Method for improving fermentation yield of glutamine

Abstract

The invention provides a method for improving fermentation yield of glutamine, which is characterized in that a coding gene aspB of aspartate transaminase in a microorganism with glutamine production capacity is weakened by utilizing a genetic engineering means to obtain a modified microorganism, and the modified microorganism is used for fermentation production of glutamine. The aspB gene of corynebacterium glutamicum is modified, so that the glutamine generation capacity of corynebacterium glutamicum is higher than that of an unmodified strain.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a method for increasing glutamine fermentation yield. Background Art

[0002] Glutamine is a non-essential amino acid. Its chemical name is 2-amino-4-carbamoylbutyric acid. Glutamine is a coded amino acid in protein synthesis. It can promote protein synthesis and inhibit protein decomposition. It can be used to treat gastric and duodenal ulcers and plays an important role in the pharmaceutical industry.

[0003] At present, the most commonly used production method of glutamine is fermentation, which mainly uses Corynebacterium glutamicum as the production bacteria to produce glutamine. Corynebacterium glutamicum is a heterotrophic aerobic type, a Gram-positive bacterium, with the characteristics of fast growth rate, non-pathogenicity, and weak degradation ability of its own metabolites. The fermentation method has the advantages of wide source of raw materials, low production cost, controllable product quality, and single product. However, the fermentation performance of the strains currently producing glutamine is still poor, the glutamine conversion rate is not ideal, the industrial demand for glutamine is extremely high, and the existing strains cannot meet the needs of large-scale industrial production at all. Summary of the invention

[0004] The purpose of the present invention is to provide a method for increasing the yield of glutamine by fermentation.

[0005] The concept of the present invention is as follows: by modifying the aspB gene (encoding aspartate aminotransferase) of Corynebacterium glutamicum, the ability of the recombinant bacteria to produce glutamine is stronger than that of the unmodified strain.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for increasing the yield of glutamine fermentation, using genetic engineering means to weaken the encoding gene of aspartate aminotransferase in a microorganism with glutamine production ability, obtain a modified microorganism, and use it for the fermentation production of glutamine.

[0007] Preferably, the weakening is selected from any one of the following 1)-3):

[0008] 1) The start codon ATG of the gene encoding aspartate aminotransferase was mutated to TTG;

[0009] 2) inserting a terminator at -24 bp upstream of the start codon of the gene encoding aspartate aminotransferase;

[0010] 3) Delete bp 503 to 835 of the ORF region of the gene encoding aspartate aminotransferase.

[0011] In the present invention, the reference sequence number of the gene encoding aspartate aminotransferase on NCBI is CEY17_01410.

[0012] Furthermore, the method further comprises: weakening the genes encoding α-ketoglutarate dehydrogenase E1o subunit, adenylyltransferase, and glutaminase in the microorganism, and enhancing the gene encoding pyruvate carboxylase.

[0013] In the present invention, the reference sequence numbers of the genes encoding α-ketoglutarate dehydrogenase E1o subunit (odhA gene), adenylyltransferase (ATase gene), glutaminase (glsA gene), and pyruvate carboxylase (pyc gene) on NCBI are CEY17_06180, CEY17_01255, CEY17_13230, and CEY17_03780, respectively.

[0014] In the present invention, the microorganism is Corynebacterium, Enterobacterium, Bacillus subtilis, preferably Corynebacterium glutamicum.

[0015] In a second aspect, the present invention provides a modified microorganism, wherein the activity of aspartate aminotransferase is reduced compared to an unmodified microorganism, and the microorganism has an enhanced glutamine production capacity compared to an unmodified microorganism.

[0016] Preferably, the reduction of aspartate aminotransferase activity is achieved by any of the following methods 1)-3):

[0017] 1) The start codon ATG of the gene encoding aspartate aminotransferase was mutated to TTG;

[0018] 2) inserting a terminator at -24 bp upstream of the start codon of the gene encoding aspartate aminotransferase;

[0019] 3) Delete bp 503 to 835 of the ORF region of the gene encoding aspartate aminotransferase.

[0020] Preferably, the microorganism is Corynebacterium glutamicum MHZ-0513-3, or Corynebacterium glutamicum engineered bacteria ATCC14067-glnA Y405F .

[0021] Among them, the engineered bacteria ATCC 14067-glnA Y405F The construction method includes: using Corynebacterium glutamicum ATCC 14067 as a starting bacterium, introducing a mutation in the glnA gene by genetic engineering means, so that the 405th amino acid of the glutamine synthetase encoded by the glnA gene is mutated from Y to F.

[0022] In a third aspect, the present invention provides use of the modified microorganism in the fermentation production of glutamine or its derivatives or in increasing the fermentation yield of glutamine or its derivatives.

[0023] In a fourth aspect, the present invention provides a method for producing glutamine or a derivative thereof, the method comprising the following steps:

[0024] a) culturing the modified microorganism to obtain a culture;

[0025] b) collecting the produced glutamine from the culture obtained in step a).

[0026] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0027] Strain MHZ-0513-3-aspB after the start codon of aspB gene was weakened to TTG a1t Compared with the starting strain MHZ-0513-3, the glutamine production increased from 28.9 g / L to 31.2 g / L, and the byproduct isoleucine decreased by 1.9 g / L. The strain MHZ-0513-3-aspB* after the terminator was inserted at -24 bp upstream of the start codon of the aspB gene, and the strain MHZ-0513-3-ΔaspB with inactivated aspB gene were both aspartate deficient. After supplementing 1.5 g / L aspartate in the fermentation medium, the glutamine production was 33.0 g / L and 32.8 g / L, respectively. The activity of aspartate aminotransferase after all the aspB gene modifications was tested, and the results showed that the activity was weakened. It can be seen that the weakening of the aspB gene is conducive to the production of glutamine. DETAILED DESCRIPTION

[0028] The present invention provides Corynebacterium glutamicum with improved glutamine production, and a construction method and application thereof.

[0029] The present invention adopts the following technical solution:

[0030] The present invention provides a Corynebacterium glutamicum, wherein an intracellular aspartate aminotransferase (aspB encoding, NCBI sequence number CEY17_01410) site is weakened and / or inactivated, and the specific modification means are: (1) the start codon ATG is mutated to TTG; (2) a terminator is inserted at -24 bp upstream of the start codon; and (3) 333 bp (503 bp to 835 bp) is deleted in the ORF region.

[0031] By modifying the aspB gene, the ability of Corynebacterium glutamicum to produce glutamine is stronger than that of the unmodified strain.

[0032] The starting bacteria used were Corynebacterium glutamicum MHZ-0513-3 and Corynebacterium glutamicum engineered bacteria ATCC14067-glnA Y405F .

[0033] Among them, MHZ-0513-3 has been disclosed in CN106701649A, and the deposit number is CGMCC No.13405.

[0034] Corynebacterium glutamicum engineered bacteria ATCC 14067-glnA Y405F Corynebacterium glutamicum ATCC 14067 was used as the model bacterium, and a point mutation glnA was introduced Y405F Thus, the adenylylation modification is eliminated, which is beneficial to the accumulation of glutamine. The specific construction method can be found in CN100392075C.

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0036] The names and sequences of the primers involved in the following examples are shown in Table 1.

[0037] Table 1 Primer information

[0038]

[0039]

[0040] Sequence Description:

[0041] SEQ ID NO: 1: Nucleotide sequence of the aspB gene of Corynebacterium glutamicum.

[0042] SEQ ID NO: 2: The amino acid sequence encoded by the aspB gene start codon after mutation from ATG to TTG.

[0043] SEQ ID NO:3: rrnB terminator sequence. The rrnB sequence was inserted at -24 bp upstream of the start codon of the aspB gene. The rrnB sequence was cloned from the genome of Escherichia coli K-12MG1655, and the strain K-12MG1655 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0044] SEQ ID NO: 4: ΔaspB deletion sequence, i.e., 333 bp (503 bp to 835 bp) is deleted from the ORF region.

[0045] Example 1 Construction of aspB gene-modified plasmid and screening of modified bacteria

[0046] 1. Strain MHZ-0513-3-aspB a1t and ATCC 14067-glnA Y405F -aspB a1t How to build

[0047] Phusion ultra-fidelity polymerase (New England BioLabs) was used, the genome of Corynebacterium glutamicum ATCC 14067 was used as a template, P58-UP-F / P59-UP-R was used as primers, the recombinant fragment UP was prepared, and P60-DN-F / P61-DN-R was used as primers to prepare the recombinant fragment DN, and the obtained fragment was purified by agarose gel recovery kit (Tiangen). At the same time, pK18-mobsacB (Wuhan Miaoling Biotechnology Co., Ltd.) was digested with XbaI / HindIII, and the UP, DN fragments and pK18-mobsacB (Wuhan Miaoling Biotechnology Co., Ltd.) vector were assembled with a seamless cloning kit (Nanjing Novi Biotechnology Co., Ltd.), and transformed into Trans1T1 competent cells (Quanshijin Biotechnology Co., Ltd.), and kanamycin-resistant clones were selected for PCR identification using P01 / P02 primers. After the plasmid was correctly extracted, it was sequenced using P01 / P02 primers (Jinweizhi Biotechnology Co., Ltd.). The resulting plasmid was named pK18-aspB a1t . a1t MHZ-0513-3 and ATCC 14067-glnA were respectively transferred into Y405F The recombinants were selected on a selective medium containing 15 mg / L kanamycin. The culture temperature was 33°C and the culture was inverted. The selected transformants were cultured overnight in a normal liquid brain heart infusion 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 solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 hours. SThe recombinant was verified by P64-id-f / P63-ID-R to verify the point mutation recombinant. By exploring the annealing temperature, the recombinant containing the point mutation was obtained. The positive recombinant was amplified and sequenced by P62-ID-F / P63-ID-R to verify the mutant strain obtained, which was named MHZ-0513-3-aspB. a1t , ATCC 14067-glnA Y405F -aspB a1t .

[0048] 2. Strains MHZ-0513-3-aspB* and ATCC 14067-glnA Y405F -aspB* construction method

[0049] The construction of plasmid and recombinant strains is with reference to the above method, with the genome of Corynebacterium glutamicum MHZ-0513-3 as template, with P65-UP-F / P66UP-R as primer, prepare recombinant fragment UP, with P69-DN-F / P70-DN-R as primer, prepare recombinant fragment DN. With Escherichia coli K-12MG1655 genome (Wuhan Miaoling Biotechnology Co., Ltd.) as template, with P67-Ter-F / P68-Ter-R as primer, prepare recombinant fragment Ter. UP, DN, Ter fragment and pK18-mobsacB vector digested with XbaI / HindIII are assembled, Trans1T1 competent cells are transformed, and kana resistance clones are picked and PCR identified with P01 / P02 primers, and XbaI / HindIII enzyme digestion is performed after correct plasmid extraction, and further the inserted fragment is correct by sequencing with P01 / P02 primers. The obtained plasmid was named pK18-aspB*. pK18-aspB* was transformed into Corynebacterium glutamicum MHZ-0513-3 and ATCC 14067-glnA Y405F The primers P71-ID-F / P72-ID-R were used to amplify the fragments and sequence them to obtain the target mutant strains, which were named MHZ-0513-3-aspB*, ATCC 14067-glnA, and Y405F -aspB*.

[0050] 3. Strains MHZ-0513-3-ΔaspB and ATCC 14067-glnA Y405F -ΔaspB construction method

[0051] The construction of plasmids and recombinant strains refers to the above method. The genome of Corynebacterium glutamicum MHZ-0513-3 is used as a template, P73-UP-F / P74-UP-R is used as primers to prepare the recombinant fragment UP, and P75-DN-F / P76-DN-R is used as primers to prepare the recombinant fragment DN. The UP and DN fragments are assembled with the pK18-mobsacB vector digested with XbaI / HindIII, and the Trans1T1 competent cells are transformed. The kanamycin-resistant clones are picked and PCR identified with P01 / P02 primers. After the plasmid is correctly extracted, XbaI / HindIII enzyme digestion is performed for identification, and the inserted fragment is further identified by sequencing with P01 / P02 primers. The resulting plasmid is named pK18-ΔaspB. pK18-ΔaspB is transferred into Corynebacterium glutamicum MHZ-0513-3 and ATCC14067-glnA, respectively. Y405F The primers P77-ID-F / P78-ID-R were used to amplify the fragments and sequence them to obtain the target mutant strains, which were named MHZ-0513-3-ΔaspB, ATCC 14067-glnA, and ATCC 14067-glnA. Y405F -ΔaspB.

[0052] Example 2 Performance investigation of engineering bacteria producing glutamine

[0053] Fermentation verification method for glutamine production: The strain frozen in a -80°C glycerol tube was inoculated into the following slant medium for activation, and after culturing at 33°C for 24 hours, a bacterial moss grew. The bacterial moss was picked from the freshly activated slant and inoculated into the following seed medium. The culture was shaken at 33°C and 100rpm until the middle and late logarithmic growth period. The culture time was 5 hours to prepare the seed solution. The above seed solution was inoculated into a 500ml shake flask containing 20ml fermentation medium at an inoculation rate of 10%, and the culture was shaken at 33°C and 150rpm for 48 hours. The results are shown in Table 2, where OD 562 The turbidity of the culture solution at 562 nm indicates the cell mass, and Gln (g / L) indicates the amount of accumulated glutamine.

[0054] The culture medium formula is as follows:

[0055] Slant culture medium: brain heart infusion 37 g / L, agar 1.8%, sterilized at 121℃ 0.1 MPa for 20 min.

[0056] Seed culture medium: glucose 50 g / L, urea 5 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.0 g / L, corn steep liquor 30 g / L, pH 7.0.

[0057] Fermentation medium: glucose 90 g / L, (NH4)2SO4 40 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O1.0 g / L, corn steep liquor 10 g / L, CaCO3 50 g / L, pH 7.0.

[0058] Table 2 Glutamine content detection of mutant strains

[0059]

[0060]

[0061] Note: 1.5 g / L aspartic acid was added to the fermentation medium of aspB* and ΔaspB.

[0062] From the above experimental results, it can be seen that after the start codon of the aspB gene in MHZ-0513-3 was mutated from ATG to TTG, the strain MHZ-0513-3-aspB was obtained. a1t , the glutamine production in shake flask fermentation increased from 28.9g / L to 31.2g / L, and the byproduct isoleucine decreased from 4.6g / L to 2.7g / L. The strain MHZ-0513-3-aspB* and the strain MHZ-0513-3-ΔaspB with inactivated aspB gene are aspartate deficient. After supplementing with 1.5g / L aspartate, the glutamine production was 33.0g / L and 28.5g / L, respectively.

[0063] To confirm the effect of aspB gene modification in different starting strains, the above aspB modification was further introduced into strain ATCC 14067-glnA Y405F The results in Table 2 show that aspB weakening is beneficial to the accumulation of glutamine in different strains. Similarly, integrating the aspB gene into other strains that can synthesize glutamine, such as Enterobacter and Corynebacterium, is beneficial to the accumulation of glutamine or its derivatives.

[0064] Example 3 Aspartate aminotransferase activity assay

[0065] The method for determining transaminase activity is as follows: collect cells by centrifugation at 4°C, 12,000 rpm for 20 minutes, suspend in 50 mM Tris-HCl at pH 8.0, and treat with 20 mg / mL lysozyme at 37°C for 1 hour. Then, the cell suspension is sonicated on ice for 15 minutes, and then centrifuged at 4°C, 12,000 rpm for 20 minutes. The resulting supernatant is the crude enzyme solution. The reaction mixture is 100 mM Tris-HCl (pH 8.0), 0.25 mM pyridoxal phosphate, 5 mM oxaloacetic acid, and 10 mM glutamic acid. Add 10 μL of the crude enzyme solution to 1 mL of the reaction mixture, and draw multiple 50 μL samples within 20 minutes. Add 30 μL of 5% perchloric acid and 38% ethanol to each sample to stop the reaction. After adding 20 μL of 20 mM Tris-HCl (pH 8.0) and 20 mM K2CO3 buffer, the sample was centrifuged at 4°C, 12,000 rpm for 10 minutes to remove proteins and salts. High performance liquid chromatography was used to quantitatively detect aspartate, and the aspartate measured was proportional to the time and protein concentration. The unit of aspartate aminotransferase specific activity was μmol / min / mg protein.

[0066] Table 3 Aspartate aminotransferase specific activity

[0067]

[0068]

[0069] The results of enzyme activity are shown in Table 3. After the start codon of the aspB gene was replaced from ATG to TTG, the enzyme specific activity decreased by 5.38 μmol / min / mg protein, and almost no enzyme activity was detected in the strains of MHZ-0513-3-aspB* and MHZ-0513-3-ΔaspB. The weakening or even inactivation of the aspB gene leads to a weakening of the diversion of oxaloacetate to other metabolites, which is beneficial to glutamine synthesis.

[0070] 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 can be made 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 method for increasing glutamine fermentation yield, characterized in that: By using genetic engineering methods, the coding gene of aspartate aminotransferase in microorganisms with glutamine production capacity is weakened to obtain modified microorganisms, which are then used for the fermentation production of glutamine.

2. The method according to claim 1, characterized in that: The weakening is selected from any one of the following 1)-3): 1) The start codon ATG of the gene encoding aspartate aminotransferase was mutated to TTG; 2) inserting a terminator at -24 bp upstream of the start codon of the gene encoding aspartate aminotransferase; 3) Delete bp 503 to 835 of the ORF region of the gene encoding aspartate aminotransferase; The reference sequence number of the gene encoding aspartate aminotransferase on NCBI is CEY17_01410.

3. The method according to claim 1, characterized in that The method further comprises: weakening the genes encoding α-ketoglutarate dehydrogenase E1o subunit, adenylyltransferase, and glutaminase in the microorganism, and enhancing the gene encoding pyruvate carboxylase; The reference sequence numbers of the genes encoding α-ketoglutarate dehydrogenase E1o subunit, adenylyltransferase, glutaminase, and pyruvate carboxylase on NCBI are CEY17_06180, CEY17_01255, CEY17_13230, and CEY17_03780, respectively.

4. The method according to any one of claims 1 to 3, characterized in that: The microorganism is Corynebacterium, Enterobacterium, Bacillus subtilis, preferably Corynebacterium glutamicum.

5. A modified microorganism, characterized in that The microorganism has reduced aspartate aminotransferase activity compared to an unmodified microorganism, and has enhanced glutamine production capacity compared to an unmodified microorganism.

6. The microorganism according to claim 5, characterized in that The reduction of aspartate aminotransferase activity is achieved by any of the following methods 1)-3): 1) The start codon ATG of the gene encoding aspartate aminotransferase was mutated to TTG; 2) inserting a terminator at -24 bp upstream of the start codon of the gene encoding aspartate aminotransferase; 3) Delete bp 503 to 835 of the ORF region of the gene encoding aspartate aminotransferase; The reference sequence number of the gene encoding aspartate aminotransferase on NCBI is CEY17_01410.

7. The microorganism according to claim 5 or 6, characterized in that The microorganism is Corynebacterium, Enterobacterium, Bacillus subtilis, preferably Corynebacterium glutamicum.

8. The microorganism according to claim 7, characterized in that The microorganism is Corynebacterium glutamicum MHZ-0513-3 or Corynebacterium glutamicum engineered bacteria ATCC 14067-glnA Y405F ; Among them, the engineered bacteria ATCC 14067-glnA Y405F The construction method includes: using Corynebacterium glutamicum ATCC 14067 as a starting bacterium, introducing a mutation in the glnA gene by genetic engineering means, so that the 405th amino acid of the glutamine synthetase encoded by the glnA gene is mutated from Y to F.

9. Use of the microorganism according to any one of claims 5 to 8 in the fermentation production of glutamine or its derivatives or in increasing the fermentation yield of glutamine or its derivatives.

10. A method for producing glutamine or a derivative thereof, characterized in that: The method comprises the following steps: a) culturing the microorganism according to any one of claims 5 to 8 to obtain a culture; b) collecting the produced glutamine from the culture obtained in step a).

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