Corynebacterium glutamicum and application thereof

Abstract

The invention discloses corynebacterium glutamicum and application thereof. A gene for coding an exogenous saccharifying enzyme is introduced into the corynebacterium glutamicum, and the gene for coding the exogenous saccharifying enzyme is selected from a saccharifying enzyme gene glaA of Aspergillus niger, a saccharifying enzyme gene CauloGA of Caulobacter crenatum or a saccharifying enzyme gene GlucaM of Corallococcus coralloides, and the gene for coding the exogenous saccharifying enzyme is selected from a saccharifying enzyme gene glaA of the Aspergillus niger, a saccharifying enzyme gene CauloGA of the Caulobacter crenatum and a saccharifying enzyme gene GlucaM of the Corallococcus coralloides. When the corynebacterium glutamicum is used for preparing the L-lysine and the derivatives thereof, oligosaccharide residues can be reduced under the condition of not influencing cell growth and product production intensity, the oligosaccharide residues are extremely low, the product separation cost is reduced, the production efficiency of the L-lysine and the derivatives thereof is improved, and the corynebacterium glutamicum has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to Corynebacterium glutamicum and application thereof. Background Art

[0002] L-Lysine is an essential amino acid belonging to the aspartic acid family of amino acids. Depending on the optical rotation, lysine can exist in two forms, L- or D-stereoisomers, but the latter is biologically inactive. L-Lysine is a colorless and odorless flaky crystal, easily soluble in water, slightly soluble in methanol and ethanol, and insoluble in organic solvents such as ether, acetone and chloroform. It is widely used in food, animal feed, medicine, cosmetics and other industries. Due to the strong hygroscopicity of L-lysine, L-lysine on commodities mainly exists in the form of L-lysine hydrochloride or sulfate. The production methods of L-lysine include protein hydrolysis, enzymatic method, chemical method and microbial fermentation method. Since the microbial fermentation method is low in cost, renewable raw materials can be used, there are few highly polluted residues, and the environmental pollution is very small. It is an efficient and environmentally friendly method for producing L-lysine, so this method is more valued and accepted.

[0003] There are two methods for breeding L-lysine, namely traditional breeding and metabolic breeding. Initially, the stable and high-yield amino acid production strains used in industry were obtained through traditional breeding methods, that is, exposing microorganisms to mutagens to induce random DNA damage in microorganisms, and then screening out survivors showing relevant phenotypes to increase the production of L-lysine. However, the efficiency of this technology is unstable and is affected by many factors. Metabolic breeding has gradually become the mainstream, that is, the strains are modified through genetic engineering technology, such as increasing the supply of precursor substances, reducing the generation of by-products, or optimizing energy utilization.

[0004] Wild-type Corynebacterium glutamicum utilizes carbon sources through the phosphoenolpyruvate-sugar transferase system, mainly glucose, followed by fructose, sucrose and maltose, but cannot use oligosaccharides containing α-1,4 glycosidic bonds and α-1,6 glycosidic bonds and starch directly as carbon sources to produce lysine. Research by Karim Kazi Muhammad Rezaul et al. (Protein Expr Purif, 2019, 164: 105462) shows that more than 70% of raw starch is used to produce glucose. The industrial production of glucose from starch as raw material mainly includes two steps, namely liquefaction and saccharification. Liquefaction is the conversion of starch into soluble dextrin and maltose solutions with different degrees of polymerization by amylase, and saccharification is the further hydrolysis of the above solution by saccharifying enzymes to produce glucose. However, this method will obtain a glucose content of about 97% due to incomplete hydrolysis, and there will still be about 3% of oligosaccharides remaining, which will affect the separation of subsequent products.

[0005] Therefore, how to reduce oligosaccharide residues without affecting cell growth and L-lysine production intensity, and thereby improve the production efficiency of L-lysine and its derivatives, is a technical problem that needs to be solved urgently. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the problem of oligosaccharide residues in the production of L-lysine by metabolic breeding in the prior art, and provide a glutamicum corynebacterium and its application. When L-lysine and its derivatives are produced by using the glutamicum corynebacterium of the present invention, the oligosaccharide residues are extremely low, which greatly reduces the separation cost of the product and improves the production efficiency of L-lysine and its derivatives.

[0007] The oligosaccharide described in the present invention is an oligosaccharide composed of 2 to 10 monosaccharide units.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] The first aspect of the present invention provides a Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is introduced with a gene encoding an exogenous saccharifying enzyme, wherein the gene encoding the exogenous saccharifying enzyme is selected from the saccharifying enzyme gene glaA of Aspergillus niger, the saccharifying enzyme gene CauloGA of Caulobacter crescentus, or the saccharifying enzyme gene GlucaM of Corallococcus coralloides. Wherein, the Corynebacterium glutamicum has a reduced amount of oligosaccharide residues when producing L-lysine and its derivatives; the derivative is cadaverine.

[0010] In some specific embodiments of the present invention, the amino acid sequence of the exogenous saccharifying enzyme is shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0011] In the present invention, the GenBank accession numbers corresponding to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 are ADX86749.1, BAO51876.1 and AKM12417.1 respectively.

[0012] In some specific embodiments of the present invention, the Corynebacterium glutamicum is a Corynebacterium glutamicum corresponding to the deposit number CCTCC NO: M20211495. The Corynebacterium glutamicum has been disclosed in CN116410883A and deposited in the China Center for Type Culture Collection (CCTCC) on November 29, 2021, with a deposit address of: Wuhan University, Wuhan, China, zip code 430072, the culture name is CathS141, and the classification name is Corynebacterium glutamicumCathS141.

[0013] In some embodiments of the present invention, the Corynebacterium glutamicum does not reduce or eliminate the expression of the sigH gene.

[0014] In some embodiments of the present invention, the Corynebacterium glutamicum CathS141 does not reduce or eliminate the expression of the sigH gene.

[0015] In some specific embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is selected from the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0016] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is integrated into a recombinant vector. The recombinant vector is introduced by, for example, electroporation.

[0017] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is integrated into the genome of the Corynebacterium glutamicum. In the present invention, when the gene encoding the saccharifying enzyme is integrated into the genome of the Corynebacterium glutamicum by means of secondary homologous recombination, for example, it is achieved by inserting a promoter-saccharifying enzyme gene into the genome.

[0018] In some embodiments of the present invention, the recombinant vector is a recombinant plasmid, and the plasmid backbone is selected from any one of pEC-XK99E, pXMJ19 or pDXW-8 or a combination of several thereof.

[0019] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme has an exogenous promoter upstream, and the exogenous promoter includes an inducible promoter, a constitutive promoter and a tissue-specific promoter.

[0020] In some preferred embodiments of the present invention, the exogenous promoter is a constitutive promoter, and the constitutive promoter is selected from P eftu , P lacM , P cspB or P porB .

[0021] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme contains an RBS upstream.

[0022] In some specific embodiments of the present invention, the RBS nucleotide sequence is as shown in SEQ ID NO: 29 (GAAAGGAGATATACC).

[0023] In some embodiments of the present invention, the recombinant plasmid is selected from any one of the following:

[0024] (1) pEC-XK99E-glucoamylase gene plasmid: connect the glucoamylase gene to the restriction sites BamHI and XbaI of the plasmid vector pEC-XK99E;

[0025] (2) pEC-XK99E-RBS-glucoamylase gene plasmid: the glucoamylase gene and the universal RBS of Corynebacterium glutamicum were connected to the restriction sites BamHI and XbaI of the plasmid vector pEC-XK99E;

[0026] (3) pEC-XK99E-constitutive promoter-saccharifying enzyme gene plasmid: The Ptrc promoter and lactose operon of the plasmid vector pEC-XK99E were removed and replaced with the constitutive promoter Peftu or PlacM.

[0027] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is integrated into the genome of the Corynebacterium glutamicum using a suicide plasmid secondary homologous recombination method. The vector of the suicide plasmid is preferably plasmid pK18mobsacB or pK18mobrpsL.

[0028] In some preferred embodiments of the present invention, the homologous fragment of the saccharifying enzyme gene is first connected to the vector of the suicide plasmid to obtain a recombinant suicide plasmid, and then the recombinant suicide plasmid is electroporated into the competent state of the starting bacteria to complete the first homologous recombination; then the secondary homology is completed by using the sucrose lethality principle, and the suicide plasmid backbone is removed to obtain a recombinant Corynebacterium glutamicum, whose genome is integrated with the gene encoding the saccharifying enzyme.

[0029] For example, the plasmid pK18mobsacB carries the sucrose lethal gene sacB, and the plasmid is electroporated into Corynebacterium glutamicum to complete the primary homology. After that, a secondary homology screening is performed, and the secondary homology is completed using the sucrose lethal principle, thereby obtaining a recombinant strain capable of expressing the target saccharifying enzyme amino acid.

[0030] In some embodiments of the present invention, the Corynebacterium glutamicum overexpresses a gene encoding lysine decarboxylase.

[0031] In some preferred embodiments of the present invention, the lysine decarboxylase is CadA lysine decarboxylase.

[0032] In the present invention, the oligosaccharide residues produced by using the Corynebacterium glutamicum described in the first aspect of the present invention to produce L-lysine and its derivatives are significantly reduced compared with using an unmodified host, with the maximum reduction being 81%.

[0033] The present invention also provides a genetically engineered bacterium, which overexpresses a gene encoding an exogenous saccharifying enzyme, and the amino acid sequence of the exogenous saccharifying enzyme is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; the host bacteria of the genetically engineered bacterium is selected from Escherichia coli, Thermusthermophilus, Hafnia alvei, Bacillus subtilis or Corynebacterium glutamicum.

[0034] In some specific embodiments of the present invention, the Escherichia coli is the Escherichia coli M11-A3 strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2018456, see Chinese invention patent CN110964682B.

[0035] The second aspect of the present invention provides a method for preparing Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is used to produce L-lysine and its derivatives, the method comprising the step of introducing a gene encoding an exogenous saccharifying enzyme or a recombinant vector comprising a gene encoding an exogenous saccharifying enzyme into Corynebacterium glutamicum; the exogenous saccharifying enzyme and Corynebacterium glutamicum are respectively defined as in the Corynebacterium glutamicum described in the first aspect; and the derivative is cadaverine.

[0036] In some preferred embodiments of the present invention, the exogenous saccharifying enzyme is selected from the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0037] In some specific embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is selected from the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0038] In some embodiments of the present invention, the recombinant vector is a recombinant plasmid, and its backbone plasmid is selected from pEC-XK99E, pXMJ19 and pDXW-8.

[0039] In some embodiments of the present invention, the gene encoding the exogenous saccharifying enzyme is integrated into the genome of the Corynebacterium glutamicum.

[0040] In some embodiments of the present invention, the recombinant vector comprises an RBS or a promoter.

[0041] In some specific embodiments of the present invention, the RBS has a nucleotide sequence as shown in SEQ ID NO:29.

[0042] In some preferred embodiments of the present invention, the promoter is a constitutive promoter.

[0043] In some specific embodiments of the present invention, the promoter is P eftu , P lacM , P cspB or P porB .

[0044] The third aspect of the present invention provides a method for preparing L-lysine and its derivatives, comprising: fermenting the Corynebacterium glutamicum described in the first aspect in a fermentation medium to obtain L-lysine and its derivatives from the fermentation broth; the derivative is cadaverine.

[0045] In some embodiments of the present invention, the fermentation medium contains a carbon source, a nitrogen source, an inorganic salt, trace metal elements and / or a surfactant.

[0046] In some embodiments of the present invention, before fermentation culture, the method further comprises the step of inoculating the Corynebacterium glutamicum into a seed culture medium for seed culture.

[0047] In some embodiments of the present invention, the seed culture medium contains a carbon source, a nitrogen source, an inorganic salt, trace metal elements and / or a surfactant.

[0048] In the present invention, the carbon source includes but is not limited to one or more of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, preferably glucose, fructose and sucrose.

[0049] In the present invention, the nitrogen source includes but is not limited to one or more of yeast extract, beef extract, peptone, corn steep liquor, urea, ammonium salt and nitrate.

[0050] In the present invention, the inorganic salt includes but is not limited to one or more of sulfates, hydrochlorides, nitrates and phosphates; preferably includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride and potassium nitrate.

[0051] In the present invention, the trace metal element source includes but is not limited to one or more of sulfates, hydrochlorides and nitrates of potassium, calcium, magnesium, iron, copper, zinc and manganese; preferably includes one or more of zinc sulfate, magnesium sulfate, manganese sulfate, ferrous sulfate, boric acid, ferric chloride and copper sulfate.

[0052] In some embodiments of the present invention, the seed culture medium includes: 20-25 g / L glucose, 10-15 g / L peptone, 0.02-0.04 g / L zinc sulfate heptahydrate, 1-2 g / L dipotassium hydrogen phosphate trihydrate, 1-2 g / L potassium dihydrogen phosphate, 3-5 g / L urea, 0.5-1 g / L magnesium sulfate, and 0.5-1 g / L corn steep liquor.

[0053] In some specific embodiments of the present invention, the seed culture medium includes: 20 g / L glucose, 10 g / L peptone, 0.02 g / L zinc sulfate heptahydrate, 1 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L potassium dihydrogen phosphate, 3 g / L urea, 0.5 g / L magnesium sulfate, and 0.5 g / L corn steep liquor.

[0054] In some embodiments of the present invention, the inoculation amount of the seed culture is 5-15%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%.

[0055] In some embodiments of the present invention, the fermentation medium includes: 10-12 g / L beef extract, 15-18 g / L corn steep liquor, 10-12 g / L yeast powder, 15-18 g / L peptone, 5-8 g / L dipotassium hydrogen phosphate trihydrate, 2.5-5 g / L potassium dihydrogen phosphate, 5-6 g / L urea, 25-30 g / L ammonium sulfate, 0.6-0.8 g / L magnesium sulfate, 5-6 g / L calcium carbonate, and 50-60 g / L glucose.

[0056] In some specific embodiments of the present invention, the fermentation medium includes: 10g / L beef extract, 15g / L corn steep liquor, 10g / L Angel yeast powder, 15g / L peptone, 5g / L dipotassium hydrogen phosphate trihydrate, 2.5g / L potassium dihydrogen phosphate, 5g / L urea, 25g / L ammonium sulfate, 0.6g / L magnesium sulfate, 5g / L light calcium carbonate, and the initial glucose concentration is 50g / L.

[0057] In some embodiments of the present invention, the fermentation culture and / or the seed culture is cultured at a temperature of 28-37°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C and 37°C.

[0058] In some embodiments of the present invention, the fermentation culture and / or seed culture time is 4 hours to 72 hours, for example, 4 hours, 12 hours, 24 hours, 48 ​​hours and 72 hours.

[0059] In some embodiments of the present invention, the seed culture time is 4 hours to 24 hours, for example, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours and 24 hours.

[0060] The fourth aspect of the present invention provides use of the Corynebacterium glutamicum as described in the first aspect of the present invention in the preparation of L-lysine and its derivatives.

[0061] In some embodiments of the invention, the derivative is cadaverine (also known as pentamethylenediamine).

[0062] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0063] The reagents and raw materials used in the present invention are commercially available.

[0064] The positive and progressive effects of the present invention are:

[0065] When the Corynebacterium glutamicum of the present invention is used to prepare L-lysine, oligosaccharide residues are reduced without affecting cell growth and product production intensity (for example, oligosaccharide residues are significantly reduced by up to 81% compared with using an unmodified host), and the oligosaccharide residues are extremely low, which reduces the separation cost of the product and improves the production efficiency of L-lysine and its derivatives. The Corynebacterium glutamicum provided by the present invention can be used as an industrial strain and can also provide theoretical support for further improving the industrial production level of L-lysine and its derivatives, and has good application prospects. DETAILED DESCRIPTION

[0066] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0067] 1. Strains and vectors

[0068] Starting bacteria: Corynebacterium glutamicum CathS141, which has been disclosed in CN116410883A and deposited in the China Center for Type Culture Collection (CCTCC) on November 29, 2021, with a deposit address of: Wuhan University, Wuhan, China, zip code 430072, deposit number: CCTCC NO: M 20211495, culture name is CathS141, classification name is Corynebacterium glutamicum CathS141. Hereinafter referred to as SYB (sigH gene expression is not reduced or eliminated).

[0069] Plasmid vectors pEC-XK99E and pK18mobsacB were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0070] 2. Enzymes and other biochemical reagents

[0071] Restriction endonucleases were used to cut plasmids or gene fragments to produce sticky ends and were purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase was used to amplify gene fragments and was purchased from Takara (Otsu, Japan). Seamless cloning kits were used to connect gene fragments and plasmid vectors containing homologous fragments and were purchased from Hanheng Biotechnology (Nanjing, China). Plasmid extraction kits, PCR product purification and recovery kits, and gel recovery kits were all purchased from Shanghai Jierui Biotechnology (Shanghai, China). Other reagents can be purchased from general biochemical reagent companies.

[0072] 3. Culture medium

[0073] Seed culture medium of recombinant Corynebacterium glutamicum: 20 g / L glucose, 10 g / L peptone, 0.02 g / L zinc sulfate heptahydrate, 1 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L potassium dihydrogen phosphate, 3 g / L urea, 0.5 g / L magnesium sulfate, and 0.5 g / L corn steep liquor.

[0074] Fermentation medium of recombinant Corynebacterium glutamicum: 10g / L beef extract, 15g / L corn steep liquor, 10g / L Angel yeast powder, 15g / L peptone, 5g / L dipotassium hydrogen phosphate trihydrate, 2.5g / L potassium dihydrogen phosphate, 5g / L urea, 25g / L ammonium sulfate, 0.6g / L magnesium sulfate, 5g / L light calcium carbonate, initial glucose concentration of added liquid sugar 50g / L.

[0075] 4. Product detection method: High performance liquid chromatography was used to detect the concentration of L-lysine and oligosaccharides in the fermentation broth.

[0076] Example 1 Construction of an expression plasmid containing the saccharifying enzyme gene glaA (SEQ ID NO: 4)

[0077] Plasmid 1: The saccharifying enzyme gene glaA (its nucleotide sequence is shown in SEQ ID NO: 4) from Aspergillus niger was connected to the restriction sites BamHI and XbaI of the plasmid vector pEC-XK99E. The promoter used was the inducible promoter carried by the plasmid itself, and IPTG was required to induce expression. The codon-optimized glaA was used as a template, primers glaA-F and glaA-R were designed, and the target fragment gene glaA was amplified using a PCR instrument. The plasmid empty vector pEC-XK99E was extracted using a plasmid extraction kit; the extracted plasmid empty vector and the purified target fragment were treated with restriction endonucleases and treated in a metal bath at 37°C for 2h. Then the plasmid: pEC-XK99E-glaA was obtained by chemical transformation of Escherichia coli.

[0078] Plasmid 2: Add a universal RBS of Corynebacterium glutamicum before gene glaA (SEQ ID NO: 29). Using plasmid pEC-XK99E-glaA as a template, design primers glaA-RBS-F and glaA-RBS-R and use a PCR instrument to amplify the target fragment gene glaA carrying RBS, and use a plasmid extraction kit to extract the empty plasmid pEC-XK99E. The extracted empty plasmid vector and the purified target fragment are digested and ligated by enzymes. The specific operation steps are as described above for plasmid 1 to obtain plasmid 2: pEC-XK99E-RBS-glaA.

[0079] Plasmid 3: Using plasmid pEC-XK99E-glaA as template, design primers glaA-P eftu -F and primers glaA-P eftu -R and use PCR instrument to amplify the promoter P eftu The target fragment gene glaA; the laboratory-preserved plasmid pEC-XK99E-P eftu As template, design primer pEC-P eftu -F and primer pEC-P eftu -R and use PCR instrument to amplify the plasmid backbone carrying the excision inducible promoter to obtain plasmid 3: pEC-XK99E-P eftu -glaA.

[0080] Plasmid 4: The construction method is the same as that of plasmid 3, except that the promoter is P lacM , the primer is pEC-P lacM -F, pEC-P lacM -R,glaA-P lacM -F,glaA-P lacM -R.

[0081] The plasmid information and primers are shown in Tables 1 and 2:

[0082] Table 1 Expression plasmids

[0083]

[0084] Table 2 Primers used to construct expression plasmids

[0085]

[0086] Example 2 Construction of a recombinant host bacterium containing a recombinant plasmid

[0087] The empty plasmid pEC-XK99E and the four plasmids constructed in Example 1, pEC-XK99E-glaA, pEC-XK99E-RBS-glaA, pEC-XK99E-P eftu -glaA, pEC-XK99E-P lacM -glaA was electroporated into the starting strain SYB.

[0088] Specific steps:

[0089] (1) First, activate the monoclonal antibody of the starting bacteria, then place it in an ice bath for 30-40 minutes, collect the bacterial solution, wash it 3-4 times with 10% cold glycerol in a refrigerated centrifuge, and collect the bacterial solution to obtain SYB competent state.

[0090] (2) Add the empty plasmid pEC-XK99E, plasmids 1-4 and the SYB competent cells from step (1) into a sterile 1 mm electroporation cuvette and electroporate twice at a high voltage of 1.8 kV on an electroporator;

[0091] (3) Add sterile LBH liquid culture medium to the electroporation cup in step (2) to wash the competent cell and transfer it to a centrifuge tube, heat shock in a 46°C water bath for 6 min, and resuscitate in a 30°C shaker for 2 h; centrifuge and aspirate 50 μL of supernatant, mix the bacteria, spread them on a kanamycin-resistant LBH plate, and culture them in a constant temperature incubator at 30°C. Verify the correct recombinant host bacteria by colony PCR, as shown in Table 3 below.

[0092] Table 3 Information of plasmid recombinant strains constructed in this experiment

[0093] Strain name Strain information SYB-P Empty plasmid pEC-XK99E was introduced into the starting bacteria SYB; inducible expression SYB-A Plasmid pEC-XK99E-glaA was introduced into the starting bacteria SYB; inducible expression SYB-B Plasmid pEC-XK99E-RBS-glaA was introduced into the starting bacteria SYB; inducible expression SYB-C <![CDATA[Plasmid pEC-XK99E-P eftu -glaA was introduced into the starting strain SYB; constitutive expression]]> SYB-D <![CDATA[Plasmid pEC-XK99E-P lacM -glaA was introduced into the starting strain SYB; constitutive expression]]>

[0094] Example 3 Shake flask fermentation results of recombinant host bacteria containing recombinant plasmids

[0095] The starting strain SYB and the recombinant strains SYB-P, SYB-A, SYB-B, SYB-C and SYB-D were cultured overnight on LBG plates, and the monoclonal transformants were picked for liquid culture medium activation, and then transferred to a 500mL Erlenmeyer flask containing 50mL seed culture medium, and cultured at 30°C, 200rpm for 12h. The seed liquid was inoculated into a 500mL Erlenmeyer flask containing 50ml fermentation medium at an inoculum of 10%, and cultured at 30°C, 200rpm for 24h. The concentrations of L-lysine and oligosaccharides in the fermentation broth were detected, and the results are shown in Table 4.

[0096] Table 4

[0097]

[0098] The experimental results showed that ① the starting bacteria SYB and the empty plasmid strain SYB-P had no degradation effect on oligosaccharides, indicating that they could not utilize oligosaccharides themselves. ② After heterologously expressing the saccharifying enzyme gene glaA in the starting bacteria SYB, the growth of the recombinant engineering strains SYB-A, SYB-B, SYB-C and SYB-D was not affected, which was basically the same as the starting bacteria; ③ the degradation rate of oligosaccharides by the recombinant strain SYB-C was the highest (2.1g / L-0.3g / L), reaching 86%, and the other recombinant strains SYB-A, SYB-B and SYB-D also had a certain degradation ability for oligosaccharides, and their degradation rates were 29%, 43% and 62% respectively.

[0099] Example 4 Construction of a genomic integration plasmid containing the saccharifying enzyme gene glaA

[0100] ①Using the genome of the starting bacterium SYB as a template, primers glaA-LF, glaA-LR, glaA-RF and glaA-RR were designed for PCR to obtain the homologous fragment of the gene glaA; ②The plasmid pEC-XK99E-P eftu -glaA was used as a template to design primer pk18-P eftu -glaA-F and pk18-P eftu -glaA-R was used for PCR to obtain the target fragment constitutive promoter P eftu and gene glaA; ③ Use a plasmid extraction kit to extract the suicide plasmid empty vector pK18mobsacB. Use fusion PCR to fuse the target fragment and its homologous fragment, and then perform restriction enzyme ligation on the fused fragment and the extracted plasmid empty vector. The specific restriction enzyme ligation system is shown in Example 1, and the primers used are shown in Table 5 below.

[0101] Table 5 Primers used in constructing gene insertion plasmids in this experiment

[0102]

[0103] Example 5 Obtaining a recombinant engineered bacterium containing the genome-integrated plasmid pK18-mobsacb-glaA

[0104] The suicide plasmid pK18-mobsacb-glaA constructed in Example 4 was electrotransformed into the starting strain SYB. The electrotransformation experiment was performed according to the steps in Example 2 to complete the first homologous recombination of the strain. The strain that completed the first homologous recombination was inoculated into LBG liquid medium (LBGS) containing 20% ​​sucrose to create a sucrose-lethal environment, forcing the strain to start the second homologous recombination. The liquid culture medium was placed in a 30°C shaker for 24 to 48 hours, and then diluted and coated or streaked on LBGS solid medium for monoclonal isolation, cultured for about 48 hours, and the colonies on the plate were selected from solid medium LBG (kanamycin resistance) and LBGS medium, and finally the transformants that did not grow on the LBG plate but grew on the LBGS plate were selected for PCR verification, and the correct transformants were screened. The obtained transformants were named SYB-E.

[0105] Example 6 Shake flask fermentation results of genotype recombinant host bacteria

[0106] The starting strain SYB and the recombinant strain SYB-E were fermented in shake flasks in the same manner as in Example 3. The fermentation results are shown in Table 6 below:

[0107] Table 6 Fermentation results of genotype engineering bacteria

[0108]

[0109] The fermentation results showed that ① the constitutive promoter P eftu ① After the optimized gene glaA was inserted into the genome of Corynebacterium glutamicum, the growth of the recombinant strain SYB-E was not affected; ② After fermentation, the oligosaccharide concentration in the fermentation broth of the recombinant strain SYB-E was significantly decreased compared with the control (2.1g / L-0.4g / L), a decrease of 81%, indicating that heterologous expression of the saccharification enzyme gene glaA in Corynebacterium glutamicum can enable the starting strain to have the ability to utilize oligosaccharides.

[0110] Embodiment 7:

[0111] The genes of saccharifying enzyme CauloGA from Caulobacter crescentus CB15 and saccharifying enzyme GlucaM from Corallococcus sp. strain EGB were codon optimized based on Corynebacterium glutamicum and gene synthesis was performed; the nucleotide sequences of CauloGA gene and GlucaM gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0112] The two genes were overexpressed using plasmid pEC-XK99E based on the promoter P eftu The gene glaA has the best expression ability, so the promoter P is directly selected in the subsequent eftu Replaced into vector pEC-XK99E, and then connected to genes CauloGA and GlucaM respectively. The primer sequences used are shown in Table 7 below:

[0113] Table 7 Primer sequences

[0114] Primer name SEQ ID NO: Primer sequences CauloGA-F 25 gagctcggtacccggggatccATGCGCACCCTCAAGACCTT CauloGA-R 26 tgcctgcaggtcgactctagattaGCGTGCGTAGCGGGCCTT GlucaM-F 27 acccggggatcctaatctagaATGGATCTGTACAGCGCCGC GlucaM-R 28 cttgcatgcctgcaggtcgacttaTTCGATGGATGGCGGTT

[0115] The plasmids pEC-XK99E-Peftu-CauloGA and pEC-XK99E-Peftu-GlucaM were constructed using the primers listed in Table 7. The plasmid construction method was the same as in Example 1. The two plasmids were then electroporated into SYB to obtain strains SYB-F and SYB-G. The strain construction method was the same as in Example 2. The strain details are shown in Table 8 below:

[0116] Table 8 Plasmid recombinant strain information

[0117] Strain name Strain information SYB-F <![CDATA[Plasmid pEC-XK99E-P eftu -CauloGA was introduced into the starting strain SYB; constitutive expression]]> SYB-G <![CDATA[Plasmid pEC-XK99E-P eftu -GlucaM was introduced into the starting strain SYB; constitutive expression]]>

[0118] The starting strain SYB and the recombinant strains SYB-P, SYB-F, and SYB-G were subjected to shake flask fermentation experiments. The fermentation conditions were the same as those in Example 3. The concentrations of L-lysine and oligosaccharides in the fermentation broth were detected. The results are shown in Table 9.

[0119] Table 9 Plasmid strain fermentation results

[0120]

[0121] The experimental results showed that ① after heterologously expressing the saccharification enzyme genes CauloGA and GlucaM from Caulobacter crescentus and Coral Globodera in the starting bacteria SYB, the growth of the recombinant strains SYB-F and SYB-G was not affected, which was basically consistent with the starting bacteria; ② the recombinant strain SYB-F had the highest degradation rate of oligosaccharides (2.2g / L-0.4g / L), reaching 82%. The oligosaccharide degradation ability of this strain in the form of plasmid was similar to that of strain SYB-C, so the oligosaccharide degradation ability of the two genotypes in the form of genome insertion was compared.

[0122] A genomic integration plasmid containing the saccharifying enzyme gene CauloGA of Caulobacter crescentus was constructed. The method for constructing the genomic integration plasmid was the same as that in Example 4. The primers used were shown in Table 10 below.

[0123] Table 10 Primers used for gene insertion plasmid

[0124]

[0125] The constructed suicide plasmid pK18-mobsacb-CauloGA was electroporated into the starting strain SYB, and the recombinant strain SYB-H containing the genome-integrated plasmid pK18-mobsacb-CauloGA was obtained using the experimental steps of Example 5. The starting strain SYB, the recombinant strains SYB-E and SYB-H were fermented in shake flasks, and the fermentation method was the same as that of Example 3. The fermentation results are shown in Table 11 below:

[0126] Table 11 Genotype engineering bacteria fermentation results

[0127]

[0128] The fermentation results showed that ① the growth of recombinant strains SYB-E and SYB-H was not affected; ② after fermentation, the oligosaccharide concentration in the fermentation broth of the recombinant strain SYB-H was significantly reduced compared with the control (2.1g / L-0.3g / L), a decrease of 86%; the oligosaccharide degradation of strain SYB-E was 81%, indicating that strain SYB-H has a stronger ability to utilize oligosaccharides. ③ From the results, it can be seen that although the oligosaccharide degradation ability of the plasmid bacteria containing glaA is better than that containing CauloGA, the oligosaccharide ability of the bacteria containing the CauloGA gene from Caulocolumbacillus is better, presumably because the bacterial saccharification enzyme is more conducive to expression in the genome of Corynebacterium glutamicum.

[0129] The sequences of the exogenous saccharifying enzymes in the examples of the present invention are shown in Table 12.

[0130] Table 12 Exogenous saccharifying enzyme sequences

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A Corynebacterium glutamicum, characterized in that The gene encoding exogenous saccharifying enzyme is introduced into the Corynebacterium glutamicum, and the gene encoding exogenous saccharifying enzyme is selected from the saccharifying enzyme gene glaA of Aspergillus niger, the saccharifying enzyme gene CauloGA of Caulobacter crescentus or the saccharifying enzyme gene GlucaM of Corallococcus coralloides.

2. A Corynebacterium glutamicum, characterized in that The gene encoding exogenous saccharifying enzyme is introduced into the Corynebacterium glutamicum, and the amino acid sequence of the exogenous saccharifying enzyme is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

3. The Corynebacterium glutamicum according to claim 1 or 2, characterized in that The gene encoding the exogenous saccharifying enzyme is selected from the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

4. The Corynebacterium glutamicum according to claim 1 or 2, characterized in that The gene encoding the exogenous saccharifying enzyme is integrated into the recombinant vector; And / or, the gene encoding the exogenous saccharifying enzyme is integrated into the genome of the Corynebacterium glutamicum; Preferably, the recombinant vector is a recombinant plasmid, and the plasmid backbone is selected from any one of pEC-XK99E, pXMJ19 or pDXW-8 or a combination of several thereof.

5. The Corynebacterium glutamicum according to any one of claims 1 to 4, characterized in that The gene encoding the exogenous saccharifying enzyme has an exogenous promoter upstream, and the exogenous promoter includes an inducible promoter, a constitutive promoter and a tissue-specific promoter; preferably, the exogenous promoter is a constitutive promoter, and the constitutive promoter is selected from P eftu , P lacM , P cspB or P porB ; And / or, the gene encoding the exogenous saccharifying enzyme contains an RBS upstream, preferably an RBS having a nucleotide sequence as shown in SEQ ID NO:

29.

6. The Corynebacterium glutamicum according to any one of claims 1 to 4, characterized in that The Corynebacterium glutamicum overexpresses a gene encoding lysine decarboxylase; Preferably, the lysine decarboxylase is CadA lysine decarboxylase.

7. A method for preparing Corynebacterium glutamicum for producing L-lysine, characterized in that: The method comprises the step of introducing a gene encoding an exogenous saccharifying enzyme or a recombinant vector comprising a gene encoding an exogenous saccharifying enzyme into Corynebacterium glutamicum; the exogenous saccharifying enzyme is defined in the Corynebacterium glutamicum described in any one of claims 1 to 6.

8. A method for preparing L-lysine and its derivatives, characterized in that: include: The Corynebacterium glutamicum according to any one of claims 1 to 6 is fermented in a fermentation medium to obtain L-lysine and its derivatives from the fermentation broth; the derivative is cadaverine.

9. The method according to claim 8, characterized in that The temperature of the fermentation culture and / or the seed culture is 28-37° C.; and / or the time of the fermentation culture is 4 h-72 h.

10. Use of the Corynebacterium glutamicum according to any one of claims 1 to 6 in the preparation of L-lysine and its derivatives; the derivative is cadaverine.

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