Deacetylase mutants and uses thereof

Abstract

The application provides a deacetylase mutant and application thereof. By mutating the 119th amino acid of the deacetylase from A to D, T, P, V or S, the obtained mutant is introduced into a glutamine production strain (such as corynebacterium glutamicum) by a genetic engineering method, the obtained recombinant strain has enhanced ability of producing L-glutamine compared with an unmodified strain, and effectively reduces a byproduct N-acetyl-L-glutamine. The application provides an effective means for glutamine fermentation production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically, to a deacetylase mutant and its applications. Background Technology

[0002] Glutamine is a non-essential amino acid. Its chemical name is 2-amino-4-carbamoylbutyric acid. Glutamine is the amino acid that encodes proteins, promoting protein synthesis and inhibiting protein breakdown. It plays an important role in the pharmaceutical industry and is used to treat gastric and duodenal ulcers.

[0003] Currently, the most common method for producing glutamine is fermentation, primarily using *Corynebacterium glutamicum* as the producing bacterium. *Corynebacterium glutamicum* is a heterotrophic aerobic, Gram-positive bacterium characterized by rapid growth, non-pathogenicity, and weak degradation of its own metabolites. Fermentation offers advantages such as a wide availability of raw materials, low production costs, controllable product quality, and a single product. The main byproduct of glutamine fermentation is N-acetyl-L-glutamine, the synthesis mechanism of which is currently unknown. How to convert this byproduct into glutamine remains unclear; therefore, researching ways to reduce byproduct production is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a deacetylase mutant and its application.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a deacetylase mutant, said mutant comprising a mutation in the 119th amino acid of the deacetylase from A to D, T, P, V or S, preferably A119V or A119D.

[0006] The deacetylases of the present invention are derived from Escherichia coli (BL21) and contain or consist of the amino acid sequence shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the deacetylase mutant or biological material containing the nucleic acid molecule.

[0008] The biomaterials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0009] Thirdly, the present invention provides any of the following applications of the nucleic acid molecule or biological materials containing the nucleic acid molecule:

[0010] (1) Increase the yield, production intensity or conversion rate of glutamine in microorganisms;

[0011] (2) Reduce the yield, production intensity or conversion rate of N-acetyl-L-glutamine in microorganisms.

[0012] Fourthly, the present invention provides a recombinant microorganism expressing the aforementioned deacetylase mutant.

[0013] Furthermore, the originating bacteria are species of the genus Corynebacterium, Escherichia, or Bacillus, such as Corynebacterium glutamicum, Escherichia coli, or Bacillus subtilis.

[0014] Fifthly, the present invention provides a method for constructing the recombinant microorganism, wherein the gene encoding the deacetylase mutant is introduced into the starting bacteria via plasmid or integrated into the chromosome of the starting bacteria via genetic engineering.

[0015] In a sixth aspect, the present invention provides the application of the recombinant microorganism in the fermentation production of glutamine.

[0016] In a seventh aspect, the present invention provides a method for increasing glutamic acid fermentation yield and / or decreasing N-acetyl-L-glutamine yield, comprising the step of fermenting and culturing the recombinant microorganism.

[0017] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0018] The mutant strain MHZ-0513-3-cobB constructed in this invention A119D Compared with the control strain MHZ-0513-3-cobB, glutamine production increased from 30.1 g / L to 31.8 g / L, while the byproduct N-acetyl-L-glutamine decreased from 9.4 g / L to 7.5 g / L. This indicates that the point mutation A119D in the deacetylase CobB promotes glutamine accumulation and reduces the byproduct N-acetyl-L-glutamine. When the amino acid at position 119 of the deacetylase containing the point mutation A119D was mutated from alanine (A) to threonine (T), proline (P), valine (V), or serine (S), glutamine production increased in all cases. Specifically, after mutating to valine (V), glutamine production increased to 32.7 g / L, and N-acetyl-L-glutamine accumulation reached 6.8 g / L. Furthermore, this mutation can be applied to other host bacteria such as Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis, for the production of glutamine, effectively reducing the byproduct N-acetyl-L-glutamine. Detailed Implementation

[0019] The present invention aims to provide a strain of Corynebacterium glutamicum that reduces the byproduct N-acetyl-L-glutamine and increases the yield of L-glutamine, as well as its construction method and application.

[0020] This invention provides a strain of *Corynebacterium glutamicum* whose genome integrates the deacetylase encoding gene *cobB* from *Escherichia coli* BL21. During fermentation, an unexpected strain was found to exhibit significantly increased glutamine production and a marked decrease in the byproduct N-acetyl-L-glutamine. Sequencing analysis revealed a point mutation at amino acid position 119 of *cobB*, where GCC is mutated to GAC, i.e., A119D. By introducing A119D into a strain of *cobB* without the point mutation using genetic engineering methods, the resulting recombinant strain exhibited enhanced L-glutamine production compared to the unmodified *Corynebacterium glutamicum*.

[0021] Corynebacterium glutamicum was constructed using Corynebacterium glutamicum MHZ-0513-3, with accession number CGMCC No. 13405, as the starting strain. Corynebacterium glutamicum MHZ-0513-3 has been disclosed in CN106701649A and deposited on November 30, 2016, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 13405.

[0022] The primer names and sequence information involved in the following examples are shown in Table 1.

[0023] Table 1 Primer sequences

[0024]

[0025]

[0026] Unless otherwise specified in the following examples, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified can be obtained through legitimate commercial channels.

[0027] The amino acid sequence of the wild-type deacetylase from *Escherichia coli* BL21 involved in this invention is shown in SEQ ID NO:2, and the nucleotide sequence of the gene cobB is shown in SEQ ID NO:1. The amino acid sequence of the deacetylase mutant containing A119D is shown in SEQ ID NO:4, and its encoding nucleic acid sequence is shown in SEQ ID NO:3.

[0028] Example 1: Construction of plasmid pK18-cobB and construction of recombinant strain MHZ-0513-3-cobB

[0029] The main byproduct of shake-flask fermentation in strain MHZ-0513-3 is N-acetyl-L-glutamine, but its synthesis and degradation mechanisms are currently unclear. It is known that the E. coli deacetylase CobB has no preference for either enzyme-catalyzed or non-enzymatic lysine acetylation substrate sites, but whether it can act on N-acetyl-L-glutamine remains unknown. Therefore, cobB was integrated into the genome of MHZ-0513-3 to construct strain MHZ-0513-3-cobB.

[0030] The specific construction process is as follows:

[0031] Using Phusion superfidelity polymerase (New England BioLabs), recombinant fragment UP was prepared using the genome of Corynebacterium glutamicum MHZ-0513-3 as a template and UP-F / UP-R as primers; recombinant fragment DN was prepared using DN-F / DN-R as primers; recombinant fragment Psod was prepared using the genome of Corynebacterium glutamicum 13032 as a template and Psod-F / Psod-R as primers; and recombinant fragment cobB was prepared using the genome of Escherichia coli BL21 as a template and cobB-F / cobB-R as primers. The obtained fragments were purified using an agarose gel extraction kit (Tiangen). Then, using UP, Psod, cobB, and DN as templates and UP-F / DN-R as primers, recombinant fragments were prepared. These recombinant fragments were purified again using the same kit. The resulting fragments were then digested with XbaI / HindIII, while pK18-mobsacB (Wuhan Miaoling Biotechnology Co., Ltd.) was also digested with XbaI / HindIII. The fragments were then ligated to the vector using T4 DNA ligase (TransGen Biotech). The ligation was performed on Trans1T1 competent cells (TransGen Biotech), and kanamycin-resistant clones were selected. XbaI / HindIII digestion confirmed the insertion of pK18mobsacB into positive clones. Further sequencing using P82 / P85 primers (Invitrogen) confirmed the correct insertion. The resulting plasmid was named pK18-cobB. pK18-cobB was transformed into Corynebacterium glutamicum MHZ-0513-3, and recombinants were selected on 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 ordinary liquid brain heart extract medium at 33°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (10⁻⁶ oz / mL). -2 Continuous dilution to 10 -4The diluted solution was spread onto ordinary solid brain and heart extract culture medium containing 10% sucrose and incubated at 33°C for 48 hours. The selected strains were further subjected to phenotypic verification, PCR identification, and sequencing of the correct mutant strain, which was named MHZ-0513-3-cobB.

[0032] Example 2 Performance of MHZ-0513-3-cobB strain in producing glutamine

[0033] Method for verifying glutamine yield through fermentation: The strain stored in glycerol tubes at -80℃ was inoculated into the following slant culture medium for activation. After culturing at 33℃ for 24 hours, bacterial growth occurred. Bacterial growth was picked from the freshly activated slant and inoculated into the following seed culture medium. The culture was then incubated at 33℃ with shaking at 100 rpm until the mid-to-late logarithmic growth stage, for 5 hours to obtain the seed culture. A 10% inoculum of the seed culture was inoculated into a 500 ml shake flask containing 20 ml of fermentation medium and incubated at 33℃ with shaking at 150 rpm for 48 hours. The results are shown in Table 2.

[0034] The culture medium formula is as follows:

[0035] Slant culture medium: brain heart extract 37g / L, agar 1.8%, sterilized at 121℃ and 0.1MPa for 20min.

[0036] The seed culture medium consisted of: 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, and pH 7.0.

[0037] The fermentation medium was: glucose 90 g / L, (NH4)2SO4 40 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.0 g / L, corn steep liquor 10 g / L, CaCO3 50 g / L, pH 7.0.

[0038] Table 2. Detection of glutamine content in mutant strains.

[0039]

[0040] Note: OD 562 The value indicates the turbidity of the culture medium at 562 nm and the cell count. Gln (g / L) indicates the amount of accumulated L-glutamine.

[0041] As shown in Table 2, heterologous expression of cobB in MHZ-0513-3 increased glutamine production from 28.9 g / L to 30.1 g / L, while the byproduct N-acetyl-L-glutamine decreased from 10.3 g / L to 9.4 g / L. During repeated fermentation, one strain showed a significant increase in glutamine production. A second sequencing of the integrated cobB from this strain revealed a point mutation A199D, i.e., a GCC to GAC mutation. To confirm the effectiveness of this site, a point mutation was performed on strain MHZ-0513-3-cobB to introduce cobB. A119D Construct mutant strain MHZ-0513-3-cobB A119D .

[0042] Example 3: Plasmid pK18-cobB A119D Construction and recombinant strain MHZ-0513-3-cobB A119D Construction

[0043] plasmid pK18-cobB A119D The construction method is described in Example 1, using primers UP1-F / UP1-R and DN1-F / DN1-R. The strain is MHZ-0513-3-cobB. A119D The construction process is the same as above, except that during the screening of secondary recombinants, Kan is selected. S The recombinants were identified using the Identification-F / DN-R method to verify the point mutation recombinants. By adjusting the annealing temperature, recombinants containing point mutations were obtained, followed by PCR identification. The correctly sequenced mutant strain was named MHZ-0513-3-cobB. A119D .

[0044] Example 4 MHZ-0513-3-cobB A119D Performance of strains in producing glutamine

[0045] MHZ-0513-3-cobB A119D The fermentation method for the strain is described in Example 2.

[0046] As shown in Table 2, when cobB undergoes the point mutation A119D, strain MHZ-0513-3-cobB A119D Glutamine production increased from 30.1 g / L to 31.8 g / L, while the byproduct N-acetyl-L-glutamine decreased from 9.4 g / L to 7.5 g / L. The acid production was better than that of MHZ-0513-3-cobB, and the byproduct was less, indicating that the point mutation of cobB is conducive to the accumulation of glutamine and the reduction of byproducts.

[0047] Example 5: Performance of deacetylase at position 119, when mutated to another amino acid, in the production of glutamine.

[0048] Given that the mutation of amino acid 119 from alanine (A) to aspartic acid (D) in the deacetylase resulted in increased glutamine production and reduced byproducts, we then investigated the mutation of amino acid 119 from alanine (A) to threonine (T), proline (P), valine (V), or serine (S). The strain construction method is as described in Example 3, and the fermentation results of the mutant strains are shown in Table 3.

[0049] Table 3. Glutamine content detection in mutant strains

[0050]

[0051] Fermentation results showed that mutating the amino acid at position 119 of the deacetylase from alanine (A) to threonine (T), proline (P), valine (V), or serine (S) increased glutamine production and reduced the byproduct N-acetyl-L-glutamine. Specifically, mutating to valine (V) increased glutamine production to 32.7 g / L and reduced byproducts to 6.8 g / L. Clearly, this mutation can be applied to other host bacteria such as Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis to improve glutamine conversion and reduce byproducts.

[0052] Example 6 ATCC 14067-cobB A119D Construction of mutant strains and their performance in producing glutamine

[0053] Corynebacterium glutamicum ATCC 14067 is a wild-type bacterium. After modification, strain MHZ-0513-3 (glutamine-producing bacterium) was obtained. This was used to confirm the presence of cobB... A119D It has shown effectiveness in different starting strains. The strain ATCC14067-cobB has now been constructed. A119D The strain construction method is the same as MHZ-0513-3-cobB A119D .

[0054] Fermentation results are shown in Table 4:

[0055] Table 4 Glutamine Content Detection

[0056] ATCC 14067 43.6±0.13 0.3±0.011 0.15±0.002 <![CDATA[ATCC 14067-cobB A119D ]]> 44.3±0.09 0.35±0.008 0.09±0.003

[0057] Shake-flask fermentation results showed that the wild-type strain ATCC 14067 introduced the point mutation cobB. A119D This promotes the accumulation of glutamine, indicating that cobB A119D The cobB point mutation promotes the accumulation of glutamine and reduces byproducts.

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A deacetylase mutant, characterized in that, The mutant is a mutant obtained by mutating amino acid A to D at position 119 of the deacetylase. The deacetylase is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the deacetylase mutant of claim 1 or biological material containing said nucleic acid molecule; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

3. Any of the following applications of nucleic acid molecules encoding deacetylase mutants or biological materials containing said nucleic acid molecules: (1) Increase the yield, production intensity or conversion rate of glutamine in microorganisms; (2) Reduce the yield, production intensity or conversion rate of N-acetyl-L-glutamine in microorganisms; The deacetylase mutant is a mutant obtained by mutating the amino acid at position 119 of the deacetylase from A to D, T, P, V, or S; The deacetylase is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO:

2.

4. A recombinant microorganism, characterized in that, The microorganism expresses the deacetylase mutant of claim 1.

5. The recombinant microorganism according to claim 4, characterized in that, The originating bacteria are Corynebacterium spp. ( Corynebacterium ), Escherichia coli ( Escherichia ) or Bacillus spp. ( Bacillus ) bacterial strains.

6. The recombinant microorganism according to claim 5, characterized in that, The originating bacteria is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Escherichia coli ( Escherichia coli ) or Bacillus subtilis ( Bacillus subtilis ).

7. The method for constructing recombinant microorganisms according to any one of claims 4-6, characterized in that, The gene encoding the deacetylase mutant of claim 1 is introduced into the starting bacteria via plasmid or integrated into the chromosome of the starting bacteria via genetic engineering. The originating bacteria are the same as those described in claim 5 or 6.

8. The application of the recombinant microorganisms according to any one of claims 4-6 in the fermentation production of glutamine.

9. A method for increasing glutamine fermentation yield and / or decreasing N-acetyl-L-glutamine yield, characterized in that, It includes the step of fermenting and culturing the recombinant microorganisms according to any one of claims 4-6.

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