiated by a strong promoter.

4. The genetically engineered bacterium for producing L-histidine according to claim 2, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to at least two gene loci on the genome and is initiated by a strong promoter.

5. The genetically engineered bacterium for producing L-histidine according to claim 1, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to a tdcD gene locus and a ylbE gene locus on the genome respectively and is initiated by a promoter P.sub.trc; the histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI of the Escherichia coli are successively integrated to a yghX gene locus on the genome in sequence and are initiated by the promoter P.sub.trc; the encoding gene lysE of the arginine/lysine transport protein is integrated to a yjiT gene locus on the genome and is initiated by the promoter P.sub.trc; and the encoding gene rocG of the glutamate dehydrogenase is integrated to a yjhE gene locus on the genome of the Eschericia coli and is initiated by the promoter P.sub.trc.

6. The genetically engineered bacterium for producing L-histidine according to claim 2, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to a tdcD gene locus and a ylbE gene locus on the genome respectively and is initiated by a promoter P.sub.trc; the histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI of the Eschericia coli are successively integrated to a yghX gene locus on the genome in sequence and are initiated by the promoter P.sub.trc; the encoding gene lysE of the arginine/lysine transport protein is integrated to a yjiT gene locus on the genome and is initiated by the promoter P.sub.trc; and the encoding gene rocG of the glutamate dehydrogenase is integrated to a yjhE gene locus on the genome of the Eschericia coli and is initiated by the promoter P.sub.trc.

7. A method for fermentation production of L-histidine, comprising the step of using the genetically engineered bacterium according to claim 1.

8. The method according claim 7, wherein, the Eschericia coli is E. coli W3110.

9. The method according to claim 7, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to at least two gene loci on the genome and is initiated by a strong promoter.

10. The method according to claim 8, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to at least two gene loci on the genome and is initiated by a strong promoter.

11. The method according to claim 7, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to a tdcD gene locus and a ylbE gene locus on the genome respectively and is initiated by a promoter P.sub.trc; the histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI of the Escherichia coli are successively integrated to a yghX gene locus on the genome in sequence and are initiated by the promoter P.sub.trc; the encoding gene lysE of the arginine/lysine transport protein is integrated to a yjiT gene locus on the genome and is initiated by the promoter P.sub.trc; and the encoding gene rocG of the glutamate dehydrogenase is integrated to a yjhE gene locus on the genome of the Eschericia coli and is initiated by the promoter P.sub.trc.

12. The method according to claim 8, wherein, the mutant encoding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG is integrated to a tdcD gene locus and a ylbE gene locus on the genome respectively and is initiated by a promoter P.sub.trc; the histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI of the Escherichia coli are successively integrated to a yghX gene locus on the genome in sequence and are initiated by the promoter P.sub.trc; the encoding gene lysE of the arginine/lysine transport protein is integrated to a yjiT gene locus on the genome and is initiated by the promoter P.sub.trc; and the encoding gene rocG of the glutamate dehydrogenase is integrated to a yjhE gene locus on the genome of the Escherichia coli and is initiated by the promoter P.sub.trc.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A is a pREDCas9 plasmid profile.

[0024] FIG. 1B is a pGRB plasmid profile.

[0025] FIG. 2 is an integrating fragment construction and verification electrophoretogram when hisG* is integrated to the tdcD gene locus, wherein, M: 1 kb DNA marker; 1: upstream homologous arm; 2: hisG* gene fragment; 3: downstream homologous arm; 4: overlapped fragment; 5: original bacterium contrast; 6: authenticating fragment for positive bacterium.

[0026] FIG. 3 is an integrating fragment construction and verification electrophoretogram when hisG* is integrated to the ylbE gene locus, wherein, M: 1 kb DNA marker; 1: upstream homologous arm; 2: hisG* gene fragment; 3: downstream homologous arm; 4: overlapped fragment; 5: original bacterium contrast; 6: authenticating fragment for positive bacterium.

[0027] FIG. 4 is a hisD integrating fragment construction and verification electrophoretogram, wherein, M: 1 kb DNA marker; 1: upstream homologous arm; 2: hisG* gene fragment; 3: downstream homologous arm; 4: overlapped fragment; 5: original bacterium contrast; 6: authenticating fragment for positive bacterium.

[0028] FIG. 5 is a hisC-hisB integrating fragment construction and verification electrophoretogram, wherein, M: 1 kb DNA marker; 1: hisC-upstream sequence-hisC-hisB fragment; 2: downstream homologous arm; 3: overlapped fragment; 4: original bacterium contrast; 5: authenticating fragment for positive bacterium.

[0029] FIG. 6 is a hisH-hisA-hisF-hisI integrating fragment construction and verification electrophoretogram, wherein, M: 1 kb DNA marker; 1: hisH-upstream sequence-hisH-hisA-hisF-hisI fragment; 2: downstream homologous arm; 3: overlapped fragment; 4: original bacterium contrast; 5: authenticating fragment for positive bacterium.

[0030] FIG. 7 is a Ptrc-lysE integrating fragment construction and verification electrophoretogram, wherein, M: 1 kb DNA marker; 1: upstream homologous arm; 2: lysE gene fragment; 3: downstream homologous arm; 4: overlapped fragment; 5: original bacterium contrast; 6: authenticating fragment for positive bacterium.

[0031] FIG. 8 is an integrating fragment construction and verification electrophoretogram when rocG is integrated to the yjhE gene locus, wherein, M: 1 kb DNA marker; 1: upstream homologous arm; 2: downstream homologous arm; 3: rocG gene fragment; 4: overlapped fragment; 5: original bacterium contrast; 6: authenticating fragment for positive bacterium.

[0032] FIG. 9 is a fermentation result of E. coli WHY3-1 in a 5 L tank.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present disclosure is described below through specific embodiments. Unless otherwise specified, the technical means used in the present disclosure are all methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the disclosure, which is only limited by the scope of the claims. For those skilled in the art, without departing from the spirit and scope of the present disclosure, various changes or modifications to the material composition and amount used in these embodiments also belong to the protection scope of the present disclosure.

[0034] Without public definition in the prior art and specific description, per cent “%” in the embodiments below means percent by volume. Per cent “% (m/v)” of a solution means gram of a solute in a 100 mL solution.

Embodiment 1

[0035] Construction of the strain E. coli WHY3-1

[0036] 1. Gene Editing Method

[0037] The gene editing method adopted in the present disclosure was carried out with reference to literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Eschericia coli using CRISPR—Cas9 meditated genome editing. Metabolic engineering, 2015, 31: 13-21.), the two plasmid profiles used by the method are shown in the FIGS. 1A-1B, wherein, pREDCas9 carried an eliminating system of a gRNA expression plasmid pGRB, a Red recombination system of a X, and a Cas9 protein expression system, spectinomycin resistance (the working concentration was 100 mg/L), culture was conducted at 32° C.; pGRB, taking pUC18 as a framework, included a promoter J23100, a gRNA-Cas9 binding region sequence and a terminator sequence, ampicillin resistance (the working concentration was 100 mg/L), and culture was conducted at 37° C.

[0038] The method includes the following specific steps:

[0039] 1.1 pGRB Plasmid Construction

[0040] The construction of plasmid pGRB aims to transcribe corresponding gRNA to form a complex with a Cas9 protein, and recognize a targeted gene target site is recognized by base pairing and PAM, so as to realize double chain breaking of a target DNA. The pGRB plasmid was constructed by adopting a method of recombining a DNA fragment containing a target sequence and a linear carrier fragment.

[0041] 1.1.1 Target Sequence Design

[0042] A target sequence (PAM:5′-NGG-3′) was designed by using CRISPR RGEN Tools.

[0043] 1.1.2 Preparation of the DNA Fragment Containing the Target Sequence

[0044] Designing a primer: 5′-linear carrier terminal sequence (15 bp)-restriction enzyme cutting site-target sequence(excluding PAM sequence)-linear carrier terminal sequence(15 bp)-3′ and a reverse complementary primer thereof were used for preparing the DNA fragment containing the target sequence by annealing of a single-strand DNA. A reaction condition: initial denaturation at 95° C. for 5 min; and annealing at 30-50° C. for 1 min. An annealing system is as follows:

[0045] Annealing System

TABLE-US-00001 Reaction system Volume (20 μL) Primer (10 μmol/L) 10 μL Reverse complementary primer (10 μmol/L) 10 μL

[0046] 1.1.3 Preparation of a Linear Carrier

[0047] The carrier was linearized by way of reverse PCR amplification.

[0048] 1.1.4 Recombination Reaction

[0049] A recombination system is shown in a table below. All used recombinases were ClonExpress® II One Step Cloning Kit serial enzymes, and a recombining condition was 37° C. for 30 min.

Recombination System

[0050]

TABLE-US-00002 Reaction system Volume (20 μL) 5*CE II Buffer 4 μL Linearized cloning vector 1 μL Insert fragment cloning vector 1 μL Exnase ® II 2 μL ddH2O 12 μL

[0051] 1.1.5 Transformation of Plasmid

[0052] 10 μL of a reaction liquid was taken and added into 100 μL, of DH5α competent cell, was then subjected to an ice-bath for 20 min after being mixed uniformly slightly, heat shock was conducted for 45-90 s at 42° C., the ice-bath was then conducted immediately for 2-3 min, and 900 μL of SOC was added for revival at 37° C. for 1 h. Centrifugation was conducted for 2 min at 8000 rpm, part of supernatant was abandoned, about 200 μL of the supernatant was reserved to resuspend thalli and then coated on a panel containing 100 mg/L of ampicillin, the panel was placed reversely and culture was performed overnight at 37° C. After a single colony grew on the panel, PCR authentication of the colony was performed and positive recons were selected.

[0053] 1.1.6 Cloning Authentication

[0054] A PCR positive bacterial colony was inoculated to the LB culture medium containing 100 mg/L of ampicillin, was cultured overnight and preserved, and then the plasmid was extracted for digestion authentication.

[0055] 1.2 Preparation of a Recombinant DNA Fragment

[0056] A recombinant fragment for knockout was comprised of upstream and downstream homologous arms (upstream homologous arm-downstream homologous arm) of gene to be knocked out, and a recombinant fragment for integration was comprised of upstream and downstream homologous arms on the integration site and gene fragment to be integrated (upstream homologous arm-target gene-downstream homologous arm). By using primer design software primer 5, upstream and downstream homologous arm primers (an amplification length was about 400-500 bp) was designed by taking upstream and downstream sequences of the gene to be knocked out or the site to be integrated as a template; and an amplification primer of the integrated gene was designed by taking the gene to be integrated as a template. After amplifying the upstream and downstream homologous arms and the target gene fragment by a PCR method, the recombinant fragment was prepared through overlapped PCR. A PCR system and method is as shown in the table below:

[0057] PCR Amplification System

TABLE-US-00003 Components Volume (50 μL) DNA template 1 μL Upstream primer (10 μmol/L) 1 μL Downstream primer (10 μmol/L) 1 μL dNTP mixture(10 mmol/L) 4 μL 5*Buffer 10 μL HS enzyme (5 U/μL) 0.5 μL ddH2O 32.5 μL

[0058] The overlapped PCR system is as shown in the table below:

[0059] Overlapped PCR amplification system

TABLE-US-00004 Components Volume (50 μL) Template 2 μL Upstream primer of upstream 1 μL homologous arm (10 μmol/L) Downstream primer of downstream 1 μL homologous arm (10 μmol/L) dNTP mixture(10 mmol/L) 4 μL 5*Buffer 10 μL HS enzyme (5 U/μL) 0.5 μL ddH2O 31.5 μL Note: the template was comprised of the amplification fragments of upstream and downstream homologous arms and the target gene at equal mole, and the total amount did not exceed 10 mg.

[0060] PCR reaction condition (Takara Bio PrimeSTAR HS enzyme): initial denaturation was conducted for 5 min at 95° C.; then 30 cycles were conducted: denaturation for 10 min at 98° C., annealing for 15 s ((Tm-3/5)° C.), and extension at 72° C. (the enzyme activity extends about 1 kb every 1 min); and extension was further conducted for 10 min at 72° C.; and was kept at 4° C.

[0061] 1.3 Transformation of Plasmid and Recombinant DNA Fragment

[0062] 1.3.1 Transformation of pREDCas9

[0063] The pREDCas9 plasmid was electrotransformed to an electrotransformed competence of W3110 by an electrotransformation method, the thalli was coated on the LB panel containing spectinomycin after revival culture, and was cultured overnight at 32° C. The single colony grew on a resistant panel was conducted colony PCR by authenticating primer, and the positive recons were selected.

[0064] 1.3.2 Electrotransformed Competence Preparation of Target Strain Containing pREDCas9

[0065] When it was cultured to OD.sub.600=0.1-0.2 at 32° C., 0.1 M of IPTG (enabling the final concentration thereof to be 0.1 mM) was added, and culture was further conducted till OD.sub.600=0.6-0.7 to prepare competence. The purpose of adding IPTG was to induce expression of the recombinase on the pREDCas9 plasmid. A culture medium needed for preparing competence and a preparation process were operated with reference to a conventional standard.

[0066] 1.3.3 Transformation of pGRB and Recombinant DNA Fragment

[0067] pGRB and the donor DNA fragment were electrotransformed to the electrotransformed competent cell containing the pREDCas9 simultaneously. The electrotransformed thalli after revival culture was coated on the LB panel containing ampicillin and spectinomycin and was cultured overnight at 32° C. Colony PCR authentication was conducted by using the upstream primer of the upstream homologous arm and the downstream primer of the downstream homologous arm, or a designed special authenticating primer, and the positive recons were selected and the bacterium was preserved.

[0068] 1.4 Elimination of Plasmid

[0069] 1.4.1 Elimination of pGRB

[0070] The positive recons were placed in the LB culture medium containing 0.2% arabinose and was cultured overnight, then was coated on the LB panel containing spectinomycin resistance after being diluted properly and was cultured overnight at 32° C. The sample points on the LB panel containing ampicillin and spectinomycin resistance were contrasted, and the single-colonies not growing on the panel containing ampicillin and growing on the panel containing spectinomycin were selected for bacterium preservation.

[0071] 1.4.2 Elimination of pREDCas9 Plasmid

[0072] The positive recons were transferred to the resistance-free LB liquid medium and was cultured overnight at 42° C., then was coated on the resistance-free LB panel after being diluted properly and was cultured overnight at 37° C. The sample points on the LB panel containing spectinomycin and the resistance-free LB panel were contrasted, and the single-colonies not growing on the panel containing spectinomycin resistance and growing on the resistance-free panel were selected for bacterium preservation.

[0073] 2. All primers in the strain construction process are as shown in the table below:

TABLE-US-00005 SEQ ID Primers NO: Sequences (5′-3′) UP-tdcD-S  2 TATTCAAAACAGAAAAACCGTCAGT UP-tdcD-A  3 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCC GGATGATTAATTGTCAAGGCAAATCGCGAAGAAGTACA G hisG*-S  4 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGACCATGTTGAAAATCGCTGTC CCA hisG*-A  5 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT TCGACTGAGCCTTTCGTTTTATTTGCTAGTTCTGCGCGTG CAAAA DN-tdcD-S  6 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCCTGAGTAGGACAAATTGAATAATCGCTCTAA CTCCTGTG DN-tdcD-A  7 CGCCCTGGTTATGGGTTTT UP-ylbE-S  8 ACCCAACCTTACGCAACCAG UP-ylbE-A  9 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCC GGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT DK-ylbE-S 10 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGA A DN-ylbE-A 11 GGCGTAACTCAGCAGGCAG UP-yghX-S 12 TTTGTTCTCTTCGACCTGATGAC UP-yghX-A 13 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCC GGATGATTAATTGTCAACTGTTCTACGTTGCGCTTTTT hisD-S 14 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGACCATGAGCTTTAACACAATC ATTGACT hisD-A 15 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGT TCATTTAAATATGATGTTGTTCAG TCATGCTTGCTCCTTAAGGG DN-yghX- 16 CTGAACAACATCATATTTAAATGAACATAACTCAATTTG S1 TAGGCTAGCATAACCCCTTGGGGC GTCATAGTAATCCAGCAACTCTTGTG DN-yghX-A 17 GAGCAGGTATTTACGTGAACCG UP-hisCB-S 18 ATTTCGTGGCTTCTGATTTGCT UP-hisCB-A 19 AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAG CGCATCAGAAAGTCTCCTGTGCATTTACAGCACTCCTTTC GACGAG DK-yghX- 20 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCT S2 AGGGTCATAGTAATCCAGCAACT GTCATAGTAATCCAGCAACTCTTGTG UP- 21 GTGACCGTTACGCTCACGTAGT hisHAFI-S UP- 22 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT hisHAFI-A TCGACTGAGCCTTTCGTTTTATTTGTCACTGATGCCGTTT ACGCA DN-yghX- 23 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA S3 ACGCTCTCCTGAGTAGGACAAAT GTCATAGTAATCCAGCAACTCTTGTG UP-yjiT-S 24 AATAGTTGTTGCCGCCTGAGT UP-yjiT-A 25 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCC GGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCCC lysE-S 26 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGACCATGGTGATCATGGAAATC TTCATTA lysE-A 27 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT TCGACTGAGCCTTTCGTTTTATTTGCTAACCCATCAACAT CAGTTTGATG DN-yjiT-S 28 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCCTGAGTAGGACAAATAAGCACTACCTGTGAA GGGATGT DN-yjiT-A 29 CAGGGCTTCCACAGTCACAAT gRNA- 30 AGTCCTAGGTATAATACTAGTAAAAGAGATGAATGAATT tdcDS TCGTTTTAGAGCTAGAA gRNA- 31 TTCTAGCTCTAAAACGAAATTCATTCATCTCTTTTACTAG tdcD-X TATTATACCTAGGACT gRNA-ylbE- 32 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCA S ACGTTTTAGAGCTAGAA gRNA-ylbE- 33 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTA A GTATTATACCTAGGACT gRNA- 34 AGTCCTAGGTATAATACTAGTGGTGCCTGACGACCATAA yghX-S AAGTTTTAGAGCTAGAA gRNA- 35 TTCTAGCTCTAAAACTTTTATGGTCGTCAGGCACCACTA yghX-A GTATTATACCTAGGACT gRNA-his1- 36 AGTCCTAGGTATAATACTAGTATGAACATAACTCAATTT S GTGTTTTAGAGCTAGAA gRNA-his1- 37 TTCTAGCTCTAAAACACAAATTGAGTTATGTTCATACTA A GTATTATACCTAGGACT gRNA-his2- 38 AGTCCTAGGTATAATACTAGTTGCGCTGGTTGATTTCTTC S TGTTTTAGAGCTAGAA gRNA-his2- 39 TTCTAGCTCTAAAACAGAAGAAATCAACCAGCGCAACTA A GTATTATACCTAGGACT gRNA-yjiT- 40 AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAA S TGGTTTTAGAGCTAGAA gRNA-yjiT- 41 TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAG A TATTATACCTAGGACT UP-yjhE-S 42 GTCAGGCACTGGCGAAAGAT UP-yjhE-A 43 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCC GGATGATTAATTGTCAACGCAAGCCATAAACCCACA rocG-S 44 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGACCATGTCAGCAAAGCAAGT CTCGA rocG-A 45 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT TCGACTGAGCCTTTCGTTTTATTTGTTAGACCCATCCGCG GAAA DN-yjhE-S 46 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCT CTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGT DN-yjhE-A 47 AGGCGTTGTTGTGGCAGATT

[0074] 3. Specific Process of Strain Construction

[0075] 3.1 Relieving a Feedback Inhibition of HisG and making it Strongly Expressed

[0076] 3.1.1 Integrating P.sub.trc-hisG* to the tdcD gene locus

[0077] By taking a genome of E. coli W3110 (ATCC 27325) as a template, the upstream homologous arm primers (UP-tdcD-S and UP-tdcD-A) and downstream homologous arm primers (DN-tdcD-S and DN-tdcD-A) were designed according to upstream and downstream sequences of the tdcD gene of the genome, and PCR amplification was conducted on the upstream and downstream homologous arm fragments; the primers (hisG*-S and hisG*-A) were designed according to the hisG* gene (the nucleotide sequence is as shown in SEQ ID NO: 1) and then the hisG* gene fragment was amplified. The promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the hisG* gene. An integrated fragment (the upstream homologous arm-P.sub.trc-hisG*-downstream homologous arm) of the hisG* gene was obtained by performing overlapped PCR method on the above fragments, and the DNA fragment containing a target sequence used for constructing pGRB-tdcD was prepared by annealing the primers gRNA-tdcD-S and gRNA-tdcD-A. The preparation of competent cell of E. coli W3110 was operated according to the method as shown in 1.3 and 1.4, the strain E. coli WHY1-1 was finally obtained. The electrophoretogram of construction of the P.sub.trc-hisG* integrated fragment and PCR verification of the positive strain is as shown in the FIG. 2. Wherein, the length of the upstream homologous arm is 496 bp, the length of the amplified hisG* gene fragment shall be 627 bp, the length of the downstream homologous arm is 1902 bp, and the total length of the integrated fragment is 3024 bp, when PCR verification was conducted by using the authenticating primers, the length of the PCR amplification fragment of the positive strain is 627 bp, and the original strain is free of band.

[0078] 3.1.2 Integrating P.sub.trc-hisG* to the ylbE Gene Locus

[0079] By taking a genome of E. coli W3110 (ATCC 27325) as a template, the upstream homologous arm primers (UP-ylbE-S and UP-ylbE-A) and downstream homologous arm primers (DN-ylbE-S and DN-ylbE-A) were designed according to upstream and downstream sequences of the ylbE gene of the genome, and PCR amplification was conducted on the upstream and downstream homologous arm fragments; and the hisG* gene fragment was amplified by using the primers (hisG *-S and hisG*-A). The integrated fragment (the upstream homologous arm-Ptrc-hisG*-downstream homologous arm) of the hisG* gene was obtained by performing overlapped PCR method on the above fragments, and the DNA fragment containing a target sequence used for constructing pGRB-ylbE was prepared by annealing the primers gRNA-ylbE-S and gRNA-ylbE-A. The preparation of competent cell of E. coli WHY1-1 was operated according to the method as shown in 1.3 and 1.4, the strain E. coli WHY1-2 was finally obtained. The electrophoretogram of constructiion of the P.sub.trc-hisG* integrated fragment and PCR verification of the positive strain is as shown in the FIG. 3. Wherein, the length of the upstream homologous arm is 601 bp, the length of the amplified hisG* gene fragment is 627 bp, the length of the downstream homologous arm is 547 bp, and the total length of the integrated fragment is 1815 bp, when PCR verification was conducted by using the authenticating primers, the length of the PCR amplification fragment of the positive strain is 903 bp, and the original strain is free of band.

[0080] 3.2 Integrating the Histidine Operon Gene of E. coli W3110 to the yghX Gene Locus

[0081] In the present disclosure, the histidine operon genes (hisDBCHAFI, including seven genes: hisD, hisB, hisC, hisH, hisA, hisF and hisI) in E. coli W3110 were successively integrated to a pseudogene yghX locus on the genome of E. coli WHY1-2 in sequence, and the promoter Ptrc initiates transcriptional expression of the operon to construct a strain E. coli HIS3-3.

[0082] Integration of the histidine operon genes was divided into three segments.

[0083] 3.2.1 Integration of Ptrc-hisD

[0084] By taking a genome of E. coli W3110(ATCC 27325) as a template, the upstream homologous arm primers (UP-yghX-S and UP-yghX-A) and downstream homologous arm primers (DN-yghX-S1 and N-yghX-A) were designed according to upstream and downstream sequences of the yghX gene of the genome, and PCR amplification was conducted on the upstream and downstream homologous arm fragments; the primers (hisD-S and hisD-A) were designed according to the hisD gene sequence and the hisD gene fragment was PCR amplified; and the promoter Pfrc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the hisD gene. The above fragments were fused by the overlapped PCR method to obtain the integrated fragment (the upstream homologous arm-Ptrc-hisD-downstream homologous arm) of the Ptrc-hisD gene, the DNA fragment containing a target sequence used for constructing pGRB-yghX was prepared by annealing the primers gRNA-yghX-S and gRNA-yghX-A. The competent cell of E. coli WHY1-2 was according to the method as shown in 1.3 and 1.4, and the strain E. coli WHY2-1 was finally obtained. In the P.sub.trc-hisD fragment integrating process, the electrophoretogram of construction of the integrated fragment and PCR verification of the positive strain is as shown in the FIG. 4. Wherein, the length of the upstream homologous arm is 602 bp, the length of the amplified hisD gene fragment is 1305 bp, the length of the downstream homologous arm is 561 bp, and the length of the overlapped fragment is 2542 bp, when PCR verification was conducted by using the authenticating primers, the length of the fragment amplified by the positive recons is 1208 bp, and the original strain is free of band.

[0085] 3.2.2 Integration of hisB-hisC

[0086] By taking a genome of E. coli W3110 (ATCC 27325) as a template, the upstream homologous arm primers (UP-hisCB-S and UP-hisCB-A) were designed according to hisC-hisB and upstream sequences thereof, and PCR amplification was conducted on the upstream homologous arm fragment; by taking a genome of E. coli HIS3-1 as a template, the downstream homologous arm primers (DN-yghX-S2 and DN-yghX-A) were designed according to downstream sequences of the yghX gene of the genome, and PCR amplification was conducted on the downstream homologous arm fragment. The fragments were integrated by the overlapped PCR method to obtain the integrated fragment (upstream fragment of hisC-hisC-hisB-downstream homologous arm) of hisC-hisB. The DNA fragment containing the target sequence used for constructing pGRB-hisI was prepared by annealing the primers gRNA-hisI-S and gRNA-his1-A. The competent cell of E. coli WHY2-1 was prepared according to the method as shown in 1.3 and 1.4, and the strain E. coli WHY2-2 was finally obtained. In the hisB-hisC fragment integrating process, the electrophoretogram of construction of the integrated fragment and PCR verification of the positive strain is as shown in the FIG. 5. Wherein, the total length of the upstream fragment of hisC-hisC-hisB is 2696 bp, the length of the downstream homologous arm is 561 bp, and the length of the overlapped fragment is 3317 bp, when PCR verification was conducted by using the authenticating primers, the length of the fragment amplified by the positive recons is 1118 bp, and the original strain is free of band.

[0087] 3.2.3 Integration of hisH-hisA-hisF-hisI

[0088] By taking a genome of E. coli W3110 (ATCC 27325) as a template, the upstream homologous arm primers (UP-hisHAFI-S and UP-hisHAFI-A) were designed according to hisH-hisA-hisF-hisI and upstream sequences thereof, and PCR amplification was conducted on the upstream homologous arm fragment; by taking a genome of E. coli HIS3-2 as a template, the downstream homologous arm primers (DN-yghX-S3 and DN-yghX-A) were designed according to downstream sequences of the yghX gene of the genome, and PCR amplification was conducted on the downstream homologous arm fragment. The fragments were fused by the overlapped PCR method to obtain the integrated fragment (upstream fragment of hisH-hisH-hisA-hisF-hisI-downstream homologous arm) of hisH-hisA-hisF-hisI. The DNA fragment containing the target sequence used for constructing pGRB-his2 was prepared by annealing the primers gRNA-his2-S and gRNA-his2-A. The competent cell of E. coli WHY2-2 was prepared according to the method as shown in 1.3 and 1.4, and the strain E. coli WHY2-3 was finally obtained. In the integrating process of hisH-hisA-hisF-hisI, the electrophoretogram of contruction of the integrated fragment and PCR verification of the positive strain is as shown in the FIG. 6. The total length of the upstream fragment of hisH-hisH-hisA-hisF-hisI is 3265 bp, the length of the downstream homologous arm is 561 bp, and the length of the overlapped fragment is 3317 bp, when PCR verification was conducted by using the authenticating primers, the length of the fragment amplified by the positive recons is 1136 bp, and the original strain is free of band.

[0089] 3.3 Integration of P.sub.trc-lysE

[0090] By taking a genome of E. coli W3110 (ATCC ATCC27325) as a template, the upstream homologous arm primers (UP-yjiT-S and UP-yjiT-A) and the downstream homologous arm primers (DN-yjiT-S and DN-yjiT-A) were designed according to upstream and downstream sequences of the yjiT gene of the genome and PCR amplification was conducted on the upstream and downstream homologous arm fragments; by taking a genome of corynebacterium glutamicum (ATCC 13032) as a template, the primers (lysE-S and lysE-A) were designed according to the sequence of lysE (NCBI-GeneID: 1019244) gene and PCR amplification was conducted on the lysE fragment; and the promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the lysE gene. The fragments were fused by the overlapped PCR method to obtain the integrated fragment (the upstream homologous arm-P.sub.trc-lysE-downstream homologous arm) of P.sub.trc-lysE, and the DNA fragment containing a target sequence used for constructing pGRB-yjiT was prepared by annealing the primers gRNA-yjiT-S and gRNA-yjiT-A. The competent cell of E. coli WHY2-3 was prepared according to the method as shown in 1.3 and 1.4, and the strain E. coli WHY3 was finally obtained. In the P.sub.trc-lysE fragment integrating process, the electrophoretogram of construction of the integrated fragment and PCR verification of the positive strain is as shown in the FIG. 7. Wherein, the length of the upstream homologous arm is 372 bp, the length of the lysE gene fragment is 834 bp, the length of the downstream homologous arm 530 bp, and the length of the overlapped fragment is 1655 bp, when PCR verification was conducted by using the authenticating primers, the length of the fragment amplified by the positive recon is 1429 bp, and the original strain is free of band.

[0091] 3.4 Integration of P.sub.trc-rocG

[0092] By taking a genome of E. coli W3110 as a template, the upstream homologous arm primers (UP-yjhE-S and UP-yjhE-A) and the downstream homologous arm primers (DN-yjhE-S and DN-yjhE-A) were designed according to upstream and downstream sequences of the yjhE gene of the genome and PCR amplification was conducted on the upstream and downstream homologous arm fragments; by taking a genome of B. subtilis 168 as a template, the primers (rocG-S and rocG-A) were designed according to the sequence of rocG (NCBI-GeneID: 937066) gene and PCR amplification was conducted on the rocG fragment; and the promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the rocG gene. The fragments were fused by the overlapped PCR method to obtain the integrated fragment (the upstream homologous arm-P.sub.trc-rocG-downstream homologous arm) of the P.sub.trc-rocG gene, and the DNA fragment containing a target sequence used for constructing pGRB-yjhE was prepared by annealing the primers gRNA-yjhE-S and gRNA-yjhE-A. The competent cell of E. coli WHY3 was prepared according to the method as shown in 1.3 and 1.4, and the strain E. coli WHY3-1 was finally obtained. In the P.sub.trc-rocG fragment integrating process, the electrophoretogram of construction of the integrated fragment and PCR verification of the positive strain is as shown in the FIG. 8. Wherein, the total length of the upstream homologous arm is 602 bp, the length of the rocG gene fragment is 1398 bp, the length of the downstream homologous arm 491 bp, and the length of the overlapped fragment is 2419 bp, when PCR verification was conducted by using the primers UP-yjhE-S and DN-yjhE-A, the length of the fragment amplified by the positive recons is 2419 bp, and the original strain is 1547 bp.

Embodiment 2

[0093] Fermentation production of the histidine by using the genetically engineered bacterium E. coli WHY3-1 is as follows:

[0094] (1) Adopting Shake-Flask Culture

[0095] slant culture: a −80° C. preserved strain was taken and inoculated on an activating slant through streak inoculation, and was cultured for 12 h at 37° C., subculture was conducted once;

[0096] shake-flask seed culture: an annular slant seed was scratched by using an inoculating loop and was inoculated in a 500 mL triangular flask filled with 30 mL of culture medium, the triangular flask was sealed with nine-layered gauze, and culture was conducted for 6-8 h at 200 rpm under 37° C.;

[0097] shake-flask fermental culture: 10-15% of the seed solution was inoculated in the 500 mL triangular flask (the final volume was 30 mL) filled with a fermentation medium, the triangular flask was sealed with nine-layered gauze, shake culture was conducted at 37° C. under 200 r/min, pH was maintained at 7.0-7.2 by replenishing ammonia water in a fermenting process, and a 60% (m/v) glucose solution was replenished to maintain fermentation;

[0098] a slant culture medium was composed of 1-5 g/L of glucose, 5-10 g/L of peptone, 5-10 g/L of beef extract, 1-5 g/L of yeast powder, 1-2.5 g/L of NaCl, 20-25 g/L of agar and the balance water, wherein pH was 7.0-7.2;

[0099] a seed culture medium was composed of 15-30 g/L of glucose, 5-10 g/L of yeast extract, 5-10 g/L of peptone, 5-15 g/L of KH.sub.2PO.sub.4, 2-5 g/L of MgSO.sub.4.7H.sub.2O, 5-20 mg/L of FeSO.sub.4.7H.sub.2O, 5-20 mg/L of MnSO.sub.4.HO, 1-3 mg/L of V.sub.B1, 0.1-1 mg/L of VH, two drops of a defoamer and the balance water, wherein pH was 7.0-7.2;

[0100] a fermentation medium was composed of 20-30 g/L of glucose, 2-5 g/L of yeast extract, 2-4 g/L of peptone, 1-3 g/L of KH.sub.2PO4, 1-2 g/L of MgSO.sub.4.7H.sub.2O, 5-20 mg/L of FeSO.sub.4.7H.sub.2O, 5-20 mg/L of MnSO.sub.4.7H.sub.2O, 1-3 mg/L of V.sub.B1, 1-3 mg/L of V.sub.B2, 1-3 mg/L of V.sub.B3, 1-3 mg/L of V.sub.B5, 1-3 mg/L of V.sub.B12, 1-3 mg/L of VH and the balance water, wherein pH was 7.0-7.2.

[0101] (2) or Adopting a Fermentation Tank for Culture

[0102] slant activating culture: a ring of strain was scraped from a strain preservation tube of a refrigerator at −80° C., and uniformly coated on the activating slant, was cultured for 12-16 h at 37° C., and was then transferred to an eggplant-shaped flask to be further cultured for 12-16 h;

[0103] seed culture: a proper amount of sterile water was taken and added into the eggplant-shaped flask, a bacterium suspension was inoculated in the seed culture medium, the pH was stabilized at about 7.0, the temperature was constant at 37° C., the content of dissolved oxygen was between 25-35%, and the bacterium suspension was cultured till the OD.sub.600 value of fermentation liquor reached 10-15;

[0104] the seed solution was inoculated into a fresh fermentation medium according to an inoculation amount of 15-20%, fermentation was started, pH was controlled stably at about 7.0 in the fermenting process, temperature was maintained at 37° C. and dissolved oxygen was maintained between 25-35%; after glucose in the fermentation medium was exhausted, a 80% (m/v) glucose solution was fed to maintain the glucose concentration in the fermentation medium at 0.1-5 g/L;

[0105] a slant culture medium was composed of 1-5 g/L of glucose, 5-10 g/L of peptone, 5-10 g/L of beef extract, 1-5 g/L of yeast powder, 1-2.5 g/L of NaCl, 20-25 g/L of agar and the balance water, wherein pH was 7.0-7.2;

[0106] a seed culture medium was composed of 15-30 g/L of glucose, 5-10 g/L of yeast extract, 5-10 g/L of peptone, 5-15 g/L of KH.sub.2PO.sub.4, 2-5 g/L of MgSO.sub.4.7H.sub.2O, 5-15 mg/L of FeSO.sub.4.7H.sub.2O, 5-15 mg/L of MnSO.sub.4.H.sub.2O, 1-3 mg/L of V.sub.B1, 0.1-1 mg/L of V.sub.H, two drops of a defoamer and the balance water, wherein pH was 7.0-7.2;

[0107] a fermentation medium was composed of 10-25 g/L of glucose, 1-5 g/L of yeast extract, 1-5 g/L of peptone, 1-5 g/L of KH.sub.2PO.sub.4, 1-3 g/L of MgSO.sub.4.7H.sub.2O, 10-30 mg/L of FeSO.sub.4.7H.sub.2O, 10-30 mg/L of MnSO.sub.4.H.sub.2O, 1-3 mg/L of V.sub.B1, 1-3 mg/L of V.sub.B2, 1-3 mg/L of V.sub.B3, 1-3 mg/L of V.sub.B5, 1-3 mg/L of V.sub.B12, 1-3 mg/L of VH and the balance water, wherein pH was 7.0-7.2.

Embodiment 3

[0108] A fermentation experiment of E. coli WHY3-1 in the 5L tank.

[0109] Producing the histidine by taking the strain E. coli WHY3-1 constructed in the Embodiment 1 as a production strain:

[0110] slant activation: a glycerinum preserved strain was taken and streak inoculation was conducted on the slant culture medium of a test tube, and culture was conducted for 12 h at 37° C.; and then a slant preserved strain was inoculated to an eggplant-shaped flask slant culture medium through streak inoculation, and culture was conducted for 14 h at 37 ° C.;

[0111] seed culture: an activated fresh eggplant-shaped flask slant was taken, and the strain was washed by 150 mL of sterile water, then was inoculated to the fermentation tank under protection of a flame, the temperature was controlled at 37° C., ammonia water was fed automatically to control the pH at 7.0, the initial ventilating rate was 2 L/min and the initial stirring rotating speed was 200 rpm, in the culture process, the DO value was maintained between 20% and 30%, and seed was cultured till OD.sub.600 was about 15;

[0112] fermentation tank culture: the seed in the fermentation tank was inoculated to the seed solution according to an inoculation amount of 15% (the seed solution was discharged to 450 mL, and was poured into the sterilized fermentation medium under protection of flame), the temperature was controlled at 35° C., ammonia water (or 20% sulfuric acid) was automatically fed to control pH at 7.0, the initial ventilating rate was 2 L/min, the ventilating ratio was 0.667 vvm and the initial stirring rotating speed was 400 rpm, the dissolved oxygen was controlled at 20-30% by adjusting the rotating speed and the air quantity, GPE was dropwise added manually to deform, and in the fermentation process, 80% of glucose solution was fed, so that sufficient sugar supply was ensured and sugar concentration was not higher than 5 g/L;

[0113] a slant culture medium was composed of 1 g/L of glucose, 10 g/L of peptone, 10 g/L of beef extract, 5 g/L of yeast powder, 2.5 g/L of NaCl, 25 g/L of agar and the balance water, wherein pH was 7.0-7.2;

[0114] a seed culture medium was composed of 10 g/L of glucose, 5 g/L of yeast extract, 5 g/L of peptone, 5 g/L of KH.sub.2PO.sub.4, 2 g/L of MgSO.sub.4.7H.sub.2O, 10 mg/L of FeSO.sub.4.7H.sub.2O, 10 mg/L of MnSO.sub.4H.sub.2O, 2 mg/L of V.sub.B1, 1 mg/L of V.sub.H, two drops of a defoamer and the balance water, wherein pH was 7.0-7.2;

[0115] a fermentation medium was composed of 10 g/L of glucose, 5 g/L of yeast extract, 4 g/L of tryptone, 3 g/L of KH.sub.2PO.sub.4, 1.5 g/L of MgSO.sub.4.7H.sub.2O, 20 mg/L of FeSO.sub.4.7H.sub.2O, 20 mg/L of MnSO.sub.4H.sub.2O, 2 mg/L of V.sub.B1, 2 mg/L of V.sub.B3, 2 mg/L of V.sub.B5, 2 mg/L of V.sub.B12, 2 mg/L of V.sub.H and the balance water, wherein pH was 7.0-7.2.

[0116] A fermentation curve of E. coli WHY3-1 on the 5 L fermentation tank is as shown in the FIG. 9.

[0117] It can be seen from the fermentation curve that the thalli start to enter a logarithmic phase after being fermented for 4 hours and enter a stable phase in 32 hours after the OD.sub.600 reaches the maximum value. After fermentation for 8 hours, a quick accumulating stage of histidine is started, and the highest output of histidine after fermentation for 44 hours is 65 g/L. The final conversion ratio is 0.223 g histidine/g glucose. Compared with WHY3 under a same fermentation condition, the biomass of WHY3-1 is reduced, but the output and the production intensity of histidine are improved by 18.2% and the conversion ratio is increased by 11.5%. It can be seen that E. coli WHY3-1 as a producing strain for histidine has extremely obvious advantages.