A ncgl0081 gene mutant and application thereof in preparation of l-lysine

Abstract

The application discloses an NCgl0081 gene mutant and application thereof in preparation of L-lysine, and belongs to the technical field of microorganisms. The nucleotide sequence of the NCgl0081 gene mutant is shown in SEQ ID NO. 3 in a sequence listing, and the amino acid sequence coded by the NCgl0081 gene mutant is shown in SEQ ID NO. 4 in the sequence listing. Compared with the NCgl0081 gene, the amino acid at the 143th position in the amino acid sequence coded by the NCgl0081 gene mutant is changed from histidine to glutamine. The NCgl0081 gene mutant is introduced into a L-lysine-producing strain through a homologous recombination method, so that a high-yield L-lysine Corynebacterium glutamicum engineering strain can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a NCgl0081 gene mutant and application thereof in preparation of L-lysine. BACKGROUND

[0002] L-lysine is one of the essential amino acids for human and mammals, which cannot be synthesized by the body and must be supplemented from food. L-lysine has physiological functions in balancing amino acid composition, regulating metabolic balance in the body, improving the absorption and utilization rate of cereal protein by the body and promoting the growth and development of the body, and thus is widely used in food additives, drug synthesis, feed and other industries.

[0003] So far, there are mainly three methods for industrial production of L-lysine: protein hydrolysis method, chemical synthesis method and microbial fermentation method. Among them, the microbial fermentation method has become the most widely used method for industrial production of L-lysine due to its low production cost, high production intensity, high specificity and small environmental pollution. The strains used for industrial production of L-lysine at home and abroad are mainly modified strains of corynebacterium glutamicum and escherichia coli. For the microbial fermentation method, the key to the cost of fermentation production lies in the performance of the lysine-producing strain, therefore, it is of great significance to breed high-yield L-lysine strains with good production performance. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a NCgl0081 gene mutant and application thereof in preparation of L-lysine. After replacing the histidine at the 143th amino acid coded by the NCgl0081 gene with glutamine, a NCgl0081 gene mutant is obtained, which is then introduced into a L-lysine-producing strain by homologous recombination to obtain a corynebacterium glutamicum engineering strain with high yield of L-lysine.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a NCgl0081 gene mutant, the nucleotide sequence of which is shown in SEQ ID NO. 3 in the sequence listing, and the amino acid sequence coded by the NCgl0081 gene mutant is shown in SEQ ID NO. 4 in the sequence listing;

[0007] The 143th amino acid in the amino acid sequence coded by the NCgl0081 gene mutant is changed from histidine to glutamine compared with the NCgl0081 gene;

[0008] The nucleotide sequence of the NCgl0081 gene is shown in SEQ ID NO. 1 in the sequence listing, and the amino acid sequence encoded by the NCgl0081 gene is shown in SEQ ID NO. 2 in the sequence listing.

[0009] The present application provides a recombinant bacterium containing the NCgl0081 gene mutant.

[0010] The starting strain of the recombinant bacterium is Corynebacterium glutamicum strain M7, the preservation number is CGMCC No. 8184, the preservation time is September 13, 2013, and the preservation center is China General Microbiological Culture Collection Center.

[0011] The present application provides the use of the NCgl0081 gene mutant or the recombinant bacterium in the preparation of L-lysine.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] The present application replaces the histidine at the 143rd amino acid encoded by the NCgl0081 gene with glutamine, and then introduces the mutation into an L-lysine-producing strain by homologous recombination, to obtain a mutant strain with improved L-lysine production capacity. Compared with the existing Corynebacterium glutamicum, the present application can further improve the yield and conversion rate of L-lysine and reduce the production cost. DETAILED DESCRIPTION

[0014] In order to have a clearer understanding of the technical features, objectives and effects of the present application, the specific embodiments of the present application will be described below.

[0015] According to the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI, the NCgl0081 gene is searched, the nucleotide sequence of the NCgl0081 gene is shown in SEQ ID NO. 1 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID NO. 2 in the sequence listing.

[0016] The nucleotide sequence of the NCgl0081 gene mutant of the present application is shown in SEQ ID NO. 3 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID NO. 4 in the sequence listing; compared with SEQ ID NO. 2, the 143rd amino acid in SEQ ID NO. 4 is changed from histidine to glutamine.

[0017] The culture medium involved in the examples is as follows:

[0018] The formula of LB solid culture medium is as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, agar 20 g / L, pH 7.0.

[0019] The formula of LB liquid culture medium is as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0020] The formula of LBG liquid culture medium is as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, glucose 5 g / L, pH 7.0.

[0021] The formula of LBG solid culture medium is as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, glucose 5 g / L, agar 20 g / L, pH 7.0.

[0022] Example 1 Construction of recombinant plasmid pK18-m-0081 containing point mutation of NCgl0081 gene

[0023] 1. According to the NCgl0081 gene sequence of ATCC 13032 disclosed in NCBI, the following primers are designed:

[0024] P1: the nucleotide sequence is shown as SEQ ID NO. 5 in the sequence listing;

[0025] P2: the nucleotide sequence is shown as SEQ ID NO. 6 in the sequence listing;

[0026] P3: the nucleotide sequence is shown as SEQ ID NO. 7 in the sequence listing;

[0027] P4: the nucleotide sequence is shown as SEQ ID NO. 8 in the sequence listing;

[0028] 2. Taking the genome of ATCC 13032 as a template, primers P1 (upstream primer) and P2 (downstream primer), P3 (upstream primer) and P4 (downstream primer) are used for PCR amplification, respectively.

[0029] The PCR amplification system is as follows:

[0030]

[0031] The PCR amplification conditions are as follows: pre-denaturation: 95℃, 5 min; denaturation: 98℃, 10 s; annealing: 55℃, 15 s; extension: 72℃, 30 s; 35 cycles; post-extension: 72℃, 10 min.

[0032] After PCR amplification, the amplified products were subjected to agarose gel electrophoresis, and the required DNA fragments were purified by using Omega D2500 column type DNA gel recovery kit to obtain a 477 bp (nucleotide sequence as SEQ ID NO. 9 in the sequence listing) fragment UP and a 473 bp (nucleotide sequence as SEQ ID NO. 10 in the sequence listing) fragment DOWN.

[0033] 3. The fragment UP and the fragment DOWN were subjected to fragment fusion, and the PCR amplification system was as follows:

[0034]

[0035] The PCR amplification conditions were as follows: pre-denaturation: 95℃, 5 min; denaturation: 98℃, 10 s; annealing: 58℃, 15 s; extension: 72℃, 1 min; 7 cycles; post-extension: 72℃, 10 min.

[0036] Then, the obtained PCR product was used as a template, and primers P1 (upstream primer) and P4 (downstream primer) were used for PCR amplification. The PCR amplification system was as follows:

[0037]

[0038] The PCR amplification conditions were as follows: pre-denaturation: 95℃, 5 min; denaturation: 98℃, 10 s; annealing: 58℃, 15 s; extension: 72℃, 1 min; 35 cycles; post-extension: 72℃, 10 min.

[0039] After the PCR reaction, the amplified products were subjected to agarose gel electrophoresis, and the required DNA fragments were purified by using Omega D2500 column type DNA gel recovery kit to obtain a 929 bp fragment UD (nucleotide sequence as SEQ ID NO. 11 in the sequence listing). The fragment contained EcoR I and Sal I enzyme cutting sites at both ends, and the fragment could cause the 429th nucleotide of the NCgl0081 gene coding region to be mutated from C to A.

[0040] 4. The fragment UD was purified and recovered after double digestion with EcoR I and Sal I, and ligated with the shuttle plasmid pk18mobsacB which was also double digested (EcoR I and Sal I). The DNA ligation kit of Takara was used for ligation, and the molar ratio of vector DNA and inserted target DNA fragment was 0.03 pmol:0.2 pmol; the ligation temperature was 16°C, and the reaction time was 4 hours; the ligation product was transformed into the competent cells of Escherichia coli DH5a (purchased from TAKARA Company) by heat shock transformation; after the transformed Escherichia coli was cultured at 37°C for 1 hour, it was inoculated on the LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL; after being cultured in the incubator at 37°C for 24 hours, it was inoculated in the LB liquid culture medium containing kanamycin with a final concentration of 50 μg / mL, and cultured at 37°C for 24 hours; the recombinant plasmid was extracted by using the plasmid extraction kit of OMEGA Company, and the recombinant plasmid pK18-m-0081 containing the kanamycin resistance marker was obtained. The recombinant plasmid pK18-m-0081 was sent to a sequencing company for sequencing identification, and the recombinant plasmid pK18-m-0081 containing the correct point mutation was preserved for later use.

[0041] Example 2 Construction of strain M7-0081 containing point mutation of NCgl0081 gene

[0042] 1. The recombinant plasmid pK18-m-0081 was transformed into the strain M7 of Corynebacterium glutamicum by electroporation. The strain M7 of Corynebacterium glutamicum is a known bacterium which has been disclosed in the Chinese patent with the patent publication number CN103992964B and the patent name of a method for producing lysine by a high-pH-resistant strain and fermentation method, the strain M7 has the preservation number CGMCC No.8184, the preservation time is September 13, 2013, and the preservation center is China General Microbiological Culture Collection Center. After the transformed bacterial liquid was incubated at 30°C for 3 hours, it was inoculated on the LBG solid culture medium containing kanamycin with a final concentration of 50 μg / mL, and cultured at 30°C for 24 hours. The single colonies produced after the electroporation and culture were identified by primers P1 and M13F, and the strain with a 964 bp band (the nucleotide sequence is shown in nucleotide sequence 12) was a positive strain.

[0043] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s; annealing at 55°C for 15 s; extension at 72°C for 1 min; 35 cycles; and post-extension at 72°C for 10 min.

[0044] 2. The positive strain was inoculated into LBG liquid medium and cultured at 30°C for 16 h, and the culture was spread on LB solid medium containing 10% sucrose and cultured. The single colonies produced in the culture were cultured on LBG solid medium containing kanamycin and LBG solid medium not containing kanamycin, respectively, to screen the strain that did not grow on the LBG solid medium containing kanamycin but grew on the medium not containing kanamycin. The strain was further subjected to PCR amplification using P5 (nucleotide sequence as shown in SEQ ID NO. 13 in the sequence listing) as an upstream primer and P6 (nucleotide sequence as shown in SEQ ID NO. 14 in the sequence listing) as a downstream primer;

[0045] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 35 cycles, and post-extension at 72°C for 10 min.

[0046] The 914 bp long fragment (nucleotide sequence as shown in SEQ ID NO. 15 in the sequence listing) amplified by PCR was sequenced after purification of the PCR product. Through sequence alignment, the sequence in which the base sequence was correctly mutated proved that the homologous recombination of the strain was successful, and the strain was named M7-0081.

[0047] Example 3 Lysine Fermentation Test

[0048] The strain M7-0081 constructed in Example 2 was first subjected to seed culture, and then subjected to fermentation culture. Each strain was repeated 3 times.

[0049] The seed culture conditions were as follows: 37°C, pH 6.9, initial rotation speed 200 rpm, air volume 0.5 vvm, tank pressure 0.5 MPa, dissolved oxygen correction 100%, controlled dissolved oxygen 20%, inoculum size 5%, culture for 24 h, and OD growth to 0.4 (600 nm, diluted 26 times) when transferring to a fermenter.

[0050] The formula of the seed culture medium used in the seed culture is shown in the following table:

[0051]

[0052] The fermentation culture conditions are as follows: 37℃, pH 6.9, initial rotation speed 200 rpm, air volume 0.5 vvm, tank pressure 0.5 MPa. Before inoculation, the dissolved oxygen is corrected to 100%, and the dissolved oxygen is controlled to 15% by adjusting the rotation speed and air volume. The seed culture solution is inoculated into the fermentation medium at an inoculation amount of 5% for fermentation culture. When the culture is to 4.5 h, the dissolved oxygen begins to rise, and the glucose solution with a mass concentration of 75% is started to be added, so that the reducing sugar concentration is controlled at 1-3 g / L to the end of fermentation. When the culture is to 9 h, the ammonium sulfate solution with a mass concentration of 50% is started to be added, and the ammonia nitrogen is controlled at 0.15-0.25 g / L at 9-30 h of fermentation. After 30 h of fermentation, the ammonia nitrogen is controlled at 0.1-0.15 g / L to the end of fermentation. The pH is adjusted to 6.9 by using liquid ammonia during the whole fermentation process, and the fermentation cycle is 48 h.

[0053] The formula of the fermentation medium used in the fermentation culture is shown in the following table:

[0054]

[0055] After the culture is completed, the L-lysine content in the supernatant of the fermentation liquor is determined by using an amino acid analyzer, and the L-lysine conversion rate is calculated. The calculation formula of the L-lysine conversion rate is as follows:

[0056] L-lysine conversion rate (%) = lysine content * end volume / (initial volume * initial sugar concentration + added sugar volume * added sugar concentration).

[0057] The L-lysine contents in the supernatants of the fermentation liquors of the three repeated fermentations are 11.31%, 11.38% and 11.34% respectively, and the L-lysine conversion rates are 45.04%, 45.08% and 45.04% respectively.

[0058] Comparative Example 1

[0059] Referring to the fermentation method of Example 3, the original strain M7 used in Example 2 is subjected to small tank fermentation for 3 times. After completion, the L-lysine content and the L-lysine conversion rate in the supernatant of the fermentation liquor are detected by using the same method as that of Example 3.

[0060] The L-lysine contents in the supernatants of the fermentation liquors of the three repeated fermentations are 9.73%, 9.75% and 9.76% respectively, and the L-lysine conversion rates are 39.27%, 39.52% and 39.34% respectively.

[0061] Result analysis

[0062] The comparison results of the L-lysine contents and the L-lysine conversion rates of Example 3 and Comparative Example 1 are shown in the following table:

[0063]

[0064] As can be seen from the data in the above table, site mutation of the NCgl0081 gene in C. glutamicum helps to improve the L-lysine production ability.

Claims

1. An NCgl0081 gene mutant, characterized by, The nucleotide sequence of the NCgl0081 gene mutant is shown as SEQ ID NO. 3 in the sequence listing, and the amino acid sequence encoded by the NCgl0081 gene mutant is shown as SEQ ID NO. 4 in the sequence listing; The amino acid at position 143 in the amino acid sequence encoded by the NCgl0081 gene mutant is changed from histidine to glutamine as compared with the NCgl0081 gene.

2. The NCgl0081 gene mutant according to claim 1, characterized by The nucleotide sequence of the NCgl0081 gene is shown as SEQ ID NO. 1 in the sequence listing, and the amino acid sequence encoded by the NCgl0081 gene is shown as SEQ ID NO. 2 in the sequence listing.

3. A recombinant bacterium, characterized in that, The recombinant bacteria contain the NCgl0081 gene mutant of claim 1.

4. Use of the NCgl0081 gene mutant of claim 1 or 2 or the recombinant bacteria of claim 3 in the preparation of L-lysine.

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