CN102191287B - Cis-fermentation preparation method of lysine - Google Patents

Abstract

The invention provides a cis-fermentation preparation method of L-lysine. The method comprises the following steps of: introducing polynucleotide of encoded pyridine nucleotide transhydrogenase variant into an L-lysine generating bacterium so that the obtained bacterium expresses the pyridine nucleotide transhydrogenase variant; and culturing the bacterium under fermentation conditions to obtain a bacterium. In addition, the invention also provides an intermediate product used in the fermentation method and a product further produced by the fermentation method.

Description

Production of lysine by cis-fermentation

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 variant of pyridine nucleotide transhydrogenase into an L-lysine-producing bacterium, Thereby making the obtained bacteria express the pyridine nucleotide transhydrogenase variant; and cultivating 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.

Pyridine nucleotide transhydrogenase is an important enzyme in the metabolic pathway of L-lysine. Although subunits of wild-type pyridine nucleotide transhydrogenase have been published (see NCBI (http://www.ncbi.nlm.nih.gov) protein and gene accession numbers NP416120.1 and AAC74674.1; See Chinese Patent ZL94194707), but the research on this enzyme variant has not been reported.

After long-term arduous research, the inventor unexpectedly discovered a new pyridine nucleotide transhydrogenase, which significantly improved the activity of the corresponding enzyme, and also obtained higher lysine production when it was 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 pyridine nucleotide transhydrogenase variant into an L-lysine-producing bacterium, so that the obtained bacteria expressing the pyridine nucleotide transhydrogenase variant, wherein the activity of the pyridine nucleotide transhydrogenase variant is increased relative to the wild-type pyridine nucleotide transhydrogenase. 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 polynucleotides encoding pyridine nucleotide transhydrogenase variants into L-lysine-producing bacteria, so that the obtained bacteria express the pyridine nucleotide transhydrogenase variants, wherein the The pyridine nucleotide transhydrogenase variant has increased activity relative to wild-type pyridine nucleotide transhydrogenase; and

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

Wherein, the wild-type pyridine nucleotide transhydrogenase is known to those skilled in the art, including α subunit and β subunit, wherein the sequences of α subunit and β subunit are respectively as in NCBI (http://www.ncbi .nlm.nih.gov) protein and gene accession numbers NP416120.1 and AAC74674.1. The methods for measuring enzyme activity are known in the art. For example, the method described in the specific embodiments of the present invention can be used to measure enzyme activity, so that it can be confirmed whether the activity of the pyridine nucleotide transhydrogenase variant is improved.

Preferably, in the fermentation method of the first aspect of the present invention, the polynucleotide sequentially encodes a pyridine nucleotide transhydrogenase α subunit variant and a pyridine nucleotide transhydrogenase β subunit variant, that is, encodes a pyridine nucleotide The polynucleotide for the transhydrogenase alpha subunit variant is located upstream of the polynucleotide encoding the pyridine nucleotide transhydrogenase beta subunit variant. In a specific embodiment of the present invention, the nucleotide sequence of the polynucleotide is shown in SEQ ID No: 1.

Wherein, the pyridine nucleotide transhydrogenase α subunit variant is replaced by other natural amino acids at the position of M102, L127 or Q322, preferably replaced by M102L, L127R and Q322K, most preferably its amino acid sequence such as SEQ ID No : 2 shown. Those skilled in the art can deduce its encoding nucleotide sequence based on the amino acid sequence of the pyridine nucleotide transhydrogenase α subunit variant, preferably a codon-optimized nucleotide sequence, such as the codon usage of bacteria used for fermentation Situation optimized. In a specific embodiment of the present invention, the variant of the alpha subunit of pyridine nucleotide transhydrogenase is encoded by a polynucleotide as shown in SEQ ID No: 3.

In addition, wherein the pyridine nucleotide transhydrogenase β subunit variant is replaced by other natural amino acids at the position of A398 or D400, preferably the replacement is selected from A398S and D400H, most preferably its amino acid sequence is as shown in SEQ ID No: 4 Show. Those skilled in the art can deduce its encoding nucleotide sequence based on the amino acid sequence of the pyridine nucleotide transhydrogenase α subunit variant, preferably a codon-optimized nucleotide sequence, such as the codon usage of bacteria used for fermentation Situation optimized. In a specific embodiment of the present invention, the variant of the beta subunit of pyridine nucleotide transhydrogenase is encoded by a polynucleotide as shown in SEQ ID No:5.

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 pyridine nucleotide transhydrogenase variants, which include pyridine nucleotide transhydrogenase α subunit variants and pyridine nucleotide transhydrogenase β subunit variants. Preferably, the pyridine nucleotide transhydrogenase α subunit variant is replaced by other natural amino acids at the position of M102, L127 or Q322, preferably the replacement is selected from M102L, L127R and Q322K, most preferably its amino acid sequence is as SEQ ID No: 2. In addition, it is also preferred that the pyridine nucleotide transhydrogenase beta subunit variant is replaced by other natural amino acids at the position of A398 or D400, preferably the replacement is selected from A398S and D400H, most preferably its amino acid sequence is as shown in SEQ ID No: 4 shown. The present invention also provides the above-mentioned pyridine nucleotide transhydrogenase α subunit variants and pyridine nucleotide transhydrogenase β subunit variants respectively.

Correspondingly, the present invention provides polynucleotides encoding pyridine nucleotide transhydrogenase variants, that is, sequentially encoding pyridine nucleotide transhydrogenase α subunit variants and pyridine nucleotide transhydrogenase β subunit variants The polynucleotide, preferably its nucleotide sequence is as shown in SEQ ID No: 1. The present invention also provides polynucleotides encoding the above-mentioned pyridine nucleotide transhydrogenase α subunit variants and pyridine nucleotide transhydrogenase β subunit variants, preferably their nucleotide sequences are respectively as SEQ ID No : 3 and 5.

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.

The preparation of embodiment 1 pyridine nucleotide transhydrogenase variant gene construct

According to the sequence we designed, Shanghai Sangon Biotechnology Co., Ltd. was commissioned to synthesize a pyridine nucleotide transhydrogenase variant gene construct encoding two pyridine nucleotide transhydrogenase subunit variants through commercial channels and constructed it into Escherichia coli - The Corynebacterium shuttle plasmid pMS2 (available from the American Type Microorganisms Collection (ATCC), item 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:

The nucleic acid fragments of the pyridine nucleotide transhydrogenase variant gene construct were synthesized by an automatic DNA synthesizer, and the 5' ends of these nucleic acid fragments were phosphorylated with T4 polynucleotide kinase (purchased from TaKaRa Company), and then equimolar After mixing the 5 nucleic acid fragments, denature at 65°C for 5 minutes, anneal and cool down to 16°C, and add T4 DNA ligase (purchased from TaKaRa Company) for 12 hours of ligation. 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: 6 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: 7 (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 2.9kb 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'. The positive clones were picked out, and the plasmids were extracted. After sequencing verification, the corresponding nucleotide sequence was shown in SEQ ID No: 1 of the sequence table, and the sequences were encoded as shown in SEQ ID No: 2 and SEQ ID No: 4. The complete ORFs of the two pyridine nucleotide transhydrogenase subunit variants will be sent back by the company with the constructed plasmid (named pMS2-cispnt) and the corresponding Escherichia coli transformant (named E.coli-cispnt).

The activity assay of embodiment 2 escherichia coli and the fermentation experiment of coryneform bacterium

According to the existing assay method for transhydrogenase activity (see Clarke DM et al. Cloning and expression of the transhydrogenase gene of Escherichia coli. J. Bacteriol., 162: 367-373), E.coli transformed with pMS2-cispnt plasmid -cispnt bacterial strain and the negative Top10F' bacterial strain as a control measure the transhydrogenase activity respectively, and the specific enzyme activity of the found E.coli-cispnt bacterial strain is 163% higher than that of the negative Top10F' bacterial strain, much higher than the prior art Enzyme specific activity increase rate with wild-type pyridine nucleotide transhydrogenase expressed in .

The pMS2-cispnt 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-cispnt plasmid, mix well, transfer to 0.1cm electric shock cup, conduct electric shock at 2kV for 5ms, then immediately add 1mL liquid LB medium containing 0.5% (M/M) glucose , incubated at 42°C for 5 minutes, and then spread on solid LB medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin and cultured 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. Agarose gel electrophoresis found a fragment of about 2.9 kb in 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 bacterium 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 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 14.8g/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 23.3%, 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 (17)

1.L-the fermentation process of Methionin, it comprises:

(1) bacterium that the polynucleotide of pyridine nucleotide transhydrogenase variant import to produce L-Methionin of will encoding; Thereby make the bacterium that is obtained express said pyridine nucleotide transhydrogenase variant; Wherein said polynucleotide encode successively pyridine nucleotide transhydrogenase α subunit variant and pyridine nucleotide transhydrogenase β subunit variant; The aminoacid sequence of wherein said pyridine nucleotide transhydrogenase α subunit variant is shown in SEQ ID No:2, and the aminoacid sequence of said pyridine nucleotide transhydrogenase β subunit variant is shown in SEQ ID No:4; With

(2) bacterium of culturing step (1) acquisition under fermentation conditions.

2. the described fermentation process of claim 1, the nucleotide sequence of wherein said polynucleotide is shown in SEQ ID No:1.

3. the described fermentation process of claim 1, wherein said polynucleotide are to import the bacterium that produces L-Methionin through shuttle plasmid.

4. the described fermentation process of claim 1, wherein said bacterium is a coryneform bacteria.

5. the described fermentation process of claim 1, the leavening temperature of wherein said fermentation condition is 28-35 ℃.

6. the described fermentation process of claim 5, the leavening temperature of wherein said fermentation condition is 29-33 ℃

7. the described fermentation process of claim 6, the leavening temperature of wherein said fermentation condition is 30-32 ℃.

8. the described fermentation process of claim 7, the leavening temperature of wherein said fermentation condition is 30 ℃.

9. the described fermentation process of claim 4, the substratum of wherein said fermentation condition comprises sugar, NH ₄Cl, CaCl ₂, KH ₂PO ₄, peptone, MgSO ₄, FeSO ₄, MnSO ₄, vitamin H, and folic acid.

10.L-the preparation method of lysine additives for forage, it comprises:

(1) step (2) of arbitrary described fermentation process of enforcement claim 1-9; With

(2) collecting fermented liquid carries out membrane sepn successively and filtrating is carried out spraying drying;

(3) particle that step (2) spraying drying is obtained sieves;

(4) particle diameter that screening is obtained is more than or equal to the grain breakage of 1.5mm, and the particle diameter that itself and screening are obtained mixes less than the particle of 1.5mm, carries out spraying drying once more; With

(5) particle that step (4) spraying drying is obtained sieves, and collection cut size is less than the particle of 1.5mm.

11. pyridine nucleotide transhydrogenase α subunit variant, its aminoacid sequence is shown in SEQ ID No:2.

12. the gene of the described pyridine nucleotide transhydrogenase α of coding claim 11 subunit variant.

13. the described gene of claim 12, its nucleotide sequence is shown in SEQ ID No:3.

14. pyridine nucleotide transhydrogenase β subunit variant, its aminoacid sequence is shown in SEQ ID No:4.

15. the gene of the described pyridine nucleotide transhydrogenase β of coding claim 14 subunit variant.

16. the described gene of claim 15, its nucleotide sequence is shown in SEQ ID No:5.

17. the polynucleotide of coding pyridine nucleotide transhydrogenase variant, its nucleotide sequence is shown in SEQ ID No:1.