CN102191289B - Fermentation preparation method of lysine - Google Patents

Abstract

The present invention provides a fermentation method of L-lysine, which includes introducing a polynucleotide encoding a dihydrodipicolinate synthase variant into an L-lysine-producing bacterium, so that the obtained bacterium expresses the a dihydrodipicolinate synthase variant; and a bacterium obtained by culturing under fermentation conditions. In addition, the present invention also provides intermediate products used in the fermentation method, and further products produced by the fermentation method.

Description

Fermentation of lysine

technical field

The invention belongs to the field of amino acid fermentation, in particular, the invention relates to a fermentation method of L-lysine, which comprises introducing a polynucleotide encoding a dihydrodipicolinate synthase variant into an L-lysine-producing bacterium , so that the obtained bacteria express the dihydrodipicolinate synthetase variant; and culturing the obtained bacteria under fermentation conditions. In addition, the present invention also provides intermediate products used in the fermentation method, and further products produced by the fermentation method.

Background technique

L-lysine is an important amino acid raw material, which can be used as condiment, food, feed additive, and also as an effective or auxiliary ingredient in health care products and medicines. It is widely used in food industry, feed industry, pharmaceutical industry and other chemical industries. in industry. At present, the production of L-lysine is mainly produced by fermentation of microorganisms, such as coryneform bacteria.

The microorganisms used for fermentation production can be wild-type microorganisms, but most of them are auxotrophs, drug-resistant mutants and metabolic mutants with higher yields obtained through mutagenesis or genetic engineering. For the microorganisms with improved traits obtained by genetic engineering, the most important thing is the genes with excellent properties.

Dihydrodipicolinate synthase variants are important enzymes in the L-lysine metabolic pathway. Although wild-type dipicolinate synthase and some variants thereof have been published (see NCBI (http://www.ncbi.nlm.nih.gov) protein and gene accession number AAC75531.1; also available See Chinese patent ZL94194962), but the study of other variants of the enzyme has not been reported, nor has it been suggested to mutate at a new site.

After long-term arduous research, the inventor unexpectedly discovered a new dihydrodipicolinate synthetase, which also obtained higher lysine yield when introduced into engineering bacteria for fermentation.

Contents of the invention

The technical problem to be solved by the present invention is to provide a fermentation method for L-lysine, which includes introducing a polynucleotide encoding a dihydrodipicolinate synthetase variant into an L-lysine-producing bacterium, so that the obtained bacteria expressing the dihydrodipicolinate synthase variant. In addition, the present invention also provides the intermediate product used in the fermentation method, and the further production of products using the fermentation method, etc.

Specifically, in the first aspect, the present invention provides a fermentation method of L-lysine, which includes:

(1) introducing a polynucleotide encoding a dihydrodipicolinate synthase variant into an L-lysine-producing bacterium, so that the obtained bacterium expresses the dihydrodipicolinate synthase variant, wherein The dihydrodipicolinate synthase variant is replaced by another natural amino acid at the M13, V60 or A83 position of the wild-type dihydrodipicolinate synthase; and

(2) Cultivate the bacteria obtained in step (1) under fermentation conditions.

Wherein, the wild-type dihydrodipicolinate synthase is known to those skilled in the art, and its sequence is shown in NCBI (http://www.ncbi.nlm.nih.gov) protein and gene accession number AAC75531.1 .

Preferably, in the fermentation method of the first aspect of the present invention, the polynucleotide encodes a dihydrodipicolinate synthase variant, as shown in SEQ ID No: 2 in its nucleotide sequence.

Preferably, in the fermentation method of the first aspect of the present invention, the dihydrodipicolinate synthase variant is replaced by other natural amino acids at the positions of M13, V60 and A83 of the wild-type dihydrodipicolinate synthase, preferably The replacement is selected from M13K, V60L and A83G. In a specific embodiment of the present invention, the amino acid sequence of the dihydrodipicolinate synthase variant is shown in SEQ ID No: 1.

Those skilled in the art can deduce its encoding nucleotide sequence according to the amino acid sequence of the dihydrodipicolinate synthase variant, preferably a codon-optimized nucleotide sequence, such as optimizing the codon usage of bacteria used for fermentation of. In a specific embodiment of the present invention, the dihydrodipicolinate synthetase variant is encoded by a polynucleotide as shown in SEQ ID No:2.

The polynucleotide can be introduced into the L-lysine-producing bacteria by various methods well known to those skilled in the art, as long as the L-lysine-producing bacteria can express the pyridine nucleotide transhydrogenase Just body. The polynucleotide can be introduced directly, for example, into cells by microsomes, gene guns, etc.; it can also be introduced indirectly, for example, it can be introduced into cells by constructing it on a plasmid vector. The introduced polynucleotide can be integrated and expressed in the genome of the cell, or can be expressed freely. Usually, since the L-lysine-producing bacteria itself is not suitable as a cloning host, it is preferred that the polynucleotide is introduced into the L-lysine-producing bacteria through a shuttle plasmid. Wherein, the shuttle plasmid is preferably a shuttle plasmid of Escherichia coli and L-lysine-producing bacteria. In this way, DNA recombination operations can be conveniently performed in E. coli hosts.

There are many kinds of L-lysine-producing bacteria, those known to those skilled in the art include Escherichia, Corynebacterium and Serratia, preferably Corynebacterium. In a specific embodiment of the present invention, the L-lysine-producing bacterium is Corynebacterium glutamicum.

Preferably, in the fermentation method of the first aspect of the present invention, the fermentation temperature of the fermentation condition is 28-35°C, preferably 29-33°C, more preferably 30-32°C, such as 30°C.

It is also preferred that in the fermentation method of the first aspect of the present invention, the medium of the fermentation condition comprises sugar, NH ₄ Cl, CaCl ₂ , KH ₂ PO ₄ , peptone, MgSO ₄ , FeSO ₄ , MnSO ₄ , biotin, and folic acid . In a specific embodiment of the present invention, the formula of the medium is: 40g sucrose, 20g NH ₄ Cl, 2g CaCl ₂ , 1gKH ₂ PO ₄ , 1g peptone, 500mg MgSO ₄ 7H ₂ O, 15mg FeSO ₄ 7H ₂ O, 10 mg MnSO ₄ ·7H ₂ O, 200 μg biotin, and 50 μg folic acid, pH 7.3, made up to 1 liter with water.

In a second aspect, the present invention provides a method for preparing an L-lysine feed additive, comprising:

(1) implementing step (2) in the fermentation method of the first aspect of the present invention; and

(2) collecting the fermented liquid to carry out membrane separation successively and carrying out spray drying to the filtrate;

(3) sieving the particles obtained by spray drying in step (2);

(4) crushing particles with a particle size greater than or equal to 1.5mm obtained through sieving, and mixing them with particles with a particle size smaller than 1.5mm obtained through sieving, and spray drying again; and

(5) Sieve the particles obtained by spray drying in step (4), and collect particles with a particle diameter less than 1.5 mm.

Among them, in order to make the shape of the final granular feed additive more uniform, the temperature of the tail gas in the fluidized bed dryer should not be too high, usually not higher than 85°C, preferably kept at 80±3°C.

The preparation method of the second aspect of the present invention may also include implementing step (1) of the fermentation method of the first aspect of the present invention before implementing step (2) of the fermentation method of the first aspect of the present invention. However, this is generally not preferred, so the preparation method of the second aspect of the present invention does not include implementing step (1) in the fermentation method of the first aspect of the present invention, but includes cultivating L-lysine-producing bacteria under fermentation conditions (especially high L-lysine-producing bacteria), this is also covered within the scope of the present invention.

Since the preparation method of the second aspect of the present invention can already produce granular feed additives that meet national standards, it is preferable to not include a coating process, which can reduce costs and facilitate commercial promotion.

In a third aspect, the invention provides products obtained according to the invention and intermediates used in the invention.

Specifically, the present invention provides the L-lysine feed additive obtained according to the preparation method of the second aspect of the present invention, preferably without coating agent (such as cyclodextrin, etc.).

In addition, the present invention provides dihydrodipicolinate synthase variants, which are replaced by other natural amino acids at positions M13, V60 or A83 of wild-type dihydrodipicolinate synthase. Preferably the dihydrodipicolinate synthase variant is replaced by other natural amino acids at positions M13, V60 and A83 of the wild-type dihydrodipicolinate synthase, preferably the substitutions are selected from M13K, V60L and A83G. In a specific embodiment of the present invention, the amino acid sequence of the dihydrodipicolinate synthase variant is shown in SEQ ID No: 1.

Correspondingly, the present invention provides a polynucleotide encoding a dihydrodipicolinate synthase variant, preferably its nucleotide sequence is shown in SEQ ID No: 2.

The invention has the following beneficial effects: the fermentation yield of lysine is effectively improved; the quality of the fermentation liquid is stable; the structural difference between the variant enzyme and the wild-type enzyme is not large, and both can be degraded smoothly without potential safety hazard; the fermentation liquid can be directly used in production Granular feed additive: The steps of producing granular feed additive are simple, and the requirements of national standards can be met without the introduction of coating agents.

In order to facilitate understanding, the present invention will be described in detail below through specific examples. It should be pointed out that these descriptions are only exemplary descriptions and do not limit the scope of the present invention. Many variations and modifications of the present invention will be apparent to those skilled in the art from the discussion of this specification.

In addition, the present invention refers to published documents, which are for the purpose of more clearly describing the present invention, the entire contents of which are incorporated herein by reference as if they had been recited herein in their entirety.

Detailed ways

The content of the present invention is further illustrated below by way of examples. If not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available common instruments and reagents, which can be found in "Molecular Cloning Experiment Guide (3rd Edition)" (Science Press), "Microbiological Experiments (4th Edition)" (Higher Education Press) and the manufacturer's instructions of corresponding instruments and reagents.

Embodiment 1 Preparation of dihydrodipicolinate synthase variant gene

According to the sequence we designed, we commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize the variant gene encoding dihydrodipicolinate synthase through commercial channels and constructed it into the Escherichia coli-Corynebacterium shuttle plasmid pMS2 (available from the American Type Microorganisms Collection (ATCC), article number ATCC 67189). The cloning process was carried out according to the "Molecular Cloning Experiment Guide" and the operation guide of the commercial reagents used. The brief process is as follows:

Synthesize nucleic acid fragments of dihydrodipicolinate synthase variant genes by an automatic DNA synthesizer, phosphorylate the 5' ends of these nucleic acid fragments with T4 polynucleotide kinase (purchased from TaKaRa Company), and then equimolar ratio The five nucleic acid fragments were mixed and denatured at 65°C for 5 minutes, annealed and cooled to 16°C, and T4 DNA ligase (purchased from TaKaRa Company) was added for ligation for 12 hours. Then, take 1 μL of the above ligation product and carry out PCR amplification in a 50 μL reaction volume, wherein the forward primer is shown in SEQ ID No: 3 of the sequence listing (the EcoR I endonuclease site is introduced), and the reverse primer is as shown in the sequence listing The SEQ ID No: 4 (introduced the Xba I endonuclease site), the reaction conditions are: denaturation at 94°C for 4 minutes, then denaturation at 94°C for 30 seconds, annealing at 63°C for 60 seconds and extension at 72°C for 30 seconds 35 cycles were performed, with a final extension at 72°C for 4 minutes and cooling to 4°C.

The above PCR product was electrophoresed on agarose gel, and a fragment of about 0.9kb size was recovered, and the fragment was double-digested with EcoR I and Xba I, and connected with the pMS2 plasmid digested by these two endonucleases with T4DNA ligase, Transformed into E. coli Top10F'. Pick out the positive clones, extract the plasmids therein, and verify by sequencing, the corresponding nucleotide sequence is shown in SEQ ID No: 1 of the sequence table, and the sequence encodes the dihydrodipicolinic acid shown in SEQ ID No: 2 For the complete ORF of the synthetase variant, the company will send back the constructed plasmid (named pMS2-dap) and the corresponding Escherichia coli transformant (named E.coli-cispnt).

The fermentation experiment of embodiment 2 coryneform bacteria

The pMS2-dap plasmid is transferred into L-lysine-fermenting coryneform bacteria engineering bacteria (available from the American Type Microorganism Collection (ATCC), product number ATCC 31269) by electroporation, and its brief process is: the coryneform bacteria Shake culture in 50mL LB liquid medium until OD500 reaches 0.7, collect the bacteria by centrifugation, wash with 0°C pre-cooled 10% (V/V) glycerol solution, and resuspend the bacteria in 200 μL pre-cooled 10% (V/V) V) In glycerol solution, add pMS2-dap plasmid, mix well, transfer to 0.1cm electric shock cup, conduct electric shock at 1.8kV for 5ms, then immediately add 1mL liquid LB containing 0.5% (M/M) glucose for culture Incubate at 42°C for 5 minutes, spread on solid LB medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin, and culture at 30°C for 36 hours. After the total DNA was extracted from the transformed bacterial strain that grew out, it was amplified by PCR with the above-mentioned forward primer and reverse primer, and agarose gel electrophoresis found a fragment of about 0.9kb size, indicating that it had been prepared as shown in SEQ ID No: 1 of the sequence table. The gene construct shown was introduced into the Corynebacterium engineering bacteria. At the same time, the pMS2 plasmid was electrotransformed into L-lysine fermented coryneform bacteria to form negative control bacteria.

The above-mentioned electrotransformed positive coryneform bacteria engineering bacteria and negative control bacteria were shaken and cultured in liquid LB medium until the OD500 reached 0.5, and were inserted into the lysine fermentation medium with a 5% inoculum (the formula per liter of medium was: 40 g sucrose, 20 g NH ₄ Cl, 2 g CaCl ₂ , 1 g KH ₂ PO ₄ , 1 g peptone, 500 _mg MgSO ₄ 7H ₂ O, 15 mg FeSO 4 7H ₂ O, 10 mg MnSO ₄ 7H ₂ O, 200 μg biotin, and 50 μg Folic acid, adjusted to pH 7.3 with Tris-HCl) and cultured at 30° C. with shaking (150 rpm) for 72 hours. The culture supernatant (ie, fermentation broth) was collected by centrifugation, and L-lysine in the culture medium was separated and quantified by paper chromatography. As a result, it was found that the content of L-lysine in the fermentation medium of positive coryneform bacteria engineering bacteria reached 19.6g/L, while the content of L-lysine in the fermentation medium of negative control bacteria was only 12.0g/L, It shows that the gene construct shown in SEQ ID No: 1 of the sequence table has been introduced, and the yield has been increased by 63.3%, which is higher than the yield increase ratio of the engineering bacteria introduced with wild-type pyridine nucleotide transhydrogenase in the prior art.

The preparation of embodiment 3L-lysine feed additive

The fermentation broth prepared in Example 2 was separated by a Suntar-III ultrafiltration membrane (available from Suntar Membrane Technology Co., Ltd.). Then, spray the filtrate directly into the fluidized bed dryer in the form of mist and keep the tail gas temperature at 80±3°C, and collect the discharged particles. Sieve the discharged particles, send the particles with a particle size greater than or equal to 1.5mm to the crusher for crushing, and then mix the crushed particles with the particles with a particle size smaller than 1.5mm obtained by screening and spray them into the fluidized bed again Keep the tail gas temperature in the dryer at 80±3°C, collect the discharged particles and sieve out the particles with a particle size of less than 1.5mm, which are L-lysine feed additives. After the particles with a particle size greater than 1.5mm are crushed again, they are mixed with the discharged particles obtained by drying the filtrate of the next batch.

The above-prepared L-lysine feed additive has been tested and found to have a water content of <1%; a particle size of less than 1.5 mm, without visible dust; a mixing coefficient of variation of 7 to 9; a bulk density of 550 to 630 g/L; Other additives have fully met the national standard.

Claims (10)

1. The fermentation method of L-lysine, it comprises: (1) introducing a polynucleotide encoding a dihydrodipicolinate synthase variant into an L-lysine-producing bacterium, so that the obtained bacterium expresses the dihydrodipicolinate synthase variant, wherein The dihydrodipicolinate synthase variant is encoded by the nucleotide sequence shown in SEQ ID No: 2; and (2) Cultivate the bacteria obtained in step (1) under fermentation conditions. 2. The fermentation method according to claim 1, wherein the polynucleotide is introduced into the L-lysine-producing bacteria through a shuttle plasmid. 3. The fermentation method of claim 1, wherein the bacterium is a coryneform bacterium. 4. The fermentation method according to claim 1, wherein the fermentation temperature of the fermentation condition is 28-35°C. 5. The fermentation method according to claim 4, wherein the fermentation temperature of the fermentation condition is 29-33°C. 6. The fermentation method according to claim 5, wherein the fermentation temperature of the fermentation condition is 30-32°C. 7. The fermentation method according to claim 6, wherein the fermentation temperature of the fermentation condition is 30°C. 8. The fermentation method of claim 1, wherein the culture medium of the fermentation condition comprises sugar, NH ₄ Cl, CaCl ₂ , KH ₂ PO ₄ , peptone, MgSO ₄ , FeSO ₄ , MnSO ₄ , biotin, and folic acid . 9. The preparation method of L-lysine feed additive, it comprises: (1) implementing the step (2) of the fermentation method described in any one of claims 1-8; and (2) collecting the fermented liquid to carry out membrane separation successively and carrying out spray drying to the filtrate; (3) sieving the particles obtained by spray drying in step (2); (4) crushing particles with a particle size greater than or equal to 1.5mm obtained through sieving, and mixing them with particles with a particle size smaller than 1.5mm obtained through sieving, and spray drying again; and (5) Sieve the particles obtained by spray drying in step (4), and collect particles with a particle diameter less than 1.5 mm. 10. A polynucleotide encoding a dihydrodipicolinate synthase variant, the nucleotide sequence of which is shown in SEQ ID No: 2.