CN117165540A - Method for reducing L-leucine in amino acid production strain and application thereof - Google Patents
Method for reducing L-leucine in amino acid production strain and application thereof Download PDFInfo
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- CN117165540A CN117165540A CN202110998221.2A CN202110998221A CN117165540A CN 117165540 A CN117165540 A CN 117165540A CN 202110998221 A CN202110998221 A CN 202110998221A CN 117165540 A CN117165540 A CN 117165540A
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- amino acid
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- malate dehydrogenase
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a method for reducing L-leucine in an amino acid production strain and application thereof. The method of the invention enables the amino acid producing strain to express an isopropyl malate dehydrogenase mutant, wherein the isopropyl malate dehydrogenase mutant takes the amino acid sequence of the wild type isopropyl malate dehydrogenase of the amino acid producing strain as a reference sequence and contains mutation of substitution of the 324 th proline by other amino acids. The method of the invention obviously reduces the yield of L-leucine in the industrial production strain of L-glutamic acid, lysine or proline, improves the yield and conversion rate of glutamic acid, lysine or proline, and reduces the cost for separating leucine when industrially extracting glutamic acid, lysine or proline.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for reducing L-leucine in an amino acid production strain and application thereof.
Background
L-glutamic acid (L-glutarate), chemical name of which is alpha-aminopentanedioic acid, molecular formula of which is C 5 H 9 NO 4 The molecular weight is 147.13076, and two carboxyl groups are contained in the molecule and are acidic amino acids. Glutamic acid is a major component of dietary protein and is also used as an umami additive in many foods in the form of monosodium glutamate to increase the flavor of the food. Glutamate also plays an important role in nutrition and signaling.
Corynebacterium glutamicum is an aerobic, fast-growing, non-spore-forming, short-rod, non-pathogenic gram-positive microorganism. Corynebacterium glutamicum has been widely used for the production of amino acids since it was isolated. Because of easy genetic operation and fast growth speed, the fermentation period is short, and the method is also applied to the production of organic acids, alcohols, carotenoids and the like except amino acid production, and is an important model organism in scientific research and industrial production.
Lysine is one of the essential amino acids of humans and mammals, and the body cannot synthesize itself and must be supplemented from food. Lysine is mainly present in animal foods and beans, and the content of lysine in cereal foods is very low. Lysine has positive nutritional significance in the aspects of promoting the growth and development of human bodies, enhancing the immunity of organisms, resisting viruses, promoting fat oxidation, relieving anxiety and emotion and the like, can promote the absorption of certain nutrients, can cooperate with certain nutrients, and can better exert the physiological functions of various nutrients. The preparation is carried out by the lysine-producing bacteria, and the application effect is good.
L-leucine has important physiological functions as essential amino acid in food addition, but in non-leucine-producing bacteria, excessive leucine content can affect the yield and conversion rate of target amino acid, and certain difficulty is brought to separation and purification of the target amino acid. Therefore, there is a need to develop a method for reducing the production of L-leucine in L-amino acid producing strains.
Disclosure of Invention
The invention aims to provide a method and application capable of reducing L-leucine in an amino acid production strain so as to improve the yield of target amino acid.
Specifically, the invention provides the following technical scheme:
a method for reducing L-leucine in an amino acid producing strain, which comprises expressing an isopropyl malate dehydrogenase (IPMD) mutant in the amino acid producing strain, wherein the isopropyl malate dehydrogenase mutant comprises a mutation in which proline at position 324 is substituted with another amino acid, using the amino acid sequence of a wild-type isopropyl malate dehydrogenase of the amino acid producing strain as a reference sequence.
Preferably, the isopropyl malate dehydrogenase mutant contains a mutation wherein proline at position 324 is substituted with serine or threonine, preferably serine.
The term "mutation" refers to a change in the sequence of an amino acid encoding a protein resulting from the addition, deletion or substitution of one or more bases on the gene encoding the protein, which in turn alters the functional activity of the protein. The mutation method can be selected from one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination. In the present invention, proline at position 324 of the protein encoded by the leuB gene is preferably mutated to another amino acid by a mutation method of homologous recombination.
The isopropyl malate dehydrogenase mutant (EC 1.1.1.85) has the functions of reducing the catalytic activity of isopropyl malate dehydrogenase and the synthesis of leucine, and improving the yield and conversion rate of glutamic acid and the like.
In the invention, the isopropyl malate dehydrogenase mutant has an amino acid sequence shown as SEQ ID NO.1 or 3;
and/or the nucleic acid encoding the isopropyl malate dehydrogenase mutant has a sequence as shown in SEQ ID No.2 or 4.
It will be appreciated by those skilled in the art that the addition of a tagged protein to the N-or C-terminus of the mutant sequence or fusion with other proteins to form a fusion protein is within the scope of the present invention without altering the activity of the mutant protein itself.
Based on the amino acid sequence of the isopropyl malate dehydrogenase mutant provided above, one skilled in the art can obtain the sequence of the nucleic acid encoding the same. Based on the degeneracy of the codons, more than one of the nucleic acid sequences encoding the amino acid sequences described above, all nucleic acids capable of encoding the mutants of the proteins described above are within the scope of the invention.
In the present invention, the amino acid producing strain is a microorganism of the genus Corynebacterium, preferably Corynebacterium glutamicum (Corynebacterium glutamicum).
Based on the function of the isopropyl malate dehydrogenase mutant, the invention provides the application of the isopropyl malate dehydrogenase mutant or the encoding nucleic acid thereof:
(1) Reducing the content of L-leucine in the amino acid producing strain;
(2) The content of L-leucine in the amino acid derivative producing strain is reduced.
The present invention also provides a recombinant microorganism expressing an isopropyl malate dehydrogenase mutant as described above; the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0112-8 with the preservation number of CGMCC No.11941, or the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0912-6 with the preservation number of CGMCC No.11942, or the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0701 with the preservation number of CGMCC No.13757.
Preferably, the original gene encoding isopropyl malate dehydrogenase on the chromosome of the starting strain is mutated into the gene encoding the isopropyl malate dehydrogenase mutant.
The recombinant microorganism expressing the isopropyl malate dehydrogenase mutant provided by the invention has the advantages that the yield of L-leucine in an industrial production strain of L-glutamic acid/lysine/proline is obviously reduced, the yield and conversion rate of glutamic acid/lysine/proline are improved, and the cost of separating leucine in industrialized extraction of glutamic acid/lysine/proline is reduced.
Likewise, based on the function of the isopropyl malate dehydrogenase mutant, the person skilled in the art will understand that the recombinant microorganism can also be used for the fermentative production of amino acid derivatives.
The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: the gene encoding isopropyl malate dehydrogenase in the starting strain was mutated to a gene encoding isopropyl malate dehydrogenase mutant as described above.
As a specific construction method, it comprises: a step of introducing a recombinant plasmid carrying a gene encoding the isopropyl malate dehydrogenase mutant into the starting strain.
Preferably, the recombinant plasmid is a plasmid which can generate homologous recombination in thalli, and the coding gene of the isopropyl malate dehydrogenase mutant on the plasmid is exchanged with a homologous gene on chromosome.
Further preferably, the construction method includes:
(1) Constructing a recombinant plasmid containing a gene encoding the isopropyl malate dehydrogenase mutant;
(2) Transforming the recombinant plasmid into a starting strain, and screening transformants by using a selection medium containing antibiotics;
(3) Screening the positive transformant for the recombinant microorganism having the mutation of interest.
The invention also provides a method for producing glutamic acid, which comprises the step of fermenting and culturing the recombinant microorganism, wherein an original strain of the recombinant microorganism is corynebacterium glutamicum MHZ-0112-8.
In the present invention, when glutamic acid fermentation is performed, the fermentation medium comprises the following components: glucose 50-60g/L, ammonium sulfate 10-20g/L, KH 2 PO 4 0.5-1.5g/L，MgSO 4 ·7H 2 O 0.2-0.5g/L，FeSO 4 ·7H 2 O 0.5-1.5mg/L，MnSO 4 ·5H 2 O 0.8-1.2mg/L，VB 1 150-250 mug/L, 250-350 mug/L biotin, 0.3-0.6g/L soybean hydrolysate, 40-80g/L calcium carbonate and pH 7.2-7.5.
The invention also provides a method for producing lysine, which comprises the step of fermenting and culturing the recombinant microorganism, wherein an original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0912-6, and the preservation number of the recombinant microorganism is CGMCC No.11942.
In the present invention, when fermentation culture of lysine is performed, the fermentation medium comprises the following components: glucose 55-65g/L, (NH) 4 ) 2 SO 4 22-28g/L，KH 2 PO 4 1.5-2.5g/L，MgSO 4 ·7H 2 0.8-1.2g/L of O, 8-12g/L of yeast powder and CaCO 3 20-30g/L，pH 6.8-7.2。
The invention also provides a method for producing proline, which comprises the step of fermenting and culturing the recombinant microorganism, wherein an original strain of the recombinant microorganism is corynebacterium glutamicum MHZ-0701, and the preservation number of the recombinant microorganism is CGMCC No.13757.
In the invention, when the fermentation culture of proline is carried out, the fermentation culture medium comprises the following components in percentage by mass: corn steep liquor 0.5-0.7%, glucose 11-13%, ammonium sulfate 3.5-4%, magnesium sulfate 0.04-0.06%, potassium dihydrogen phosphate 0.09-0.11%, caCO 3 3.8-4.2％，V H 65-75μg/L，V B1 ·HCl 75-85μg/L，pH 7.2。
Specifically, the method for reducing L-leucine in an amino acid-producing strain comprises the following steps: inoculating the recombinant microorganism to a slant culture medium for slant culture, picking a lawn on the slant culture medium, inoculating the lawn to a seed culture medium for seed culture, and transferring the seed culture to a fermentation culture medium for fermentation.
When glutamic acid production is performed, preferably, the slant medium is: 5g/L of yeast powder, 10g/L of beef extract, 10g/L of peptone, 10g/L of sodium chloride, 2.5g/L of agar powder and pH of 7.0-7.2.
The seed culture medium is as follows: glucose 25g/L, urea 3.0g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 O0.9 g/L, corn steep liquor 33mL/L, bean cake hydrolysate 22mL/L, pH 7.0-7.2.
The fermentation medium is as follows: 60g/L glucose, 15g/L ammonium sulfate and KH 2 PO 4 1.0g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 1.0mg/L，MnSO 4 ·5H 2 O 1.0mg/L，V B1 200 mug/L, 300 mug/L biotin, 0.48g/L soybean hydrolysate, 50g/L calcium carbonate and pH 7.2-7.5.
The seed culture is carried out at 31.5 ℃ and 220rpm until the culture is carried out at the middle and late stage of logarithmic growth, and the culture time is 10-14h.
The fermentation is carried out at 31.5 ℃ for 12-20h under shaking at 220 rpm.
The invention has the beneficial effects that:
isopropyl malate dehydrogenase is a key enzyme in the leucine biosynthetic pathway. According to the invention, the 324 th proline of isopropyl malate dehydrogenase leuB in the L-leucine biosynthesis pathway is subjected to point mutation, and is mutated into other amino acids, so that the catalytic activity of the isopropyl malate dehydrogenase is obviously reduced, the synthesis of L-leucine is reduced, the accumulation of leucine in the fermentation production of other amino acids (such as glutamic acid, lysine, proline and the like) is reduced, the yield of the amino acids is improved, and experiments show that the point mutation of the 324 th proline of the isopropyl malate dehydrogenase leuB has less influence on cell growth.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art.
The names and sequences of the primers SEQ ID NOS.5-16 referred to in the examples below are shown in Table 1.
TABLE 1 primer sequences
| Primer(s) | Sequence (5 '-3') |
| leuB-UP-1F | GGTACCCGGGGATCCTCTAGAGCGTTATTCGCTACGCATTCG |
| leuB-UP-1R | GAAATCGACTGAGAGTTATCTCGG |
| leuB-DN-2F | GATAACTCTCAGTCGATTTCTACAACTG |
| leuB-DN-2R | ACGACGGCCAGTGCCAAGCTTCTAAACACCAAGCGCTTGAAAC |
| leuB-DN-3F | GATAACTCTCAGACGATTTCTACAACTG |
| leuB-UP-3R | GAAATCGTCTGAGAGTTATCTCGG |
| test-leuB-F | TGGCCGAGATAACTCTCATT |
| test-leuB-F2 | TGGCCGAGATAACTCTCAAA |
| leuB-ID-F | GATTTCGTTGTGGTCCGCG |
| leuB-ID-R | CGTCACTCAACGTGCATTACC |
| P82 | CTCGTATGTTGTGTGGAATTGTG |
| P85 | CGCCCTGAGTGCTTGCGGCA |
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The strains involved in the following examples were as follows:
the original strain MHZ-0112-8 is Corynebacterium glutamicum, pure cultures of the Corynebacterium glutamicum (Corynebacterium glutamicum) MHZ-0112-8 are preserved in China general microbiological culture Collection center (CGMCC, address: north Xiyu No.1, 3 of Beijing Kogyo, university of China, and Proc. Number 100101) of China general microbiological culture Collection center (CGMCC No. 11941) at 12 months and 25 days of 2015. The corynebacterium glutamicum MHZ-0112-8 (CGMCC No. 11941) of the present invention is disclosed in Chinese patent publication No. CN 105695383A.
In some embodiments, the coryneform bacterium used in the method of constructing the point mutation of the leuB gene introduced into the lysine-producing strain is Corynebacterium glutamicum (Corynebacterium glutamicum) MHZ-0912-6 having a preservation number of CGMCC No.11942, which has been preserved in China general microbiological culture Collection center, having an address of China center for culture Collection of microorganisms, having a preservation number of CGMCC No.11942, which has been disclosed in patent CN105734004B, at 12 months 25 of 2015.
In some embodiments, the coryneform bacterium used in the method for constructing the point mutation of the leuB gene introduced by the proline producing strain is Corynebacterium glutamicum (Corynebacterium glutamicum) MHZ-0701 with the preservation number of CGMCC No.13757, and a pure culture of the strain is preserved in China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No.1, 3, academy of sciences of China, and the accession number of the strain is CGMCC No. 13757) on the 15 th month of 2017. The corynebacterium glutamicum MHZ-0701 (CGMCC No. 13757) of the present invention is disclosed in Chinese patent publication No. CN 107227283A.
EXAMPLE 1 construction of recombinant plasmids pK18-leuB (P324S) and pK18-leuB (P324T)
Primers were designed based on the sequence of the leuB (GenBank NO: BBD29_ 06845) gene in NCBI database, and the primer sequences are shown in Table 1. The recombinant fragments leuB-P324S-UP, leuB-P324S-DN, leuB-P324T-UP and leuB-P324T-DN were prepared respectively by using Phusion super fidelity polymerase (New England BioLabs), leuB-UP-1F/leuB-UP-3R, leuB-DN-3F/leuB-DN-2R as primer pairs and the genome of Corynebacterium glutamicum MHZ-0112-08 as templates, and the PCR procedures were: denaturation at 98 ℃ for 10s, renaturation at 50 ℃ for 20s, extension at 72 ℃ for 15s, circulation for 30 times, thorough extension at 72 ℃ for 10min, purification of the obtained fragment by agarose gel recovery kit (radix angelicae sinensis), and freezing storage at-20 ℃ for standby. The pK18mobsacB was digested with XbaI/HindIII, amplified leuB-P324S-UP/leuB-P324S-DN was mixed with the vector after double digestion in a certain ratio using ClonExpress MultiS One Step Cloning Kit kit (Nuo Wenyujin' S Biotech), and after mixing, the mixture was placed in a metal bath (Ginko Biotech) at 37℃for 30min, after the reaction was completed, trans1T1 competent cells (TransGen Biotech) were transformed, and the Canada resistant clones were picked UP, primers P82/P85, and transformants grown on the Canada resistant plates were identified by PCR with the following PCR parameters: a positive clone with a length of 1305bp was amplified for the insertion of the fragment pK18mobsacB, which was 94℃30s,50℃30s,72℃45s for a total of 30 cycles, and extended thoroughly at 72℃for 10min. The correct transformants were further identified by XbaI/HindIII digestion, and the inserted fragment was identified by sequencing (Invitrogen company) and the resulting plasmid sequenced correctly was designated pK18-leuB (P324S), pK18-leuB (P324T).
Example 2 MHZ-0112-8 Strain introduced LeuB (P324S) and (P324T) Gene Point mutations
MHZ-0112-8 competent cells were prepared according to the classical Corynebacterium glutamicum method (C.glutamicum Handbook, charpter 23), and the recombinant plasmid pK18-leuB (P324S)/pK 18-leuB (P324T) was transformed into Corynebacterium glutamicum MHZ-0112-8 competent cells by the electrotransformation method, and the crossover recombinants were selected on selection medium containing 15mg/L kanamycin. Colony PCR identification of Kan using Fast Taq DNA polymerase (TransGen Biotech) with leuB-UP-1F/P85, P82/leuB-DN-2R primers for the leuB (P324S) and leuB (P324T) Gene point mutant recombinants, respectively R Cloning, PCR parameters are: 94℃for 30s,50℃for 30s and 72℃for 35s, and a total of 30 cycles, 72℃for 10min. The clones of the fragments of 1226bp and 1121bp amplified by the two primer pairs are positive clones. Inoculating the selected positive clone into a non-anti BHI culture medium, culturing for 12-14h, diluting bacterial liquid by 100-1000 times, coating on a solid BHI culture medium containing 10% sucrose, culturing for 36h, further performing kana resistance phenotype verification on the screened bacterial strain, selecting a KanS recombinant, verifying a leuB (P324S) point mutation recombinant by using an identification primer test-leuB-F/leuB-DN-2R, and verifying a leuB (P324T) point mutation recombinant by using an identification primer test-leuB-F2/leuB-DN-2R. The recombinant containing the point mutation is obtained by searching annealing temperature, the obtained positive recombinant is amplified and sequenced by using leuB-ID-F/leuB-ID-R, the correct recombinant is 1210bp long, and the strains with correct sequencing are named MHZ-0112-24 and MHZ-0112-25.
The nucleic acid sequence of the isopropyl malate dehydrogenase mutant leuB (P324S) is shown in SEQ ID NO. 2. The amino acid sequence is shown as SEQ ID NO. 1. The nucleic acid sequence of the isopropyl malate dehydrogenase mutant leuB (P324T) is shown in SEQ ID NO. 4. The amino acid sequence is shown as SEQ ID NO. 3.
Example 3 MHZ-0912-6 Strain LeuB (P324S) and LeuB (P324T) Gene Point mutation
MHZ-0912-6 competent cells were prepared according to the classical Corynebacterium glutamicum method (C.glutamicum Handbook, charpter 23), and the recombinant plasmid pK18-leuB (P324S)/pK 18-leuB (P324T) was transformed into Corynebacterium glutamicum MHZ-0912-6 competent cells by the electrotransformation method, and the crossover recombinants were selected on selection medium containing 15mg/L kanamycin. Colony PCR identification of Kan using Fast Taq DNA polymerase (TransGen Biotech) with leuB-UP-1F/P85, P82/leuB-DN-2R primers for the leuB (P324S) and leuB (P324T) Gene point mutant recombinants, respectively R Cloning, PCR parameters are: 94℃for 30s,50℃for 30s and 72℃for 35s, and a total of 30 cycles, 72℃for 10min. The clones of the fragments of 1226bp and 1121bp amplified by the two primer pairs are positive clones. Inoculating the selected positive clone into a non-anti BHI culture medium, culturing for 12-14h, diluting bacterial liquid by 100-1000 times, coating on a solid BHI culture medium containing 10% sucrose, culturing for 36h, further performing kana resistance phenotype verification on the screened bacterial strain, selecting a KanS recombinant, verifying a leuB (P324S) point mutation recombinant by using an identification primer test-leuB-F/leuB-DN-2R, and verifying a leuB (P324T) point mutation recombinant by using an identification primer test-leuB-F2/leuB-DN-2R. The recombinant containing the point mutation is obtained by searching annealing temperature, the obtained positive recombinant is amplified and sequenced by using leuB-ID-F/leuB-ID-R, the correct recombinant is 1210bp long, and the strains with correct sequencing are named MHZ-0912-10 and MHZ-0912-11.
Example 4 MHZ-0701 Strain introduced LeuB (P324S) and LeuB (P324T) Gene Point mutation
MHZ-0701 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C.glutamicum Handbook, charpter 23), and the recombinant plasmid pK18-leuB (P324S)/pK 18-leuB (P324T) was transformed into Corynebacterium glutamicum MHZ-0701 competent cells by the electrotransformation method, and the crossover recombinants were selected on selection medium containing 15mg/L kanamycin. Colony PCR identification of Kan using Fast Taq DNA polymerase (TransGen Biotech) with leuB-UP-1F/P85, P82/leuB-DN-2R primers for the leuB (P324S) and leuB (P324T) Gene point mutant recombinants, respectively R Cloning, PCR parameters are: 94℃for 30s,50℃for 30s and 72℃for 35s, and a total of 30 cycles, 72℃for 10min. The two primer pairs amplify 1226bp,The clone of 1121bp fragment was a positive clone. Inoculating the selected positive clone into a non-anti BHI culture medium, culturing for 12-14h, diluting bacterial liquid by 100-1000 times, coating on a solid BHI culture medium containing 10% sucrose, culturing for 36h, further performing kana resistance phenotype verification on the screened bacterial strain, selecting a KanS recombinant, verifying a leuB (P324S) point mutation recombinant by using an identification primer test-leuB-F/leuB-DN-2R, and verifying a leuB (P324T) point mutation recombinant by using an identification primer test-leuB-F2/leuB-DN-2R. The recombinant containing the point mutation is obtained by searching annealing temperature, the obtained positive recombinant is amplified and sequenced by using leuB-ID-F/leuB-ID-R, the correct recombinant is 1210bp long, and the strains with correct sequencing are named MHZ-0704 and MHZ-0705.
Example 5 Performance verification of the mutant strains MHZ-0112-24 and MHZ-0112-25 for producing glutamic acid and leucine
The recombinant corynebacterium glutamicum constructed in example 2 was fermented to verify the productivity of glutamic acid and leucine, and the production performance was specifically as follows:
inoculating strain frozen in glycerol pipe at-80deg.C into slant culture medium for activation, culturing at 31.5deg.C for 24 hr, collecting lawn, inoculating into seed culture medium, shake culturing at 220rpm at 31.5deg.C for 12 hr to obtain seed solution, inoculating into 500ml shake flask containing 20ml fermentation culture medium, and shake culturing at 220rpm for 16 hr at 31.5deg.C. After glucose was completely consumed, the concentrations of L-glutamic acid and leucine accumulated in the medium were determined by HPLC method.
The specific culture medium formula is as follows:
slant culture medium: 5.0g/L of yeast powder, 10g/L of beef extract, 10g/L of peptone, 10g/L of sodium chloride, 2.5g/L of agar powder, pH of 7.0-7.2 and sterilization at 121 ℃ under 0.1MPa for 30min;
seed culture medium: glucose 25g/L, urea 3.0g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 0.9g/L of O, 33mL/L of corn steep liquor, 22mL/L of bean cake hydrolysate, pH 7.0-7.2 and sterilization at 121 ℃ and 0.1MPa for 15min;
fermentation medium: 60g/L glucose, 15g/L ammonium sulfate and KH 2 PO 4 1.0g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 1.0mg/L，MnSO 4 ·5H 2 O 1.0mg/L，V B1 200. Mu.g/L, 300. Mu.g/L biotin, 0.48g/L soybean hydrolysate, 50g/L calcium carbonate, and sterilizing with NaOH to pH 7.2-7.5 at 121deg.C under 0.1MPa for 15min. The results are shown in Table 2.
TABLE 2 detection of glutamic acid and leucine content of mutant strains
| Strain | OD 600 (×100) | Glu(g/L) | Conversion% | leuB(g/L) |
| MHZ-0112-8 | 0.421±0.02 | 34.3±0.12 | 57.1±0.6 | 1.97±0.01 |
| MHZ-0112-24 | 0.379±0.03 | 36.8±0.05 | 61.3±0.03 | 0.12±0.03 |
| MHZ-0112-25 | 0.386±0.03 | 35.9±0.13 | 59.8±0.08 | 0.23±0.12 |
The results are shown in Table 2 (OD 600 The turbidity of the culture medium diluted 100 times at 600nm indicates the cell amount, glu (g/L) indicates the amount of accumulated L-glutamic acid, and leuB (g/L) indicates the amount of accumulated L-leucine. In MHZ-0112-24, the 324 th amino acid of leuB is mutated from proline (P) to serine (S), namely, after CCG is mutated to TCG, the leucine content is reduced from 1.97g/L to 0.12g/L, the glutamic acid is increased from 34.3g/L to 36.8g/L, and the glutamic acid conversion rate is increased by 4.2%. In MHZ-0112-25, the 324 th amino acid of leuB is mutated from proline (P) to threonine (T), namely, after CCG is mutated to ACG, the leucine content is reduced from 1.97g/L to 0.23g/L, the glutamic acid is increased from 34.3g/L to 35.9g/L, and the glutamic acid conversion rate is increased by 2.7%. The mutation of amino acid at position 324 of isopropyl malate dehydrogenase can reduce the content of leucine in glutamic acid producing strain and raise the conversion rate of glutamic acid. By analysis of cell growth, the OD of the mutant strains MHZ-0112-24 and MHZ-0112-25 decreased slightly compared to the control strain, indicating that mutation of amino acid 324 of leuB had less effect on strain growth. EXAMPLE 6 Performance verification of the mutant strains of MHZ-0912-10 and MHZ-0912-11 for producing lysine and leucine
The recombinant Corynebacterium glutamicum constructed in example 3 was fermented to verify its lysine productivity, as follows:
inoculating strain frozen in glycerol pipe at-80deg.C into slant culture medium for activation, culturing at 31.5deg.C for 24 hr, collecting lawn, inoculating into seed culture medium, shake culturing at 220rpm at 31.5deg.C for 12 hr to obtain seed solution, inoculating into 500ml shake flask containing 20ml fermentation culture medium, and shake culturing at 220rpm for 16 hr at 31.5deg.C. After glucose was completely consumed, the concentrations of lysine and leucine accumulated in the medium were determined by HPLC method. The results are shown in Table 3.
The specific culture medium formula is as follows:
slant culture medium: 5.0g/L of yeast powder, 10g/L of beef extract, 10g/L of peptone, 10g/L of sodium chloride, 2.5g/L of agar powder, pH of 7.0-7.2 and sterilization at 121 ℃ under 0.1MPa for 30min;
seed culture medium: glucose 25g/L, urea 3.0g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 0.9g/L of O, 33mL/L of corn steep liquor, 22mL/L of bean cake hydrolysate, pH 7.0-7.2 and sterilization at 121 ℃ and 0.1MPa for 15min;
fermentation medium: glucose 60g/L, (NH) 4 ) 2 SO 4 25g/L，KH 2 PO 4 2.0g/L，MgSO 4 ·7H 2 O1.0 g/L, yeast powder 10g/L, caCO 3 30g/L, naOH was used to adjust pH7.0.
TABLE 3 detection of lysine and leucine content of mutant strains
| Strain | OD 600 (×100) | Lys(g/L) | Conversion% | LeuB(g/L) |
| MHZ-0912-6 | 0.486±0.021 | 7.5±0.10 | 12.5±0.01 | 1.52±0.011 |
| MHZ-0912-10 | 0.478±0.032 | 9.9±0.11 | 16.5±0.03 | 0.32±0.015 |
| MHZ-0912-11 | 0.475±0.015 | 9.4±0.08 | 15.6±0.02 | 0.35±0.006 |
The results are shown in Table 3 (OD 600 The turbidity of the culture medium diluted 100 times at 600nm and representing the cell amount, lys (g/L) representing the amount of accumulated L-lysine, and leuB (g/L) representing the amount of accumulated L-leucine. In MHZ-0912-10, the 324 th amino acid of leuB is mutated from proline (P) to serine (S), namely, after CCG is mutated to TCG, the leucine content is reduced from 1.52g/L to 0.32g/L, the lysine is increased from 7.5g/L to 9.9g/L, and the lysine conversion rate is increased by 4%. Similarly, when the 324 th amino acid of leuB is mutated from proline (P) to threonine (T), namely CCG is mutated to ACG, the leucine content is reduced from 1.52g/L to 0.35g/L, the lysine is increased from 7.5g/L to 9.4g/L, and the lysine conversion rate is increased by 3.1%. The result shows that the leuB gene mutation has promoting effect on lysine synthesis and can increase the content of lysine in lysine producing bacteria. By analyzing the cell growth, the mutant strains MHZ-0912-10 and MHZ-0912-11 were not significantly different from the OD of the control strain, indicating that mutation of amino acid 324 of leuB had little effect on the strain growth.
Example 7 Performance verification of MHZ-0704 and MHZ-0705 variant strains for production of proline and leucine
The recombinant corynebacterium glutamicum constructed in example 4 was fermented to verify the productivity of proline, and the production performance was as follows:
inoculating strain frozen in glycerol pipe at-80deg.C into slant culture medium for activation, culturing at 31.5deg.C for 24 hr, collecting lawn, inoculating into seed culture medium, shake culturing at 31.5deg.C and 220rpm for 12 hr to obtain seed solution, inoculating into 500ml shake flask containing 20ml fermentation culture medium, and shake culturing at 33 deg.C and 220rpm for 72 hr. After glucose was completely consumed, the concentrations of proline and leucine accumulated in the medium were determined by HPLC method. The results are shown in Table 4.
The specific culture medium formula comprises the following components in percentage by mass:
slant culture medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.2.
Seed culture medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, urea 0.1%, caCO 3 0.5％，pH 7.2。
Fermentation medium: corn steep liquor 0.6%, glucose 12.0%, ammonium sulfate 3.7%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, caCO 3 4％，V H 70μg/L，V B1 ·HCl 80μg/L，pH 7.2。
TABLE 4 detection of proline and leucine content in mutant strains
| Strain | OD 600 (×100) | Pro(g/L) | Conversion% | LeuB(g/L) |
| MHZ-0701 | 0.486±0.021 | 37.2±0.018 | 31.0±0.001 | 0.98±0.021 |
| MHZ-0704 | 0.479±0.032 | 40.9±0.022 | 34.1±0.0011 | 0.22±0.014 |
| MHZ-0705 | 0.482±0.015 | 39.4±0.011 | 32.8±0.003 | 0.43±0.005 |
The results are shown in Table 4 (OD 600 The turbidity of the culture medium diluted 100 times at 600nm and expressed the cell amount, pro (g/L) expressed the amount of accumulated L-proline, and leuB (g/L) expressed the amount of accumulated L-leucine. In MHZ-0704, the 324-position amino acid of leuB is mutated from proline (P) to serine (S), namely, after CCG is mutated to TCG, the leucine content is reduced from 0.98g/L to 0.22g/L, the proline content is increased from 37.2g/L to 40.9g/L, and the proline conversion rate is increased by 3.1%. Similarly, when the 324 th amino acid of leuB is mutated from proline (P) to threonine (T), namely CCG is mutated to ACG (strain MHZ-0705), the leucine content is reduced from 0.98g/L to 0.43g/L, the proline content is increased from 37.2g/L to 39.4g/L, and the proline conversion rate is increased by 1.8%. Shows that the leuB gene mutation has promoting effect on proline synthesis and can raise the proline content in proline producing bacteria. By analysis of cell growth, mutant strains MHZ-0704 and MHZ0705 differ little from the OD of the control strain, indicating that mutation of amino acid 324 of leuB has little effect on strain growth.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Hudan
<120> a method for reducing L-leucine in amino acid-producing strains and use thereof
<130> KHP211117895.5
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 340
<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Met Lys Leu Ala Val Ile Gly Gly Asp Gly Ile Gly Pro Glu Val Thr
1 5 10 15
Ala Glu Ala Leu Lys Val Leu Asn Ala Val Arg Asp Asp Ile Glu Thr
20 25 30
Thr Asp Tyr Asp Leu Gly Ala Arg Arg Tyr Leu Lys Asn Gly Glu Leu
35 40 45
Leu Thr Asp Glu Asp Leu Ala Ser Leu Arg Glu His Asp Ala Ile Leu
50 55 60
Leu Gly Ala Ile Gly Ala Pro Gly Ser Val Pro Pro Gly Ile Leu Glu
65 70 75 80
Arg Gly Leu Leu Leu Lys Met Arg Phe Ala Leu Asp His His Val Asn
85 90 95
Leu Arg Pro Ser Lys Leu Tyr Asp Gly Val Glu Ser Pro Leu Arg Asn
100 105 110
Pro Gly Lys Ile Asp Phe Val Val Val Arg Glu Gly Thr Glu Gly Ala
115 120 125
Tyr Thr Gly Asn Gly Gly Ala Ile Arg Val Gly Thr Pro His Glu Ile
130 135 140
Ala Asn Glu Thr Ser Val Asn Thr Arg Tyr Gly Ala Glu Arg Val Ile
145 150 155 160
Arg Tyr Ala Phe Glu Leu Ala Gln Ser Arg Arg Lys Lys Leu Thr Leu
165 170 175
Val His Lys Thr Asn Val Leu Val His Gly Gly Gly Leu Trp Gln Arg
180 185 190
Thr Val Asp Glu Val Ala Lys Glu Tyr Pro Glu Val Ala Val Asp Tyr
195 200 205
Asn His Ile Asp Ala Ala Thr Ile Tyr Leu Val Thr Asp Pro Ser Arg
210 215 220
Phe Asp Val Ile Val Thr Asp Asn Leu Phe Gly Asp Ile Leu Thr Asp
225 230 235 240
Glu Ala Gly Ala Val Ser Gly Gly Ile Gly Leu Ala Ala Ser Gly Asn
245 250 255
Ile Asp Ala Thr Gly Thr Asn Pro Ser Met Phe Glu Pro Val His Gly
260 265 270
Ser Ala Pro Asp Ile Ala Gly Gln Gly Ile Ala Asp Pro Thr Ala Ala
275 280 285
Ile Leu Ser Ala Ala Met Leu Leu Arg His Leu Gly Asp Glu Asp Asn
290 295 300
Ala Val Arg Ile Glu Thr Ala Ile Ala Ala Asp Val Ala Gly Arg Asp
305 310 315 320
Asn Ser Gln Ser Ile Ser Thr Thr Glu Val Gly Asp Arg Val Val Arg
325 330 335
Ala Leu Gln Ser
340
<210> 2
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atgaaacttg ctgttattgg tggagacggt atcggcccag aggttactgc agaagccctc 60
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cgttacctca aaaatggcga gctgctcacc gacgaggatc tggcatccct gcgcgagcat 180
gacgcgatcc ttcttggcgc tatcggtgca ccaggttccg tccctccagg aattctcgag 240
cgcggtttgc tgctgaagat gcgattcgca ctggatcacc acgtgaacct gcgcccatcc 300
aagctgtacg acggcgtgga gtccccactg cgtaacccag gcaagattga tttcgttgtg 360
gtccgcgaag gtaccgaagg cgcctacact ggcaacggtg gagcaatccg cgtgggaacc 420
cctcacgaga ttgccaatga aacctccgtg aacactcgct acggcgctga gcgcgttatt 480
cgctacgcat tcgagctggc acagagccgc cgcaagaagc tcaccctcgt gcacaagacc 540
aacgtcctgg ttcacggtgg tggcctgtgg cagcgcaccg tagatgaggt tgcaaaggaa 600
tacccagagg tagccgtcga ttacaaccac atcgatgcag caaccatcta tctggtcact 660
gatccttccc gcttcgatgt gattgttacc gataacctct tcggcgacat cctcaccgat 720
gaggcaggcg cagtctctgg cggaattggc ctcgcagcat ccggcaacat cgatgccacg 780
ggcaccaacc cttccatgtt cgagccagtc cacggctctg caccagatat cgcaggccag 840
ggaatcgcag acccaacggc agcaatccta tccgctgcga tgttgctgcg tcacttaggt 900
gatgaggaca acgcagtacg tattgaaaca gccatcgcgg ctgatgtggc tggccgagat 960
aactctcagt cgatttctac aactgaggtg ggagaccgcg tcgttagggc gctgcaaagc 1020
taa 1023
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Met Lys Leu Ala Val Ile Gly Gly Asp Gly Ile Gly Pro Glu Val Thr
1 5 10 15
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Thr Asp Tyr Asp Leu Gly Ala Arg Arg Tyr Leu Lys Asn Gly Glu Leu
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Leu Thr Asp Glu Asp Leu Ala Ser Leu Arg Glu His Asp Ala Ile Leu
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Arg Gly Leu Leu Leu Lys Met Arg Phe Ala Leu Asp His His Val Asn
85 90 95
Leu Arg Pro Ser Lys Leu Tyr Asp Gly Val Glu Ser Pro Leu Arg Asn
100 105 110
Pro Gly Lys Ile Asp Phe Val Val Val Arg Glu Gly Thr Glu Gly Ala
115 120 125
Tyr Thr Gly Asn Gly Gly Ala Ile Arg Val Gly Thr Pro His Glu Ile
130 135 140
Ala Asn Glu Thr Ser Val Asn Thr Arg Tyr Gly Ala Glu Arg Val Ile
145 150 155 160
Arg Tyr Ala Phe Glu Leu Ala Gln Ser Arg Arg Lys Lys Leu Thr Leu
165 170 175
Val His Lys Thr Asn Val Leu Val His Gly Gly Gly Leu Trp Gln Arg
180 185 190
Thr Val Asp Glu Val Ala Lys Glu Tyr Pro Glu Val Ala Val Asp Tyr
195 200 205
Asn His Ile Asp Ala Ala Thr Ile Tyr Leu Val Thr Asp Pro Ser Arg
210 215 220
Phe Asp Val Ile Val Thr Asp Asn Leu Phe Gly Asp Ile Leu Thr Asp
225 230 235 240
Glu Ala Gly Ala Val Ser Gly Gly Ile Gly Leu Ala Ala Ser Gly Asn
245 250 255
Ile Asp Ala Thr Gly Thr Asn Pro Ser Met Phe Glu Pro Val His Gly
260 265 270
Ser Ala Pro Asp Ile Ala Gly Gln Gly Ile Ala Asp Pro Thr Ala Ala
275 280 285
Ile Leu Ser Ala Ala Met Leu Leu Arg His Leu Gly Asp Glu Asp Asn
290 295 300
Ala Val Arg Ile Glu Thr Ala Ile Ala Ala Asp Val Ala Gly Arg Asp
305 310 315 320
Asn Ser Gln Thr Ile Ser Thr Thr Glu Val Gly Asp Arg Val Val Arg
325 330 335
Ala Leu Gln Ser
340
<210> 4
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atgaaacttg ctgttattgg tggagacggt atcggcccag aggttactgc agaagccctc 60
aaggttctaa acgctgtccg cgacgacatc gagaccaccg attatgacct tggcgcacgc 120
cgttacctca aaaatggcga gctgctcacc gacgaggatc tggcatccct gcgcgagcat 180
gacgcgatcc ttcttggcgc tatcggtgca ccaggttccg tccctccagg aattctcgag 240
cgcggtttgc tgctgaagat gcgattcgca ctggatcacc acgtgaacct gcgcccatcc 300
aagctgtacg acggcgtgga gtccccactg cgtaacccag gcaagattga tttcgttgtg 360
gtccgcgaag gtaccgaagg cgcctacact ggcaacggtg gagcaatccg cgtgggaacc 420
cctcacgaga ttgccaatga aacctccgtg aacactcgct acggcgctga gcgcgttatt 480
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aacgtcctgg ttcacggtgg tggcctgtgg cagcgcaccg tagatgaggt tgcaaaggaa 600
tacccagagg tagccgtcga ttacaaccac atcgatgcag caaccatcta tctggtcact 660
gatccttccc gcttcgatgt gattgttacc gataacctct tcggcgacat cctcaccgat 720
gaggcaggcg cagtctctgg cggaattggc ctcgcagcat ccggcaacat cgatgccacg 780
ggcaccaacc cttccatgtt cgagccagtc cacggctctg caccagatat cgcaggccag 840
ggaatcgcag acccaacggc agcaatccta tccgctgcga tgttgctgcg tcacttaggt 900
gatgaggaca acgcagtacg tattgaaaca gccatcgcgg ctgatgtggc tggccgagat 960
aactctcaga cgatttctac aactgaggtg ggagaccgcg tcgttagggc gctgcaaagc 1020
taa 1023
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ggtacccggg gatcctctag agcgttattc gctacgcatt cg 42
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<213> Artificial sequence (Artificial Sequence)
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gaaatcgact gagagttatc tcgg 24
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<213> Artificial sequence (Artificial Sequence)
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gataactctc agtcgatttc tacaactg 28
<210> 8
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<213> Artificial sequence (Artificial Sequence)
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acgacggcca gtgccaagct tctaaacacc aagcgcttga aac 43
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<213> Artificial sequence (Artificial Sequence)
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gataactctc agacgatttc tacaactg 28
<210> 10
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<213> Artificial sequence (Artificial Sequence)
<400> 10
gaaatcgtct gagagttatc tcgg 24
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tggccgagat aactctcatt 20
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tggccgagat aactctcaaa 20
<210> 13
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gatttcgttg tggtccgcg 19
<210> 14
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
cgtcactcaa cgtgcattac c 21
<210> 15
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ctcgtatgtt gtgtggaatt gtg 23
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
cgccctgagt gcttgcggca 20
Claims (10)
1. A method for reducing L-leucine in an amino acid producing strain, wherein the amino acid producing strain is caused to express an isopropyl malate dehydrogenase mutant comprising a mutation in which proline at position 324 is substituted with another amino acid, using the amino acid sequence of a wild-type isopropyl malate dehydrogenase of the amino acid producing strain as a reference sequence.
2. The method according to claim 1, characterized in that the isopropyl malate dehydrogenase mutant contains a mutation in which proline at position 324 is substituted by serine or threonine, preferably by serine;
and/or the isopropyl malate dehydrogenase mutant has an amino acid sequence shown as SEQ ID NO.1 or 3;
and/or the nucleic acid encoding the isopropyl malate dehydrogenase mutant has a sequence as shown in SEQ ID No.2 or 4;
and/or the amino acid producing strain is a microorganism of the genus Corynebacterium, preferably Corynebacterium glutamicum (Corynebacterium glutamicum).
3. A recombinant microorganism expressing an isopropyl malate dehydrogenase mutant according to any one of claims 1-2; the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0112-8 with the preservation number of CGMCC No.11941, or the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0912-6 with the preservation number of CGMCC No.11942, or the original strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0701 with the preservation number of CGMCC No.13757.
4. A method of constructing a recombinant microorganism according to claim 3, comprising: mutating a gene encoding isopropyl malate dehydrogenase in a starting strain to a gene encoding an isopropyl malate dehydrogenase mutant as described in any one of claims 1-2.
5. A method for producing glutamic acid, comprising the step of fermentation-culturing the recombinant microorganism according to claim 3, wherein the starting strain of the recombinant microorganism is corynebacterium glutamicum MHZ-0112-8.
6. The method according to claim 5, wherein the fermentation medium during fermentation comprises the following components: glucose 50-60g/L, ammonium sulfate 10-20g/L, KH 2 PO 4 0.5-1.5g/L，MgSO 4 ·7H 2 O 0.2-0.5g/L，FeSO 4 ·7H 2 O 0.5-1.5mg/L，MnSO 4 ·5H 2 O 0.8-1.2mg/L，VB 1 150-250 mug/L, 250-350 mug/L biotin, 0.3-0.6g/L soybean hydrolysate, 40-80g/L calcium carbonate and pH 7.2-7.5.
7. A method for producing lysine, characterized by comprising the step of fermenting and culturing the recombinant microorganism according to claim 3, wherein the starting strain of the recombinant microorganism is Corynebacterium glutamicum MHZ-0912-6, and the preservation number is CGMCC No.11942.
8. The method according to claim 7, wherein the fermentation medium during fermentation comprises the following components: glucose 55-65g/L, (NH) 4 ) 2 SO 4 22-28g/L，KH 2 PO 4 1.5-2.5g/L，MgSO 4 ·7H 2 0.8-1.2g/L of O, 8-12g/L of yeast powder and CaCO 3 20-30g/L，pH 6.8-7.2。
9. A method for producing proline, comprising the step of fermenting and culturing the recombinant microorganism according to claim 3, wherein the recombinant microorganism has an initial strain of corynebacterium glutamicum MHZ-0701 and a preservation number of CGMCC No.13757.
10. The method according to claim 9, wherein the fermentation medium during fermentation comprises the following components in mass percent: corn steep liquor 0.5-0.7%, glucose 11-13%, ammonium sulfate 3.5-4%, magnesium sulfate 0.04-0.06%, potassium dihydrogen phosphate 0.09-0.11%, caCO 3 3.8-4.2％，V H 65-75μg/L，V B1 ·HCl 75-85μg/L，pH 7.2。
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