WO2021037190A1 - 一种2-异丙基苹果酸合成酶及其工程菌与应用 - Google Patents

一种2-异丙基苹果酸合成酶及其工程菌与应用 Download PDF

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WO2021037190A1
WO2021037190A1 PCT/CN2020/112038 CN2020112038W WO2021037190A1 WO 2021037190 A1 WO2021037190 A1 WO 2021037190A1 CN 2020112038 W CN2020112038 W CN 2020112038W WO 2021037190 A1 WO2021037190 A1 WO 2021037190A1
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leucine
gene
leua
genetically engineered
ilvbn
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张成林
徐庆阳
李燕军
张宇
李英滋
朱福周
芦楠
韩世宝
董解荣
王子申
徐昊
李子翼
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天津科技大学
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Priority claimed from CN201910820591.XA external-priority patent/CN110540976B/zh
Priority claimed from CN201910886078.0A external-priority patent/CN110551670B/zh
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Priority to US17/137,327 priority Critical patent/US11866737B2/en
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    • C12Y101/01085—3-Isopropylmalate dehydrogenase (1.1.1.85)
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    • C12Y203/03013—2-Isopropylmalate synthase (2.3.3.13)

Definitions

  • the invention relates to a 2-isopropylmalate synthase, a genetic engineering bacterium for producing L-leucine and applications thereof, and belongs to the field of metabolic engineering.
  • L-leucine is a branched-chain amino acid and is one of the eight essential amino acids for the human body.
  • L-leucine is a raw material for synthesizing protein and hormones, and it plays a vital role in human life activities. Therefore, L-leucine has a very broad market and application prospects in food and medicine industries.
  • L-leucine Industrial synthesis methods of L-leucine include hair extraction methods and fermentation methods.
  • the extraction method has limited raw material sources, high production costs, and environmental pollution. Therefore, the fermentation method is the mainstream method for producing L-leucine.
  • the industrial production strains of L-leucine are mainly obtained by mutagenesis, which have deficiencies such as nutritional deficiency, slow growth, and unstable genetic traits, which cause problems such as long fermentation cycle, unstable fermentation performance, low yield and conversion rate, etc. .
  • the present invention provides a method to relieve L-leucine.
  • Leucine feedback-inhibited isopropylmalate synthase mutant and its coding gene and use the gene to construct a genetically engineered bacteria producing L-leucine.
  • One of the technical solutions of the present invention to solve the above-mentioned problem is to provide a 2-isopropylmalate synthase mutant LEUA M that relieves L-leucine feedback inhibition, and has the amino acid sequence shown in SEQ ID NO.1, so said 2-isopropyl malate synthase gene encoding the mutant form of leuA M, the nucleotide sequence of SEQ ID NO.2 as shown in the sequence Listing.
  • the isopropylmalate synthase mutant is derived from a mutant strain of Corynebacterium glutamicum, and the screening process of the mutant strain is as follows: Corynebacterium glutamicum ATCC13032 is used as the starting strain, through atmospheric pressure and room temperature plasma Mutagenesis, and then the strain LEU262 was selected on the minimal medium containing 50mg/L leucine hydroxamate; LEU262 was used as the starting strain, and plasma mutagenesis was carried out at room temperature at atmospheric pressure, and then the strain containing 50mg/L ⁇ -hydroxyl brightening The strain LEU741 was selected on the minimal medium containing amino acids.
  • the LEU741 genome was extracted, and the gene encoding 2-isopropylmalate synthase was amplified by PCR by designing primers, and the PCR product was recovered and sequenced. It was found that the 2-isopropylmalate synthase encoded by the gene was relative to that derived from glutamine.
  • the wild-type 2-isopropylmalate synthase of Corynebacterium glutamicum ATCC13032 has the following amino acid mutations: F7L, I14F, I51S, G127D, I197V, F370L, K380M, R529H, G561D, V596A.
  • amino acid replaced by the original amino acid position is used to refer to the mutated amino acid in the 2-isopropylmalate synthase mutant.
  • F7L it means that the amino acid at position 7 is replaced by Phe of wild-type 2-isopropylmalate synthase with Leu
  • F7 means that the amino acid at position 7 is Phe
  • the position number corresponds to wild type 2 in SEQ ID NO.3 -The amino acid sequence number of isopropylmalate synthase.
  • leuA represents the wild-type 2-isopropylmalate synthase encoding gene (shown in SEQ ID NO. 4), and LEUA represents the wild-type 2-isopropylmalate synthase (shown in SEQ ID NO. 3). ); leuA M is a mutant gene of 2-isopropylmalate synthase (shown in SEQ ID NO. 2); LEUA M is a mutant of 2-isopropylmalate synthase (shown in SEQ ID NO. 1) .
  • the amino acid comparison before and after mutation is as follows:
  • the 2-isopropylmalate synthase mutant LEUA M has the following enzymatic properties: under the condition of L-leucine concentration of 0-15 mmol/L, the enzyme activity of LEUA M has no significant change, that is, the mutant The feedback inhibition effect of L-leucine on it is lifted; and the enzyme activity of LEUA M is the same as that of the wild-type 2-isopropylmalate synthase LEUA under the condition of L-leucine concentration of 0-15mmol/L.
  • the leucine concentration is 0mmol/L compared with no significant decrease.
  • the second technical solution of the present invention to solve the above-mentioned problem is to provide a genetically engineered bacterium for producing L-leucine, the genetically engineered bacterium is to eliminate L-leucine feedback by overexpressing the L-leucine in the host cell.
  • Inhibited isopropylmalate synthase encoding gene leuA M , release of L-isoleucine feedback inhibition acetohydroxy acid synthase encoding gene ilvBN M , 3-isopropylmalate dehydrogenase encoding gene leuB, 3-iso Propylmalate dehydratase encoding gene leuCD was obtained.
  • the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, and sodium-requiring bacteria. Vibrio natriegens or Saccharomyces cerevisiae, etc.;
  • the acetohydroxy acid synthase encoded by the ilvBN M gene relieves the feedback inhibition of L-isoleucine, and the nucleotide sequence is shown in SEQ ID NO.5 in the sequence table.
  • the leuB gene can be from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus megaterium, etc., such as the leuB gene of Genbank numbering b0073, JW5807, NCgl1237, BSU28270, BAMF_2634.
  • the leuCD gene can be from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus megaterium, etc., such as the leuCD gene of Genbank numbering b0071, b0072, JW0070, JW0071, NCgl1262, NCgl1263, BSU28250, BSU28260, BAMF_2632, BAMF_2633 .
  • the genetically engineered bacteria producing L-leucine is TE03, which uses Escherichia coli W3110 as a host cell and overexpresses the leuA M gene shown in SEQ ID NO. 2, SEQ ID NO.
  • the ilvBN M gene shown in 5, and the leuBCD shown in SEQ ID NO.6 (leuB and leuCD in Escherichia coli form an operon leuBCD) were obtained;
  • construction method is specifically as follows:
  • the nucleotide sequence of the UHF is shown in SEQ ID NO. 7;
  • the nucleotide sequence of the DHF is shown in SEQ ID NO. 8;
  • the nucleotide sequence of the UHFA is shown in SEQ ID NO. 9;
  • the nucleotide sequence of the DHFB is shown in SEQ ID NO. 10;
  • the leuBCD gene was amplified by PCR, and ligated with the plasmid pTrc99a to obtain the recombinant plasmid pTR-leuBCD;
  • PG-1 and PG-2, PG-3 and PG-4 were annealed at 52°C, and then ligated to plasmid pGRB to obtain pGRB1 and pGRB2; using E. coli W3110 as the starting strain, pGRB1 and UHF-leuA M- DHF was transformed into E. coli W3110 to obtain recombinant strain TE01; with strain TE01 as the starting strain, pGRB2 and UHFA-ilvBN M- DHFB were transformed into TE01 respectively to obtain strain TE02; pTR-leuBCD was transformed into TE02 to obtain TE03.
  • the present invention also provides a method for fermenting and synthesizing L-leucine by using the genetically engineered bacteria, which is specifically as follows:
  • the dissolved oxygen is maintained at 20-40%, the pH is maintained at 6.5-7.5, the culture temperature is 30-35°C, and the fermentation period is 40-48h. , Maintain the residual sugar concentration at 0-0.4% (W/V) during the fermentation process;
  • the fermentation medium components are: glucose 25g/L, peptone 12g/L, yeast powder 4g/L, KH 2 PO 4 3.5 g/L, MgSO 4 1.5 g/L, FeSO 4 15 mg/L, MnSO 4 15 mg/L L, VB1 0.01mg/L, pH7.0, 0.075MPa autoclave sterilization for 15min.
  • the 2-isopropylmalate synthase encoded by the leuA M gene of the present invention has the following characteristics: the enzyme relieves the feedback inhibition effect of L-leucine on it ( Figure 1). At a concentration of 0-15mmol/L, the activity of LEUA M enzyme did not change significantly, and its activity was not significantly lower than that of the 2-isopropylmalate synthase encoded by wild-type leuA ( Figure 2)
  • the L-leucine genetically engineered bacteria TE03 of the present invention has no auxotrophs, fast growth, short fermentation cycle, high yield, and high conversion rate. After 40-48h fermentation, the L-leucine in the fermentation broth The concentration reached 60.5-69.6g/L ( Figure 3).
  • FIG. 1 The effect of L-leucine on the activity of 2-isopropylmalate synthase encoded by leuA and leuA M genes;
  • Figure 3 The effect of L-isoleucine on the activity of acetohydroxy acid synthase encoded by ilvBN and ilvBN M genes;
  • the genetically engineered bacteria for the production of L-leucine constructed by the present invention is obtained by overexpressing the L-leucine feedback inhibition isopropylmalate synthase encoding gene leuA M and the release L-leucine obtained by the present invention in host cells.
  • -Isoleucine feedback inhibition of acetohydroxy acid synthase encoding gene ilvBN M , 3-isopropylmalate dehydrogenase encoding gene leuB, and 3-isopropylmalate dehydratase encoding gene leuCD were obtained;
  • the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens), Natriegens (Vibrio natriegens) or Saccharomyces cerevisiae (Saccharomyces cerevisiae), etc.;
  • the ilvBN M gene is derived from Corynebacterium glutamicum resistant to the structural analogs of L-isoleucine ⁇ -aminobutyric acid and thioisoleucine;
  • the leuB gene is a leuB gene with Genbank numbering b0073, JW5807, NCgl1237, BSU28270, or BAMF_2634.
  • the leuCD gene is a leuCD gene with Genbank numbering b0071, b0072, JW0070, JW0071, NCgl1262, NCgl1263, BSU28250, BSU28260, BAMF_2632, or BAMF_2633.
  • the above-mentioned host cells, the ilvBN M gene, the leuB gene and the leuCD gene can all achieve the effects of the present invention.
  • Escherichia coli W3110 is used as the host cell to overexpress the sequence shown in SEQ ID NO.2.
  • the leuA M gene shown in the following example, the ilvBN M gene shown in SEQ ID NO.5, and the leuBCD shown in SEQ ID NO.6 (leuB and leuCD form an operon leuBCD in E. coli) are used as examples to construct and produce L-leucine
  • the genetically engineered strain of is TE03, which exemplifies the present invention.
  • Example 1 Obtaining the isopropylmalate synthase encoding gene leuA M that relieves feedback inhibition of L-leucine
  • the mutagenesis parameters are: the slide is placed at the airflow port 2mm, the power is 120W, the airflow is 10SLM, and the action time is 20s.
  • step 2 Spread the mutagenized bacterial suspension in step 2 on a minimal medium containing 50 mg/L leucine hydroxamate, and after culturing at 35°C for 48 hours, select strains with larger colonies.
  • the strain selected in step 3 was cultured in a 96-well plate with a seed medium, and then inoculated to a 96-well plate containing a fermentation medium with an inoculum of 5% for fermentation experiments.
  • the strain LEU262 had the highest L-leucine yield.
  • LEU262 as the mutagenesis object, repeat steps 1 and 2, spread the mutagenized bacterial suspension on the basic medium containing 50mg/L ⁇ -hydroxyleucine, cultivate at 35°C for 48h, select the larger colony Strains.
  • Repeat step 4 LEU741 has the highest yield of L-leucine.
  • Seed medium glucose 20g/L, yeast powder 5g/L, (NH 4 ) 2 SO 4 4g/L, KH 2 PO 4 2.5g/L, MnSO 4 0.5g/L, corn steep liquor 30mL/L, pH 6.5 -7.0, high-pressure steam sterilization at 115°C for 15 minutes.
  • Fermentation medium glucose 70g/L, (NH 4 ) 2 SO 4 4g/L, KH 2 PO 4 1g/L, MgSO 4 ⁇ 7H 2 O 0.6g/L, MnSO 4 0.02g/L, V B1 0.002g /L, corn steep liquor 30mL/L. pH 6.5-7.0, high-pressure steam sterilization at 115°C for 15 minutes.
  • PCR conditions are: 94°C 5min 1 cycle, 94°C 30s, 50°C 30s, 72°C 2min 30 cycles, 72°C 10min 1
  • the reaction system is 100 ⁇ L. Take 10 ⁇ L of PCR product and detect it by 1.5% agarose gel electrophoresis.
  • the PCR-amplified target fragment was recovered and ligated to pMD TM 18-T Vector and transformed into E.coli DH5 ⁇ competent cells, and then spread on the solid culture of LB containing ampicillin (100 ⁇ g/mL) at 37 Incubate inverted at °C for 24h. Pick 3 single clones, extract the recombinant plasmid and determine its sequence.
  • the genomes of Corynebacterium glutamicum ATCC13032 and LEU741 were used as templates, and primers LA-1 and LA-2 were used for PCR amplification. After the product was recovered, it was connected to the pET-His plasmid digested with BamH I, and then transformed into Escherichia Escherichia coli BL21(DE3), the strains E.coli-leuA and E.coli-leuA M were obtained .
  • IPTG IPTG to induce the expression of recombinant proteins LEUA and LEUA M in E.coli-leuA and E.coli-leuA M , collect the bacteria, resuspend in 50mmol/L Tris-HCl buffer (pH 7.5), ultrasonically break and centrifuge. Take the supernatant.
  • the change value of OD 412 per minute is measured by spectrophotometry and the generated Coenzyme A is calculated, thereby calculating the enzyme activity.
  • the results are shown in Figure 2.
  • the activities of LEUA M and LEUA were 12.1 and 13.5 nmol/(min ⁇ mg total protein), respectively, and there was no significant difference between the two.
  • the method for determining the effect of L-leucine on the enzyme activity of LEUA M and LEUA is as follows: Add 0, 2, 4, 6, 8, 10, 12, 15 mmol/L L-leucine to the above reaction solution, and then determine The amount of coenzyme A produced was used to investigate the feedback inhibition effect of LEUA M to release L-leucine.
  • the enzyme activity when the L-leucine addition concentration is 0 is defined as 100%, and the enzyme activities of LEUA M and LEUA under the conditions of the remaining L-leucine concentration conditions are compared as relative enzyme activities.
  • Example 2 Obtaining the acetohydroxy acid synthase encoding gene ilvBN M that relieves feedback inhibition of L-isoleucine
  • the mutagenesis parameters are: the slide is placed at the airflow port 2mm, the power is 120W, the airflow is 10SLM, and the action time is 25s.
  • step 2 Spread the mutagenized bacterial suspension in step 2 on a minimal medium containing 50 mg/L ⁇ -aminobutyric acid, and after culturing at 35°C for 48 hours, select strains with larger colonies.
  • strains selected in step 3 were cultured in 96-well plates with seed medium, and then inoculated into 96-well plates with fermentation medium at a 10% inoculum for fermentation experiments.
  • ILE396 had the highest L-isoleucine production.
  • ILE396 Take ILE396 as the mutagenesis object, repeat steps 1 and 2, spread the mutagenized bacterial suspension on a minimal medium containing 50mg/L thioisoleucine, culture at 35°C for 48h, and select larger colonies Strains.
  • ILE693 has the highest yield of L-isoleucine.
  • Seed medium glucose 25g/L, yeast powder 5g/L, (NH 4 ) 2 SO 4 5g/L, KH 2 PO 4 2g/L, MnSO 4 0.6g/L, corn steep liquor 40mL, pH 6.8-7.2, High-pressure steam sterilization at 115°C for 15 minutes.
  • Fermentation medium (g/L): glucose 80g/L, (NH 4 ) 2 SO 4 3g/L, KH 2 PO 4 1.5g/L, MgSO 4 ⁇ 7H 2 O 0.6g/L, MnSO 4 0.015g/ L, V B1 0.001g/L, corn steep liquor 30mL. pH 6.8-7.2, autoclave at 115°C for 15min.
  • Extract the ILE693 genome use primers ilvBN-1 and ilvBN-2 for PCR amplification, PCR conditions are: 94°C 5min 1 cycle, 94°C 30s, 56°C 30s, 72°C 1min 30 cycles, 72°C 10min 1 cycle ,
  • the reaction system is 100 ⁇ L. Take 10 ⁇ L of PCR product and detect it by 1.5% agarose gel electrophoresis.
  • the PCR-amplified target fragment was recovered and ligated to pMD TM 18-T Vector and transformed into E.coli DH5 ⁇ competent cells, and then spread on the solid culture of LB containing ampicillin (100 ⁇ g/mL) at 37 Incubate inverted at °C for 24h. Pick 3 single clones, extract the recombinant plasmid and determine its sequence.
  • Corynebacterium glutamicum ATCC13032 and ILE693 were used as templates, and primers IV-1 and IV-2 were used for PCR amplification. After the product was recovered, it was connected to the pET-His plasmid digested with BamH I, and then transformed into Escherichia Escherichia coli BL21(DE3), the strains E.coli-ilvBN and E.coli-ilvBN M were obtained .
  • the enzyme activity determination method of ILVBN M and ILVBN is as follows: Take 100 ⁇ L of the above supernatant to 1mL potassium phosphate buffer (100mmol/L, pH 7.8 containing 100mmol/L sodium pyruvate, 100mmol/L 2-ketobutyric acid, 10mmol/LMgCl 2 , 0.2mmol/L thiamine pyrophosphate), after reacting at 37°C for 1h, add 100 ⁇ L of sulfuric acid (3mol/L), and treat at 65°C for 15min to stop the reaction.
  • 1mL potassium phosphate buffer 100mmol/L, pH 7.8 containing 100mmol/L sodium pyruvate, 100mmol/L 2-ketobutyric acid, 10mmol/LMgCl 2 , 0.2mmol/L thiamine pyrophosphate
  • the method for determining the effect of L-isoleucine on the enzyme activity of ILVBN M and ILVBN is as follows: add 0, 2, 4, 6, 8, 10, 12 mmol/LL-isoleucine to the above reaction solution, and then measure 2 -The amount of keto-2-hydroxybutyric acid produced to investigate the feedback inhibition effect of ILVBN M to release L-isoleucine.
  • the enzyme activity when the added concentration of L-isoleucine is 0 is defined as 100%, and the enzyme activities of ILVBN M and ILVBN under the conditions of the other L-isoleucine concentrations are compared as relative enzyme activities. The results are shown in Figure 3.
  • the relative enzyme activity of ILVBN decreases rapidly with the increase of L-isoleucine concentration.
  • the acetohydroxy acid synthase mutant ILVBN M relieved the feedback inhibition of L-isoleucine, and its activity was not lower than that of wild-type ILVBN.
  • UHF and DHF are respectively the upper and downstream homology arms of the lacI gene; UHF, DHF and leuA M are used as templates, and the primers LEUA-1 and LEUA-6 are used for PCR amplification. After recovery, the recombinant fragment UHF-leuA M- DHF is obtained.
  • a synthetic plasmid containing the ilvBN M gene was used as a template, and IlvB-3 and IlvB-4 were used as primers to perform PCR amplification to obtain ilvBN M ; the E. coli W3110 genome was used as a template, and primers IlvB-1 and IlvB- were used respectively.
  • IlvB-5 and IlvB-6 are amplified to obtain fragments UHFA and DHFB, UHFA and DHFB are the upper and downstream homology arms of lacZ gene respectively; UHFA, DHFB and ilvBN M are used as templates, using primers IlvBN-1 and IlvBN- 6 Carry out PCR amplification and recover the recombinant fragment UHFA-ilvBN M- DHFB.
  • leuBCD leuB and leuCD form an operon leuBCD in E. coli.
  • the plasmid pTrc99a was digested with BamH I. After electrophoresis, gel cutting and recovery, it was connected with leuBCD to obtain the recombinant plasmid pTR-leuBCD.
  • PG-1 and PG-2 and PG-3 and PG-4 were annealed at 52°C, respectively, and then ligated to plasmid pGRB to obtain pGRB1 and pGRB2.
  • PG-1 and PG-2 and PG-3 and PG-4 are the single-stranded DNA guide sequences used by Cas9 to recognize the lacI and lacZ gene sequences of the W3110 genome. After the two are annealed, they become double-stranded DNA and can be connected to pGRB.
  • the pREDCas9 plasmid was transformed into E. coli W3110, and positive clones were picked to obtain the W3110-pREDCas9 strain.
  • the pGRB1 and UHF-leuA M- DHF were transformed into W3110-pREDCas9, the positive clones were picked, and the pGRB-gRNA and pREDCas9 plasmids were eliminated to obtain the TE01 strain.
  • pGRB2 and UHFA-ilvBN M -DHFB were transformed into TE01 containing pREDCas9 to obtain TE02.
  • Example 4 Fermentation experiment of L-leucine producing strain TE03 in fermentor
  • the seed culture of step (1) was connected to a 5L fermentor containing 3L of fermentation medium with 5% inoculum for growth tank culture.
  • the fermentation temperature was 35°C
  • the ventilation volume was 3-5m 3 /h
  • the stirring speed was 600rpm.
  • the oxygen is maintained at 20-40%
  • the glucose solution with a concentration of 80% (W/V) is fed
  • the residual sugar concentration is maintained at 0.1-0.5% (W/V)
  • the ammonia is fed with 25% (W/V).
  • the pH of the fermentation broth is 6.5-7.5
  • the fermentation cycle is 48h (the fermentation process curve is shown in Figure 5).
  • the method is the same as that in Example 1(1)7. After testing, the yield of L-leucine was the highest at 44 hours of fermentation, reaching 69.6 g/L, and the conversion rate was 19.1%.
  • the seed culture is composed of:
  • composition of the fermentation medium is:
  • Example 4 Using the same method in Example 1, to construct 1 ilvBN M and leuBCD overexpression strain TE04, 2 ilvBN, leuA and leuBCD overexpression strain TE05, 3 ilvBN M , leuA and leuBCD overexpression strain TE06, 4 ilvBN, leuA M and leuBCD overexpression strain TE07 .
  • the fermentation experiment was carried out using the same method as in Example 4. After 44 hours of fermentation, TE03 has the highest L-leucine production (69.2g/L), followed by TE07 (35.37g/L) and TE06 (18.16g/L), TE04 and TE05 L-leucine The lowest yields were 0.12 and 2.15g/L, respectively (Figure 6).

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Abstract

提供一种2-异丙基苹果酸合成酶及生产L-亮氨酸的基因工程菌及其应用，属于代谢工程领域。所述基因工程菌是通过在宿主细胞中过表达解除L-亮氨酸反馈抑制异丙基苹果酸合成酶编码基因leuA M、解除L-异亮氨酸反馈抑制乙酰乳酸合酶编码基因ilvBN M、3-异丙基苹果酸脱氢酶编码基因leuB、3-异丙基苹果酸脱水酶编码基因leuCD获得。所述的L-亮氨酸基因工程菌无营养缺陷、生长快、发酵周期短、产量高、转化率高。

Description

一种2-异丙基苹果酸合成酶及其工程菌与应用
本申请要求于2019年8月29日提交中国专利局、申请号为CN201910820591X、发明名称为“一种异丙基苹果酸合成酶及其应用”以及2019年9月19日提交中国专利局、申请号为CN2019108860780、发明名称为“一种生产L-亮氨酸的基因工程菌及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种2-异丙基苹果酸合成酶及生产L-亮氨酸的基因工程菌及其应用，属于代谢工程领域。
背景技术
L-亮氨酸属于分支链氨基酸，是人体必需的八种氨基酸之一。L-亮氨酸是合成蛋白质和激素原料，在人体生命活动中扮演着至关重要角色。因此，L-亮氨酸在食品和医药等行业具有非常广泛的市场及应用前景。
工业上L-亮氨酸的合成方法包括毛发提取法和发酵法。然而提取法具有原料来源受限制、生产成本高、污染环境等不足，由此发酵法是生产L-亮氨酸的主流方法。目前，L-亮氨酸的工业生产菌种主要由诱变获得，具有营养缺陷、生长慢、遗传性状不稳定等不足，从而引起发酵周期长、发酵性能不稳定、产量和转化率低等问题。
发明内容
为了克服目前野生型异丙基苹果酸合成酶受L-亮氨酸反馈抑制以及现有L-亮氨酸生产菌株生长慢、营养缺陷、发酵不稳定等不足，本发明提供一种解除L-亮氨酸反馈抑制的异丙基苹果酸合成酶突变体及其编码基因，并利用该基因构建生产L-亮氨酸的基因工程菌。
本发明解决述问题的技术方案之一是：提供一个解除L-亮氨酸反馈抑制的2-异丙基苹果酸合成酶突变体LEUA M，具有SEQ ID NO.1所示的氨基酸序列，所述2-异丙基苹果酸合成酶突变体的编码基因为leuA M，核苷酸序列如序列表SEQ ID NO.2所示。
所述异丙基苹果酸合成酶突变体来自一株谷氨酸棒杆菌突变株，所述突变株筛选过程如下：以谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032为出发菌株，通过常压室温等离子诱变，然后在含50mg/L亮氨酸氧肟酸盐的基本培养基上筛选出菌株LEU262；以LEU262为出发菌株，通过常压室温等离子诱变，然后在含50mg/L β-羟基亮氨酸的基本培养基上筛选出菌株LEU741。
提取LEU741基因组，通过设计引物进行PCR扩增2-异丙基苹果酸合成酶编码基因，将PCR产物回收后进行测序，发现该基因编码的2-异丙基苹果酸合成酶相对于来自谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032的野生型2-异丙基苹果酸合成酶发生如下氨基酸突变：F7L，I14F，I51S，G127D，I197V，F370L，K380M，R529H，G561D，V596A。
在本发明中采用如下定义：
1、异丙基苹果酸合成酶突变体的标识
采用“原始氨基酸位置替换的氨基酸”来表示2-异丙基苹果酸合成酶突变体中突变的氨基酸。如F7L，表示位置7的氨基酸由野生型2-异丙基苹果酸合成酶的Phe替换成Leu，F7表示第7位的氨基酸为Phe，位置的编号对应于SEQ ID NO.3中野生型2-异丙基苹果酸合成酶的氨基酸序列编号。
本发明中，leuA代表野生型2-异丙基苹果酸合成酶编码基因(SEQ ID NO.4所示)，LEUA代表野生型2-异丙基苹果酸合成酶(SEQ ID NO.3所示)；leuA M为2-异丙基苹果酸合成酶突变体基因(SEQ ID NO.2所示)；LEUA M为2-异丙基苹果酸合成酶突变体(SEQ ID NO.1所示)。突变前后的氨基酸对照如下表：
Figure PCTCN2020112038-appb-000001
所述2-异丙基苹果酸合成酶突变体LEUA M，具有如下酶学特性：在L-亮氨酸浓度为0-15mmol/L条件下，LEUA M酶活性无明显变化，即该突变体解除了L-亮氨酸对其反馈抑制作用；且在L-亮氨酸浓度为0-15mmol/L条件下LEUA M的酶活性与野生型2-异丙基苹果酸合成酶LEUA在L-亮氨酸浓度为0mmol/L条件下相比无明显降低。
本发明解决述问题的技术方案之二是：提供一种生产L-亮氨酸的基因工程菌，所述基因工程菌是通过在宿主细胞中过表达本发明所述解除L-亮氨酸反馈抑制的异丙基苹果酸合成酶编码基因leuA M、解除L-异亮氨酸反馈抑制乙酰羟酸合成酶编码基因ilvBN M、3-异丙基苹果酸脱氢酶编码基因leuB、3-异丙基苹果酸脱水酶编码基因leuCD获得的。
所述宿主细胞可以是大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、需钠弧菌(Vibrio natriegens)或酿酒酵母(Saccharomyces cerevisiae)等；
所述ilvBN M基因编码的乙酰羟酸合成酶解除了L-异亮氨酸的反馈抑制，核苷酸序列如序列表SEQ ID NO.5所示。
所述leuB基因可以来自大肠杆菌、谷氨酸棒杆菌、枯草芽孢杆菌或巨大芽孢杆菌等，如Genbank编号为b0073、JW5807、NCgl1237、BSU28270、BAMF_2634的leuB基因。
所述leuCD基因可以来自大肠杆菌、谷氨酸棒杆菌、枯草芽孢杆菌或巨大芽孢杆菌等，如Genbank编号为b0071、b0072、JW0070、JW0071、NCgl1262、NCgl1263、BSU28250、BSU28260、BAMF_2632、BAMF_2633的leuCD基因。
优选地，所述生产L-亮氨酸的基因工程菌为TE03，是以大肠杆菌杆菌(Escherichia coli)W3110为宿主细胞，过表达SEQ ID NO.2所示的leuA M基因，SEQ ID NO.5所示的ilvBN M基因，SEQ ID NO.6所示的leuBCD(大肠杆菌里leuB和leuCD组成一个操纵子leuBCD)获得；
进一步地，上述基因工程菌的构建方法如下：
(1)分别扩增异丙基苹果酸合成酶编码基因leuA M、乙酰羟酸合成酶编码基因ilvBN M基因，并分别构建基因组整合片段；
(2)扩增leuBCD基因，并与质粒连接，获得重组质粒；
(3)采用CRISPR/Cas9基因编辑技术将上述基因组整合片段和重组质粒依次在宿主细胞内表达；
进一步地，所述构建方法具体如下：
(1)以大肠杆菌W3110基因组为模板，分别PCR扩增获得异丙基苹果酸合成酶编码基因leuA M、以及UHF和DHF片段(分别为lacI基因上、下游同源臂)，并 通过重叠PCR获得重组片段UHF-leuA M-DHF；
所述UHF的核苷酸序列如SEQ ID NO.7所示；
所述DHF的核苷酸序列如SEQ ID NO.8所示；
(2)利用相同的原理获得lacZ基因上、下游同源臂UHFA和DHFB片段以及ilvBN M基因片段，通过重叠PCR将UHFA、DHFB和ilvBN M构建重组片段UHF-ilvBN M-DHF；
所述UHFA的核苷酸序列如SEQ ID NO.9所示；
所述DHFB的核苷酸序列如SEQ ID NO.10所示；
(3)以大肠杆菌W3110基因组为模板，PCR扩增获得leuBCD基因，并与质粒pTrc99a连接，获得重组质粒pTR-leuBCD；
(4)L-亮氨酸基因工程菌TE03的构建
分别将PG-1和PG-2及PG-3和PG-4在52℃条件下退火，然后连接至质粒pGRB，获得pGRB1和pGRB2；以大肠杆菌W3110为出发菌株，分别将pGRB1和UHF-leuA M-DHF转化至大肠杆菌W3110，获得重组菌株TE01；以菌株TE01为出发菌株，分别将pGRB2和UHFA-ilvBN M-DHFB转化至TE01，获得菌株TE02；将pTR-leuBCD转化至TE02获得TE03。
本发明还提供利用上述基因工程菌发酵合成L-亮氨酸的方法，具体如下：
以5％-10％接种量将种子培养物接至发酵培养基中进行发酵培养，溶氧维持在20-40％，pH维持在6.5-7.5，培养温度30-35℃，发酵周期40-48h，发酵过程中维持残糖浓度为0-0.4％(W/V)；
发酵结束时，发酵液中的L-亮氨酸浓度达到60.5-69.6g/L。
所述发酵培养基成分为：葡萄糖25g/L，蛋白胨12g/L，酵母粉4g/L，KH 2PO 43.5g/L，MgSO 4 1.5g/L，FeSO 415mg/L，MnSO 415mg/L，VB1 0.01mg/L，pH7.0，0.075MPa高压蒸汽灭菌15min。
有益效果：
1、本发明所述leuA M基因编码的2-异丙基苹果酸合成酶具有如下特点：该酶解除了L-亮氨酸对其的反馈抑制作用(图1)，在L-亮氨酸浓度为0-15mmol/L条件下，LEUA M酶活性无明显变化，且其活性较野生型leuA编码的2-异丙基苹果酸合成酶未见明显降低(图2)
2、本发明所述的L-亮氨酸基因工程菌TE03无营养缺陷、生长快、发酵周期短、产 量高、转化率高，经过40-48h发酵后，发酵液中的L-亮氨酸浓度达到60.5-69.6g/L(图3)。
附图说明：
图1 L-亮氨酸对leuA和leuA M基因编码的2-异丙基苹果酸合成酶活性的影响；
图2 leuA M与leuA编码的2-异丙基苹果酸合成酶活性对比；
图3 L-异亮氨酸对ilvBN和ilvBN M基因编码的乙酰羟酸合酶活性的影响；
图4 ilvBN M与ilvBN编码的乙酰羟酸合酶活性对比；
图5 L-亮氨酸基因工程菌TE03发酵过程曲线；
图6过表达leuA M对L-亮氨酸合成的影响。
具体实施方式：
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本专利，并不用于限定本发明。
本发明所构建的生产L-亮氨酸的基因工程菌，是通过在宿主细胞中过表达本发明获得的解除L-亮氨酸反馈抑制异丙基苹果酸合成酶编码基因leuA M、解除L-异亮氨酸反馈抑制乙酰羟酸合成酶编码基因ilvBN M、3-异丙基苹果酸脱氢酶编码基因leuB、3-异丙基苹果酸脱水酶编码基因leuCD获得；
在一些实施例方式中，宿主细胞可以是大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、需钠弧菌(Vibrio natriegens)或酿酒酵母(Saccharomyces cerevisiae)等；
在一些实施方式中，ilvBN M基因来源于抗L-异亮氨酸结构类似物α-氨基丁酸和硫代异亮氨酸的谷氨酸棒杆菌；
在一些实施方式中，leuB基因是Genbank编号为b0073、JW5807、NCgl1237、BSU28270或BAMF_2634的leuB基因。
在一些实施方式中，leuCD基因是Genbank编号为b0071、b0072、JW0070、JW0071、NCgl1262、NCgl1263、BSU28250、BSU28260、BAMF_2632或BAMF_2633的leuCD基因。
上述来源的宿主细胞、ilvBN M基因、leuB基因及leuCD基因均可实现本发明所 述的效果，以下实施例，以大肠杆菌杆菌(Escherichia coli)W3110为宿主细胞，过表达SEQ ID NO.2所示的leuA M基因、SEQ ID NO.5所示的ilvBN M基因，SEQ ID NO.6所示的leuBCD(大肠杆菌里leuB和leuCD组成一个操纵子leuBCD)为例，构建生产L-亮氨酸的基因工程菌为TE03，对本发明做示例性说明。
以下实施例所用引物序列列表：
Figure PCTCN2020112038-appb-000002
Figure PCTCN2020112038-appb-000003
实施例1：解除L-亮氨酸反馈抑制的异丙基苹果酸合成酶编码基因leuA M的获得
(1)抗L-亮氨酸结构类似物突变株的筛选
①谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032菌悬液的制备
将谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032接种至LB液体培养基，于32℃，200rpm，培养12h，离心收集菌体，无菌生理盐水洗涤3次后重悬，使得OD 600＝0.6-0.8，取10μL菌悬液涂在载片上。
②常压室温等离子诱变
诱变参数为：载片置于气流端口2mm处，功率120W，气流量10SLM，作用时间20s。
③抗L-亮氨酸结构类似物α-氨基丁酸突变株的筛选
将步骤②诱变后的菌悬液涂布在含50mg/L亮氨酸氧肟酸盐的基本培养基上，35℃培养48h后，选取菌落较大的菌株。
④菌株产L-亮氨酸能力测定
将步骤③筛选的菌株利用种子培养基进行96孔板培养，然后以5％的接种量接种至含发酵培养基的96孔板进行发酵实验，菌株LEU262的L-亮氨酸产量最高。
⑤抗L-亮氨酸结构类似物硫代异亮氨酸突变株的筛选及产L-亮氨酸能力测定
以LEU262为诱变对象，重复步骤①和②，将诱变后的菌悬液涂布在含50mg/Lβ-羟基亮氨酸的基本培养基上，35℃培养48h后，选取菌落较大的菌株。重复步骤④，LEU741的L-亮氨酸产量最高。
⑥培养基
种子培养基：葡萄糖20g/L，酵母粉5g/L，(NH 4) 2SO 4 4g/L，KH 2PO 4 2.5g/L，MnSO 4 0.5g/L，玉米浆30mL/L，pH 6.5-7.0，115℃高压蒸汽灭菌15min。
发酵培养基：葡萄糖70g/L，(NH 4) 2SO 4 4g/L，KH 2PO 4 1g/L，MgSO 4·7H 2O 0.6g/L， MnSO 40.02g/L，V B1 0.002g/L，玉米浆30mL/L。pH 6.5-7.0，115℃高压蒸汽灭菌15min。
⑦检测方法
将发酵液于8000g离心5min后取上清液，使用0.8％(V/V)2，4-二硝基氟苯对其进行衍生反应，采用高效液相色谱测定L-亮氨酸含量，其检测条件为：Agilent C18(15mm×4.6mm，5μm)，采用乙腈/醋酸钠二元梯度洗脱，柱温33℃，检测波长360nm，根据高效液相法的测定结果，根据与标准品出峰时间及峰面积对比，确定L-亮氨酸产量。
(2)解除L-亮氨酸反馈抑制异丙基苹果酸合成酶编码基因leuA M突变体的获得
提取LEU741基因组，利用引物leuA-1’和leuA-2’进行PCR扩增，PCR条件为：94℃ 5min 1个循环，94℃ 30s、50℃ 30s、72℃ 2min 30个循环，72℃ 10min 1个循环，反应体系为100μL。取10μL PCR产物经1.5％琼脂糖凝胶电泳检测。将PCR扩增的目的片段回收后连接至pMD TM18-T Vector并转化至大肠杆菌E.coliDH5α感受态细胞中，然后涂布于含氨苄青霉素(100μg/mL)的LB固体培养上，于37℃倒置培养24h。挑取3个单克隆，提取重组质粒并测定其序列。
测序结果表明，与野生型leuA相比，突变后基因编码的2-异丙基苹果酸合成酶发生了F7L，I14F，I51S，G127D，I197V，F370L，K380M，R529H，G561D，V596A突变，将该突变体命名为LEUA M，编码基因命名为leuA M。
(3)异丙基苹果酸合成酶突变体LEUA M与野生型异丙基苹果酸合成酶LEUA的酶学性质比较
分别以谷氨酸棒杆菌ATCC13032和LEU741基因组为模板，利用引物LA-1和LA-2进行PCR扩增，产物回收后连接至经BamH I酶切的pET-His质粒，然后转化至大肠杆菌Escherichia coli BL21(DE3)，获得菌株E.coli-leuA和E.coli-leuA M。利用IPTG对E.coli-leuA和E.coli-leuA M诱导表达重组蛋白LEUA和LEUA M，收集菌体，用50mmol/L Tris-HCl缓冲液(pH 7.5)重悬后进行超声破碎并离心后取上清液。
LEUA M和LEUA的酶活性测定方法如下：
取10μL上述上清液至990μL Tris-HCl缓冲液(50mmol/L，pH 7.5含400mmol/L谷氨酸钾、20μL 5,5'-二硫代双(2-硝基苯甲酸)、3mmol/L乙酰辅酶A、4mmol/L酮异戊酸)。于30℃反应1h后，加入100μL硫酸(3mol/L)，并于65℃处理15min以终止反应。在反应过程中，2-异丙基苹果酸合成酶可催化乙酰辅酶A生成辅酶A，后者在 OD 412处有最大吸光度。根据该原理利用分光光度测定每分钟OD 412的变化值并计算生成的辅酶A，从而计算酶活性。结果如图2所示，LEUA M和LEUA的活性分别为12.1和13.5nmol/(min·mg总蛋白)，二者无明显差异。
L-亮氨酸对LEUA M和LEUA的酶活性影响测定方法如下：向上述反应液中分别加入0、2、4、6、8、10、12、15mmol/L L-亮氨酸，然后测定辅酶A生成量，以考察LEUA M解除L-亮氨酸的反馈抑制作用。
将L-亮氨酸添加浓度为0时的酶活性定义为100％，其余L-亮氨酸浓度条件下的LEUA M和LEUA的酶活性与之相比即为相对酶活性。
结果如图1所示，LEUA的相对酶活性随L-亮氨酸浓度增加迅速降低，L-亮氨酸浓度高于6mmol/L时，几乎无活性，表明该酶受L-亮氨酸反馈抑制作用；而突变体LEUA M的相对活性随L-亮氨酸浓度的增加无明显变化，表明其解除了L-亮氨酸的反馈抑制作用。
综合以上结果，2-异丙基苹果酸合成酶突变体LEUA M解除了L-亮氨酸的反馈抑制作用，同时其活性较野生型LEUA相比未见明显降低。
实施例2解除L-异亮氨酸反馈抑制的乙酰羟酸合成酶编码基因ilvBN M的获得
(1)抗L-异亮氨酸结构类似物突变株的筛选
①谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032菌悬液的制备
将谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032接种至LB液体培养基，于32℃，200rpm，培养12h，离心收集菌体，无菌生理盐水洗涤3次后重悬，使得OD 600＝0.6-0.8，取10μL菌悬液涂在载片上。
②常压室温等离子诱变
诱变参数为：载片置于气流端口2mm处，功率120W，气流量10SLM，作用时间25s。
③抗L-异亮氨酸结构类似物α-氨基丁酸突变株的筛选
将步骤②诱变后的菌悬液涂布在含50mg/Lα-氨基丁酸的基本培养基上，35℃培养48h后，选取菌落较大的菌株。
④菌株产L-异亮氨酸能力测定
将步骤③筛选的菌株利用种子培养基进行96孔板培养，然后以10％的接种量接种至含发酵培养基的96孔板进行发酵实验，ILE396的L-异亮氨酸产量最高。
⑤抗L-异亮氨酸结构类似物硫代异亮氨酸突变株的筛选及产L-异亮氨酸能力测定
以ILE396为诱变对象，重复步骤①和②，将诱变后的菌悬液涂布在含50mg/L硫代异亮氨酸的基本培养基上，35℃培养48h后，选取菌落较大的菌株。重复步骤④，ILE693的L-异亮氨酸产量最高。
⑥培养基
种子培养基：葡萄糖25g/L，酵母粉5g/L，(NH 4) 2SO 45g/L，KH 2PO 42g/L，MnSO 40.6g/L，玉米浆40mL，pH 6.8-7.2，115℃高压蒸汽灭菌15min。
发酵培养基(g/L)：葡萄糖80g/L，(NH 4) 2SO 4 3g/L，KH 2PO 4 1.5g/L，MgSO 4·7H 2O 0.6g/L，MnSO 40.015g/L，V B1 0.001g/L，玉米浆30mL。pH 6.8-7.2，115℃高压蒸汽灭菌15min。
⑦检测方法
将发酵液于8000g离心5min后取上清液，使用0.8％(V/V)2，4-二硝基氟苯对其进行衍生反应，采用高效液相色谱测定L-异亮氨酸含量，其检测条件为：Agilent C18(15mm×4.6mm，5μm)，采用乙腈/醋酸钠二元梯度洗脱，柱温33℃，检测波长360nm，根据高效液相法的测定结果，根据与标准品出峰时间及峰面积对比，确定L-异亮氨酸产量。
(2)解除L-异亮氨酸反馈抑制乙酰羟酸合成酶编码基因ilvBN M突变体的获得
提取ILE693基因组，利用引物ilvBN-1和ilvBN-2进行PCR扩增，PCR条件为：94℃ 5min 1个循环，94℃ 30s、56℃ 30s、72℃ 1min 30个循环，72℃ 10min 1个循环，反应体系为100μL。取10μL PCR产物经1.5％琼脂糖凝胶电泳检测。将PCR扩增的目的片段回收后连接至pMD TM18-T Vector并转化至大肠杆菌E.coliDH5α感受态细胞中，然后涂布于含氨苄青霉素(100μg/mL)的LB固体培养上，于37℃倒置培养24h。挑取3个单克隆，提取重组质粒并测定其序列。
测序结果表明，与野生型ilvBN相比，突变后基因编码的乙酰羟酸合成酶发生了K30Q，A84T，G128S，A226S，K227R，Y252H，T362S，H674L突变，将该突变体命名为ILVBN M，编码基因命名为ilvBN M(SEQ ID NO.5)。
(3)乙酰羟酸合成酶突变体ILVBN M与野生型乙酰羟酸合成酶ILVBN的酶学性质比较
分别以谷氨酸棒杆菌ATCC13032和ILE693基因组为模板，利用引物IV-1和IV-2进行PCR扩增，产物回收后连接至经BamH I酶切的pET-His质粒，然后转化至大肠杆菌Escherichia coli BL21(DE3)，获得菌株E.coli-ilvBN和E.coli-ilvBN M。利用IPTG对 E.coli-ilvBN和E.coli-ilvBN M诱导表达重组蛋白ILVBN和ILVBN M，收集菌体，用100mmol/L磷酸钾缓冲液(pH 7.8)重悬后进行超声破碎并离心后取上清液。
ILVBN M和ILVBN的酶活性测定方法如下：取100μL上述上清液至1mL磷酸钾缓冲液(100mmol/L，pH 7.8含100mmol/L丙酮酸钠、100mmol/L 2-酮丁酸、10mmol/LMgCl 2，0.2mmol/L焦磷酸硫胺素)，于37℃反应1h后，加入100μL硫酸(3mol/L)，并于65℃处理15min以终止反应。将上述反应液与1mL 0.5％肌酸和1mLα-萘酚溶液(含2.5mol/LNaOH)混合，于65℃处理20min后冷却取室温，利用分光光度测定2-酮基-2-羟基丁酸生成量(OD 525)。结果如图4所示，ILVBN M和ILVBN的活性分别为16.7和16.9nmol/(min·mg总蛋白)，二者无明显差异。
L-异亮氨酸对ILVBN M和ILVBN的酶活性影响测定方法如下：向上述反应液中分别加入0、2、4、6、8、10、12mmol/LL-异亮氨酸，然后测定2-酮基-2-羟基丁酸生成量，以考察ILVBN M解除L-异亮氨酸的反馈抑制作用。将L-异亮氨酸添加浓度为0时的酶活性定义为100％，其余L-异亮氨酸浓度条件下的ILVBN M和ILVBN的酶活性与之相比即为相对酶活性。结果如图3所示，ILVBN的相对酶活性随L-异亮氨酸浓度增加迅速降低，L-异亮氨酸浓度高于8mmol/L时，几乎无活性，表明该酶受L-异亮氨酸反馈抑制作用；而突变体ILVBN M的相对活性随L-异亮氨酸浓度的增加无明显变化，表明其解除了L-异亮氨酸的反馈抑制作用。
综合以上结果，乙酰羟酸合成酶突变体ILVBN M解除了L-异亮氨酸的反馈抑制作用，同时其活性较野生型ILVBN相比未见降低。
实施例3：L-亮氨酸生产菌TE03的构建
(1)重组片段UHF-leuA M-DHF的构建
以人工合成的包含leuA M基因的质粒为模板、以LEUA-3和LEUA-4为引物，进行PCR扩增，获得leuA M；
以大肠杆菌W3110基因组为模板，分别利用引物LEUA-1和LEUA-2以及LEUA-5和LEUA-6扩增获得片段UHF和DHF，UHF和DHF分别为lacI基因的上、下游同源臂；以UHF、DHF和leuA M为模板，利用引物LEUA-1和LEUA-6进行PCR扩增，回收后即为重组片段UHF-leuA M-DHF。
(2)重组片段UHFA-ilvBN M-DHFB的构建
以人工合成的包含ilvBN M基因的质粒为模板、以IlvB-3和IlvB-4为引物，进行PCR扩增，获得ilvBN M；以大肠杆菌W3110基因组为模板，分别利用引物IlvB-1 和IlvB-2以及IlvB-5和IlvB-6扩增获得片段UHFA和DHFB，UHFA和DHFB分别为lacZ基因的上、下游同源臂；以UHFA、DHFB和ilvBN M为模板，利用引物IlvBN-1和IlvBN-6进行PCR扩增，回收后即为重组片段UHFA-ilvBN M-DHFB。
(3)重组质粒pTR-leuBCD的构建
以大肠杆菌W3110基因组为模板、以leuBCD-1和leuBCD-2为引物，进行PCR扩增，获得leuBCD(在大肠杆菌里leuB和leuCD组成一个操纵子leuBCD)，将质粒pTrc99a经BamH I酶切，经电泳、切胶回收后与leuBCD连接，获得重组质粒pTR-leuBCD。
(4)L-亮氨酸基因工程菌TE03的构建
分别将PG-1和PG-2以及PG-3和PG-4于52℃条件下退火，然后分别连接至质粒pGRB，获得pGRB1和pGRB2。其中PG-1和PG-2及PG-3和PG-4为用于Cas9识别W3110基因组lacI和lacZ基因序列的引导序列单链DNA，二者退火后成为双链DNA，可与pGRB连接。将pREDCas9质粒转化入大肠杆菌W3110，挑取阳性克隆菌，获得W3110-pREDCas9菌株。分别将pGRB1和UHF-leuA M-DHF转化至W3110-pREDCas9，挑取阳性克隆菌，进行pGRB-gRNA和pREDCas9质粒的消除，即获得TE01菌株。同理将pGRB2和UHFA-ilvBN M-DHFB转化至含pREDCas9的TE01，获得TE02。将pTR-leuBCD转化至TE02获得TE03。
实施例4：L-亮氨酸生产菌TE03的发酵罐发酵实验
(1)种子培养
用接种环将3-5支经新鲜斜面活化的TE03全部接种至装有1L种子培养基的5L发酵罐，流加25％(W/V)的氨水调节发酵液pH至6.5-7.5，溶氧维持在20-50％，通风量3-5m 3/h，搅拌转速400-500rpm，32℃培养6-8h。
(2)发酵罐发酵
以5％接种量将步骤(1)的种子培养物接至装有3L发酵培养基的5L发酵罐进行发罐培养，发酵温度35℃，通风量3-5m 3/h，搅拌转速600rpm，溶氧维持在20-40％，流加浓度为80％(W/V)的葡萄糖溶液，维持残糖浓度为0.1-0.5％(W/V)，流加25％(W/V)的氨水调节发酵液pH至6.5-7.5，发酵周期48h(发酵过程曲线如图5)。
(3)发酵液中L-亮氨酸的检测
方法同实施例1(1)⑦，经检测，发酵44h时L-亮氨酸产量最高，达到69.6g/L，转化率为19.1％。
其中：种子培养组成为：
葡萄糖14g/L，蛋白胨5g/L，酵母粉3g/L，KH 2PO 4 2g/L，MgSO 4 1g/L，FeSO 410mg/L，MnSO 410mg/L，pH7.0，0.075MPa高压蒸汽灭菌15min。
发酵培养基组成为：
葡萄糖25g/L，蛋白胨12g/L，酵母粉4g/L，KH 2PO 4 3.5g/L，MgSO 4 1.5g/L，FeSO 415mg/L，MnSO 415mg/L，VB1 0.01mg/L，pH7.0，0.075MPa高压蒸汽灭菌15min。
实施例5过表达leuA M对L-亮氨酸合成的影响
采用实例1中相同的方法，分别构建①ilvBN M和leuBCD过表达菌TE04，②ilvBN、leuA和leuBCD过表达菌株TE05，③ilvBN M、leuA和leuBCD过表达菌株TE06，④ilvBN、leuA M和leuBCD过表达菌株TE07，采用实施例4相同的方法进行发酵实验。经检测，经发酵44h，TE03的L-亮氨酸产量最高(69.2g/L)，其次为TE07(35.37g/L)和TE06(18.16g/L)，TE04和TE05的L-亮氨酸产量最低分别为0.12和2.15g/L(图6)。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (10)

  1. 一种2-异丙基苹果酸合成酶，其特征在于，所述2-异丙基苹果酸合成酶具有SEQ ID NO.1所示的氨基酸序列。
  2. 一种生产L-亮氨酸的基因工程菌，其特征在于，所述基因工程菌是通过在宿主细胞中过表达权利要求1所述2-异丙基苹果酸合成酶的编码基因leuA M、解除L-异亮氨酸反馈抑制乙酰羟酸合成酶编码基因ilvBN M、3-异丙基苹果酸脱氢酶编码基因leuB、3-异丙基苹果酸脱水酶编码基因leuCD获得的；
    所述leuA M核苷酸序列如序列表SEQ ID NO.2所示。
  3. 如权利要求2所述的一种生产L-亮氨酸的基因工程菌，其特征在于，所述宿主细胞是大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、需钠弧菌(Vibrio natriegens)或酿酒酵母(Saccharomyces cerevisiae)。
  4. 如权利要求2所述的一种生产L-亮氨酸的基因工程菌，其特征在于，所述ilvBN M基因编码的乙酰羟酸合成酶解除了L-异亮氨酸的反馈抑制，核苷酸序列如序列表SEQ ID NO.5所示。
  5. 如权利要求2所述的一种生产L-亮氨酸的基因工程菌，其特征在于，所述leuB基因是Genbank编号为b0073、JW5807、NCgl1237、BSU28270或BAMF_2634的leuB基因；
    所述leuCD基因是Genbank编号为b0071、b0072、JW0070、JW0071、NCgl1262、NCgl1263、BSU28250、BSU28260、BAMF_2632或BAMF_2633的leuCD基因。
  6. 如权利要求2所述的一种生产L-亮氨酸的基因工程菌，其特征在于，所述基因工程菌是以大肠杆菌杆菌(Escherichia coli)W3110为宿主细胞，过表达SEQ ID NO.2所示的leuA M基因，SEQ ID NO.5所示的ilvBN M基因，SEQ ID NO.6所示的leuBCD基因获得的。
  7. 如权利要求2所述的一种生产L-亮氨酸的基因工程菌，其特征在于，构建方法如下：
    (1)分别扩增基因leuA M、leuB、leuCD以及ilvBN M，并分别构建基因组整合片段；
    (2)采用CRISPR/Cas9基因编辑技术将上述基因组整合片段和重组质粒依次在宿主细胞内表达。
  8. 权利要求2-7任意一项所述基因工程菌在生产L-亮氨酸中的应用。
  9. 如权利要求8所述的应用，其特征在于，利用上述基因工程菌发酵合成L-亮氨酸的方法具体如下：
    以5％-10％接种量将种子培养物接至发酵培养基中进行发酵培养，溶氧维持在20-40％，pH维持在6.5-7.5，培养温度30-35℃，发酵周期40-45h，发酵过程中维持残糖浓度为0-0.4％；
    所述发酵培养基成分为：葡萄糖25g/L，蛋白胨12g/L，酵母粉4g/L，KH 2PO 4 3.5g/L，MgSO 4 1.5g/L，FeSO 415mg/L，MnSO 415mg/L，VB1 0.01 mg/L，pH7.0，0.075MPa高压蒸汽灭菌15min。
  10. 权利要求1所述2-异丙基苹果酸合成酶在生产L-亮氨酸中的应用。
PCT/CN2020/112038 2019-08-29 2020-08-28 一种2-异丙基苹果酸合成酶及其工程菌与应用 WO2021037190A1 (zh)

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