Patent Publication Number: US-2021189354-A1

Title: 2-isopropylmalate synthetase and engineering bacteria and application thereof

Description:
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (SEQUENCE-LISTING-20200709-DTJKJUSN.txt; Size: 33,000 bytes; and Date of Creation: Mar. 14, 2021) is herein incorporated by reference in its entirety. 
     CROSS REFERENCE TO RELATED APPLICATION 
     The application claims priority to Chinese Patent Application No. CN201910820591X, filed on Aug. 29, 2019, and entitled “Isopropyl Malate Synthase and Application thereof”, and Chinese Patent Application No. CN2019108860780, filed on Sep. 19, 2019, and entitled “Genetically Engineered Bacterium for Producing L-leucine and Application thereof”, the entire contents of which are incorporated herein by reference. 
     TECHNICAL FIELD 
     The invention relates to a 2-isopropyl malate synthase, a genetically engineered bacterium for producing L-leucine and application thereof and belongs to the field of metabolic engineering. 
     BACKGROUND ART 
     L-leucine belongs to branched chain amino acids and is one of the eight amino acids essential to human body and a raw material for synthesizing proteins and hormones, playing a vital role in the life activities of human body. Therefore, the L-leucine has a very broad marketing and application prospect in the industries such as food and medicine. 
     Industrial methods for synthesizing L-leucine include a hair extraction method and a fermentation method, wherein the hair extraction method, however, has the shortcomings of limited raw material resources, high production costs, environmental pollution and the like. Accordingly, the fermentation method is the mainstream method for producing the L-leucine. Existing industrial production strains of the L-leucine are mainly obtained through mutagenesis and have the shortcomings of nutritional deficiency, slow growth, unstable hereditary characters, causing the problems long fermentation period, unstable fermentation performance, low yield and conversion rate, and the like. 
     SUMMARY OF THE INVENTION 
     The present disclosure provides an isopropyl malate synthase for relieving the feedback inhibition by the L-leucine and a coding gene thereof, and constructs a genetically engineered bacterium for producing the L-leucine by the coding gene. The present disclosure overcome the shortcomings that existing wild type isopropyl malate synthases are subjected to feedback inhibition by the L-leucine and existing L-leucine production strains are slow in growth, deficient in nutrition, unstable in fermentation and the like. 
     One of the technical solutions of the present invention is to provide an isopropyl malate synthase mutant LEUA M  for relieving the feedback inhibition by the the L-leucine, of which the amino acid sequence is shown as SEQ ID NO. 1, and the coding gene of the isopropyl malate synthase mutant is leuA M , of which the nucleotide sequence is shown as SEQ ID NO. 2. 
     The isopropyl malate synthase mutant originates from a  Corynebacterium glutamicum  mutant strain, of which the artificial mutation process comprises taking  Corynebacterium glutamicum  ATCC13032 as an original strain, performing plasma mutagenesis at atmospheric pressure and room temperature, and screening out a strain LEU262 on a minimal medium containing 50 mg/L leucine hydroxamate; then taking the strain LEU262 as an original strain, performing plasma mutagenesis at atmospheric pressure and room temperature, and screening out a strain LEU741 on a minimal medium containing 50 mg/L beta-hydroxyleucine. 
     The genome of the strain LEU741 is extracted, primers are designed for performing PCR (polymerase chain reaction) amplification of the 2-isopropyl malate synthase coding gene, and PCR products are recovered and sequenced; the 2-isopropyl malate synthase encoded by the gene is discovered to have the following amino acid mutations compared with the wild type 2-isopropyl malate synthase from the  Corynebacterium glutamicum  ATCC13032: F7L, I14F, I51S, G127D, I197V, F370L, K380M, R529H, G561D and V596A. 
     The present invention adopts the following definitions: 
     1. Identification of the isopropyl malate synthase mutant 
     ‘original amino acid+position+amino acid after substituted’ is used to represent the mutated amino acids in the 2-isopropyl malate synthase mutant. For example, F7L represents that the amino acid at the position 7 is Leu substituted from Phe in the wild type 2-isopropyl malate synthase, F7 represents that the amino acid at the position 7 is Phe, and the number of the position corresponds to that in the amino acid sequence of the wild type 2-isopropyl malate synthase in SEQ ID No. 3. 
     According to the present invention, leuA represents a wild type 2-isopropyl malate synthase coding gene (as shown in SEQ ID NO. 4), LEUA represents a wild type 2-isopropyl malate synthase (as shown in SEQ ID NO. 3), leuA M  represents a mutated 2-isopropyl malate synthase gene (as shown in SEQ ID NO. 2), and LEUA M  represents the 2-isopropyl malate synthase mutant (as shown in SEQ ID NO. 1). Comparison of the amino acids before and after the mutation is as follows: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 2- 
                   
               
               
                   
                 isopropyl 
                   
               
               
                   
                 malate 
                   
               
               
                   
                 synthase 
                 Amino acids 
               
               
                   
                   
               
             
            
               
                   
                 LEUA 
                 F7, I14, I51, G127, I197, F370, 
               
               
                   
                   
                 K380, R529, G561, V596 
               
               
                   
                   LEUA M   
                 F7L, I14F, I51S, G127D, I197V, F370L,  
               
               
                   
                   
                 K380M, R529H, G561D, V596A 
               
               
                   
                   
               
            
           
         
       
     
     The 2-isopropyl malate synthase mutant LEUA M  has the following enzymatic characteristics that, under the condition that the concentration of L-leucine ranges from 0-15 mmol/L, the enzymatic activity of the LEUA M  has no significant change, which means that the mutant of the present invention relieves the feedback inhibition of the L-leucine. Meanwhile, the enzymatic activity of the LEUA M  under the condition that the concentration of L-leucine ranges from 0-15 mmol/L has no significant decrease compared with that of the wild type 2-isopropyl malate synthase LEUA under the condition that the concentration of L-leucine is 0-mmol/L. 
     Another technical solution of the present invention to the problem is to provide a genetically engineered bacterium for producing L-leucine, wherein the genetically engineered bacterium is obtained by overexpressing the isopropyl malate synthase coding gene leuA M  for relieving the feedback inhibition of the L-leucine, an acetohydroxy acid synthase coding gene ilvBN M  for relieving the feedback inhibition of L-isoleucine, a 3-isopropyl malate dehydrogenase coding gene leuB and a 3-isopropyl malate dehydratase coding gene leuCD in host cells. 
     The host cells can be  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Vibrio natriegens, Saccharomyces cerevisiae  and the like. 
     An acetohydroxy acid synthase encoded by the gene ilvBN M  in the present disclosure relieves the feedback inhibition of the L-isoleucine, and the nucleotide sequence of the gene ilvBN M  is shown as SEQ ID NO. 5. 
     The gene leuB in the present disclosure can be obtained from  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium  and the like, such as those with Genbank accession numbers of b0073, JW5807, NCg11237, BSU28270 and BAMF_2634. 
     The gene leuCD in the present disclosure can be obtained from  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium  or the like, such as those with Genbank accession numbers of b0071, b0072, JW0070, JW0071, NCg11262, NCg11263, BSU28250, BSU28260, BAMF_2632 and BAMF_2633. 
     In the preferred embodiments, the genetically engineered bacterium in the present disclosure is obtained by taking  Escherichia coli  W3110 as the host cells to overexpress the gene leuA M  as shown in SEQ ID NO. 2, the gene ilvBN M  as shown in SEQ ID NO. 5 and the gene leuBCD (an operon composed of the leuB and the leuCD in  Escherichia coli ) as shown in SEQ ID NO.6. The preferred genetically engineered bacterium in the present disclosure producing the L-leucine is strain TE03. 
     Further, the construction method of the genetically engineered bacterium is as follows:
         (4) performing amplification of the isopropyl malate synthase coding gene leuA M  and the acetohydroxy acid synthase coding gene ilvBN M  separately, and constructing genome integration fragments separately;   (5) performing amplification of the gene leuBCD, and connecting it with a plasmid to obtain a recombinant plasmid;   (6) performing expression of the genome integration fragments and the recombinant plasmid in previous steps subsequently in the host cells by the CRISPR/Cas9 gene editing technology.       

     Further, the construction method of the genetically engineered bacterium specifically comprises:
         (5) taking the genome of  Escherichia coli  W3110 as a template to perform PCR amplification to obtain the isopropyl malate synthase coding gene leuA M  and UHF and DHF fragments (respectively the upstream homologous arm and the downstream homologous arm of gene lacI), and then performing overlapping PCR to obtain a recombinant fragment UHF-leuA M -DHF;
           The nucleotide sequence of the UHF is shown as SEQ ID NO. 7;   The nucleotide sequence of the DHF is shown as SEQ ID NO. 8;   
           (6) obtaining UHFA and DHFB fragments (respectively the upstream homologous arm and the downstream homologous arm of gene lacZ) and ilvBN M  gene segment by the same principle of previous step, and performing overlapping PCR on those fragments to obtain a recombinant fragment UHF -ilvBN M -DHF;
           The nucleotide sequence of the UHFA is shown as SEQ ID NO. 9;   The nucleotide sequence of the DHFB is shown as SEQ ID NO. 10;   
           (7) taking the genome of the  Escherichia coli  W3110 as a template to perform PCR amplification to obtain the gene leuBCD, and connecting the gene leuBCD with a plasmid pTrc99a to obtain a recombinant plasmid pTR-leuBCD;   (8) performing construction of the L-leucine genetically engineered bacterium TE03; annealing PG-1 and PG-2 as well as PG-3 and PG-4 respectively at 52 ° C. and then connecting PG-1 and PG-2 as well as PG-3 and PG-4 to a plasmid pGRB to obtain pGRB1 and pGRB2; taking  Escherichia coli  W3110 as an original strain, and performing transformation of pGRB1 and UHF-leuA M -DHF respectively into the  Escherichia coli  W3110 to obtain a recombinant strain TE01; taking the strain TE01 as an original strain and performing transformation of pGRB2 and UHFA-ilvBN M -DHFB respectively into TE01 to obtain a strain TE02; and then performing transformation of pTR-leuBCD into strain TE02 to obtain strain TE03.       

     The invention also provides a method for synthesizing L-leucine with the genetically engineered bacterium through fermentation. The method specifically includes: 
     inoculating a seed culture at an inoculum size of 5-10% onto a fermentation culture medium for fermentation culture, wherein the content of dissolved oxygen is maintained at 20-40%, the pH is maintained at 6.5-7.5, the culture temperature is 30-35 ° C., the fermentation period is 40-48 h, and the residual sugar concentration is maintained at 0-0.4% (W/V) during the fermentation. 
     At the end of the fermentation, the concentration of the L-leucine in the fermentation liquid reaches 60.5-69.6 g/L. 
     The fermentation culture medium is composed of 25 g/L glucose, 12 g/L peptone, 4 g/L yeast powder, 3.5 g/L KH 2 PO 4 , 1.5 g/L MgSO 4 , 15 mg/L FeSO 4 , 15 mg/L MnSO 4  and 0.01 mg/L VB1 (vitamin B1). The pH of the fermentation culture medium is 7.0, the pressure is 0.075 MPa, and the fermentation culture medium is subjected to high-pressure steam sterilization for 15 min. 
     The present disclosure possesses the following advantages: 
     1. The 2-isopropyl malate synthase encoded by the gene leuA M  of the present disclosure has the characteristics that the 2-isopropyl malate synthase relieves the feedback inhibition effects of L-leucine (as shown in  FIG. 1 ). Under the condition that the concentration of L-leucine ranges from 0-15 mmol/L, the enzymatic activity of the LEUA M  has no significant change and meanwhile has no significant decrease compared with that of the wild type 2-isopropyl malate synthase encoded by the gene leuA (as shown in  FIG. 2 ). 
       2 . The L-leucine genetically engineered bacterium strain TE03 has the advantages of no nutritional deficiency, rapid growth, short fermentation period, high yield and high conversion rate. After 40-48 h of fermentation by the strain TE03, the concentration of L-leucine in the fermentation liquid reaches 60.5-69.6 g/L (as shown in  FIG. 3 ). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Influence of L-leucine on the activity of the 2-isopropyl malate synthase encoded by the genes leuA and leuA M . 
         FIG. 2 : Comparison of the activity of the 2-isopropyl malate synthases encoded by leuA M  and leuA. 
         FIG. 3 : Influence of L-isoleucine on the activity of the acetohydroxy acid synthases encoded by the genes ilvBN and ilvBN M . 
         FIG. 4 : Comparison of the activity of the acetohydroxy acid synthases(AHAS) encoded by ilvBN M  and ilvBN. 
         FIG. 5 : The process curve of fermentation of the L-leucine genetically engineered bacterium strain TE03. 
         FIG. 6 : Influence of overexpression of leuA M  on L-leucine synthesis. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In order to make the objects, technical solutions and advantages of the present invention clearer and more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described herein are only intended to illustrate of the present invention but not to limit the present invention. 
     The present embodiment provides a genetically engineered bacterium for producing L-leucine, which is constructed by overexpressing an isopropyl malate synthase coding gene leuA M  for relieving the feedback inhibition by L-leucine, an acetohydroxy acid synthase coding gene ilvBN M  for relieving the feedback inhibition by L-isoleucine, a 3-isopropyl malate dehydrogenase coding gene leuB and a 3-isopropyl malate dehydratase coding gene leuCD in host cells. 
     In some embodiments, the host cells can be  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Vibrio natriegens, Saccharomyces cerevisiae  and the like. 
     In some embodiments, the gene ilvB1Vm is derived from  Corynebacterium glutamicum  which is resistant to such L-isoleucine-structured analogues as α-aminobutyric acid and thioisoleucine. 
     In some embodiments, the gene leuB is selected from those with Genbank accession numbers of b0073, JW5807, NCg11237, BSU28270 or BAMF_2634. 
     In some embodiments, the gene leuCD is selected from those with Genbank accession numbers of b0071, b0072, JW0070, JW0071, NCg11262, NCg11263, BSU28250, BSU28260, BAMF_2632 or BAMF_2633. 
     The host cells, the gene ilvBN M , the gene leuB and the gene leuCD from the above sources can all achieve the effects of the present invention. In the following embodiments,  Escherichia coli  W3110 is taken as the host cells to overexpress the gene leuA M  shown in SEQ ID NO. 2, the gene ilvBN M  shown in SEQ ID NO. 5 and leuBCD (an operon composed of the leuB and the leuCD in the  Escherichia coli ) shown in SEQ ID NO. 6 to construct the genetically engineered bacterium strain TE03 for producing L-leucine to illustrate the present invention in an exemplary manner. 
     Sequence table of primers applied in the following embodiments: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                 Names 
                 Sequences 
                 NO. 
               
               
                   
               
             
            
               
                 LEUA-1 
                 GTGAAACCAGTAACGTTATACG 
                 11 
               
               
                   
               
               
                 LEUA-2 
                 CCACACATTATACGAGCCGGATGATTAATTGTCAA 
                 12 
               
               
                   
                 CCGTCTTCATGGGAGAA 
                   
               
               
                   
               
               
                 LEUA-3 
                 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATA 
                 13 
               
               
                   
                 ACAATTTCACACAAGGAGATATACATGTCTCCTAA 
                   
               
               
                   
                 CGATGCATT 
                   
               
               
                   
               
               
                 LEUA-4 
                 CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCG 
                 14 
               
               
                   
                 ACTGAGCCTTTCGTTTTATTTGCTTAAACGCCGCC 
                   
               
               
                   
                 AGC 
                   
               
               
                   
               
               
                 LEUA-5 
                 TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC 
                 15 
               
               
                   
                 CTGAGTAGGACAAATGCTGTTAGCGGGC 
                   
               
               
                   
               
               
                 LEUA-6 
                 TCACTGCCCGCTTTCCAG 
                 16 
               
               
                   
               
               
                 leuA-1′ 
                 ATGTCTCCTAACGATGCATT 
                 17 
               
               
                   
               
               
                 leuA-2′ 
                 TTAAACGCCGCCAGC 
                 18 
               
               
                   
               
               
                 IlvB-1 
                 ATGACCATGATTACGGATTCAC 
                 19 
               
               
                   
               
               
                 IlvB-2 
                 CCACACATTATACGAGCCGGATGATTAATTGTCAA 
                 20 
               
               
                   
                 CGGGTTTTCGACGTTCAGACGTA 
                   
               
               
                   
               
               
                 IlvB-3 
                 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATA 
                 21 
               
               
                   
                 ACAATTTCACACAAGGAGATATACCATGAATGTGG 
                   
               
               
                   
                 CAGCTTCTC 
                   
               
               
                   
               
               
                 IlvB-4 
                 CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCG 
                 22 
               
               
                   
                 ACTGAGCCTTTCGTTTTATTTGTTAGATCTTGGCC 
                   
               
               
                   
                 GGAGCCATGGTC 
                   
               
               
                   
               
               
                 IlvB-5 
                 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGT 
                 23 
               
               
                   
                 GAACGCTCTCCTGAGTAGGACAAATTTGATGGTAG 
                   
               
               
                   
                 TGGTCAAATGG 
                   
               
               
                   
               
               
                 IlvB-6 
                 TTATTTTTGACACCAGACCAA 
                 24 
               
               
                   
               
               
                 LA-1 
                 ATCATCACAGCAGCGGCCTGGTGCCGCGCATGTCT 
                 25 
               
               
                   
                 CCTAACGATGCATT 
                   
               
               
                   
               
               
                 LA-2 
                 TGATGATGTTAGCTAGCGCTGAATTCTGCTTAAAC 
                 26 
               
               
                   
                 GCCGCCAGC 
                   
               
               
                   
               
               
                 leuBCD-1 
                 GACCATGGAATTCGAGCTCGGTACCCGGATGTCGA 
                 27 
               
               
                   
                 AGAATTACCATATTGCC 
                   
               
               
                   
               
               
                 leuBCD-2 
                 CTTGCATGCCTGCAGGTCGACTCTAGAATAATTCA 
                 28 
               
               
                   
                 TAAACGCAGGTTGTTTTG 
                   
               
               
                   
               
               
                 PG-1 
                 AGTCCTAGGTATAATACTAGTTTCTCCCATGAAGA 
                 29 
               
               
                   
                 CGGGTTTTAGAGCTAGAA 
                   
               
               
                   
               
               
                 PG-2 
                 TTCTAGCTCTAAAACCCGTCTTCATGGGAGAAACT 
                 30 
               
               
                   
                 AGTATTATACCTAGGACT 
                   
               
               
                   
               
               
                 PG-3 
                 AGTCCTAGGTATAATACTAGTAAACTGTGGAGCGC 
                 31 
               
               
                   
                 CGAAATCCGTTTTAGAGCTAGAA 
                   
               
               
                   
               
               
                 PG-4 
                 TTCTAGCTCTAAAACGGATTTCGGCGCTCCACAGT 
                 32 
               
               
                   
                 TTACTAGTATTATACCTAGGACT 
                   
               
               
                   
               
               
                 IV-1 
                 ATCATCACAGCAGCGGCCTGGTGCCGCGCATGACC 
                 33 
               
               
                   
                 ATGATTACGGATTCAC 
                   
               
               
                   
               
               
                 IV-2 
                 TGATGATGTTAGCTAGCGCTGAATTCTGCTTAGAT 
                 34 
               
               
                   
                 CTTGGCCGGAGCCATGG 
                   
               
               
                   
               
               
                 ilvBN-1 
                 ATGACCATGATTACGGATTCAC 
                 35 
               
               
                   
               
               
                 ilvBN-2 
                 TTAGATCTTGGCCGGAGCCATGG 
                 36 
               
               
                   
               
            
           
         
       
     
     Embodiment 1: Acquisition of the isopropyl malate synthase coding gene leuA M  for relieving the feedback inhibition by L-leucine
         (4) Screening of mutant strains resistant to structural analogues of L-leucine
           1.1 Preparation of a suspension of a  Corynebacterium glutamicum  ATCC13032 The  Corynebacterium glutamicum  ATCC13032 is inoculated into an LB (Luria broth) liquid medium for culture at 32 DEG C and 200 rpm for 12 h, centrifugation is performed for collecting bacterial cells, which are then washed with sterile normal saline for 3 times and then resuspended until OD 600  is 0.6-0.8, and 10 uL of the suspension is applied onto a slide glass.   1.2 Plasma mutagenesis at room pressure and temperature Applied mutagenesis parameters include that the slide is arranged 2 mm away from an air flow port, the power is 120 W, the air flow velocity is 10 SLM (standard liter per minute), and the action period is 20 s.   
           1.3 Screening of the mutant strains resistant to the L-leucine-structured analogue-α-aminobutyric acid
           The suspension subjected to mutagenesis in the step 1.2 is spread onto a minimal medium containing 50 mg/L leucine hydroxamate for culture at 35 DEG C for 48 h, and then the strains with a large bacterial colony are selected.   1.4 Determination of L-leucine producing capacity of the strains The strains screened in the step 1.3 are subjected to 96-well plate culture through a seed culture medium and then inoculated at an inoculum size of 5% into a 96-well plate containing a fermentation culture medium for a fermentation experiment, according to which the strain LEU262 is the highest in the yield of L-leucine.   1.5 Screening of the mutant strains resistant to the L-leucine-structured analogue-thioisoleucine and determination of L-leucine producing capacity of the strains The LEU262 is taken as a mutagenesis object. The steps 1.1 and 1.2 are repeated. The mutagenized suspension is applied onto a minimal medium containing 50 mg/L (3-hydroxy leucine for culture at 35 DEG C for 48 hours, then the strains with a large bacterial colony are selected, and the step 4) is repeated to determine that the strain LEU741 is the highest in the yield of L-leucine.   1.6 Culture mediums   The seed culture medium is composed of 20 g/L glucose, 5 g/L yeast powder, 4 g/L (NH 4 ) 2 SO 4 , 2.5 g/L KH 2 PO 4 , 0.5 g/L MnSO 4  and 30 mL/L corn steep liquor, the pH is 6.5-7.0, and the seed culture medium is subjected to high-pressure steam sterilization at 115 DEG C for 15 min.   The fermentation culture medium is composed of 70 g/L glucose, 4 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 0.6 g/L MgSO 4 .7H 2 O, 0.02 g/L MnSO 4 , 0.002 g/L VB1 and 30 mL/L corn steep liquor, the pH is 6.5-7.0, and the fermentation culture medium is subjected to high-pressure steam sterilization at 115 DEG C for 15 min.   1.7 Determination method   8000 g of the fermentation liquor is centrifuged for 5 min, then the supernatant is extracted and subjected to derivatization reaction with 0.8% (V/V) 2, 4-dinitrofluorobenzene, and the content of L-leucine is detected by high performance liquid chromatography under the conditions that Agilent C18 (15 mm*4.6 mm, 5 mum) is subjected to acetonitrile/sodium acetate binary gradient elution, the column temperature is 33 DEG C and the detection wavelength is 360 nm. According to the detection result of the high performance liquid chromatography and comparison with the peak appearance time and the peak area of a standard product, the yield of L-leucine can be determined.   
           (5) Acquisition of the mutant of the isopropyl malate synthase coding gene leuA M  for relieving the feedback inhibition of L-leucine
           The genome of the strain LEU741 is extracted, primers leuA-1′ and leuA-2′ are applied to perform PCR amplification under the conditions: 94 DEG C, 5 min, 1 cycle; 94 DEG C, 30 s, 50 DEG C, 30 s, 72 DEG C, 2 min, 30 cycles; 72 DEG C,10 min,1 cycle. The volume of the reaction system is 100 uL. 10 uL of the PCR products is detected through 1.5% agarose gel electrophoresis. A target fragment amplified by PCR is recovered and connected to a pMD™18-T Vector and is then transformed into  Escherichia coli  ( E. coli  DH5a) competent cells, the cells are applied onto an LB solid culture medium containing ampicillin (100 ug/mL) for inverted culture at 37 DEG C for 24 h. 3 single colonies are picked, and recombinant plasmids are extracted and sequenced.   Sequencing results show that, compared with the wild type leuA, the 2-isopropyl malate synthase encoded by the mutated gene has mutations of F7L, I14F, I51S, G127D, I197V, F370L, K380M, R529H, G561D and V596A, the mutant is named as LEUA M , and the coding gene is named as leuA M      
           (6) Comparison of the enzymatic characteristics of the isopropyl malate synthase mutant LEUA M  and the wild type isopropyl malate synthase LEUA   The genomes of the  Corynebacterium glutamicum  ATCC13032 and the strain LEU741 are taken as templates respectively, primers LA-1 and LA-2 are applied to perform PCR amplification. The products are recovered and connected to pET-His plasmids digested by BamH I and are then transformed into  Escherichia coli  BL21 (DE3) to obtain strains  E. coli -leuA and  E. coli -leuA M , which are induced by IPTG (isopropyl-beta-thiogalactoside) to express recombinant proteins LEUA and LEUA M , bacteria are collected, resuspended in 50 mmol/L Tris-HCl buffer solution (pH=7.5), subjected to ultrasonic disruption and centrifuged, and then the supernatant is collected.
           The enzymatic activities of the LEUA M  and the LEUA are determined by the following method:   adding 10 uL of the above-described supernatant into 990 uL of Tris-HCl buffer solution (50 mmol/L, pH=7.5 and composed of 400 mmol/L potassium glutamate, 20 uL of 5, 5′-dithiobis (2-nitrobenzoic acid), 3 mmol/L acetyl-CoA and 4 mmol/L ketoisovaleric acid) for reaction at 30 DEG C for 1 h, and then adding 100 uL of sulfuric acid (3 mol/L) for treatment at 65 DEG C for 15 min to terminate the reaction, wherein, during the reaction, the 2-isopropyl malate synthase can catalyze the acetyl-CoA to produce coenzyme A, which has the maximum absorbance at OD 412 . Therefore, according to the principle, the change value per minute of OD412 can be measured through spectrophotometry to calculate the production of the coenzyme A and accordingly calculate the enzymatic activity. As results shown in  FIG. 2 , the activities of the LEUA M  and the LEUA are 12.1 and 13.5 nmol/(min*mg*total protein), respectively, presenting no significant difference between them.   The influence of the L-leucine on the enzymatic activity of LEUA M  and LEUA is determined by the following method:   0, 2, 4, 6, 8, 10, 12 and 15 mmol/L of L-leucine are respectively added into the above reaction solution, and then the amount of the produced coenzyme A is measured to study the performance of the LEUA M  on relieving the feedback inhibition by the L-leucine. The enzymatic activity when the addition concentration of the L-leucine is 0 is defined as 100%. Compared with which the enzymatic activity of the LEUA M  or the LEUA under other L-leucine concentration conditions is the relative enzymatic activity. As shown in  FIG. 1 , the relative activity of the LEUA decreases rapidly with increasing L-leucine concentration, and almost decreases to 0 when the L-leucine concentration is higher than 6 mmol/L. This indicates that the LEUA is subjected to the feedback inhibition by the L-leucine. While the relative enzymatic activity of the mutant LEUA M  has no significant change with increasing L-leucine concentration, indicating that the LEUA M  can relieve the feedback inhibition by the L-leucine.   It can be seen from the above results, the 2-isopropyl malate synthase mutant LEUA M  relieves the feedback inhibition by the L-leucine and has no significant decrease in the activity compared with the wild type LEUA.   
               

     Embodiment 2: Acquisition of the acetohydroxy acid synthase coding gene ilvBN M for relieving the feedback inhibition by L-isoleucine
         (4) Screening of mutant strains resistant to L-isoleucine-structured analogues
           1.1 Preparation of a suspension of a  Corynebacterium glutamicum  ATCC13032   The  Corynebacterium glutamicum  ATCC13032 is inoculated into an LB (Luria-Bertani) liquid medium for culture at 32 DEG C and 200 rpm for 12 h, centrifugation is performed for collecting bacterial cells, which are then washed with sterile normal saline for 3 times and then resuspended until OD600 is 0.6-0.8, and 10 uL of the suspension is applied onto a slide.   1.2 Plasma mutagenesis at room pressure and temperature Applied mutagenesis parameters include that the slide is arranged 2 mm away from an air flow port, the power is 120 W, the air flow velocity is 10 SLM, and the action period is 25 s.   1.3 Screening of the mutant strains resistant to the L-isoleucine-structured analogue of α-aminobutyric acid   The suspension subjected to mutagenesis in the step 1.2 is applied onto a minimal medium containing 50 mg/L a-aminobutyric acid for culture at 35 DEG C for 48 h, and then the strains with a large bacterial colony are selected.   1.5 Determination of L-isoleucine producing capacity of the strains   The strains screened in the step 1.3 are subjected to 96-well plate culture through a seed culture medium and then inoculated at an inoculum size of 10% into a 96-well plate containing a fermentation culture medium for a fermentation experiment, according to which the strain ILE396 is the highest in the yield of L-isoleucine.   1.5 Screening of the mutant strains resistant to the L-isoleucine-structured analogue of thioisoleucine and determination of L-leucine producing capacity of the strains The ILE396 is taken as a mutagenesis object, the steps 1.1 and 1.2 are repeated, the mutagenized suspension is applied onto a minimal medium containing 50 mg/L thioisoleucine for culture at 35 DEG C for 48 hours, then the strains with a large bacterial colony are selected, and the step 4) is repeated to determine that the strain ILE693 is the highest in the yield of L-isoleucine.   1.6 Culture mediums   The seed culture medium is composed of 25 g/L glucose, 5 g/L yeast powder, 5 g/L (NH 4 ) 2 SO 4 , 2 g/L KH 2 PO 4 , 0.6 g/L MnSO 4  and 40 mL/L corn steep liquor, the pH is 6.8-7.2, and the seed culture medium is subjected to high-pressure steam sterilization at 115 DEG C for 15 min.   The fermentation culture medium is composed of 80 g/L glucose, 3 g/L (NH4)2504, 1.5 g/L KH 2 PO 4 , 0.6 g/L MgSO 4 .7H 2 O, 0.015 g/L MnSO 4 , 0.001 g/L VB1 and 30 mL/L corn steep liquor, the pH is 6.8-7.2, and the fermentation culture medium is subjected to high-pressure steam sterilization at 115 DEG C for 15 min.   1.7 Determination method   8000 g of the fermentation liquor is centrifuged for 5 min, then the supernatant is extracted and subjected to derivatization reaction with 0.8% (V/V) 2, 4-dinitrofluorobenzene, and the content of L-isoleucine is detected by high performance liquid chromatography under the conditions that Agilent C18 (15 mm*4.6 mm, 5 mum) is subjected to acetonitrile/sodium acetate binary gradient elution, the column temperature is 33 DEG C and the detection wavelength is 360 nm. According to the detection result of the high performance liquid chromatography and comparison with the peak appearance time and the peak area of a standard product, the yield of L-isoleucine can be determined.   
           (5) Acquisition of the mutant of the acetohydroxy acid synthase coding gene ilvBN M  for relieving the feedback inhibition by L-isoleucine
           The genome of the strain ILE693 is extracted, primers ilvBN-1 and ilvBN-2 are applied to PCR amplification under the conditions that treatment at 94 DEG C is performed for 5 min and 1 cycle, treatment at 94 DEG C is performed for 30 s, treatment at 56 DEG C is performed for 30 s, treatment at 72 DEG C is performed for 1 min and 30 cycles and treatment at 72 DEG C is performed for 10 min and 1 cycle, and the volume of the reaction system is 100 uL. 10 uL of the PCR products is detected through 1.5% agarose gel electrophoresis, a target fragment amplified by PCR is recovered and connected to a pMD™18-T Vector and is then transformed into  E. coli  DH5 a competent cells, the cells are applied onto an LB solid culture medium containing ampicillin (100 ug/mL) for inverted culture at 37 DEG C for 24 h, 3 single colonies are picked, and recombinant plasmids are extracted and sequenced.   Sequencing results show that, compared with the wild type ilvBN, the acetohydroxy acid synthase encoded by the mutated gene has mutations of K30Q, A84T, G128S, A226S, K227R, Y252H, T362S and H674L, the mutant is named as ILVBN M , and the coding gene is named as ilvBN M  (as shown in SEQ ID NO. 5).   
           (6) Comparison of the enzymatic characteristics of the acetohydroxy acid synthase mutant ILVBN M  and the wild type acetohydroxy acid synthase ILVBN
           The genomes of the  Corynebacterium glutamicum  ATCC13032 and the strain ILE693 are taken as templates respectively, primers IV-1 and IV-2 are applied to PCR amplification, the products are recovered and connected to pET-His plasmids digested by BamH I and are then transformed into  Escherichia coli  BL21 (DE3) to obtain strains  E. coli -ilvBN and  E. coli -ilvBN M , which are induced by IPTG to express recombinant proteins ILVBN and ILVBN M , bacteria are collected, resuspended in 100 mmol/L potassium phosphate buffer solution (pH=7.8), subjected to ultrasonic disruption and centrifuged, and then the supernatant is collected.   The enzymatic activities of the ILVBN M  and the ILVBN are determined by the following method: adding 100 uL of the above-described supernatant into 1 mL of potassium phosphate buffer solution (100 mmol/L, pH=7.8 and composed of 100 mmol/L sodium pyruvate, 100 mmol/L L2-ketobutyric acid, 10 mmol/L MgCl 2  and 0.2 mmol/L thiamine pyrophosphate) for reaction at 37 DEG C for 1 h, adding in 100 uL of sulfuric acid (3 mol/L) for treatment at 65 DEG C for 15 min to terminate the reaction, mixing the reaction solution with 1 mL of 0.5% creatine and 1 mL of a-naphthol solution (containing 2.5 mol/L NaOH) for treatment at 65 DEG C for 20 min, cooling down to room temperature, and measuring the amount of 2-keto-2-hydroxybutyric acid produced (OD 525 ) through spectrophotometry. and accordingly calculate the enzymatic activity. As results shown in  FIG. 4 , the activities of the ILVBN M  and the ILVBN are 16.7 and 16.9 nmol/(min*mg*total protein), respectively, presenting no significant difference.   The influence of the L-isoleucine on the enzymatic activity of the ILVBN M  and the ILVBN is determined by the following method: adding 0, 2, 4, 6, 8, 10 and 12 mmol/L L-isoleucine respectively into the above reaction solution, and then measuring the amount of the produced 2-keto-2-hydroxybutyric acid to study the performance of the ILVBN M  on relieving the feedback inhibition by the L-isoleucine.   The enzymatic activity when the concentration of the added L-isoleucine is 0 is defined as 100%, compared with which the enzymatic activity of the ILVBN M  or the ILVBN under other L-leucine concentration conditions is the relative enzymatic activity. As shown in  FIG. 3 , the relative activity of the ILVBN decreases rapidly with increasing L-isoleucine concentration, and when the L-isoleucine concentration is higher than 8 mmol/L, the ILVBN almost presents no activity, indicating that the ILVBN is subject to the feedback inhibition by the L-isoleucine, while the relative enzymatic activity of the mutant ILVBN M  has no significant change with increasing L-leucine concentration, indicating that the ILVBN M  can relieve the feedback inhibition by the L-isoleucine.   It can be seen from the above results, the acetohydroxy acid synthase mutant ILVBN M  relieves the feedback inhibition by the L-leucine and has no significant decrease in the activity compared with the wild type ILVBN.   
               

     Embodiment 3: Construction of the L-leucine producing bacterium TE03
         (5) Construction of a recombinant fragment UHF-leuA M -DHF
           An artificially synthesized plasmid containing the gene leuA M  is taken as a template and LEUA-3 and LEUA-4 as primers to perform PCR amplification to obtain the leuA M ; The genome of the  Escherichia coli  W3110 is taken as a template and LEUA-1 and LEUA-2 as well as LEUA-5 and LEUA-6 as primers to perform amplification to obtain fragments UHF and DHF, which are the upstream homologous arm and the downstream homologous arm of a gene lad, respectively; UHF, DHF and the leuA M  are taken as templates and LEUA-1 and LEUA-6 as primers to perform PCR amplification, and then recovering is performed to obtain the recombinant fragment UHF-leuA M -DHF.   
           (6) Construction of a recombinant fragment UHFA-ilvBN M  DHFB
           A artificially synthesized plasmid containing the gene ilvBN M  is taken as a template and IlvB-3 and IlvB-4 as primers to perform PCR amplification to obtain the ilvBN M ; the genome of the  Escherichia coli  W3110 is taken as a template and IlvB-1 and IlvB-2 as well as IlvB-5 and IlvB-6 as primers to perform amplification to obtain fragments UHFA and DHFB, which are the upstream homologous arm and the downstream homologous arm of a gene lacZ, respectively; UHFA, DHFB and the ilvBN M  are taken as templates and IlvBN-1 and IlvBN-6 as primers to perform PCR amplification, and then recovering is performed to obtain the recombinant fragment UHFA-ilvBN M -DHFB.   
           (7) Construction of a recombinant plasmid pTR-leuBCD
           The genome of the  Escherichia coli  W3110 is taken as a template and leuBCD-1 and leuBCD-2 as primers to perform PCR amplification to obtain leuBCD (an operon composed of leuB and leuCD in the  Escherichia coli ), and a plasmid pTrc99a is subjected to digestion by BamH I, electrophoresis and gel extraction and is then connected to the leuBCD to obtain the recombinant plasmid pTR-leuBCD.   
           (8) Construction of the L-leucine genetically engineered bacterium TE03
           PG-1 and PG-2, PG-3 and PG-4 are respectively annealed at 52 DEG C and then connected to plasmids pGRB to obtain pGRB1 and pGRB2, wherein PG-1 and PG-2 as well as PG-3 and PG-4 are single-stranded DNAs of guide sequences for Cas9 to identify the lacI and lacZ gene sequences of the genome of the W3110, and the single-stranded DNAs are annealed to double-stranded DNAs which can be connected with the pGRB. The pREDCas9 plasmids are transformed into the  Escherichia coli  W3110, and positive clones are selected to obtain a W3110-pREDCas9 strain. The pGRB1 and the UHF-leuA M -DHF are respectively transformed into the W3110-pREDCas9, positive clones are selected and subjected to elimination of pGRB-gRNA and the pREDCas9 plasmids to obtain a TE01 strain. In the same way, the pGRB2 and the UHFA-ilvBN M -DHFB are transformed into the TE01 containing the pREDCas9 to obtain a TE02 strain. The pTR-leuBCD is transformed into the TE02 to obtain the TE03.   
               

     Embodiment 4: Fermentation experiment of the L-leucine producing bacterium TE03 in a fermentation tank
         (4) Seed culture
           3-5 tubes of fresh slant activated TE03 are inoculated by an inoculating loop into a 5 L fermentation tank filled with 1 L of a seed culture medium, the pH of the fermentation liquid is regulated to 6.5-7.5 by batch-feeding 25% (W/V) ammonia liquor, the content of dissolved oxygen is maintained to be 20-50%, the ventilating rate is 3-5 m3/h, the stirring velocity is 400-500 rpm, and culture is performed at 32 DEG C for 6-8 h.   
           (5) Fermentation in the fermentation tank
           The seed culture obtained in the step (1) is inoculated at an inoculum size of 5% to a 5 L fermentation tank filled with 3 L of a fermentation culture medium for tank fermentation, the fermentation temperature is 35 DEG C, the ventilating rate is 3-5 m3/h, the stirring velocity is 600 rpm, the content of dissolved oxygen is maintained to be 20-40%, an 80% (W/V) glucose solution is batch-fed to maintain the residual sugar concentration to be 0.1-0.5% (W/V), the pH of the fermentation liquid is regulated to 6.5-7.5 by batch-feeding 25% (W/V) ammonia water and the fermentation is performed for 48h (the process curve of fermentation is shown as  FIG. 5 ).   
           ( 6 ) Detection of L-leucine in the fermentation liquid       

     The detection method is the same as that in the step 1.7 of (1) of the embodiment 1, and according to the detection, after the fermentation is performed for 44 h, the yield of L-leucine reaches the highest 69.6g/L at 69.6 g/L with a conversion rate of 19.1%. 
     The seed culture medium is composed of: 
     14 g/L glucose, 5 g/L peptone, 3 g/L yeast powder, 2 g/L KH 2 PO 4 , 1 g/L MgSO 4 , 10 mg/L FeSO 4  and 10 mg/L MnSO 4 , the pH is 7.0, and the seed culture medium is subjected to high-pressure steam sterilization at 0.075 MPa for 15 min. 
     The fermentation culture medium is composed of: 
     25 g/L glucose, 12 g/L peptone, 4 g/L yeast powder, 3.5 g/L KH 2 PO 4 , 1.5 g/L MgSO 4 , 15 mg/L FeSO 4 , 15 mg/L MnSO 4  and 0.01 mg/L VB1, the pH is 7.0, and the fermentation culture medium is subjected to high-pressure steam sterilization at 0.075 MPa for 15 min. 
     Embodiment 5: Influence of overexpression of leuA M  on L-leucine synthesis 
     A method identical to that in the embodiment 1 is applied to respectively constructing strains: 1) an ilvBN M  and leuBCD overexpressing strain TE04, 2) an ilvBN, leuA and leuBCD overexpressing strain TE05, 3) an ilvBN m , leuA and leuBCD overexpressing strain TE06 and 4) an ilvBN, leuA M  and leuBCD overexpressing strain TE07. A method identical to that in the embodiment 4 is applied to performing fermentation experiments. Detection results show that, after 44 h of fermentation, the strain TE03 has the highest yield of L-leucine (69.2 g/L), followed by strain TE07 (35.37 g/L) and strain TE06 (18.16 g/L), and strain TE04 and strain TE05 are the lowest(0.12 and 2.15 g/L, respectively) (as shown in  FIG. 6 ). 
     Above-described are merely several embodiments of the present invention, which are described specifically in detail but cannot be construed as limitation to the scope of the patent. It should be noted that, for those skilled in the art, modifications, combinations and improvements can be made on the described embodiments without departing from the concept of the patent and all fall into the scope of protection of the patent. Therefore, the scope of protection of the patent should be subject to the claims.