Bacillus methylophilus for producing L-arginine as well as construction method and application of bacillus methylophilus

Abstract

The invention belongs to the field of gene engineering, and discloses bacillus methylophilus for producing L-arginine as well as a construction method and application of the bacillus methylophilus. According to the recombinant strain, bacillus methanolicus is taken as an original strain, and a repressor protein ArgR is not expressed; the amino acid sequence of the repressor protein ArgR is as shown in SEQ ID NO. 1. The recombinant strain constructed by the invention can grow by taking methanol as a unique carbon source, L-arginine is synthesized, and compared with a traditional method for producing L-arginine by fermentation, the recombinant strain has unique metabolic advantages, and the production cost can be remarkably reduced. The invention provides a brand-new and environment-friendly way for industrial fermentation production of L-arginine, and has wide application prospect and important economic value.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and discloses a methanolic bacillus producing L-arginine and a construction method and application thereof. Background Art

[0002] L-Arginine (L-Arginine, L-Arg) is also known as 2-amino-5-guanidine valeric acid or α-amino-σ-guanidine valeric acid, with a chemical formula of C 6 H 14 N 4 O 2 Its appearance is white rhombus crystal or monoclinic crystal, which is easily soluble in water, slightly soluble in ethanol, and insoluble in ether. L-arginine is widely used in medicine, feed, food, cosmetics and other industries.

[0003] In recent years, microbial fermentation has become the mainstream method for producing L-arginine. Compared with other production methods, microbial fermentation has the advantages of simple production process, mild reaction conditions, and environmental friendliness. At present, the chassis for microbial fermentation to produce L-arginine mainly includes Escherichia coli, Corynebacterium glutamicum, and Corynebacterium blunt-toothed, all of which use glucose as raw material for fermentation production. This method has the risk of "competing with people for food and competing with food for land." Summary of the invention

[0004] In view of these defects, the present invention provides a methanolic Bacillus producing L-arginine and its construction method and application. Bacillus methanolicus ) was used as the starting strain. Through genetic engineering, the feedback inhibition of the repressor protein ArgR on genes related to the L-arginine biosynthesis pathway was effectively released, and a recombinant strain that can synthesize L-arginine using methanol as the sole carbon source was constructed, providing a new way for the production of L-arginine.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a recombinant strain of Bacillus methanolicus producing L-arginine, wherein the recombinant strain is based on Bacillus methanolicus ( Bacillus methanolicus ) is the starting strain, which does not express the repressor protein ArgR; the amino acid sequence of the repressor protein ArgR is shown in SEQ ID NO.1.

[0006] Methanol, as a non-grain carbon raw material, has the advantages of abundant sources, low price and high energy density, and has gradually become the preferred raw material for biomanufacturing. Methanol Bacillus is a Gram-positive, thermophilic, facultative natural methylotrophic bacterium that assimilates methanol through NAD-dependent methanol dehydrogenase and ribulose monophosphate cycle. It can grow rapidly and efficiently synthesize the precursor L-glutamate at 50°C using methanol as the only carbon source, thus having the potential to produce L-arginine.

[0007] The present invention uses methanolic Bacillus as the starting strain, and effectively relieves the repressor protein ArgR from affecting the L-arginine synthesis operon by not expressing the repressor protein ArgR. argCJBDFR and argGH The transcriptional feedback inhibition of methanolic Bacillus enables the synthesis of L-arginine, thereby constructing a recombinant strain that synthesizes L-arginine using methanol as the sole carbon source.

[0008] The "non-expression" in the present invention means that the amount of the protein is significantly lower than the original level, for example, significantly reduced by at least 50%, 60%, 70%, 80%, 90%, 100%. Gene editing on the starting strain to make the protein non-expressed can be achieved by conventional means in the art, including but not limited to gene knockout, gene knock-in, point mutation, deletion mutation and their combination.

[0009] Preferably, the recombinant strain is based on Bacillus methanolicus and the repressor protein gene is knocked out in its genome. argR , the repressor gene argR The nucleotide sequence is shown in SEQ ID NO.2.

[0010] Preferably, the recombinant strain also overexpresses acetylglutamate kinase.

[0011] Acetylglutamate kinase is a key enzyme in the arginine biosynthetic pathway, which can catalyze the phosphorylation of N-acetylglutamate to generate N-acetylglutamate-5-phosphate. Based on the methanolic Bacillus as the starting strain, acetylglutamate kinase is overexpressed on the basis of not expressing the repressor protein ArgR, the L-arginine synthesis pathway is strengthened, and the ability of the recombinant strain to produce L-arginine is improved. The strain constructed by the present invention that does not express the repressor protein ArgR and expresses acetylglutamate kinase can produce 600-700 mg / L of L-arginine after 24 h of shake flask culture.

[0012] The "overexpression" mentioned in the present invention means that the amount of the gene expression product is significantly higher than the original level. The method of overexpressing the gene can adopt conventional means in the art, including but not limited to vector-mediated gene overexpression, inserting the corresponding gene into the genome to increase the gene copy number or replacing the promoter of the corresponding gene and other editing methods.

[0013] More preferably, the recombinant strain contains a gene that can overexpress acetylglutamate kinase argB Plasmid; the acetylglutamate kinase gene argB The nucleotide sequence is shown in SEQ ID NO.3.

[0014] When selecting a promoter, a reasonable selection can be made according to the specific expression level requirements and the characteristics of the target gene. More preferably, the acetylglutamate kinase gene argB The promoter used for overexpression was P mdh The promoter, whose nucleotide sequence is shown in SEQ ID NO.4.

[0015] More preferably, the plasmid is pNW33NC.

[0016] P mdh The promoter exhibits high promoter activity in a variety of microorganisms and can efficiently drive gene expression.

[0017] Preferably, the starting strain is Bacillus methanolicus MGA3.

[0018] Methanol Bacillus MGA3 can grow rapidly using methanol at 50-55°C and can adapt to low-cost seawater matrix. The recombinant strain constructed using it as a chassis strain can better adapt to industrial production.

[0019] In a second aspect, the present invention also provides a method for constructing a recombinant strain of Bacillus methanolicus that produces L-arginine, wherein the recombinant strain is gene-edited using Bacillus methanolicus as a starting strain; the gene editing comprises knocking out a repressor protein gene in its genome. argR , the repressor gene argR The nucleotide sequence is shown in SEQ ID NO.2.

[0020] Preferably, the knockout repressor gene argR Using CRISPR Cas9 gene editing method.

[0021] Preferably, the gene editing further comprises overexpressing the acetylglutamate kinase gene argB , the acetylglutamate kinase gene argB The nucleotide sequence is shown in SEQ ID NO.3.

[0022] Preferably, the starting strain is Bacillus methanolicus MGA3.

[0023] The present invention utilizes gene editing technology to perform overexpression and knockout operations on the starting strain of the target gene. The order of gene editing has no priority and can be flexibly adjusted according to experimental requirements. Different editing orders can achieve the expected genetic engineering goals without affecting the performance of the finally constructed genetically engineered bacteria and the production capacity of L-arginine.

[0024] More preferably, overexpression of acetylglutamate kinase gene argB Plasmid vector-mediated gene overexpression was used.

[0025] Specifically, the present invention also provides a method for constructing a recombinant strain of Bacillus methanolicus that produces L-arginine, comprising the following steps: (1) Using the MGA3 genome of Bacillus methanolicus as a template, amplify the repressor protein gene argR The upstream homology arm and the downstream homology arm of argR The upstream homology arm, downstream homology arm and argR -gRNA gene fragment is connected to the vector carrying Cas9 protein to obtain argR Gene knockout vector; argR The gene knockout vector was transformed into methanolic Bacillus MGA3 to obtain argR MGA3 knockout strain ∆argR ; (2) Based on the strain genome or acetylglutamate kinase gene argB The gene sequence was used as a template to amplify argB gene fragments; using restriction enzymes and ligases, argB The gene fragment was inserted into the plasmid vector to obtain argB The recombinant vector of the gene fragment is transformed into the MGA3 obtained in step (1). ∆argR A recombinant strain of Bacillus methanolicus producing L-arginine was obtained.

[0026] The present invention " argR -gRNA" indicates that argR The guide RNA (gRNA) designed and synthesized by the targeting sequence of the gene can be in the form of a single guide RNA (sgRNA), which is used to guide the Cas9 nuclease to recognize and bind to the target DNA sequence, thereby achieving gene editing.

[0027] In a third aspect, the present invention also provides applications of the above-mentioned methanolic Bacillus recombinant strain producing L-arginine, including: application in constructing a recombinant strain producing L-arginine; application in producing L-arginine by fermentation.

[0028] In a fourth aspect, the present invention also provides a method for producing L-arginine by fermentation, using the above-mentioned methanolic Bacillus subtilis recombinant strain producing L-arginine or the methanolic Bacillus subtilis recombinant strain producing L-arginine obtained according to the above-mentioned construction method to ferment with methanol or a culture medium containing methanol as a substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 pNW33N-U in Example 1 argR- D argR- Gel electrophoresis results of sgRNA plasmid construction; (M1: Trans 8K DNA Marker; M2: Trans 5K DNA Marker; 1: pNW33N backbone fragment; 2: upstream homology arm; 3: downstream homology arm; 4: argR -sgRNA gene fragment) Figure 2 The results of ARG01 strain screening in Example 1; (1: colony PCR verification using MGA3 wild type as template; 2: colony PCR verification using a single colony on a plate as template) Figure 3 pNW33NC-P in Example 2 mdh -gfp Gene arrangement map of plasmid; Figure 4 pNW33NC-P in Example 2 mdh - argB Gel electrophoresis results of plasmid construction; (M: Trans 5K DNA Marker; 1: pNW33NC backbone fragment; 2: argB fragment) Figure 5 The results of strain screening of ARG02 in Example 2; (1: Colony PCR verification using a single colony on a plate as a template) Figure 6 The concentration of wild-type methanolic Bacillus and the recombinant strain in the shake flask fermentation process in Example 3; Figure 7 The residual amount of methanol during the shake flask fermentation of the wild-type methanolic Bacillus and the recombinant strain in Example 3; Figure 8 The results of shake flask fermentation of wild-type methanolic Bacillus and the recombinant strain in Example 3 show the production of L-arginine. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The methanolic Bacillus used in the embodiments of the present invention ( Bacillus methanolicus ) MGA3 was purchased from German Microbial Culture Collection DSMZ.

[0033] pNW33NC-P used in the examples of the present invention mdh -gfp The plasmid was obtained by modifying the original pNW33N plasmid. A TC point mutation was introduced into the replication origin (ori) region of the pNW33N plasmid to obtain a low-copy number pNW33NC plasmid. mdh Promoter and its corresponding ribosome binding site (RBS) and green fluorescent protein gene gfp Integration was performed to construct pNW33NC-P mdh - gfp Plasmid.

[0034] The pNW33N-TCas9-sgRNA plasmid used in the embodiment of the present invention is obtained by modifying the original pNW33N plasmid, and the plasmid contains the coding gene of the Cas9 protein and the coding sequence of the sgRNA. The plasmid and its construction method have been disclosed in the patent application number CN2024103425806.

[0035] SOBsuc medium components: tryptone 20 g / L, yeast extract 5 g / L, sodium chloride 0.5 g / L, potassium chloride 0.2 g / L, magnesium chloride 1 g / L, magnesium sulfate heptahydrate 2.5 g / L, sucrose 85.5 g / L.

[0036] The detection method involved in the embodiments of the present invention is: Determination of bacterial concentration: After diluting the fermentation broth appropriately with distilled water, measure the OD with a UV spectrophotometer. 600 , through OD 600 Reflects the concentration of bacteria in the fermentation broth.

[0037] Determination of L-arginine content: After centrifuging the fermentation broth, retain the supernatant and dilute it to an appropriate multiple, then derivatize it with 2-4-dinitrofluorobenzene and detect the L-arginine content by HPLC.

[0038] Determination of methanol content: After centrifuging the fermentation broth, retain the supernatant and dilute it to an appropriate multiple, and then determine its concentration using a biosensor.

[0039] If not otherwise specified, the technical means used in the embodiments are conventional means known to those skilled in the art. The test methods in the following embodiments are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained commercially. The test materials used in the present invention are all common commercial products and can be purchased on the market. The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information of the gene or amplified using the microbial genome of the corresponding source as a template. The primers used in the embodiments of the present invention are shown in Table 1.

[0040] Table 1 Primer sequences used in the examples of the present invention

[0041] Example 1 This embodiment provides a genetically engineered bacterium for producing L-arginine using methanol, and its construction method comprises the following steps: (1) Using the genome of Bacillus methanolicus MGA3 as a template, primers argR-up-F / argR-up-R and argR-down-F / argR-down-R were used to amplify argR The upstream homology arm and downstream homology arm of the gene were amplified using the pNW33N-TCas9-sgRNA plasmid as a template and primers argR-sgRNA-F / 1212NTR. argR -sgRNA gene fragment. The PCR amplification system and conditions are shown in Table 2 below, and the obtained PCR amplification products were stored at 12°C.

[0042] Table 2 PCR amplification system and conditions

[0043] Amplification program: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, reaction for 30 cycles; extension at 72°C for 7 min.

[0044] (2) After the above PCR products were purified and recovered, they were ligated into the pNW33N plasmid backbone carrying the Cas9 protein to obtain pNW33N-U argR- D argR- sgRNA plasmid (verification results see Figure 1 ). The PCR product and vector connection system is shown in Table 3 below.

[0045] Table 3 PCR product and vector connection system

[0046] The linearized vector and the inserted fragment were mixed in proportion and reacted at 50°C for 20 min under the catalysis of ligase to obtain the ligation product. The ligation product was then chemically transformed into Escherichia coli Trans1-T1 competent cells. After a large number of cell proliferation, a high concentration of the recombinant plasmid pNW33N-U was extracted. argR- D argR- sgRNA and stored in a 4°C refrigerator.

[0047] (3) Recombinant plasmid pNW33N-U argR- D argR- sgRNA was electrotransformed into methanolic Bacillus MGA3 competent cells containing pREDcas9 plasmid, and after recovery, it was spread on SOBsuc plates containing 5 μg / mL chloramphenicol and 8 g / L methanol and inverted at 50°C for culture. After a single clone is grown, the single colony needs to be lysed with 20 μM NaOH. Subsequently, the wild-type strain MGA3 was used as a control, and the single colony on the plate was verified by PCR using primers argR-F (external) / argR-R (external). The colony PCR amplification system and conditions are shown in Table 4 below. The recombinant strains were screened by gel electrophoresis experiments, and the strains with reduced PCR product size were the correct knockout strains (verification results are shown in Figure 2 ), to obtain knockout argR The recombinant strain ARG01.

[0048] Table 4 Colony PCR amplification system and conditions

[0049] “*”: Use a pipette tip to pick up a single colony and add it directly into the prepared 20 μL PCR reaction system.

[0050] Amplification program: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, reaction for 30 cycles; extension at 72°C for 7 min.

[0051] Example 2 This embodiment provides a genetically engineered bacterium for producing L-arginine using methanol, and its construction method comprises the following steps: (1) Using the genome of Bacillus methanolicus MGA3 as a template, primers argB-F / argB-R were used to amplify argB Gene fragments, PCR amplification system and conditions are shown in Table 2; then XhoI, KpnI pNW33NC-P mdh - gfpPlasmid, 37℃, 1 h, to obtain the plasmid backbone. The enzyme digestion system is shown in Table 5.

[0052] Table 5 Enzyme digestion system

[0053] (2) Connection argB Gene fragments and plasmid backbones, according to the connection system in Table 6, mixed plasmid backbones and argB The gene fragment was reacted at 50℃ for 15 min under the catalysis of ligase to obtain the recombinant plasmid pNW33NC-P mdh - argB Plasmid (verification results see Figure 4 ) and stored at 4°C.

[0054] Table 6 Connection system

[0055] (3) pNW33NC-P mdh - argB The recombinant plasmid was electrotransformed into ARG01 (provided in Example 1) competent cells, and after recovery, it was spread on SOBsuc solid medium containing 5 μg / mL chloramphenicol and 8 g / L methanol, and inverted at 50°C. After a single clone is grown, the single colony needs to be lysed with 20 μM NaOH, and the single colony on the plate is verified by PCR with primers Cm-F / Pmdh-R. The colony PCR amplification system and conditions are shown in Table 4 below. The recombinant strain was verified and selected by gel electrophoresis experiment (the verification results are shown in Figure 5 ), obtained overexpression argB The recombinant strain ARG02.

[0056] Example 3 Shake flask fermentation The shake flask fed-batch fermentation process of wild-type strains of methanolic Bacillus MGA3, ARG01 and ARG02 was compared by shake flask fed-batch fermentation, and each strain was repeated three times. Starting from 12 h of fermentation, samples were taken every 3 h, and the cell concentration, L-arginine content, and residual methanol were determined.

[0057] The specific scheme of shake flask fermentation is as follows: (1) Seed culture: The strain stored at -80℃ was inoculated into the seed culture medium at a 1% inoculation rate and cultured in a constant temperature shaking incubator at 50℃ and 220 rpm for 12-18 h. 600 When the initial OD reaches 6-7, the activated bacterial solution is obtained; 600 The activated bacterial solution was inoculated into the seed culture medium with an inoculation volume of 0.1, and then placed in a constant temperature shaking incubator at 50°C and 220 rpm for 12 h.600 When the pH reaches 6-7, the seed solution is obtained. Seed culture medium nutrient components (g / L): tryptone 15, soy peptone 5, sodium chloride 5; adjust the pH to 7.0, sterilize at 121℃ for 20 min, and add 8 g / L of anhydrous methanol before inoculation.

[0058] (2) Shake flask fed-batch fermentation: The seed solution prepared above was fermented according to the initial OD 600 The inoculation volume was 0.1, inoculated into a 500 mL baffled shake flask containing 50 mL fermentation medium, and placed in a constant temperature shaking incubator at 50°C and 220 rpm for 24 h. When the fermentation lasted for 18 h, anhydrous methanol was added to a final concentration of 8 g / L, and the pH was adjusted to 6-7 every 3 h after 12 h of fermentation. Fermentation medium nutrient components (g / L): dipotassium hydrogen peroxide 4.09, sodium dihydrogen phosphate monohydrate 1.49, ammonium sulfate 2.11; adjust the pH to 7.2, sterilize at 121°C for 20 min; before inoculation, add 1 mL 1 M magnesium sulfate heptahydrate, 1 mL trace elements, 1 mL vitamin solution, and 8 g anhydrous methanol per liter of fermentation medium. Trace element components ( / L): 5.56 g of ferrous sulfate heptahydrate, 27.28 mg of copper sulfate dihydrate, 7.35 g of calcium chloride dihydrate, 40.5 mg of phosgene hexahydrate, 9.9 g of manganese chloride tetrahydrate, 287.54 mg of zinc sulfate heptahydrate, 48.4 mg of sodium molybdate dihydrate, 30.92 mg of boric acid, 80 mL of hydrogen chloride. Vitamin solution components ( / L): 0.1 g of biotin, 0.1 g of thiamine hydrochloride, 0.1 g of riboflavin, 0.1 g of D-calcium pantothenate, 0.1 g of pyridoxine hydrochloride, 0.1 g of nicotinamide, 0.02 g of p-aminobenzoic acid, 0.01 g of folic acid, V B12 0.01 g, lipoic acid 0.01 g.

[0059] like Figures 6 - 8 As shown in the figure, the three strains showed good growth trends during the fermentation process, especially ARG01 and ARG02 strains, whose bacterial concentrations continued to increase after 20 hours, and they showed strong methanol utilization ability. The methanol consumption rate of ARG02 strain was the fastest. ARG01 strain used methanol as the only carbon source and could produce about 100 mg / L of L-arginine in 24 hours of shake flask fermentation; while the wild-type strain methanolic Bacillus MGA3 did not produce L-arginine during the entire fermentation process; ARG02 strain could produce 600-700 mg / L of L-arginine in 24 hours of shake flask fermentation, and the L-arginine production was increased by 6-7 times compared with ARG01.

[0060] In summary, the present invention uses methanolic Bacillus as a starting strain, relieves the feedback inhibition of repressor proteins on genes related to the L-arginine synthesis pathway, and simultaneously strengthens the L-arginine synthesis pathway to construct a recombinant strain that synthesizes L-arginine using methanol as the sole carbon source.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant strain of Bacillus methanolicus producing L-arginine, characterized in that: The recombinant strain is based on Bacillus methanolicus ( Bacillus methanolicus ) is the starting strain, which does not express the repressor protein ArgR; the amino acid sequence of the repressor protein ArgR is shown in SEQ ID NO.

1.

2. The recombinant strain of Bacillus methanolica according to claim 1, characterized in that The recombinant strain uses methanolic Bacillus as the starting strain, and the repressor protein gene is knocked out in its genome argR , the repressor gene argR The nucleotide sequence is shown in SEQ ID NO.2; and / or The recombinant strain also overexpresses acetylglutamate kinase.

3. The recombinant strain of Bacillus methanolicus according to claim 2, characterized in that The recombinant strain contains a gene capable of overexpressing acetylglutamate kinase argB Plasmid; the acetylglutamate kinase gene argB The nucleotide sequence is shown in SEQ ID NO.

3.

4. The recombinant strain of Bacillus methanolica according to claim 3, characterized in that Acetylglutamate kinase gene argB The promoter used for overexpression was P mdh A promoter, the nucleotide sequence of which is shown in SEQ ID NO.4; and / or The plasmid is pNW33NC.

5. The recombinant strain of Bacillus methanolica according to any one of claims 1 to 4, characterized in that The starting strain is methanolic Bacillus MGA3.

6. A method for constructing a recombinant strain of Bacillus methanolicus producing L-arginine, characterized in that: The recombinant strain is gene-edited with methanolic Bacillus as the starting strain; the gene editing includes knocking out the repressor protein gene on its genome argR , the repressor gene argR The nucleotide sequence is shown in SEQ ID NO.

2.

7. The method for constructing a recombinant strain of Bacillus methanolica according to claim 6, characterized in that: Knockout of the repressor gene argR Using CRISPR Cas9 gene editing method.

8. The method for constructing a recombinant strain of Bacillus methanolica according to claim 6, characterized in that: The gene editing also includes overexpression of acetylglutamate kinase gene argB , the acetylglutamate kinase gene argB The nucleotide sequence is shown in SEQ ID NO.3; and / or The starting strain is methanolic Bacillus MGA3.

9. Use of the recombinant Bacillus methanolicus strain producing L-arginine according to any one of claims 1 to 5, characterized in that: include: Application in constructing a recombinant strain producing L-arginine; Application in the fermentation production of L-arginine.

10. A method for producing L-arginine by fermentation, using the recombinant strain of Bacillus methanolica producing L-arginine according to any one of claims 1 to 5 or the recombinant strain of Bacillus methanolica producing L-arginine obtained by the construction method of any one of claims 6 to 8, and fermenting with methanol or a medium containing methanol as a substrate.

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