CN1757737A - Genetically engineered bacterium for producing L-phenylalanine, construction method and application thereof - Google Patents
Genetically engineered bacterium for producing L-phenylalanine, construction method and application thereof Download PDFInfo
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- CN1757737A CN1757737A CN200510040802.6A CN200510040802A CN1757737A CN 1757737 A CN1757737 A CN 1757737A CN 200510040802 A CN200510040802 A CN 200510040802A CN 1757737 A CN1757737 A CN 1757737A
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- phenylalanine
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- 238000010276 construction Methods 0.000 title claims abstract description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 134
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Abstract
The invention discloses a genetically engineered bacterium for preparing L-phenylalanine, a construction method and production application thereof. The bacterium is obtained by connecting transaminase gene with constitutive promoter, inserting into vector to construct constitutive expression plasmid, and transforming in proper host bacteria. The genetic engineering bacteria expressing high-activity aspartate aminotransferase are used for converting the phenylketoprofen to prepare the L-phenylalanine, and the molar conversion rate can reach 65-85%. The L-phenylalanine is prepared by converting the phenylketonic acid by adopting the genetic engineering bacteria expressing the high-activity aspartate aminotransferase and the enolphosphopyruvate carboxykinase, and the molar conversion rate can reach 80-95 percent. The culture condition of the thalli is simple, the activity is stable, inducers IPTG and coenzyme pyridoxal phosphate are not needed, and the industrial large-scale production is facilitated.
Description
Technical field
The invention belongs to the amino acid preparing technical field, the genetic engineering bacterium that is specifically related to utilize composing type to start prepares the method for L-phenylalanine.
Background technology
L-phenylalanine (English name L-phenylalanine) is the die aromatischen Aminosaeuren with physiologically active, be that human body and animal one of can not the synthetic indispensable amino acids, being widely used in medical for example L-phenylalanine is the intermediate of some amino acids cancer therapy drug, as: phenylpropyl alcohol ammonia benzyl, formic acid Sarkolizin etc. (Wang Duoren. chemical intermediate 2004,1 (2): 12～20).Can be carrier with amino acid again, cancer therapy drug molecule or group are written into tumor area, reach and to suppress tumor growth, can reduce the toxic purpose of tumour medicine (Janet T Powell etc., Eur.J Biochem.1978, (87): 391～400) again.The L-phenylalanine still is the important composition of built amino-acid transfusion, also is to produce suprarenin and thyroxinic raw material.Also be widely used in the foodstuff additive industry, particularly as the raw materials for production of aspartame (Aspartame) and Neotame (Neotame), market demand is very big.
The preparation method of L-phenylalanine mainly contains microbe fermentation method and microbial enzyme method conversion method.
Microbe fermentation method is to have self amino acid needed ability of synthesizing by biology, by processing such as mutagenesis to bacterial strain, select various auxotrophs and amino acid structure analogue resistant mutant, to remove feedback inhibition and the obstruction in the metabolism adjusting, reach the purpose of excessive synthetic L-phenylalanine.The companies such as Monsanto of the U.S. produce with this method in the world.But domesticly do not see the bacterial strain report that high vigor is arranged as yet.
Genetic engineering fungus fermentation method prepares the L-phenylalanine: genetic engineering technique the L-phenylalanine produce aspect the bacterium seed selection to be applied in the fermentation method aspect more, enzyme process prepares L-phenylalanine aspect and uses less.Genetically engineered mainly is to change metabolic regulation seed selection L-phenylalanine high yield bacterium by engineered method in the application that fermentation method prepares in the seed selection of L-phenylalanine production bacterium, studies the more E.coli that has; Brevibacteriums; Three class bacterium such as C.glutamicum (Jia Honghua etc., biological processing .2004,2 (2): 9～12).Wherein of greatest concern with the research of genetic engineering modified E.coli fermentative preparation L-phenylalanine, for example, Sugimoto etc. are put into the gene of L-phenylalanine biosynthetic enzyme system on the plasmid of temperature control promotor, lead people E.coli, regulate metabolism by temperature variation and produce acid, L-phenylalanine production peak reach 28.5g/L (Sugimoto S waits .FermentBioeng.1990,70 (6): 376～380.)。The Monsanto company of the U.S. produces the L-phenylalanine by genetic engineering technique improvement Escherichia coli fermentation, and acid production rate is up to 45g/L.And gone into operation on a large scale, be maximum in the world at present L-phenylalanine manufacturing enterprise (Yang Shunkai. meticulous and specialty chemicals .2001, (2): 18～17).1998, Fudan University model is long to be won to wait and makes up new phenylalanine from strain brevibacterium flavum 3621s and produce bacterium, and the bacterium that sets out of the phenylalanine rate ratio after the fermentation exceeds 1 times, is up to 26.26g/L.But at the bottom of the domestic fermentation production rate, no industrialization report.
Enzyme process synthesizes L-phenylalanine aspect, and the step that plays a decisive role is catalytic by key enzyme.Take the lead in checking order the tyrB gene of having identified coding die aromatischen Aminosaeuren transaminase in the intestinal bacteria and the dna sequence dna of coding aspartate aminotransferase aspC gene such as Britain Fothering.ham in 1986; The WaLbr of the U.S. in 1986 is fixed in the colibacillus engineering of high expression level aspartate aminotransferase and is used for the catalysis phenyl-pyruvic acid in the gelatin.Japanese Tsutomu Takagi in 1991 etc. have cloned the gene of aspartate aminotransferase and have made up phenylalanine with it and produced bacterium; The 1994 history Yan Dong of pharmaceutical university etc. make up genetic engineering bacterium CTB2, and the transformation efficiency that the catalysis phenyl-pyruvic acid is converted into the L-phenylalanine is 81%, and generating the L-phenylalanine is 14.4g/L; Yun-PengChao etc. have made up a strain gene engineering bacterium and have expressed transaminase and L-Aspartase Fourier Series expansion technique production L-phenylalanine simultaneously, also make up another strain gene engineering bacterium and expressed transaminase and phosphoenolpyruvate carboxykinase simultaneously, improved the substrate molar yield; (Yun-Peng Chao etc., Biotechnol.Prog.1999, (15): 453～458; Enzyme and MicrobialTechnology.2000, (27): 19～25).Transform the 20g/L phenyl-pyruvic acid and generate 18.6g/L L-phenylalanine, molar yield reaches 93%.But above expression system according to bibliographical information, needs expensive deleterious inductor IPTG and expensive coenzyme coenzyme pyridoxal phosphate PLP, is unfavorable for large-scale industrial production.
At present, the external existing report that bioengineered strain adopts the glucose fermentation legal system to be equipped with the L-phenylalanine and industrialization is arranged successfully that makes up.And that phenylalanine prepares the domestic research work in aspect is backward relatively, and the efficient and cheap engineering bacteria of fermentation costs does not appear in the newspapers so far as yet.
The microbial enzyme method conversion method has three types:
(1) be that precursor prepares the L-phenylalanine through aspartate aminotransferase with the phenyl-pyruvic acid: change ammonia by the precursor substance phenyl-pyruvic acid through aspartate aminotransferase catalysis and generate the L-phenylalanine, when the L-aspartic acid is amino donor, product oxaloacetic acid instability behind the commentaries on classics ammonia, further decarboxylation generates pyruvic acid, and then reaction is constantly carried out to the direction that generates the L-phenylalanine.Substrate phenyl-pyruvic acid in this method can be by benzylidene glycolylurea hydrolysis preparation, and the L-aspartic acid can be prepared by the fumaric acid ammonification.The inventor has solved benzene feedstock pyruvic acid production technique preferably.Make the breakthrough of this phenylalanine synthesis route become possibility, the seed selection that the high yield of catalysis phenyl-pyruvic acid (PPA) preparation L-phenylalanine is produced bacterium then is another bottleneck that this operational path is badly in need of breakthrough.
(2) be precursor with the styracin through phenylalanine ammonia lyase catalytic preparation L-phenylalanine: phenylalanine ammonia lyase can be decomposed into styracin and ammonia by catalysis L-phenylalanine, thereby can be by its reversed reaction, under suitable condition by styracin and ammonia synthesis L-phenylalanine.Because of the restraining effect of substrate styracin to phenylalanine ammonia lyase, thereby the difficult high density production that realizes the L-phenylalanine.The Chengdu Inst. of Biology, Chinese Academy of Sciences poplar is along pattern etc., through strain improvement for many years, and the report that the existing trial throw of this method is produced.
(3) enzyme process splits: adopt enzyme process selectivity degraded preparation L-phenylalanine on the basis of chemosynthesis racemization intermediate (as N-acetyl DL-phenylalanine), this method cost height, no industrialization report.
Summary of the invention
The invention reside in provides a kind of and efficiently expresses, has good regenerating coenzyme system, the genetic engineering bacterium that constitutive promoter is connected with the gene of aspartate aminotransferase, its fermentation can be saved IPTG and be induced step and reach efficient startup, is beneficial to large scale fermentation, is easy to realize industrialization.
The present invention also is to provide a kind of to have that constitutive promoter is connected with the gene of aspartate aminotransferase and has the placed in-line genetic engineering bacterium of multienzyme coupling, fermentation not only can be saved IPTG and be induced step and reach efficient startup, be beneficial to large scale fermentation, can also use more inexpensive raw material and promote reaction process, be easy to realize industrialization.
The present invention also is to provide a kind of preparation to have that constitutive promoter is connected with the gene of aspartate aminotransferase, further has the method for the placed in-line genetic engineering bacterium of multienzyme coupling, and this method is easy and simple to handle, process stabilizing, good reproducibility.
The present invention also is to provide a kind of cultivation and fermentation method of utilizing engineering bacteria of the present invention to produce the L-phenylalanine, can stablize, inexpensive production, helps large-scale production.
The object of the present invention is achieved like this:
At first be the structure of engineering bacteria:
The constructing plan one of engineering bacteria: select from the gene aspC of aspartate aminotransferase or or aromatic series transaminase gene tyr B a kind of, be connected with the constitutive promoter that makes up voluntarily, insert carrier, construct the recombinant plasmid of constitutive expression, and in different host bacterium, transform, obtain having the aminotransferase gene engineering bacteria of constitutive promoter.Utilize this project bacterium in suitable medium, under the appropriate condition, to ferment and obtain fermented liquid, utilize this fermented liquid fermentative production L-phenylalanine.Its technological line can accompanying drawing 1 expression.
Construction of recombinant plasmid wherein can make up by general method in common, and for example: the design quoted passage angles the aspartate aminotransferase gene aspC that gets e. coli k12, links to each other with the constitutive promoter that makes up voluntarily, imports the T carrier.Utilize the HindIII and the EcoR I double enzyme site of T carrier, be inserted into the carrier after same enzyme is cut respectively.For example: pUC19, pSE380, pET22b is carrier preferably, makes up to obtain plasmid such as example obtains pUC/aspC; PET/aspC; The pSE/aspC plasmid.Import respectively again and obtain the range gene engineering bacteria among the different hosts.The host can select the bacterium used always, and can measure enzyme work and select according to getting fermented liquid after the fermentation culture.For example escherichia coli host JM109, DH-5 α, BL21, TOP10F, Sure, XL1-blue of host preferably, wherein E.coli BL21 is better.Carrier is selected pET, and the host selects the expression enzyme of the E.coliBL21-pET/aspC of E.coli BL21 acquisition to live higher.Utilize this engineering bacteria to ferment, do not need to use costliness and the big IPTG of toxicity to induce step, and can efficiently start, be beneficial to industrial production.
Constitutive promoter wherein can make up with usual method, is fit to import selected carrier as long as be beneficial to be connected with aminotransferase gene.Can utilize the lactose promoter engineering easily, for example: the transcription initiation position of lactose operon+base at several places of 5 to+17 suddenlys change, and it can be become the promotor of composing type, but mutating alkali yl is as follows, but there is underscore at the mutating alkali yl position.
TGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA
ACAACACACC
TTAACACTC
GCC
TA
TTGTTAAAGTGTGT
Again for example: in the lactose manipulation is given, also can insert the promotor (BautistaDS etc. that mutagenesis transform composing type as; Gene1990; 87 (1): 155).
Also can directly adopt e. coli k12 aspartate aminotransferase carry promotor.
The constructing plan two of engineering bacteria: further, in scheme one, select the appropriate host bacterium as required, the natural bacterium E.coli ATCC11303 that for example selects high reactivity L-Aspartase (aspA) is as host (Xu Hong etc., Nanjing University of Chemical Technology's journal, 1996; 18 (1): 70-73), for example pET/aspC insertion of recombinant plasmid with making up obtains genetic engineering bacterium such as example and obtains E.coli ATCC11303-pET/aspC engineering bacteria.Because high reactivity L-Aspartase (aspA) and aspartate aminotransferase coupling mutually among the natural bacterium E.coli ATCC11303, high reactivity L-Aspartase (aspA) wherein can use fumaric acid, ammonia to produce aspartic acid, therefore this project bacterium can be a raw material production L-phenylalanine with fumaric acid and ammonia, reduces production costs effectively.This.Its technological line can accompanying drawing 2 expressions.
The constructing plan three of engineering bacteria: in scheme one, further, when construction recombination plasmid, the enzyme of oxaloacetic acid, for example PCK (pckA) will be decomposed, with transaminase, for example aspC or tyrB connect, and make up the coexpression recombinant plasmid, transform by scheme one like this and obtain engineering bacteria, for example carrier is selected pET, and the host selects E.coli BL21 to obtain E.coli BL21-pET/aspCpck engineering bacteria.Since the enzyme that decomposes oxaloacetic acid for example in this embodiment PCK (pckA) can to decompose with phenyl-pyruvic acid, aspartic acid be raw material transforms preparation L-phenylalanine, conversion reaction through aspartate aminotransferase another product oxaloacetic acid, further decompose and generate pyruvic acid, can promote the conversion reaction balance to carry out along with oxaloacetic acid decomposes, therefore can further improve the productive rate of L-phenylalanine to the direction that the L-phenylalanine generates.Technological line can accompanying drawing 3 expressions.
The constructing plan four of engineering bacteria: in scheme one, on two and three the basis, connect with the transaminase recombinant plasmid of coexpression of the PCK (pekA) that the recombinant plasmid of scheme three decomposes oxaloacetic acid, the host bacterium of import plan two promptly for example transforms among the natural bacterium E.coli ATCC11303 of high reactivity L-Aspartase (aspA), making up the genetic engineering bacterium that obtains as example is E.coli ATCC11303-pET/aspCpckA, in such engineering bacteria, in example, coupling L-Aspartase (aspA), aspartate aminotransferase (aspC, tyrB) enzyme and among PCK (pckA) three, utilize this kind engineering bacteria, can be with fumaric acid, ammonia, phenyl-pyruvic acid is a substrate, high yield directly synthetic L-phenylalanine has promptly reduced the transformation efficiency that cost can improve the L-phenylalanine again.Its technological line can be represented with accompanying drawing 4.
Wherein, the aspartate aminotransferase enzyme activity determination gets final product according to a conventional method, and typical measuring method can be according to substrate phenyl-pyruvic acid and Fe
3+The principle that the complex reaction colour developing takes place is measured.Step is: get the 1ml fermented liquid, add 0.1ml CTAB (1%), constant temperature oscillator (37 ℃, 180rpm) in reaction 5min, add 1ml substrate (ammoniacal liquor is transferred PH8.5 for PPA0.1mol/L, L-Asp 0.11mol/L), reaction 1h (37 ℃, 180rpm), the ice bath termination reaction.Get 20 μ l reaction solutions to 5ml colour developing liquid (DMSD600ml, Glacial acetic acid 20ml, FeCl
30.5g distilled water is settled to 1L) in, the reaction back is surveyed its OD value with spectrophotometer at 640nm wavelength place.Enzyme is defined as the amount of the μ molPPA that per hour every milliliter of fermented liquid consumed, i.e. μ molPPAh than unit alive " μ "
-1Ml
-1Formula is calculated in enzyme work: E=334* (C
0-OD
640), C
0Be to change the 1ml fermented liquid in the system into behind the water measured value.
Plasmid detects and can be undertaken by general method, also can carry out (the soughing of the wind in forest trees etc. by literature method, Chinese biological engineering magazine .2005 (increasing): 313～315[18]), the content of L-phenylalanine and phenyl-pyruvic acid adopts HPCE (HPCE) method to measure (Tang Fang etc., the journal .2003 of Beijing University of Chemical Technology, 30 (5): 21～23).
Secondly, the fermentation of genetic engineering bacterium:
According to for example making up engineering bacteria scheme one or two or three bacterium that obtain: E.coli BL21-pET/aspC (or E.coliBL21-pET/aspCpck) is when fermenting, its substratum can be undertaken by common dosage with substratum LB substratum commonly used, the enzyme work that obtains is at 50～100u/mL, (typical LB liquid nutrient medium (g/L) is a yeast powder 5, peptone 10, NaCl 10, PH7.0; LB liquid nutrient medium (g/L) is a yeast powder 5, peptone 10, and NaCl 10, PH7.0; LB solid medium (g/L) is a yeast powder 5, peptone 10, and NaCl 10, agar powder 18, pH7.2; ) also can to adopt with corn steep liquor be the fermention medium of the Cheap highly effective of main nutrition composition, as carbon source, generally needn't limit especially, available Zulkovsky starch, glucose, sucrose etc., tret is 0～10g/L; Can use corn steep liquor, yeast extract paste, peptone, extractum carnis, fish meal as organonitrogen, tret is 0～20g/L, and corn steep liquor is better.The plasmid that this substratum helps in the fermenting process is stable, can improve expression of exogenous gene, and corn steep liquor contains moisture content, and tret is 0～60ml/L, is preferably 30～50ml/L.In the substratum that with corn steep liquor is main carbon nitrogen source,, can not add other magnesium, iron ion and phosphoric acid salt etc. because of containing each metal ion species and inorganic salt.Also can add salts such as a spot of sodium-chlor.Fermention medium be initially pH6.5～7.5.The aspartate aminotransferase that obtains is than living at 120～260u/mL like this.Do not need to add genetic engineering bacterium inductor IPTG commonly used in the fermenting process.
With the natural bacterium E.coli ATCC11303 that contains high reactivity L-Aspartase (aspA) is the host, the genetic engineering bacterium that makes up is promptly according to constructing plan two or four engineering bacterias that make up for example during E.coli ATCC11303-pET/aspC fermentation, suitably go into a small amount of fumaric acid in the substratum and carry out inducing of L-Aspartase, the fumaric acid addition adds 1～20 gram in every liter of fermented liquid of fermentating liquid volume.Like this, can use fumaric acid and ammoniacal liquor to generate aspartic acid, produce the L-phenylalanine, reduce production costs greatly.
When fermenting, the inoculum size of bacterial classification is selected by general general knowledge, needn't limit especially.Bacterial classification inoculation amount by volume per-cent meter is generally 1～10%, more preferably 3～6%, under 150～300rpm rotating speed stirs, maintain the temperature at 30～45 ℃, fermentation time was generally 4～20 hours, ferment 6～12 hours better.Fermentation is aerobic fermentation.
The 3rd, utilize engineering bacteria to produce the L-phenylalanine.
Press constructing plan one or three engineering bacterias that make up, genetic engineering bacterium E.coli BL21-pET/aspC for example, can collect thalline after the fermentation, also can directly use fermented liquid as enzyme liquid, substrate is phenyl-pyruvic acid and aspartic acid, and the hydrolyzate of also available benzylidene glycolylurea (containing phenyl-pyruvic acid) directly replaces phenyl-pyruvic acid.When directly using fermented liquid as enzyme liquid, the addition by volume enzyme liquid of enzyme liquid amasss and the substrate volume ratio is 1: 1～1: 6, is preferably 1: 2～1: 4.The content of phenyl-pyruvic acid is 10～90g/L, more preferably 30～60g/L in the fermented liquid.The content of phenyl-pyruvic acid is 10～70g/L, more preferably 30～50g/L when directly using hydrolyzed solution.Can add a small amount of broken wall agent (as the CTAB-cetyl trimethylammonium bromide) in the conversion fluid, also can not add the broken wall agent and directly transform, addition is counted 0-0.3% with volume percent.The ratio of phenyl-pyruvic acid and aspartic acid is 1: 1～1: 2, be preferably 1: 1.1～and 1.3.The pH of conversion fluid is 7.5～9.0.Needn't add the coenzyme pyridoxal phosphate in the conversion fluid.Invert point is 30～40 ℃.At this moment, transformation time was generally 2～15 hours, and more preferably 3～7 hours, through the HPLC detection computations, the L-phenylalanine content of every liter of conversion fluid was at 12～50 grams, and productive rate can reach 60～96%.
The genetic engineering bacterium that the engineering bacterias that make up by constructing plan two or four are the host with the natural bacterium E.coliATCC11303 that contains high reactivity L-Aspartase (aspA) promptly, E.coli ATCC11303-pET/aspC for example, can collect thalline after the fermentation, also can directly use fermented liquid as enzyme liquid, substrate is to use phenyl-pyruvic acid and aspartic acid, for further reducing cost, substrate also can be used phenyl-pyruvic acid, fumaric acid and ammonia, the hydrolyzate of also available benzylidene glycolylurea, promptly contain the reaction solution of phenyl-pyruvic acid, directly replace phenyl-pyruvic acid.When directly using fermented liquid as enzyme liquid, the addition by volume of enzyme liquid amasss for enzyme liquid and the substrate volume ratio is 1: 1～1: 6, is preferably 1: 1.5～1: 4.The content of phenyl-pyruvic acid is 10～90g/L, more preferably 30-60g/L in the fermented liquid.The content of phenyl-pyruvic acid is 10～70g/L, more preferably 30～50g/L when directly using hydrolyzed solution.Can add a small amount of broken wall agent (as the CTAB-cetyl trimethylammonium bromide) in the conversion fluid, also can not add the broken wall agent and directly transform, addition is counted 0-0.3% with volume percent.The ratio of phenyl-pyruvic acid and fumaric acid is 1: 1～1: 2, more preferably 1: 1.1～1: 1.5.The pH of conversion fluid is 7.5～9.0.Needn't add the coenzyme pyridoxal phosphate in the conversion fluid.Invert point is 30～40 ℃.At this moment, transformation time was generally 2～20 hours, and more preferably 4～9 hours, through the HPLC detection computations, the L-phenylalanine content of every liter of conversion fluid restrained at 12-45, and productive rate can reach 60-85%.
Adopt connect with PCK (pckA) the genetically engineered bacterium E.coliBL21-pET/aspCpck for example of coexpression of transaminase, the molar yield that phenyl-pyruvic acid is converted into the L-phenylalanine can further improve, and reaches 75～96%.
The genetic engineering bacterium E.coli ATCC11303-pET/aspC that employing is the host with natural bacterium E.coli ATCC11303, substrate can replace aspartic acid with fumaric acid and ammonia, needn't add the coenzyme pyridoxal phosphate in the conversion fluid, can further reduce cost.
Simultaneously, the culture condition of thalline is fairly simple, is easy to obtain activity stabilized thalline, therefore is beneficial to industrialization scale operation.
Description of drawings
Fig. 1 produces the technological line synoptic diagram of L-phenylalanine
Fig. 2 produces the technological line synoptic diagram of L-phenylalanine
Fig. 3 produces the technological line synoptic diagram of L-phenylalanine
Fig. 4 produces the technological line synoptic diagram of L-phenylalanine
Embodiment
The structure of embodiment 1 genetic engineering bacterium E.coli BL21-pUC/aspC
Aspartate aminotransferase aspC gene is transferred from intestinal bacteria K12 (reference culture).By the NCBI retrieval, as follows through VectorNTI8.0 software design primer according to e. coli k12 (reference culture) the aspC sequence of report:
Forward primer 1:5 ' ATGTTTGAGAACATTACCGC 3 '
Reverse primer 2:5 ' GTTTGTCATCAGTCTCAGCC 3 '.
Adopt PCR to angle the aspartate aminotransferase gene aspC that gets e. coli k12, link to each other with following constitutive promoter (being designated as P-121), (ruling as follows in the change base position of this promotor),
TGTTGTGTGGAATTGTGAGTGGCTAACAATTTCACACA
ACAACACACCTTAACACTC
ACC
GATTGTTAAAGTGTGT
And P121-aspC is inserted in the pMD-18T of the TaKaRa company carrier, identify angling the aspC gene of getting to carry out order-checking.Utilize the HindIII and the EcoR I site of T carrier, double digestion obtains the new segment P121-aspC that has restriction enzyme site.Be inserted among the carrier pUC18 after same enzyme is cut.Obtain recombinant plasmid pUC/aspC.Preparation E.coli BL21 competent cell imports recombinant plasmid pUC/aspC among the E.coli BL21, selects recon E.coli BL21-pUC/aspC.
Embodiment 2 genetic engineering bacterium E.coli BL21-pET/aspC
Aspartate aminotransferase gene aspC links to each other with following constitutive promoter (being designated as P123), and (ruling as follows in the change base position of this promotor) obtains P123-aspC.
TGTTGTGTGGAATTGTGAGCGGATTGCAATTTCACACA
ACAACACACCTTAACACTCGCCTA
ACGTTAAAGTGTGTP
P123-aspC is inserted in the pMD-18T of the TaKaRa company carrier, identifies angling the aspC gene of getting to carry out order-checking.Utilize the HindIII and the EcoR I site double digestion of T carrier to obtain the new segment P123-aspC that has restriction enzyme site.Be inserted among the carrier pET22b after same enzyme is cut.Obtain recombinant plasmid pET/aspC.Preparation E.coli BL21 competent cell imports recombinant plasmid pET/aspC among the E.coli BL21, selects recon E.coli BL21-pET/aspC.
The structure of embodiment 3 genetic engineering bacterium E.coli ATCC11303-pUC/aspC
Preparation contains the competent cell of the natural bacterium E.coli ATCC11303 of high reactivity L-Aspartase (aspA), and the recombinant plasmid pUC/aspC that makes up among the embodiment 1 is imported among the E.coli ATCC11303, selects recon E.coliATCC11303-pUC/aspC.
The structure of embodiment 4 genetic engineering bacterium E.coli ATCC11303-pET/aspC
Preparation contains the competent cell of the natural bacterium E.coli ATCC11303 of high reactivity L-Aspartase (aspA), and the recombinant plasmid pET/aspC that makes up among the embodiment 2 is imported among the E.coli ATCC11303, selects recon E.coliATCC11303-pET/aspC.
The structure of embodiment 5 genetic engineering bacterium E.coli BL21-pET/aspCpckA
Adopt PCR to angle the PCK (pckA) of getting e. coli k12, P121 links to each other with constitutive promoter, and P121-pckA is inserted in the pMD-18T of the TaKaRa company carrier, identifies angling the pckA structure gene of getting to carry out order-checking.Again P121-aspC is connected with P121-pckA, is inserted among the carrier pET22b.Obtain recombinant plasmid pET/aspCpckA.Preparation E.coli BL21 competent cell imports recombinant plasmid pET/aspCpckA among the E.coli BL21, selects recon E.coli BL21-pET/aspCpckA.
The structure of embodiment 6 genetic engineering bacterium E.coli ATCC11303-pET/aspCpckA
Preparation contains the competent cell of the natural bacterium E.coli ATCC11303 of high reactivity L-Aspartase (aspA), the recombinant plasmid pET/aspCpckA that makes up among the embodiment 5 is imported among the E.coli ATCC11303, select recon E.coli ATCC11303-pET/aspCpckA.
Embodiment 7 genetically engineered fermented liquids 1
The fermentation of genetic engineering bacterium E.coli BL21-pET/aspC (genetic engineering bacterium E.coli BL21-pUC/aspC or E.coliBL21-pET/aspCpckA) is prepared fermented liquid by following part by weight: corn steep liquor 30ml/L, yeast extract paste 3g/L, starch 2g/L, NaCl 1g/L regulates pH value 7.0.Sterilization back inoculum size is 3% (seed culture medium is identical, incubation time be 8 hours), is that 37 ℃ and rotating speed are under 180rpm stirs, to ferment 10 hours in temperature.
Embodiment 8 genetically engineered fermented liquids 2
The fermentation of genetic engineering bacterium E.coli ATCC11303-pET/aspC (or E.coli ATCC11303-pET/aspCpckA) is prepared fermented liquid by following part by weight: corn steep liquor 25ml/L, yeast extract paste 3g/L, starch 2g/L, fumaric acid 5g/L, NaCl1g/L regulates pH value 7.0.Sterilization back inoculum size is 3% (seed culture medium is identical, incubation time be 8 hours), is that 37 ℃ and rotating speed are under 180rpm stirs, to ferment 10 hours in temperature.
Embodiment 9 genetically engineered fermented liquids 3
The fermentation of genetic engineering bacterium E.coli JM109-pET/aspC (or E.coli JM109-pET/aspCpckA) is prepared fermented liquid by following part by weight: corn steep liquor 30ml/L, and yeast extract paste 3g/L, starch 2g/L, NaCl 1g/L regulates pH value 7.0.Sterilization back inoculum size is 3% (seed culture medium is identical, incubation time be 8 hours), is that 37 ℃ and rotating speed are under 180rpm stirs, to ferment 10 hours in temperature.
Embodiment 10 transformation experiments 1
Genetic engineering bacterium E.coli BL21-pET/aspC fermented liquid (genetically engineered fermented liquid 1) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid, and the molar ratio of phenyl-pyruvic acid and aspartic acid is 1: 1.2, needn't add the coenzyme pyridoxal phosphate in the conversion fluid.36 ℃ of oscillatory reactions (or stirring reaction) 8 hours can get the L-phenylalanine of 23.6g/L, the molar yield of phenyl-pyruvic acid about 79%.The L-phenylalanine is 72.8% to the molar yield of benzylidene glycolylurea.
Embodiment 11 transformation experiments 2
Genetic engineering bacterium E.coli BL21-pET/aspCpckA fermented liquid (genetically engineered fermented liquid 1) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid, the pH of conversion fluid is 8.5, and the molar ratio of phenyl-pyruvic acid and aspartic acid is 1: 1.2,36 ℃ of oscillatory reactions (or stirring reaction) 8 hours, can get the L-phenylalanine of 26.3g/L, the molar yield of phenyl-pyruvic acid about 88%.The L-phenylalanine is 79.6% to the molar yield of benzylidene glycolylurea.
Embodiment 12 transformation experiments 3
Genetic engineering bacterium E.coli BL21-pET/aspCpckA fermented liquid (genetically engineered fermented liquid 1) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid; the pH of conversion fluid is 8.5; the molar ratio of phenyl-pyruvic acid and aspartic acid is 1: 1.2; inflated with nitrogen protection phenyl-pyruvic acid is avoided oxygenolysis before transforming; 36 ℃ of oscillatory reactions (or stirring reaction) 8 hours; can get the L-phenylalanine of 28.1g/L, the molar yield of phenyl-pyruvic acid is about 94%, and the L-phenylalanine is 85.7% to the molar yield of benzylidene glycolylurea.
Embodiment 13 transformation experiments 3
The fermented liquid of genetic engineering bacterium E.coli ATCC11303-pET/aspC (genetically engineered fermented liquid 2) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid, substitutes aspartic acid with fumaric acid and ammonia, and the molar ratio of phenyl-pyruvic acid and fumaric acid is 1: 1.2, and the pH of conversion fluid is 8.5, needn't add the coenzyme pyridoxal phosphate in the conversion fluid.36 ℃ of oscillatory reactions (or stirring reaction) 12 hours can get the L-phenylalanine of 21.4g/L, and the molar yield of phenyl-pyruvic acid is about 71%, and the L-phenylalanine is 65.1% to the molar yield of benzylidene glycolylurea.
Embodiment 14 transformation experiments 4
The fermented liquid of genetic engineering bacterium E.coli ATCC11303-pET/aspCpck (genetically engineered fermented liquid 2) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid, substitute aspartic acid with fumaric acid and ammonia, the molar ratio of phenyl-pyruvic acid and fumaric acid is 1: 1.2,36 ℃ of oscillatory reactions (or stirring reaction) 12 hours, can get the L-phenylalanine of 26.1g/L, the molar yield of phenyl-pyruvic acid about 87%.The L-phenylalanine is 79.4% to the molar yield of benzylidene glycolylurea.
Embodiment 15 transformation experiments 5
The fermented liquid of genetic engineering bacterium E.coli ATCC11303-pET/aspCpck (genetically engineered fermented liquid 2) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid; substitute aspartic acid with fumaric acid and ammonia; the molar ratio of phenyl-pyruvic acid and fumaric acid is 1: 1.2; inflated with nitrogen protection phenyl-pyruvic acid is avoided oxygenolysis before transforming; 36 ℃ of oscillatory reactions (or stirring reaction) 12 hours; can get the L-phenylalanine of 27.3g/L, the molar yield of phenyl-pyruvic acid is about 91%, and the L-phenylalanine is 83.5% to the molar yield of benzylidene glycolylurea.
Embodiment 16 transformation experiments 6
Genetic engineering bacterium E.coli JM109-pET/aspC fermented liquid (genetically engineered fermented liquid 3) is directly as enzyme liquid, with the hydrolyzate (phenyl-pyruvic acid that contains 40g/L) of benzylidene glycolylurea with 1: 3 mixed.The content of phenyl-pyruvic acid is 30g/L in the conversion fluid, and the molar ratio of phenyl-pyruvic acid and aspartic acid is 1: 1.2, needn't add the coenzyme pyridoxal phosphate in the conversion fluid.36 ℃ of oscillatory reactions (or stirring reaction) 8 hours can get the L-phenylalanine of 22.5g/L, the molar yield of phenyl-pyruvic acid about 76%.The L-phenylalanine is 70.2% to the molar yield of benzylidene glycolylurea.
Claims (10)
1, a kind of genetic engineering bacterium that is used to prepare the L-phenylalanine, in the host bacterium, transform and get by expression plasmid, it is characterized in that its expression plasmid is to be connected the constitutive expression plasmid of insertion vector construction one-tenth with constitutive promoter with a kind of in aspartate aminotransferase gene aspC or the aromatic series transaminase gene tyr B.
2, a kind of genetic engineering bacterium that is used to prepare the L-phenylalanine according to claim 1, it is characterized in that wherein carrier is selected from a kind of among pUC18, pET22b or the pSE380, host bacterium wherein is selected from a kind of of escherichia coli host JM109, DH-5 α, BL21, TOP10F, Sure or XL1-blue.
3, a kind of genetic engineering bacterium that is used to prepare the L-phenylalanine according to claim 1 is characterized in that host bacterium wherein is the natural bacterium E.coli ATCC11303 with high reactivity L-Aspartase aspA.
4, a kind of genetic engineering bacterium that is used to prepare the L-phenylalanine according to claim 1 is characterized in that also having connected in the expression plasmid wherein by decomposing the enzyme of oxaloacetic acid and being connected of constitutive promoter.
5, a kind of genetically engineered that is used to prepare the L-phenylalanine according to claim 4, the enzyme that it is characterized in that decomposition oxaloacetic acid wherein are enol pyruvic acid carboxylic kinases pck.
6, a kind of genetic engineering bacterium of the production L-phenylalanine according to claim 4 is characterized in that host wherein is the natural bacterium E.coli ATCC11303 of high reactivity L-Aspartase aspA.
7, a kind of genetic engineering bacterium that utilizes claim 1 or 4 prepares the method for L-phenylalanine, obtains fermented liquid with the culture medium culturing genetic engineering bacterium, fermented liquid is added in the solution that contains phenyl-pyruvic acid and aspartic acid carry out conversion reaction.
8, a kind of method of utilizing the genetic engineering bacterium production L-phenylalanine of claim 3 or 6, obtain fermented liquid with the culture medium culturing genetic engineering bacterium, fermented liquid added to contain in fumaric acid, ammonia and the phenyl-pyruvic acid solution carry out conversion reaction, adding a small amount of fumaric acid in substratum is auxiliary carbon source, and the addition of fumaric acid is counted 1-20g/L with culture volume.
9, a kind of genetic engineering bacterium that utilizes claim 7 method of producing the L-phenylalanine, wherein substratum is selected from the LB substratum or the corn oar is the substratum of main carbon nitrogen source, and the addition of corn oar substratum is 10-60ml/L.
10, a kind of genetic engineering bacterium that utilizes claim 8 method of producing the L-phenylalanine, wherein substratum is selected from the LB substratum or the corn oar is the substratum of main carbon nitrogen source, and the addition of corn oar substratum is 10-60ml/L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| CN102181503A (en) * | 2011-04-15 | 2011-09-14 | 江苏汉光生物工程有限公司 | Method for producing L-phenylalanine (L-Phe) through fermentation |
| CN103911333A (en) * | 2014-04-16 | 2014-07-09 | 南京工业大学 | Bacterial strain for producing high-yield phenylalanine and method for producing phenylalanine by bacterial strain |
| CN104862264A (en) * | 2015-06-02 | 2015-08-26 | 江南大学 | Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency |
| CN107012179A (en) * | 2017-05-15 | 2017-08-04 | 南京医科大学 | The enzymatic conversion preparation method of 3,4 dimethoxy L phenylalanines |
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| JP2022550349A (en) | 2019-09-25 | 2022-12-01 | 味の素株式会社 | Method for producing 2-methylbutyric acid by bacterial fermentation |
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| DE3631829A1 (en) * | 1986-09-19 | 1988-07-28 | Hoechst Ag | CLONING AND USE OF THE TRANSAMINASE GENE TYRB |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| CN102181503A (en) * | 2011-04-15 | 2011-09-14 | 江苏汉光生物工程有限公司 | Method for producing L-phenylalanine (L-Phe) through fermentation |
| CN103911333A (en) * | 2014-04-16 | 2014-07-09 | 南京工业大学 | Bacterial strain for producing high-yield phenylalanine and method for producing phenylalanine by bacterial strain |
| CN103911333B (en) * | 2014-04-16 | 2016-04-13 | 南京工业大学 | Bacterial strain for producing high-yield phenylalanine and method for producing phenylalanine by bacterial strain |
| CN104862264A (en) * | 2015-06-02 | 2015-08-26 | 江南大学 | Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency |
| CN104862264B (en) * | 2015-06-02 | 2018-05-04 | 江南大学 | A kind of recombinant bacterium for converting production α-phenylpyruvic acid efficiency and improving |
| CN107012179A (en) * | 2017-05-15 | 2017-08-04 | 南京医科大学 | The enzymatic conversion preparation method of 3,4 dimethoxy L phenylalanines |
| CN107012179B (en) * | 2017-05-15 | 2020-05-29 | 南京医科大学 | Enzymatic conversion preparation method of 3, 4-dimethoxy-L-phenylalanine |
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