CN101580813A - Method for producing L-threonine by fermentation - Google Patents

Abstract

The invention provides a colon bacillus variant which is realized through the following modified colon bacilli: (1) the activity in cells of succinyl homoserine synthetase and dihydrodipicolinic acid synthetase is weakened; (2) the activity in the cells of threonine deaminase is reduced; (3) the activity in the cells of aspartokinase, aspartic acid-delta-semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine synthetase and phosphoenolpyruvate carboxykinase is enhanced; and (4) vitreoscilla globin genes are introduced, wherein the succinyl homoserine synthetase, the dihydrodipicolinic acid synthetase, the aspartokinase, the homoserine dehydrogenase, the aspartic acid-delta-semialdehyde dehydrogenase, the homoserine kinase, the threonine synthetase, the phosphoenolpyruvate carboxykinase and the threonine synthetase are respectively derived from colon bacilli. The invention also provides a method for fermenting the L-threonine, which comprises the following steps: culturing the colon bacillus variant in a proper culture medium; accumulating the L-threonine; and collecting the L-threonine from the culture medium.

Description

The method of producing L-threonine by fermentation

Technical field

The present invention relates to microbiological industry.Particularly relate to the method for using microbe production of L-threonine by fermentation, and the genetic engineering bacterium that is used for this production method.

Background technology

Threonine is that animal is kept the necessary amino acid of growth, is being in the live pig low-protein daily ration of protein main source with rapeseed meal or soybean meal, and Methionin is first limiting amino acid, and Threonine is second limiting amino acid.The L-Threonine is widely used in animal-feed and foodstuff additive, and the fluid and the synthetic materials that are used as medical science or pharmacology purposes.The L-Threonine is to use derived from wild-type e. coli, corynebacterium, kills the synthesis mutant and the auxotrophic synthesis mutant of their diaminopimelic acid, methionine(Met), Methionin or Isoleucine (the flat 2-219582 of Japanese Unexamined Patent Publication No of thunder Salmonella (Serratia), Providence (Providencia) etc., applicant Microbiolo.Biotechnol., and Korean Patent Publication No. 92-8365).Chinese patent publication number 85104604A discloses a kind of pantonine-hydroxypentanoic acid (AHV) of brevibacterium sp bacterium and method that S-(2-amino-ethyl)-the dual resistant mutant of L-halfcystine (AEC) produces the L-Threonine of adopting.Pantonine-hydroxypentanoic acid), lysine analogues (AEC:S-(2-amino-ethyl)-L-halfcystine), Isoleucine analogue (butyrine) and methionine(Met) analogue (ethionine) Korean Patent Application No. 90-22965 discloses a kind of L-Threonine and has produced bacterial strain TF4076 (KFCC10718), and it is methionine(Met) auxotroph and anti-Threonine analogue (AHV:.

The synthetic route of L-Threonine mainly is in intestinal bacteria:

In intestinal bacteria, L-Threonine synthetic from aspartic acid, aspartic acid changes aspartylphosphate under E.C. 2.7.2.4. I (AK I) catalysis, through the last homoserine that generates of reduction.E.C. 2.7.2.4. I (AK I) is a multifunctional enzyme, and the metabolism from the aspartic acid to the homoserine is by AK I catalysis.AK I is by the thrA genes encoding.Homoserine is phosphorylation under homoserine kinase catalysis, and the homoserine of phosphorylation is by the synthetic L-Threonine of threonine synthetase.Homoserine kinase is by the thrB genes encoding, and threonine synthetase is by the thrC genes encoding.

Metabolism to Threonine is regulated, and studies more clearly now.In the metabolic process of L-Threonine, the AK I of thrA genes encoding plays a decisive role.The amount of the synthetic Threonine of general intestinal bacteria is limited, because the L-Threonine suppresses the activity of AK I enzyme, and regulates transcribing of threonine operon by Threonine-tRNAs.The Threonine analogue can be used for screening Threonine and produce bacterial classification, and as in the resistant strain of pantonine-hydroxypentanoic acid, the thrA gene is undergone mutation, and the AK I enzyme of mutator gene coding is resisted the feedback inhibition of Threonine, thereby causes the accumulation of Threonine.In the intestinal bacteria with microbiotic borrelidin resistance, the conformation of Threonine-tRNAs synthetic enzyme changes, and greatly reduces the avidity with Threonine, and Threonine is accumulated in a large number.

Another principal element of a large amount of synthetic of restriction Threonine derives from Isoleucine, and the synthetic of Isoleucine is raw material with the L-Threonine.Therefore, if its route of synthesis does not cut off, can only cause the accumulation of Isoleucine, and the L-threonine content can not increase; On the other hand, Isoleucine has the intensive feedback inhibition to thrA gene transcription level, and high-load Isoleucine is blocked the synthetic of AK I enzyme.

Summary of the invention

Main purpose of the present invention is to regulate by the metabolism that changes bacterial classification, searches out a kind of genetic engineering bacterium of energy high yield Threonine.For achieving the above object, this researchist has carried out unremitting effort and diligent research, and the result has successfully obtained the intestinal bacteria through following modification: (1) succinyl homoserine synthetic enzyme and dihydrodipicolinic acid synthase's intracellular reactive weakens; (2) intracellular reactive of threonine deaminase reduces; (3) the kinase whose intracellular reactive of E.C. 2.7.2.4., homoserine dehydrogenase, homoserine kinase, threonine synthetase and phosphoenolpyruvic acid strengthens; And/or (4) have imported the hemoglobin gene from Vitreoscilla.(hereinafter also claiming " bacterium of the present invention ")

Preferably, bacterium of the present invention is the intestinal bacteria that comprise above-mentioned all modifications; Most preferred, the intestinal bacteria of modification of the present invention are genetic engineering bacterium K21P3.

In the present invention, term " intracellular reactive enhancing " expression is compared with wild type strain (for example e. coli strain bl21) or parent strain (this bacterial strain comprises that in the specific combination of the present invention the intracellular reactive of all enzymes does not all have the enhanced bacterial strain), intracellular enzymatic activity strengthens, and represents that also a kind of bacterium has a kind of wild type strain or the unexistent enzymatic activity of parent strain.

In the present invention, term " intracellular reactive reduction " expression is compared with wild type strain or parent strain, and intracellular enzymatic activity weakens.The method that is used to measure above-mentioned enzymic activity is known, and those skilled in the art can make things convenient for and confirm that exactly enzyme is to strengthen or weaken in intracellular activity.

Bacterium of the present invention is intestinal bacteria, and wherein, homoserine synthetic enzyme and dihydrodipicolinic acid synthase's intracellular reactive reduces.The homoserine synthetic enzyme, or the succinyl homoserine synthetic enzyme is the enzyme that the catalysis homoserine generates succinyl homoserine, is the key enzyme of L-methionine(Met) metabolic pathway of synthesizing.The dihydrodipicolinic acid synthase is that the catalysis aspartic-generates dihydropyridine-2, and the enzyme of 6-dicarboxylic acid is the key enzyme of L-Methionin metabolic pathway of synthesizing.Reduce the succinyl homoserine synthetic enzyme and reduce dihydrodipicolinic acid synthase's intracellular reactive, increased the metabolism stream that L-Threonine and L-Isoleucine generate approach accordingly, thereby made L-Threonine and L-Isoleucine excess accumulation.

In bacterium of the present invention, homoserine synthetic enzyme and dihydrodipicolinic acid synthase are that the method by chemomorphosis and ultraviolet mutagenesis makes it intracellular active the reduction.Use chemical mutagen ethyl sulfate (DES) and first thiamines to handle intestinal bacteria, obtain mutant strain, mutant strain is cultivated seed selection, and further it is carried out ultraviolet mutagenesis and composing type seed selection.By centrifugal to the fermented liquid of the mutant strain after the seed selection, collect supernatant liquor, measure the content of its Threonine, to come from the succinyl homoserine synthetic enzyme and the dihydrodipicolinic acid synthase of intestinal bacteria mutant strain at last and come from colibacillary wild-type succinyl homoserine synthetic enzyme and the dihydrodipicolinic acid synthase carries out the aminoacid sequence comparative analysis, find:

The sudden change of succinyl homoserine synthetic enzyme comprises: proline(Pro) replaces the 61st Serine, and Serine replaces the 107th glycine, and Histidine replaces the 165th Serine.

Dihydrodipicolinic acid synthase's sudden change comprises: L-Ala replaces the 44th Threonine, and Methionin replaces the 55th L-glutamic acid, and Histidine replaces the 65th aspartic acid.

Bacterium of the present invention is intestinal bacteria, and wherein, the intracellular reactive of threonine deaminase reduces.Threonine deaminase catalysis L-Threonine generates α-alpha-ketobutyric acid, passes through acetohydroxy acid synthetase etc. again, further generates the L-Isoleucine.Threonine deaminase is the lytic enzyme of L-Threonine in the intestinal bacteria, and its encoding gene is tdc.If above-mentioned route of synthesis does not cut off, can only cause the accumulation of Isoleucine, and the content of Threonine is not high.

The intracellular reactive of threonine deaminase reduces and can knock out the realization of threonine deaminase gene by the Red homologous recombination technique.The Red homologous recombination technique can be used for making up the genetic engineering bacterium with specific sudden change as a kind of novel gene target practice technology, to change the biological metabolism approach.The Red homologous recombination technique is one section to be carried with the target gene both wings respectively have the PCR fragment of 40-60bp homologous sequence to import host's mycetocyte, utilize phage Red recombinase (EXO, Beta, three kinds of albumen of Gam) effect, make linear DNA fragment and chromosomal particular target sequence carry out homologous recombination, make target gene be replaced by linear DNA fragment.

In bacterium of the present invention, knock out the gene or the known CadA gene of threonine deaminase by the Red homologous recombination technique, make up threonine deaminase genetic flaw strain (coli strain of Isoleucine defective), thereby the intracellular reactive of threonine deaminase is reduced.In bacterium of the present invention, utilize metabolic engineering technique construction threonine deaminase genetic flaw strain (coli strain of Isoleucine defective), be expected to reduce the decomposition of L-Threonine, accumulation L-Threonine promotes thalli growth.Use round pcr, amplify the upstream and downstream sequence homology of both wings and threonine deaminase gene, the centre is the dna fragmentation of chloramphenicol resistance gene.Transform through electricity, exogenous dna fragment is changed over to respectively in the intestinal bacteria (K16P2).Under the effect of Red recombinase, homology zone reorganization on exogenous dna fragment and the karyomit(e) with the threonine deaminase gene knockout, obtains threonine deaminase genetic flaw strain (K16P3) (coli strain of Isoleucine defective).

The method that increases the intracellular reactive of enzyme comprises following method, and the arbitrary combination of these methods, but is not limited to these methods.

The method that is usually used in increasing intracellular reactive is to improve the method for the expression amount of described enzyme.Specifically, the method for raising expression of enzymes amount comprises following method:

(1) imports the plasmid that contains the enzyme gene

As plasmid, be can use and can be in Bacillus coli cells the carrier of self-replicating, can it be imported with known method.In other words, purpose (interested) gene can be inserted described carrier, and can be with this rotaring carrier transformed into escherichia coli.This carrier is multiple copied type plasmid preferably.Described goal gene can be carried by identical plasmid or different plasmid, and some gene can be carried by identical plasmid, and the order that imports described gene is not particularly limited.

(2) number of copies of enzyme gene on the increase karyomit(e)

By with chromosomal DNA of amplification such as Mu phages, increase described number of copies.DNA on the chromosomal DNA can be that intestinal bacteria just had originally, or by using transduction, transposon (BergiD, E. and Burgc, M, biotechnology, 1,417 (1983)) method of (open No.2-109985 of Japanese patent application later stage), or the homologous recombination method is inserted in the host microorganism karyomit(e).

(3) promoter sequence of change enzyme gene

Can change promoter sequence, so that improve the amount of transcribing, and and then raising expression amount.For example, can strengthen this promotor by a sudden change is imported in the promotor, so that improve the gene transcription amount that is positioned at this promotor downstream.Import the described promotor except suddenling change, can import the new promotor that can in intestinal bacteria, work, as lac, trp, tac, trc and PL.In addition, can be by importing new enhanser to improve the gene transcription amount.Although described promoter sequence can be the promotor of enzyme gene on the promotor of enzyme gene on the karyomit(e) or the plasmid, the promotor of enzyme gene on the preferred karyomit(e).For example disclosed among the open No.1-215280 of Japanese patent application later stage and will import the method for chromosomal DNA such as the gene of promotor.

The preferred above-mentioned first kind of importing of the present invention contains the method for enzyme gene plasmid.In bacterium of the present invention,, strengthen aspartate-, homoserine kinase, the kinase whose intracellular reactive of threonine synthetase and phosphoenolpyruvic acid by increasing the expression amount of the outer plasmid of karyomit(e).

Source to above-mentioned enzyme gene is not particularly limited, therefore, can the using of various sources, as long as this gene can be at expression in escherichia coli, and its genetic products can work just in intestinal bacteria.

In addition,, also comprise importing enzyme, comprise the enzyme of removing its feedback inhibition etc. with the sudden change that strengthens specific activity as the method that improves specific enzyme activity.Here, " removal feedback inhibition " expression weakens substantially to restraining effect, rather than will be to suppressing desensitization fully, as long as the feedback inhibition degree is lower than the degree that comes from colibacillary wild enzyme.No matter and its biological wild-type or mutant enzyme that be this.Can assess the degree of feedback inhibition with known method.As be disclosed in embodiment 1 among the international publication number WO95/16042 and the method among the embodiment 2.

E.C. 2.7.2.4. (being designated hereinafter simply as AK) is a kind of aspartic acid to be changed into the enzyme of β-phosphoaspartate, and it is the main regulator site of aspartic acid and derivative amino biosynthetic pathway thereof.Intestinal bacteria have three types AK (AK I, AK II, AKIII), preceding two kinds have the active bifunctional enzyme of homoserine dehydrogenase (the following HD that abbreviates as sometimes), a kind of is AK I-HD I by the thrA genes encoding, and another kind is the AK II-HD II by MetL (M) genes encoding.

Having only AKIII is single functional enzyme, and it is the product that is called the gene of lysC, and knownly is subjected to checking and feedback inhibition of Methionin.On the other hand, AK I is subjected to the coordinate repression of Threonine and Isoleucine, and suppressed by Threonine, and AK II is checked by methionine(Met) then.The ratio of its intracellular reactive is about AK I: AK II: AKIII=5: 1: 4.

Intestinal bacteria variant of the present invention comprises also that wherein the intracellular reactive of E.C. 2.7.2.4. strengthens.

In bacterium of the present invention, by the binding site of transformation with Threonine, remove E.C. 2.7.2.4. I (AK I) the L-Threonine feedback inhibition and by transforming the binding site with Methionin, remove the feedback inhibition of the L-Methionin of E.C. 2.7.2.4. III (AKIII), to strengthen the intracellular reactive of E.C. 2.7.2.4..

Removal comprises the sudden change of E.C. 2.7.2.4. I (AK I) L-Threonine feedback inhibition: use another kind of amino acid, preferred glycine replaces the Serine on the 109th, uses another kind of amino acid, and preferred L-Ala replaces the Serine on the 163rd.

Carrying out the intestinal bacteria variant of the present invention that obtains after the above modification, is intestinal bacteria BL1126P.

Removal comprises the sudden change of E.C. 2.7.2.4. III (AKIII) L-Methionin feedback inhibition: use another kind of amino acid, preferred aspartic acid replaces the glycine on the 330th, use another kind of amino acid, preferred glycine replaces the halfcystine on the 400th, use another kind of amino acid, preferred aspartic acid replaces the glycine on the 403rd.

Carrying out the intestinal bacteria variant of the present invention that obtains after the above modification, is e. coli bl21 25P.

Well-knownly be do not influence its active aminoacid sequence difference may existing between the kind or between the bacterial strain, and those skilled in the art to discern at an easy rate in above-mentioned particular amino acid residue.

The example that is used for the preparation of DNA vitro mutagenesis processing comprises azanol etc., and azanol is by cytosine(Cyt) being become N4-hydroxyl cytosine(Cyt), the chemomorphosis treatment agent that causes cytosine(Cyt) to be replaced by thymus pyrimidine.When microorganism itself is carried out mutagenic treatment, be with ultraviolet radiation or be usually used in mutagenesis such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) and above mention mutagenic compound such as ethyl sulfate (DES), first thiamines and nitrous and carry out.

We can obtain mutant AK I, the DNA of AKIII that coding is removed the feedback inhibition of Threonine, Methionin in order to following method.For example, the DNA of the gene that contains wild-type AK I, AKIII or other AK I with sudden change, AKIII gene is carried out vitro mutagenesis handle, and will be connected with carrier DNA, prepare recombinant DNA through the DNA of mutagenic treatment with host's compatibility.The DNA of reorganization is imported in the host microorganism, and screening can be expressed the transformant of mutant AK I, AKIII, and such transformant has the gene of mutant AK I, AKIII.Perhaps, can be connected with the carrier DNA of host's compatibility, so that the preparation recombinant DNA containing wild-type AK I, AKIII gene or having other AK I of sudden change, the DNA of AKIII gene.Then, recombinant DNA is carried out vitro mutagenesis handle, and it is imported in the host microorganism, screening can be expressed the transformant of mutant AK I, AKIII, and such transformant has this mutated genes equally.

In addition, in intestinal bacteria of the present invention, the researchist preferentially selects following method for use: the microorganism that produces the wild-type E.C. 2.7.2.4. is carried out mutagenic treatment, obtain to produce the mutant strain of removing to the E.C. 2.7.2.4. of L-Threonine, L-Methionin feedback inhibition, then, from this mutant strain, obtain the mutated genes of removal to the E.C. 2.7.2.4. of L-Threonine, L-Methionin feedback inhibition.Afterwards, the DNA of mutant AK I, AKIII gene and the carrier DNA of host's compatibility are connected, and the preparation recombinant DNA imports it in host microorganism then, screening can be expressed the transformant of mutant AK I, AKIII, and such transformant has this mutated genes.

Because expression of exogenous gene in the genetic engineering bacterium, the cell that contains recombinant plasmid can need more oxygen, thereby may change the energy metabolism of cell, and be unfavorable for the growth (Khoravi of cell, M, Ryanw, webter, D, A or the like, plasmid, 1990,23:138-143) in the fermentation of large scale and high density, the cell raised growth causes dissolved oxygen concentration sharply to descend.Therefore, raising equipment leads to oxygen condition, makes cell be in the aerobic repiration state, becomes one of key issue in the genetic engineering bacterium fermentation.Existing improving one's methods mainly is to improve stirring velocity, increases air flow, adds solubility promoter, improve gas Liquid Mass Transfer Coefficient or the like, but effect is limited, and can increases production cost greatly.

Bacterium of the present invention is intestinal bacteria, by introducing the bacterial hemoglobin gene in the host bacterium, it is expressed in the host, and the oxygen that improves the bacillus coli gene engineering bacteria transmits system, increases the oxygen transmission capacity.Vitreoscilla is a kind of aerobic gram negative bacterium of obligate that grows in the oxygen deprivation environment, can induce synthetic a kind of oxyphorase, Vitreoscilla hemoglobin (Vitreocilla, Hemoglobin, VHb) be that a kind of oxygen is conjugated protein, this proteic homodimer is close to the avidity and the eukaryote of oxygen, very high oxygen dissociation constant is arranged, has very strong oxygen transmittability, especially the promotor of its autogene can start this genetic expression in mass efficient ground under the oxygen deprivation condition, can improve the transmission speed of oxygen in the fermented liquid, thereby promote cell growth and proteinic synthetic.Vgb can increase the electron transport number in colibacillary expression, strides film pH value and ATP synthesis rate.

In the present invention, hemoglobin gene in the Vitreoscilla (Vgb) is inserted plasmid PT5, make up the clone's who contains the Vgb gene plasmid PT6 (referring to embodiment 8), change in the intestinal bacteria then.

The intracellular reactive that further comprises at least a enzyme in aspartate-, homoserine dehydrogenase, homoserine kinase, threonine synthetase and/or the phosphoenolpyruvic acid kinases in the intestinal bacteria variant of the present invention strengthens.The active enhancing of wherein said desmo enzyme is to improve its intracellular reactive by increasing the plasmid expression amount.

Further imported hemoglobin gene in the intestinal bacteria variant of the present invention, described hemoglobin gene derives from the hemoglobin gene of Vitreoscilla.

In bacterium of the present invention, homoserine synthetic enzyme, dihydrodipicolinic acid synthase, E.C. 2.7.2.4., homoserine dehydrogenase, aspartate-, homoserine kinase, threonine synthetase, phosphoenolpyruvic acid kinases and threonine deaminase come from intestinal bacteria respectively, and hemoglobinase comes from Vitreoscilla.

Intestinal bacteria variant of the present invention through above modification is genetic engineering bacterium K21P3.

Another object of the present invention provides the method for producing the L-Threonine, and this method is included in cultivates intestinal bacteria of the present invention in suitable medium, and produces in its substratum and gathering L-Threonine, and therefrom collects the L-Threonine.Employed various substratum can be selected the conventional various substratum that use in the Threonine production for use, for example uses the substratum of following prescription:

1, slant medium (g/l)

Glucose 2.0, NH ₄Cl 1.0, KH ₂PO ₄1.5, Na ₂HPO ₄3.5, MgSO ₄7H2O0.1, agar 20.0, pH7.0-7.2 is transferred in the adding distil water dissolving, constant volume 1000ml, 0.8Kg/cm2 sterilization 30 minutes is cooled to adding penbritin solution about 50 ℃, and ultimate density is 50 γ/ml.

2, shake-flask seed substratum (g/l)

Glucose 40.0, KH ₂PO ₄1.0, MgSO ₄7H ₂O 0.5, (NH ₄) ₂SO ₄10.0, corn steep liquor 2.0, CaCO ₃15, penbritin 50 γ/ml add the distilled water dissolving, transfer pH7.0-7.2, constant volume 1000ml, and branch is filled to 500ml and shakes bottle, and 0.8Kg/cm2 sterilization 30 minutes adds CaCO before the inoculation ₃(121 ℃, sterilization in 60 minutes, oven dry) and penbritin.

3, shake flask fermentation substratum (g/l)

Glucose 80.0, (NH ₄) ₂SO ₄25.0, KH ₂PO ₄2.0, MgSO ₄7H ₂O 1.0, MnSO ₄5H ₂O 0.5, FeSO ₄7H ₂O 0.5, CaCO ₃30.0, add the tap water dissolving, transfer pH7.0-7.2, constant volume 1000ml, branch is filled to 500ml and shakes bottle, and 0.8Kg/cm2 sterilization 30 minutes adds CaCO3 (121 ℃ were sterilized oven dry in 60 minutes) before the inoculation

4, seed tank culture base

Glucose 4%, (NH ₄) ₂SO ₄1%, KH ₂PO ₄0.1%, MgSO ₄7H ₂O 0.05%, corn steep liquor 0.2%, bubble enemy 0.01%.The pH that is dissolved in water is natural, sterilizes 20 minutes for 121 ℃, and back constant volume 400L disappears.Add aseptic penbritin 50ug/L before the inoculation.

5, fermentation tank culture medium

Glucose 8%, (NH ₄) ₂SO ₄2.5%, KH ₂PO ₄0.2%, MgSO ₄7H ₂O 0.1%, FeSO ₄5H ₂O 0.05%, MnSO ₄5H ₂O 0.05%, bubble enemy 0.01%.Add tap water dissolving pH nature, sterilized 20 minutes for 121 ℃, back constant volume 5.1M3 disappears.1.0Kg/cm ²Sterilized 20 minutes.

Employed bacterium is the intestinal bacteria through following modification in the inventive method: (1) succinyl homoserine synthetic enzyme and dihydrodipicolinic acid synthase's intracellular reactive weakens; (2) the threonine deaminase intracellular reactive reduces; (3) E.C. 2.7.2.4., homoserine dehydrogenase, homoserine kinase, threonine synthetase and phosphoenolpyruvic acid kinases intracellular reactive strengthen; And/or (4) have imported the hemoglobin gene from Vitreoscilla.Wherein said homoserine synthetic enzyme, dihydrodipicolinic acid synthase, E.C. 2.7.2.4., homoserine dehydrogenase, aspartate-, homoserine kinase, threonine synthetase, phosphoenolpyruvic acid kinases and threonine deaminase come from intestinal bacteria respectively.

In the method for the present invention, preferred intestinal bacteria preferably have the intestinal bacteria of above-mentioned all modifications; Most preferred intestinal bacteria are genetic engineering bacterium (K21P3).

Preferably, the invention provides a kind of method of the L-of production Threonine, this method is included in and cultivates the genetic engineering bacterium (K21P3) that obtains through behind above-mentioned modification and the importing hemoglobin gene in above-mentioned suitable medium, in its substratum, produce and gathering L-Threonine, and therefrom collect the L-Threonine.Preferred, described cultivation was carried out under aerobic conditions 36～60 hours; Between incubation period temperature is controlled at about 37 ℃; The pH value is controlled between 6～8, can use inorganic or organic acid or alkaline matter and oxygen adjusting pH.

Use new genetic engineering bacterium of the present invention, can optimize the generation and the gathering of L-Threonine in the fermentation culture process, improve the output of L-Threonine by number of ways.

In addition, the following beneficial effect in addition of method of the present invention:

1, original strain is after process is genetic engineering modified, and except that having a small amount of variation on L-Threonine pathways metabolism, other physiological characteristic is constant, does not therefore influence the fermentation of bacterial strain.

2, the E.C. 2.7.2.4. I of engineering strain of the present invention is not subjected to the Threonine feedback inhibition, and therefore in L-Threonine building-up process, bacterial growth and the metabolism of L-Threonine are unaffected.

3, engineering strain of the present invention is an Isoleucine defective bacterial strain, and Isoleucine is synthetic to be to synthesize precursor with the L-Threonine, and therefore, the Isoleucine defective can make the L-Threonine accumulate in a large number.

4, genetic engineering bacterium of the present invention normal growth under the finite concentration microbiotic, plasmid wherein keep stable.

Embodiment

Below by embodiment technical scheme of the present invention is further described, but the scope that these embodiment do not limit the present invention in any way.When wherein relating to the percentage concentration of the unit of explanation not, described concentration unit is meant the solute gram number in every 100ml solution.

Embodiment 1: the weaken screening of bacterial strain of homoserine synthetic enzyme and dihydrodipicolinic acid synthase

Handle the wide spectrum carbon source coli strain (K16) that process is screened with chemical mutagen ethyl sulfate (DES) and methane amide, obtain mutant bacteria K16P1 bacterial strain.

The phosphoric acid buffer 100ml sterilization of configuration pH7.0, will (this bacterial classification be known through the wide spectrum carbon source intestinal bacteria bacterial classifications (K16) (this bacterium by this laboratory preserve) of screening, can be by having bought on the market) in broth culture, cultivate 20h, get concentration then and be about 2.5 * 10 ⁸Cultivation bacterium liquid with physiological saline centrifuge washing twice, with the about 20min of granulated glass sphere vibration, with the bottle swingging machine 20min that vibrates, make it homodisperse again.Bacterium liquid is prepared with phosphoric acid buffer.Mutagenesis reagent is to add the small amount of ethanol dissolving earlier with ethyl sulfate (DES) the solution 5ml of 0.8%-1% (g/100ml), compound method, adds the phosphoric acid buffer mixing then.The concentration of methane amide reagent is 70%, and its method for making is similar to aforesaid method.Get each 2.5ml of bacterium liquid and reagent solution, 37 ℃ of bottle swingging machine vibration 30min.Bacterium liquid 2%Na after the processing ₂S ₂O ₃Termination reaction, dilution back coating plate, with separation screening substratum separation and Culture, used substratum is as follows again: glucose 150g/l, peptone 10g/l. (NH ₄) ₂SO ₄35g/l, KH ₂PO ₄1.0g/l, MgSO ₄7H ₂O 0.5g/l, CaCO ₃30g/l, pH7.0 obtains mutant bacteria K16P1 bacterial strain.

This mutant strain is cultivated seed selection, and further it is carried out ultraviolet mutagenesis: draw the 4-5ml bacteria suspension, put into plate, bacterium liquid thickness does not surpass 2mm in the ware, ware is placed the irradiation down apart from fluorescent tube 30cm, and the rotating speed of magnetic stirring apparatus is 100r/min, mutation time 5min, inhale 0.1ml irradiation back suspension and add gravy peptone flat board, shake up.The seed selection of cultivating the constitutive mutation strain be with the bacterial strain behind the ultraviolet mutagenesis with two kinds of substratum, i.e. gravy peptone substratum and the screening that contains orange meal replaces cultured method with substratum, the exchange cultivation is 3 times repeatedly, seed selection constitutive mutation strain from colony.Can obtain bacterial strain K16P2 after the seed selection thus.

With the bacterial strain K16P2 that obtained in aforesaid liquid shake flask fermentation substratum, cultivate by described culture condition, simultaneously, with the not also cultivation in contrast in same substratum of bacterial strain K16 of mutagenic treatment, accumulation L-Threonine, from substratum, collect Threonine according to ordinary method, and compare the output of the L-Threonine of intestinal bacteria K16 and K16P2 bacterial strain.

Table 1

Embodiment 2: make up intestinal bacteria K16P3

According to colibacillary Soviet Union amino acid dehydrogenase gene, design following PCR primer:

ILVAP1：5’-ATGGCTGACTCGCAACCCCTGTCCGGTGCTCCGGAAGGTGTCAGTGGAACTCCGTCG--3’(SEQ?ID?NO：1)，

ILVAP2：5’-CTAACCCGCCAAAAAGAACCTGAACGCCGGGTTATTGGTTTGTAAAACGTCCGAAGGCC--3’(SEQ?ID?NO：2)

ILVAP1 wherein , ILVAP2Near the zone of 5 ' end and the both wings sequence homology of threonine deaminase gene, ILVAP1, ILVAP2 goes up the sequence complementation of Cat gene both sides near the zone and the plasmid PKF3 of 3 ' end, (BIO ENGINEERING INST MILITARY is so kind as to give with plasmid PKF3 (containing chloramphenicol resistance gene Cat gene), be known bacterium, can can buy from Dalian Bao Bio-Engineering Company) be template, carry out pcr amplification, the PCR reaction conditions is: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min., 16 ℃, 30min..Pcr amplification goes out both wings and threonine deaminase gene upstream and downstream sequence homology, and the centre is the 1.1kbDNA fragment of chloramphenicol resistance gene.

Single bacterium colony of picking intestinal bacteria K16P2/pKD46 is inoculated in the fresh LB substratum (containing the penbritin that final concentration is 50ug/ml), and 30 ℃ of shaken overnight are cultivated, and transfer then in the 50mLLB substratum, continue to be cultured to A ₆₀₀=0.2-0.3.Add L-arabinose in substratum, final concentration is 1mmol/l, continues to be cultured to A ₆₀₀=0.5-0.6.Bacterium liquid was placed ice bath 15-20 minute, in 4 ℃, centrifugal 10 minutes of 5000 * g.Wash thalline 3 times with 10% glycerine, thalline is resuspended in the 0.5mL10% glycerine, make competent cell through sterilization.Pipette above-mentioned PCR product 5ul and mix with competent cell 100ul, and electric shock (shock parameters 25 μ F, 200,2.5kv) back adds the LB substratum of 1ml precooling, cultivates 1 hour for 30 ℃.Pipette 200 μ l bacterium liquid and coat on the LB flat board that contains penbritin (final concentration is 50ug/ml) and paraxin (final concentration is 50ug/ml), 30 ℃ of cultivations, screening positive transformant.After above-mentioned dna fragmentation through pcr amplification changes intestinal bacteria K16P2 bacterial strain over to, under the effect of Red recombinase, homology zone reorganization on dna fragmentation and the karyomit(e) has knocked out the threonine deaminase gene, obtains the positive strain K16P3 of threonine deaminase genetic flaw.

With the recombination bacillus coli K16P3 of above-mentioned acquisition in above-mentioned culture medium culturing, simultaneously, to the bacterial strain K16P2 of threonine deaminase gene knockout not cultivated in same substratum in contrast yet, accumulation L-Threonine, from cultivate, collect the L-Threonine, and compare the output (table 2) of the L-Threonine of intestinal bacteria K16P3 and K16P2

Table 2

Embodiment 3: make up the pT1 plasmid

Make up the pT1 plasmid, be used to make up plasmid vector.

(available from Dalian Bao Bio-Engineering Company) is template with the pBR322 plasmid, with FP2 (SEQID NO:3), RP2 (SEQ ID NO:4) is primer, and the Pfu enzyme carries out the pcr amplification (reaction conditions of PCR: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 3min, 30 circulations; 72 ℃, 10min), obtain the gene fragment of 2583bp.Fragment with above-mentioned amplification is a template again, with FP1 (SEQ ID NO:5), RP1 (SEQ ID NO:6) is primer, and the EXTaq enzyme carries out the pcr amplification (reaction conditions of PCR: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 3min, 30 circulate 72 ℃, 10min), obtain the gene fragment of 2607bp.The gene fragment of this 2607bp is linked among the carrier pGEM-T (available from Dalian Bao Bio-Engineering Company), formed plasmid pGEM-TPL1.Cut pGEM-TPL1 with EcoR I enzyme, reclaim the 2601bp fragment, use T ₄This fragment of dna ligase cyclisation, transformed into escherichia coli DH5 α extracts plasmid, obtains the pT1 plasmid, and this plasmid contains EcoRI KpnI BamHI HindIII NcolI EcoR I restriction enzyme site.

Embodiment 4: make up intestinal bacteria K17P3

According to the known nucleotide sequence of intestinal bacteria aspartate-gene (asd), design and prepare two types Oligonucleolide primers ASDF1 (SQE ID NO:7) and ASDR1 (SQE ID NO:8); ASDF2 (SQE ID NO:9) and ASDR2 (SQE ID NO:10).With ASDF1, ASDR1 is primer, (is preserved by this laboratory with the pGEMT-UPM plasmid.This plasmid is known, can be by having bought on the market.Described pGEMT is a known carrier, can buy on market; UPM is the promotor that contains 79 bases) for template, carry out pcr amplification (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 72 ℃ of 30 circulations, 10min.); With ASDF2, ASDR2 is primer, with the pfu enzyme to the K type, promptly the coventional type e. coli dna increase (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min.).Each 1 μ l is a template with above-mentioned two kinds of PCR products, with ASDF1, ASDR1 is primer, with the ExTaq enzyme increase (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min), reclaim the amplified fragments of 1195bp, be connected among the carrier pGEM-T, transformed into escherichia coli DH5 α extracts and plasmid purification.This plasmid is cut with BamH I enzyme, and be connected, obtain recombinant plasmid pT 2 with same pT 1 plasmid of cutting with BamH I enzyme.

Intestinal bacteria K16P3 with among recombinant plasmid (being pT2) the conversion embodiment 3 that is obtained obtains intestinal bacteria K17P3.In minimum medium, cultivate (substratum: tryptone 1g/100ml, yeast extract 0.5g/100ml, NaCl 1g/100ml, agar 1.5-2g/100ml.Culture condition: pH7.0,0.1Mpa 20 minutes), the K16P3 bacterial strain that will not import plasmid pT2 is simultaneously also cultivated in same substratum in contrast, accumulation L-Threonine, from culture collection L-Threonine, and the output (table 3) of the L-Threonine of comparison intestinal bacteria K16P3 and K17P3.

Table 3

Intestinal bacteria K17P3 (asd) is owing to changed the plasmid pT 2 that contains aspartate-gene (asd) over to, the aspartate-gene duplicates under the effect of promotor, increased the number of copies of the aspartate-gene in the intestinal bacteria, thereby the productive rate of L-Threonine is improved.

Embodiment 5: make up intestinal bacteria K18P3

According to method similar to Example 1, (this bacterial classification is known, can be by having bought on the market to handle coli strain BL1126 with chemical mutagen ethyl sulfate (DES) and methane amide.Preserve by this laboratory.), mutant strain is cultivated seed selection, and further it is carried out ultraviolet mutagenesis and composing type seed selection.

The phosphoric acid buffer 100ml sterilization of configuration pH7.0 will be cultivated 20h through the wide spectrum carbon source intestinal bacteria bacterial classifications (K16) (this bacterium is preserved by this laboratory) of screening in broth culture, get concentration then and be about 2.5 * 10 ⁸Cultivation bacterium liquid with physiological saline centrifuge washing twice, with the about 20min of granulated glass sphere vibration, with the bottle swingging machine 20min that vibrates, make it homodisperse again.Bacterium liquid is prepared with phosphoric acid buffer, and mutagenesis reagent concentration is ethyl sulfate (DES) the solution 5ml of 0.8%-1%, and compound method is to add the small amount of ethanol dissolving earlier, adds the phosphoric acid buffer mixing then.The method for making of methane amide (70%) reagent is similarly got each 2.5ml of bacterium liquid and reagent solution, 37 ℃ of bottle swingging machine vibration 30min.Bacterium liquid 2%Na after the processing ₂S ₂O ₃Termination reaction, coating plate in dilution back in separation screening separation and Culture in the substratum, uses following substratum: glucose 150g/l, peptone 10g/l. (NH again ₄) ₂SO ₄35g/l, KH ₂PO ₄1.0g/l, MgSO ₄7H ₂O 0.5g/l, CaCO ₃30g/l.PH 7.0)), obtain mutant bacteria K16P1 bacterial strain.

Mutant strain is cultivated seed selection, and further it is carried out ultraviolet mutagenesis, draw the 4-5ml bacteria suspension, put into plate, bacterium liquid thickness does not surpass 2mm in the ware, and ware is placed the irradiation down apart from fluorescent tube 30cm, the rotating speed of magnetic stirring apparatus is 100r/min, mutation time 5min inhales 0.1ml irradiation back suspension and adds gravy peptone flat board, shakes up.The seed selection of cultivating the constitutive mutation strain is with two kinds of substratum with the bacterial strain behind the ultraviolet mutagenesis, be that gravy peptone substratum and the screening that contains orange meal replace cultured method with substratum, exchange is cultivated 3 times repeatedly, seed selection constitutive mutation strain from colony, screening accumulation Threonine output is many or to the bacterial strain of the insensitive E.C. 2.7.2.4. I of Threonine.Through screening, obtain mutant strain BL1126P, this bacterial classification has that (preserving number of this bacterial classification is: CGMCC No:2369, date saved: on January 28th, 2008) to the insensitive E.C. 2.7.2.4. I of Threonine.

According to the known nucleotide sequence of intestinal bacteria threonine operon, design and prepare two types Oligonucleolide primers THRF1 (SQE ID NO:11), THRR1 (SQE ID NO:12); THRF2 (SQE ID NO:13), THRR2 (SQE ID NO:14).With THRF1, THRR1 is primer, (is preserved by this laboratory with the pGEMT-UPM plasmid.PGEMT is a known carrier, can buy on market; UPM is the promotor that contains 79 bases) for template carry out pcr amplification (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min); With THRF2, THRR2 is primer, with the DNA of above-mentioned intestinal bacteria BL1126P be template increase (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 3min, 30 circulations, 72 ℃, 10min).With each 1 μ l of above-mentioned two PCR products is template, is primer with THRF1, THRR1, with ExTaq increase (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 5min, 30 circulations, 72 ℃, 10min).Reclaim the amplified fragments of 4775bp, be connected among the carrier pGEM-T (available from Dalian Bao Bio-Engineering Company), the plasmid that transformed into escherichia coli DH5 α and extraction and purifying are obtained.The plasmid that is obtained is cut with the HindIII enzyme, and be connected, obtain recombinant plasmid pT3 (wherein containing) the insensitive E.C. 2.7.2.4. I of Threonine with same pT 2 plasmids of cutting with the HindIII enzyme.

Intestinal bacteria K17P3 with among the recombinant plasmid pT3 conversion embodiment 4 that is obtained obtains intestinal bacteria K18P3.In minimum medium, cultivate (tryptone 1g/100ml, yeast extract 0.5g/100ml, NaCl 1g/100ml, agar 1.5-2g/100ml.Condition: pH7.0,0.1Mpa 20 minutes), the intestinal bacteria K17P3 bacterial strain and the K16P3 bacterial strain among the embodiment 3 that to not import plasmid pT3 are simultaneously also cultivated in same substratum in contrast, accumulation L-Threonine, from culture, collect the L-Threonine, and compare the output (table 4) of the L-Threonine of intestinal bacteria K16P3, K17P3 and K18P3.

Table 4

Intestinal bacteria K18P3 (asd ThrA ^*+ thrB+ThrC) owing to changed plasmid pT3 over to, and connected aspartate-gene (asd) on the plasmid pT3 and to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene (ThrA ^*), homoserine kinase gene (ThrB) and threonine synthetase gene (ThrC).Therefore, by duplicating of pT3 plasmid, aspartate-gene and the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene, homoserine kinase gene and threonine synthetase gene be enhanced, number of copies increases, thereby and the productive rate of L-Threonine is improved.

Embodiment 6: make up intestinal bacteria K19P3

According to ground method similar to Example 1, ((this bacterial classification is known to handle coli strain BL2125 with chemical mutagen ethyl sulfate (DES) and methane amide, can be by having bought on the market, preserve by this laboratory), mutant strain is cultivated seed selection and further it carried out ultraviolet mutagenesis and composing type seed selection.

The phosphoric acid buffer 100ml sterilization of configuration pH7.0 will be cultivated 20h through the wide spectrum carbon source intestinal bacteria bacterial classifications (K16) (this bacterium is preserved by this laboratory) of screening in broth culture, get concentration then and be about 2.5 * 10 ⁸Cultivation bacterium liquid with physiological saline centrifuge washing twice, with the about 20min of granulated glass sphere vibration, with the bottle swingging machine 20min that vibrates, make it homodisperse again.Bacterium liquid is prepared with phosphoric acid buffer, and mutagenesis reagent adds the small amount of ethanol dissolving earlier with 0.8%-1% (g/100ml) ethyl sulfate (DES) solution 5ml, adds the phosphoric acid buffer mixing then.The method for making of methane amide reagent 70% is similarly got each 2.5ml of bacterium liquid and reagent solution, 37 ℃ of bottle swingging machine vibration 30min, the bacterium liquid 2%Na after the processing ₂S ₂O ₃Termination reaction, coating plate in dilution back in separation screening substratum separation and Culture, uses following substratum: glucose 150g/l, peptone 10g/l. (NH again ₄) ₂SO ₄35g/l, KH ₂PO ₄1.0g/l, MgSO ₄7H ₂O 0.5g/l, CaCO ₃30g/l.PH 7.0), obtain mutant bacteria K16P1 bacterial strain, and further it is carried out ultraviolet mutagenesis, and draw the 4-5ml bacteria suspension, put into plate, bacterium liquid thickness does not surpass 2mm in the ware, ware is placed the irradiation down apart from fluorescent tube 30cm, and the rotating speed of magnetic stirring apparatus is 100r/min, mutation time 5min, inhale 0.1ml irradiation back suspension and add gravy peptone flat board, shake up.The seed selection of cultivating the constitutive mutation strain is with two kinds of substratum with the bacterial strain behind the ultraviolet mutagenesis, be that gravy peptone substratum and the screening that contains orange meal replace cultured method with substratum, exchange is cultivated 3 times repeatedly, seed selection constitutive mutation strain from colony, screening accumulation lysine production is many or to the bacterial strain of the insensitive E.C. 2.7.2.4. III of Methionin, acquisition suddenly change e. coli bl21 25P (to the bacterial strain of the insensitive E.C. 2.7.2.4. III of Methionin), the preserving number of this bacterial classification is: CGMCC No:2367 date saved: on January 28th, 2008.

According to the insensitive E.C. 2.7.2.4. III of L-Methionin gene (lysC ^*) the known nucleotide sequence, design and prepare two types Oligonucleolide primers LYSCF1 (SQE IDNO:15) and LYSCR1 (SQE ID NO:16); LYSCF2 (SQE ID NO:17) and LYSCR2 (SQEID NO:18).With LYSCF1, LYSCR1 is primer, and (preserved by this laboratory, pGEMT is a known carrier, can buy on market with pGEMT-UPM plasmid (the promotor UPM that contains 79 bases); UPM is the promotor that contains 79 bases .) for template carry out pcr amplification (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min); With LYSCF2, LYSCR2 is primer, is that template is carried out pcr amplification (PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s with above-mentioned DNA to the insensitive e. coli bl21 25P of Methionin, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min), discussing each 1 μ l of above-mentioned two kinds of PCR products is template, is primer with LYSCF1, LYSCR1, carry out pcr amplification (amplification condition: 94 ℃ of sex change 30s with ExTaq, 55 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations, 72 ℃, 10min).Reclaim the 1441bp amplified fragments, be connected among the carrier pGEM-T (this plasmid is available from Dalian Bao Bio-Engineering Company), transformed into escherichia coli DH5 α extracts and plasmid purification.The plasmid that is obtained is cut with Ncol I enzyme, and be connected, obtain recombinant plasmid pT4 with the same pT3 plasmid of cutting with Ncol I enzyme.

Intestinal bacteria K18P3 with among the recombinant plasmid pT4 conversion embodiment 5 that is obtained obtains the K19P3 bacterial strain.In minimum medium, cultivate (tryptone 1g/100ml, yeast extract 0.5g/100ml, NaCl 1g/100ml, agar 1.5-2g/100ml.Condition: pH7.0,0.1Mpa 20 minutes), intestinal bacteria K18P3, the K16P3 and the K17P3 that will not import pT4 simultaneously also cultivate in same substratum in contrast, accumulation L-Threonine, from culture, collect the L-Threonine, and compare the output (table 5) of their L-Threonine

Table 5

Intestinal bacteria K19P3 (asd ThrA ^*+ thrB+ThrC lysC ^*) owing to changed plasmid pT4 over to, and connected aspartate-gene (asd) on the plasmid pT4, to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene (ThrA ^*), homoserine kinase gene (ThrB), threonine synthetase gene (ThrC) and to the insensitive E.C. 2.7.2.4. III of L-Methionin gene (lysC ^*).Therefore, by duplicating of pT4 plasmid, the aspartate-gene, be enhanced to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene, homoserine kinase gene, threonine synthetase gene and to the insensitive E.C. 2.7.2.4. III of L-Methionin gene, the number of copies of gene increases, thereby the productive rate of L-Threonine is improved.

Embodiment 7: preparation intestinal bacteria K20P3

According to the known nucleotide sequence of intestinal bacteria PCK gene (ppc), design and prepare two types Oligonucleolide primers PCKF1 (SQE ID NO:19) and PCKR1 (SQE ID NO:20); PCKF2 (SQE ID NO:21) and PCKR2 (SQE ID NO:22).With PCKF1 and PCKR1 is primer, (preserving pGEMT by this laboratory is known carrier with pGEMT-UPM plasmid (the promotor UPM that contains 79 bases), can buy UPM on market is the promotor that contains 79 bases .) carry out pcr amplification (pcr amplification condition: 94 ℃ of sex change 30s for template, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min); With PCKF2 and PCKR2 is primer, with Bacillus coli communis BW3110DNA A carry out pcr amplification (reaction conditions: 94 sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min).With each 1 μ l of above-mentioned two kinds of P C R products is template, is primer with PCKF1 and PCKR1, with ExTaq carry out pcr amplification (the PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 2min10s, 30 circulations, 72 ℃, 10min).Reclaim the amplified fragments of 1714bp, be connected among the carrier pGEM-T (this plasmid is available from Dalian Bao Bio-Engineering Company), transformed into escherichia coli DH5 α extracts and plasmid purification.The plasmid that is obtained is cut with EcoR I enzyme, and be connected, obtain recombinant plasmid pT5 with the same pT4 plasmid of cutting with EcoR I enzyme.

Intestinal bacteria K19P3 with among the recombinant plasmid pT5 conversion embodiment 6 that is obtained obtains the K20P3 bacterial strain.In minimum medium, cultivate (tryptone 1g/100ml, yeast extract 0.5g/100ml, NaCl 1g/100ml, agar 1.5-2g/100ml.Condition: pH7.0,0.1Mpa, 20 minutes), K19P3, the K16P3, K17P3 and the K18P3 bacterial strain that to not import pT5 are simultaneously also cultivated in same substratum in contrast, accumulation L-Threonine is collected the L-Threonine from culture, and compares the output (table 6) of L-Threonine.

Table 6

Intestinal bacteria K20P3 (asd ThrA ^*+ thrB+ThrC lysC ^*Ppc) owing to changed plasmid pT5 over to, and connected aspartate-gene (asd) on the plasmid pT5, to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene (ThrA ^*)+homoserine kinase gene (ThrB), threonine synthetase gene (ThrC), to the insensitive E.C. 2.7.2.4. III of L-Methionin gene (lysC ^*) and intestinal bacteria PCK gene (ppc), by duplicating of plasmid pT5, the aspartate-gene, to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene, homoserine kinase gene, threonine synthetase gene, L-Methionin insensitive E.C. 2.7.2.4. III gene and intestinal bacteria PCK gene are enhanced, the number of copies of gene increases, thereby the productive rate of L-Threonine is improved.

Embodiment 8: preparation intestinal bacteria K21P3

According to known nucleotide sequence, design and prepare two types Oligonucleolide primers VGBF1 (SQE ID NO:23) and VGBR1 (SQE IDNO:24) to Vitreoscilla hemoglobin gene (vgb); VGBF2 (SQE ID NO:25) and VGBR2 (SQE ID NO:26).With VGBF1 and VGBR1 is primer, with pGEMT-UPM plasmid (the promotor UPM that contains 79 bases) (source of plasmid) is that template is carried out pcr amplification (PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 1min, 30 circulate 72 ℃, 10min.); With VGBF2 and VGBR2 is primer, (this bacterial classification is known to Vitreoscilla DNA with the pfu enzyme, be so kind as to give by Peking University agricultural Molecular Biology Lab) carry out pcr amplification (PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 2min10s, 30 circulations, 72 ℃, 10min is 16 ℃ of insulations).With each 1 μ l of above-mentioned two PCR products is template, is primer with VGBF1, VGBR1, carries out pcr amplification (PCR reaction conditions: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃, 10min is 16 ℃ of insulations) with ExTaq.Reclaim the amplified fragments of 532bp, be connected among the carrier pEGM-T (available from Dalian Bao Bio-Engineering Company), transformed into escherichia coli DH5 α extracts and plasmid purification.The acquisition plasmid is cut with Kpn I enzyme, and be connected, obtain recombinant plasmid pT6 with the same pT5 plasmid of cutting with Kpn I enzyme.

Recombinant plasmid pT6 transformed into escherichia coli K20P3 (preserve in this laboratory, referring to embodiment 7) with being obtained obtains the K21P3 bacterial strain.In minimum medium, cultivate (tryptone 1g/100ml, yeast extract 0.5g/100ml, NaCl 1g/100ml, agar 1.5-2g/100ml.Culture condition: pH7.0,0.1Mpa 20 minutes), K20P3, the K16P3, K17P3, K18P3 and the K19P3 bacterial strain that do not import simultaneously plasmid pT6 are also cultivated in same substratum in contrast, accumulation L-Threonine, from culture, collect the L-Threonine, and compare the output (table 7) of their L-Threonine.

Table 7

Intestinal bacteria K21P3 (asd ThrA ^*+ thrB+ThrC lysC ^*Ppc vgb) owing to changed plasmid pT6 over to, and plasmid pT6 has connected aspartate-gene (asd), to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene (ThrA ^*), homoserine kinase gene (ThrB), threonine synthetase gene (ThrC), to the insensitive E.C. 2.7.2.4. III of L-Methionin gene (lysC ^*), intestinal bacteria PCK gene (ppc) and Vitreoscilla hemoglobin gene (vgb).By duplicating of plasmid pT6, the aspartate-gene, to the insensitive E.C. 2.7.2.4. I-of L-Threonine homoserine dehydrogenase I gene, homoserine kinase gene+threonine synthetase gene, the insensitive E.C. 2.7.2.4. III of L-Methionin gene, intestinal bacteria PCK gene and Vitreoscilla hemoglobin gene are enhanced, the number of copies of gene increases, thereby the productive rate of L-Threonine is improved.

By above-mentioned specific embodiment, be more readily understood the present invention.The foregoing description is just described for example, and does not limit protection scope of the present invention in any form.

Sequence table

＜110〉applicant Changchun Dacheng Industry Group Co.,Ltd

＜120〉method of producing L-threonine by fermentation

<160>26

<170>Patent?In?Version?3.0

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<210>2

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<400>2

ctaacccgcc?aaaaagaacc?tgaacgccgg?gttattggtt?tgtaaaacgt?ccgaaggcc 59

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<400>3

gaattcggta?cctctagaga?gctccggaag?tc 32

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<400>4

tctagagagc?tccggaagtc?agcgccctgc?ac 32

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gaattcccat?ggaagcttgg?atccaaccct?gat 33

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aagcttggat?ccaaccctga?taaatgcttc?aataatattg 40

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ggatcctcac?tcattaggca?ccccaggc 28

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catttttcat?atgtatatct?ccttagtaca?t 31

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aagcttttac?tgatgattca?tcatcaat 28

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tctagatcac?tcattaggca?ccccaggc 28

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tctagattac?tcaaacaaat?tactatgc 28

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ggtacctcac?tcattaggca?ccccaggc 28

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taacgcgcat?atgtatatct?ccttagtaca?t 31

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ggtaccttac?agtttcggac?cagccgct 28

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gaattctcac?tcattaggca?ccccaggc 28

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ggtctaacat?atgtatatct?ccttagtaca?t 31

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＜213〉artificial sequence

<400>25

gagatataca?tatgttagac?caacaaaccg?t 31

<210>26

<211>28

<212>DNA

＜213〉artificial sequence

<400>26

gaattcttat?tcagcgtctt?gagcgtac 28

Claims (10)

1, a kind of intestinal bacteria variant, wherein succinyl homoserine synthetic enzyme and dihydrodipicolinic acid synthase's intracellular reactive reduces.

2, intestinal bacteria variant according to claim 1, wherein said intracellular reactive reduce to be finished by chemomorphosis and ultraviolet mutagenesis; After wherein passing through described chemistry and ultraviolet mutagenesis, the sudden change of succinyl homoserine synthetic enzyme comprises: proline(Pro) replaces the 61st Serine, and Serine replaces the 107th glycine, and Histidine replaces the 165th Serine; Dihydrodipicolinic acid synthase's sudden change comprises: L-Ala replaces the 44th Threonine, and Methionin replaces the 55th L-glutamic acid and Histidine and replaces the 65th aspartic acid; Resulting through above-mentioned mutagenesis is intestinal bacteria K16P2 strain.

3, intestinal bacteria variant according to claim 1 and 2, the intracellular reactive that wherein also comprises threonine deaminase reduces, and the intracellular reactive reduction of described threonine deaminase knocks out the threonine deaminase gene by the Red homologous recombination technique and finishes.

4, intestinal bacteria variant according to claim 1 and 2, the intracellular reactive that wherein also comprises E.C. 2.7.2.4. strengthens, the intracellular reactive of wherein said E.C. 2.7.2.4. strengthens by following method to be realized: transform and Threonine bonded site, remove the L-Threonine and remove the feedback inhibition of L-Methionin to E.C. 2.7.2.4. III to the feedback inhibition of E.C. 2.7.2.4. I with by transforming with lysine-binding site; Preferably, wherein said removal L-Threonine comprises the sudden change of E.C. 2.7.2.4. I (AK I) feedback inhibition: with the Serine on 109 of the another kind of aminoacid replacement, with the Serine in 163 of the another kind of amino replacements; Perhaps, wherein replace 109 and go up Serine with glycine; Serine on replacing 163 with L-Ala.

5, intestinal bacteria variant according to claim 4, it is intestinal bacteria BL1126P, the preserving number of this bacterial classification is: CGMCC No:2369.

6, intestinal bacteria variant according to claim 4, wherein said removal L-Methionin comprises the sudden change of the feedback inhibition of E.C. 2.7.2.4. III (AKIII): with the glycine on 330 of the another kind of aminoacid replacement, with the halfcystine on 400 of the another kind of aminoacid replacement, with the glycine on 403 of the another kind of aminoacid replacement; Perhaps, the glycine on replacing 330 with aspartic acid, the halfcystine on replacing 400 with glycine, the glycine on replacing 403 with aspartic acid.

7, intestinal bacteria variant according to claim 6, it is e. coli bl21 25P, the preserving number of this bacterial classification is: CGMCC No:2367.

8, according to any described intestinal bacteria variant among the claim 1-7, the intracellular reactive that wherein further comprises at least a enzyme in aspartate-, homoserine dehydrogenase, homoserine kinase, threonine synthetase and/or the phosphoenolpyruvic acid kinases strengthens, preferably, the active enhancing of wherein said desmo enzyme is to improve its intracellular reactive by increasing the plasmid expression amount.

9, intestinal bacteria variant according to claim 8 wherein further comprises having imported hemoglobin gene; Preferably, wherein said hemoglobin gene derives from the hemoglobin gene of Vitreoscilla; And formed intestinal bacteria variant is genetic engineering bacterium K21P3.

10, a kind of method of producing the L-Threonine, this method are included in cultivates any described intestinal bacteria variant among the claim 1-19 in suitable medium, produce in its substratum and gathering L-Threonine, and therefrom collect the L-Threonine; Wherein said bacterium is the intestinal bacteria through following modification: (1) succinyl homoserine synthetic enzyme and dihydrodipicolinic acid synthase's intracellular reactive weakens; (2) the threonine deaminase intracellular reactive reduces; (3) aspartate-, E.C. 2.7.2.4., homoserine dehydrogenase, homoserine kinase, threonine synthetase and phosphoenolpyruvic acid kinases intracellular reactive strengthen; And/or (4) have imported the hemoglobin gene from Vitreoscilla; Wherein said homoserine synthetic enzyme, dihydrodipicolinic acid synthase, E.C. 2.7.2.4., homoserine dehydrogenase, aspartate-, homoserine kinase, threonine synthetase, phosphoenolpyruvic acid kinases and threonine deaminase come from intestinal bacteria respectively; Preferably, wherein said intestinal bacteria variant is genetic engineering bacterium K21P3; Preferred, wherein said cultivation was carried out under aerobic conditions 36～60 o'clock; Temperature between incubation period is controlled at about 37 ℃; The pH value is controlled between 6～8; And can use inorganic or organic acid or alkaline matter and oxygen adjusting pH value.