Phosphoenolpyruvate carboxykinase mutant and application of phosphoenolpyruvate carboxykinase mutant in lysine production

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a phosphoenolpyruvate carboxykinase mutant and application thereof in lysine production. The research finds that the 377th amino acid of phosphoenolpyruvate carboxykinase is mutated from proline to serine, so that the yield of lysine produced by zymophyte can be increased, and therefore, the phosphoenolpyruvate carboxykinase mutant is provided, which takes an amino acid sequence as shown in SEQ ID NO.7 or 9 as a reference sequence; the phosphoenolpyruvate carboxykinase mutant contains a 377th amino acid mutation which is mutated from proline to serine. According to the phosphoenolpyruvate carboxykinase mutant provided by the invention, the yield of lysine fermentation can be increased, and a new effective method is provided for lysine production.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a phosphoenolpyruvate carboxykinase mutant and application thereof in producing lysine. Background Art

[0002] As an essential amino acid, L-lysine is widely used in food processing, medicine, and feed additives. About 90% of L-lysine is used in the feed industry, and 10% is used in the food and pharmaceutical industries. As an animal feed additive, L-lysine can help the body absorb other amino acids, thereby improving feed quality. Therefore, the production of lysine has broad prospects.

[0003] The methods for producing L-lysine include protein decomposition, chemical method, enzymatic method and microbial fermentation. At present, L-lysine is mainly produced by microbial fermentation in industry, among which Corynebacterium glutamicum ( Corynebacterium glutamicum ) is the most commonly used strain, which has the characteristics of fast growth, non-pathogenicity, and weak ability to degrade its own metabolites. It is widely used in the production of various amino acids, nucleotides and other organic acids.

[0004] It is necessary to develop more solutions to improve the fermentation production of L-lysine. Summary of the invention

[0005] One of the purposes of the present invention is to provide a new method for increasing the yield of lysine by fermentation.

[0006] The present invention provides a phosphoenolpyruvate carboxykinase mutant, which uses the amino acid sequence shown in SEQ ID NO. 7 or 9 as a reference sequence, and the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the 377th amino acid is mutated from proline to serine.

[0007] The present invention has found that some intermediates in the tricarboxylic acid cycle (TCA cycle) are precursors for the synthesis of many important organic substances. Oxaloacetate is an important substance for the synthesis of lysine. When the intermediates of the TCA cycle are consumed in large quantities for the synthesis of organic substances, the oxaloacetate replenishment reaction will be initiated to ensure the normal operation of the TCA cycle, including phosphoenolpyruvate carboxylase catalyzing phosphoenolpyruvate to irreversibly generate oxaloacetate, which is an important way for oxaloacetate replenishment in bacteria, and phosphoenolpyruvate carboxykinase ( Phosphoenolpyruvate carboxykinase,PCK ) catalyzes oxaloacetate to generate phosphoenolpyruvate and carbon dioxide, which is the reverse reaction of the anaplerotic reaction. The present invention has found in many years of research and practice that phosphoenolpyruvate carboxykinase can regulate the synthesis of lysine. The specific variant of the enzyme can significantly improve the efficiency of lysine synthesis by Corynebacterium, thereby providing a phosphoenolpyruvate carboxykinase variant, wherein the amino acid at position 377 of the phosphoenolpyruvate carboxykinase variant is mutated, and the variant can subtly disturb the metabolic balance and is conducive to improving the efficiency of lysine synthesis by Corynebacterium.

[0008] Specifically, the present invention uses two Corynebacterium glutamicum model strains ATCC 13032 and ATCC 14067 as test objects, and mutates the coding sequence of the wild-type phosphoenolpyruvate carboxykinase of the model bacteria (SEQ ID NO.7 and 9) by genetic engineering methods, so that the 377th position of its protein sequence is mutated from proline to serine (the sequence of the 377th position of the phosphoenolpyruvate carboxykinase of ATCC 13032 mutated from proline to serine is shown in SEQ ID NO. 8). The model bacteria carrying the above mutants are subjected to shake flask fermentation, and it is found that the lysine yield has an improved effect.

[0009] Corynebacterium glutamicum ATCC 13032 and ATCC 14067 are model strains of Corynebacterium glutamicum, which are well known in the art. The above strains can be purchased from public channels or obtained from relevant research institutes. The genome sequences of the relevant strains have been made public, and relevant information can be queried from the NCBI website.

[0010] The present invention further uses lysine-producing Corynebacterium CGMCC No.13407 (deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on November 30, 2016, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Corynebacterium glutamicum, which has been disclosed in patent application CN106635944A) and CGMCC No.11942 (deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on December 25, 2015, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Corynebacterium glutamicum, which has been disclosed in patent CN105734004B) as implementation objects, and applies the above-mentioned phosphoenolpyruvate carboxykinase mutants to the fermentation production of lysine by Corynebacterium, and it is found that the modified strains have a significant effect of improving the conversion rate.

[0011] The present invention also provides a nucleic acid encoding the above-mentioned phosphoenolpyruvate carboxykinase mutant.

[0012] The nucleic acid sequence encoding the phosphoenolpyruvate carboxykinase of the type strain ATCC 13032 is shown in SEQ ID NO. 5. The nucleic acid of the mutant thereof has the nucleotide sequence shown in SEQ ID NO. 6. The nucleic acid sequence encoding the phosphoenolpyruvate carboxykinase of the type strain ATCC 14067 is shown in SEQ ID NO. 10. The nucleic acid of the mutant thereof has the nucleotide sequence shown in SEQ ID NO. 11.

[0013] The present invention also provides a biological material containing the above nucleic acid, wherein the biological material is an expression cassette, a vector or a host cell.

[0014] The present invention also provides a recombinant microorganism, which expresses a phosphoenolpyruvate carboxykinase mutant, wherein the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the 377th amino acid is mutated from proline to serine; and the starting strain of the recombinant microorganism is a coryneform bacterium.

[0015] Preferably, the starting strain of the recombinant microorganism of the present invention is Corynebacterium glutamicum, and more preferably, it is Corynebacterium with the deposit number of CGMCC No.13407 and CGMCC No.11942.

[0016] The present invention also provides any of the following uses of the above-mentioned phosphoenolpyruvate carboxykinase mutant, or nucleic acid, or biological material, or recombinant microorganism: (1) Application in the fermentation production of L-lysine; (2) Application in genetic breeding of microorganisms for producing L-lysine; (3) Application in increasing the yield of L-lysine in fermentation production.

[0017] The present invention also provides the use of mutating the 377th amino acid of the phosphoenolpyruvate carboxykinase of a microorganism from proline to serine in the fermentation production of L-lysine.

[0018] The present invention also provides the use of mutating the 377th amino acid of phosphoenolpyruvate carboxykinase from proline to serine in the genetic breeding of microorganisms for producing L-lysine.

[0019] The present invention found that the above mutation of phosphoenolpyruvate carboxykinase in different chassis strains (Corynebacterium glutamicum) can increase the lysine production.

[0020] The present invention also provides a method for producing L-lysine, which comprises the step of fermenting and culturing the recombinant microorganism, wherein the recombinant microorganism is as described above.

[0021] The present invention also provides a method for constructing a recombinant microorganism, which comprises the step of causing the recombinant microorganism to express a phosphoenolpyruvate carboxykinase mutant, wherein the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the amino acid at position 377 is mutated from proline to serine; and the starting strain of the recombinant microorganism is a coryneform bacteria.

[0022] The beneficial effects of the present invention are at least: The invention provides a phosphoenolpyruvate carboxykinase mutant, which can improve the yield of lysine fermentation and provide a new and effective method for lysine production. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0025] The names and sequences of the primers involved in the embodiments of the present invention are shown in Table 1.

[0026] Table 1 Primer sequences (SEQ ID No. 1-4) Example 1 Introducing the mutant gene PCK into the model bacteria ATCC 13032 P377S (SEQ ID NO.6, the mutant gene encodes the sequence shown in SEQ ID NO.8) The genome of Corynebacterium glutamicum ATCC 13032 was used as a template and the primer pair PCK-1f / PCK-1r was used for PCR amplification to obtain the upstream homology arm fragment 13032-PCK-up; the genome of Corynebacterium glutamicum ATCC 13032 was used as a template and the primer pair PCK P377S -2f / PCK-2r primer pair was used for PCR amplification to obtain the downstream homology arm fragment 13032-PCK-dn. The mixture of 13032-PCK-up and 13032-PCK-dn was used as template and the PCK-1f / PCK-2r primer pair was used for PCR amplification to obtain the fragment 13032-up-dn. 13032-up-dn was cloned into amH I.N heI was double-digested, and the vector pK18mobsacB (GenBank: FJ437239.1; kindly donated by Professor Yang Sheng of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and can also be purchased from public channels) was double-digested with the same enzyme. The double-digested products were ligated with T4 DNA Ligase and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-13032-PCK P377S .

[0027] Prepare competent cells of ATCC 13032 according to the C. glutamicum Handbook (Charpter 23). Recombinant plasmid pK18mobsacB-13032-PCK P377S The competent cells were transformed by electroporation and the transformants were screened on BHI selective medium containing 15 mg / L kanamycin. The screened transformants were cultured overnight in ordinary BHI liquid medium at 33°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome by gene exchange. The culture was diluted in a series of gradients (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common BHI solid medium containing 10% sucrose and cultured at 33°C for 48 hours. The grown strain did not carry the inserted vector sequence in its genome. The mutant gene PCK was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis. P377S The strain was named 13032-PCK P377S .

[0028] Example 2 Introducing the mutant gene PCK into the model bacteria ATCC 14067 P377S (SEQ ID NO.11, the mutant gene encodes the amino acid in which the 377th position of the sequence shown in SEQ ID NO.9 is mutated from P to S) Referring to the method of Example 1, the genome of Corynebacterium glutamicum ATCC 14067 was used as a template for amplification, and finally the recombinant plasmid pK18mobsacB-14067-PCK was obtained. P377S The constructed recombinant plasmid pK18mobsacB-14067-PCK P377S The recombinant strain was transformed into the model bacteria ATCC 14067, and the amino acid 377 of phosphoenolpyruvate carboxykinase was mutated to serine, and the strain was named 14067-PCK. P377S .

[0029] Example 3 Shake flask test of fermentation performance of two model bacteria after introduction of PCR mutants The culture medium used in the lysine fermentation experiment is as follows: Seed activation medium: BHI 37g / L, 18g / L agar powder.

[0030] Seed culture medium: sucrose 20 g / L, yeast powder 5 g / L, peptone 10 g / L, urea 5 g / L, magnesium sulfate heptahydrate 0.4 g / L, adjust pH to 7.0.

[0031] Fermentation medium: glucose 60g / L, ammonium sulfate 25g / L, potassium dihydrogen phosphate 2.0g / L, magnesium sulfate heptahydrate 1.0g / L, soybean meal hydrolyzate 10g / L, calcium carbonate 30g / L, adjust pH to 7.0.

[0032] The lysine fermentation method is as follows: 1. Seed activation: Take the strain to be verified from the cryopreserved tube, streak it on the seed activation medium, and culture it at 33℃ for 24h; 2. Seed culture: Pick the activated seeds 1 from the plate and transfer them to a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Culture at 33℃ and 220r / min with shaking for 6h. 3. Fermentation culture: inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, culture at 33°C and 220 rpm for 14-15 h, and make three replicates for each strain.

[0033] 4.OD 562 Determination: Take 100 μl of fermentation liquid and dilute it to an appropriate multiple. Use a spectrophotometer to detect the OD at a wavelength of 562. Do three replicates for each strain and calculate the average value. 562 As shown in Table 2.

[0034] 5. Determination of lysine concentration: 2 mL of fermentation broth was centrifuged (12000 rpm, 2 min), the supernatant was collected, and the L-lysine content in the fermentation broth of the recombinant bacteria and the control bacteria was detected by HPLC. Three parallels were made for each strain, and the average value was calculated. The detected lysine concentrations are shown in Table 2.

[0035] Table 2 Lysine production and growth detection of recombinant strains The lysine content in the fermentation broth was detected, and it was found that the lysine concentration in the culture broth of Corynebacterium glutamicum carrying a phosphoenolpyruvate carboxykinase mutant was higher.

[0036] The above examples prove that the mutant phosphoenolpyruvate carboxykinase has a promoting effect on the lysine synthesis of the model strain. The present invention further verifies the effect of the mutation on lysine-producing bacteria. Two lysine-producing bacteria CGMCC No. 13407 and CGMCC No. 11942 that have been preserved and disclosed in patents are used for verification. CGMCC No. 13407 and CGMCC No. 11942 are obtained by genetic modification of the model bacteria ATCC13032, and their lysine synthesis-related genes have been enhanced.

[0037] Example 4 Introducing the mutant gene PCK into CGMCC No.13407 P377S Referring to the method of Example 1, the recombinant plasmid pK18mobsacB-13032-PCK constructed in Example 1 was P377S The result was transferred into CGMCC No.13407 to obtain a recombinant strain in which the amino acid at position 377 of phosphoenolpyruvate carboxykinase was mutated to serine, and the strain was named 13407-PCK P377S .

[0038] Example 5 Introducing the mutant gene PCK into CGMCC No.11942 P377S Referring to the method of Example 1, the recombinant plasmid pK18mobsacB-13032-PCK constructed in Example 1 was P377S The recombinant strain with amino acid 377 of phosphoenolpyruvate carboxykinase mutated to serine was obtained by transferring into CGMCC No.11942, and the strain was named 11942-PCK P377S .

[0039] Example 6 Lysine fermentation experiment CGMCC No.13407, CGMCC No.11942 and their modified recombinant bacteria 13407-PCK were treated with the method of Example 3. P377S 、11942-PCK P377S The results of the lysine fermentation experiment are shown in Table 3.

[0040] Table 3 Lysine production and growth detection of recombinant strains The fermentation results showed that in the same batch of two lysine-producing bacteria and a modified bacteria carrying a phosphoenolpyruvate carboxykinase mutant, the lysine production and sugar-acid conversion rate of the modified bacteria carrying a phosphoenolpyruvate carboxykinase mutant were significantly improved.

[0041] The above results show that the phosphoenolpyruvate carboxykinase mutant provided by the present invention has a promoting effect on the lysine production and sugar-acid conversion rate of Corynebacterium.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A phosphoenolpyruvate carboxykinase mutant, characterized in that: Taking the amino acid sequence shown in SEQ ID NO. 7 or 9 as a reference sequence, the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the amino acid at position 377 is mutated from proline to serine.

2. A nucleic acid encoding the phosphoenolpyruvate carboxykinase mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that Having a nucleotide sequence as shown in SEQ ID NO. 6 or 11.

4. A biological material containing the nucleic acid according to claim 2 or 3, wherein the biological material is an expression cassette, a vector or a host cell.

5. A recombinant microorganism, characterized in that The recombinant microorganism expresses a phosphoenolpyruvate carboxykinase mutant, wherein the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the 377th amino acid is mutated from proline to serine; and the starting strain of the recombinant microorganism is coryneformis.

6. Any one of the following uses of the phosphoenolpyruvate carboxykinase mutant according to claim 1, or the nucleic acid according to claim 2 or 3, or the biological material according to claim 4, or the recombinant microorganism according to claim 5: (1) Application in the fermentation production of L-lysine; (2) Application in genetic breeding of microorganisms for producing L-lysine; (3) Application in increasing the yield of L-lysine in fermentation production.

7. Application of mutating the 377th amino acid of phosphoenolpyruvate carboxykinase of microorganisms from proline to serine in the fermentation production of L-lysine.

8. Application of mutating the 377th amino acid of phosphoenolpyruvate carboxykinase from proline to serine in genetic breeding of microorganisms for producing L-lysine.

9. A method for producing L-lysine, characterized in that: The method comprises the step of fermenting and culturing a recombinant microorganism, wherein the recombinant microorganism is as described in claim 5.

10. A method for constructing a recombinant microorganism, characterized in that: The method comprises the steps of making the recombinant microorganism express a phosphoenolpyruvate carboxykinase mutant, wherein the phosphoenolpyruvate carboxykinase mutant contains a mutation in which the amino acid at position 377 is mutated from proline to serine; and the starting strain of the recombinant microorganism is coryneform bacteria.

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