CN108220289A - A kind of polynucleotide, transformant and its application - Google Patents
A kind of polynucleotide, transformant and its application Download PDFInfo
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Abstract
The present invention relates to a kind of polynucleotides, the invention further relates to a kind of polynucleotide, the transformants in the manipulation site containing promoter and bacteriophage lambda aporepressor CI, the invention further relates to the applications of the polynucleotide, transformant, particularly in fermenting and producing polypeptide, application in terms of producing 1,5 pentanediamines especially with the polypeptide product of fermenting and producing.A kind of first polynucleotide, the complementary base sequences thereof of the sequence of first polynucleotide sequence for the sequence as shown in SEQ ID NO 1 or as shown in SEQ ID NO 1.A kind of second polynucleotide, second polynucleotide include a promoter and the first polynucleotide as described above.A kind of transformant, the transformant include the second polynucleotide as described above.The present invention solves λ PRThe technical issues of expression effect is bad under T 41C inductive conditions provides new selection for induction recombinant expression.
Description
Technical field
The present invention relates to a kind of polynucleotides, further relate to manipulation of this polynucleotide as bacteriophage lambda aporepressor CI
The application in site；The invention further relates to a kind of polynucleotide of the manipulation sequence containing promoter and bacteriophage lambda aporepressor CI, turn
Beggar, the invention further relates to the application of the polynucleotide, transformant, particularly in fermenting and producing polypeptide, especially with fermentation
Application in terms of the polypeptide product production 1,5- pentanediamines of production.
Background technology
In the fermentation for the purpose of expressing recombinant protein, usually used constitutive promoter or inducible promoter are (again
Claim inducible promoters) start the expression of recombination.Constitutive promoter is expressed as persistent expression, without regulation and control.
Inducible promoter is divided into check type promoter and activated form promoter, and by different transcription factors, (also known as transcription is adjusted respectively
Control albumen) control.It checks type promoter to combine with aporepressor (or transcriptional repressor proteins), be suppressed；In inductive condition
Under, aporepressor is dissociated with promoter sequence, derepression.It, can when aporepressor lacks for checking type promoter
It is considered as in successive induction state, can also treats as constitutive promoter.Such as Plac promoters are inhibited by aporepressor LacI, are
Inducible promoter.But lac promoters can be with constitutive expression in the host strain that cannot express LacI albumen.Activated form starts
Son cannot be expressed under the conditions of non-induced or expression efficiency is low；Under inductive condition, activator protein is activated, and starts with activated form
Son combines, and promotes its expression.Such as PBADPromoter is activated by activator protein AraC.
There are two key element, -10 elements and -35 elements in the promoter of prokaryotes, respectively positioned at transcription initiation
Point upstream about -35 and -10 base positions, are sigma factor recognition sites.Sigma factor is prokaryotes rna polymerase holoenzyme (including RNA
Polymerase core enzyme and sigma factor) in confactor.Sigma factor promotes RNA polymerase to be combined with promoter sequence, starts transcription.
In the sequence of inducible promoter, Sequences upstream and/or sequence downstream have transcription factor (aporepressor swash
Living protein) manipulation site.Transcription factor by and manipulate site combination control promoter.Such as in bacteriophage lambda genome
Manipulation site containing 6 bacteriophage lambda aporepressor CI (abbreviation λ CI)：OL1 (TATCACCGCCAGTGGTA), OL2
(CAACACCGCCAGAGATA), OL3 (TATCACCGCAGATGGTT), OR1 (TACCTCTGGCGGTGATA), OR2
(TAACACCGTGCGTGTTG), OR3 (TATCACCGCAAGGGATA).L promoters (the abbreviation λ regulated and controled in bacteriophage lambda by λ CI
PL) and R promoters (abbreviation λ PR) -10 elements and -35 elements all manipulate Post sections with two λ CI respectively and overlap, therefore λ CI
And the combination of RNA polymerase and promoter can be hindered by manipulating the combination in site, inhibit transcription.λ CI and each combination for manipulating site
It all has a certain impact to the activity of promoter.For λ PRFor, when OR1 and OR2 act synergistically, the inhibiting effect that rises
It is maximum；The inhibiting effect that OR1 and OR3 has acted synergistically is taken second place；And OR2 and OR3 synergistic effects or each site that manipulates individually are made
It is weaker (see Lewis etc., " New insights into the phage genentic switch with risen inhibiting effect:
Effects of bacteriophage lambda operator mutations on DNA looping and
regulation of PR,PL,and PRM", Journal of Molecular Biology (2016), doi:10.1016/
j.jmb.2016.08.027)。
Wild type λ CI can be autotomyed by RecA is protein induced, release the inhibition to controlled promoter.The expression of RecA albumen
With activity by the SOS reacting activations caused by host strain DNA break.Therefore the condition of host strain DNA break is can result in, is such as changed
Learn derivant mitomycin C or nalidixic acid or UV radiation, the expression of promoter that λ CI can be induced to be controlled.With it is wild
Type λ CI are compared, and what is be used widely in recombinant expression is the thermally labile saltant type CI857 of λ CI (see Valdez-Crus
Deng " Production of recombinant proteins in E.coli by the heat inducible
Expression system based on the phage lambda pL and/or pR promoters ", Microbial
Cell Factories(2010)9:18).The thermally labile saltant type of λ CI further includes：I21S, G53S, A62T, V73A,
F141I/P153L, N207T, K224E are (see Jana etc., " Amino acid changes in the repressor of
bacteriophage lambda due to temperature-sensitive mutations in its cI gene
And the structure of a highly temperature-sensitive mutant repressor ", Protein
Engineering (1999) Volume 12, Issue 3, pp225-233).The aporepressor of thermally labile is at a lower temperature
(such as 30 DEG C or lower) have the activity for inhibiting transcription, and (such as 42 DEG C) lose inhibitory activity, thus its institute at relatively high temperatures
The promoter of control has the characteristic of thermal induction.
Composition inducible promoter and the element of manipulation sequence can be used for building Artificial promoters.For example, De Boer etc.
Sequence of the people by Plac promoters and downstream containing -10 elements and LacI operons contains the sequence of -35 elements with Ptrp promoters
With reference to constructing the expressive function Ptac promoter more more powerful than Plac promoter and Ptrp promoters (see " The tac
promoter:A functional hybrid derived from the trp and lac promoters ",
Proceedings of the National Academy of Sciences of the United States of
America (1983), Volume 80, Issue 1, pp21-25).Therefore research inducible promoter and its manipulate sequence and
Their mutant, obtain it is new can application element thereof it is particularly significant for molecular biotechnology.Jechlinger et al. is disclosed
Phageλ PRThe saltant type λ P of promoterRT-41C is (see " Modulation of gene expression by promoter
Mutants of the λ cI857/pRM/pR system ", Journal of Biotechnology (2005) Volume116,
Issue 1, pp11-20).Mutating alkali yl T-41C is located at λ PRIn the manipulation site OR2 sequences of promoter.This λ PRPromoter
Recombinant expression amount of the saltant type under inductive condition is less than wild type λ PRPromoter.
Inventor is had found in λ PRAnother point mutation G- being located in OR2 sequences is introduced in T-41C promoters
38A improves saltant type λ PRExpression quantity of the promoter under inductive condition.This case provides a new λ CI and manipulates site
Sequence.
Invention content
The first aspect of the present invention purpose is a kind of first polynucleotide of proposition, and first polynucleotide can conduct
The manipulation site of bacteriophage lambda aporepressor CI.
The first polynucleotide proposed in the present invention, sequence is for the sequence as shown in SEQ ID NO 1 or such as SEQ ID
The complementary base sequences thereof of sequence shown in NO 1.
The polynucleotide can manipulate Sites Combination with 1,2,3,4 or 5 λ CI becomes the behaviour of promoter
Vertical sequence.Described 1,2,3,4 or 5 λ CI manipulate manipulation site OL1 of the site in bacteriophage lambda genome,
OL2, OL3, OR1, OR2, OR3, the first polynucleotide, manipulation site OL1, OL2, OL3, OR1 in bacteriophage lambda genome,
The set of the saltant type of OR2, OR3 and the saltant type of the first polynucleotide composition.
The polynucleotide can be in promoter sequence upstream, downstream or embedded promoter sequence.In certain implementations
In example, -10 elements or -35 element sequences of the polynucleotide and promoter partially overlap.
The promoter can be natural promoter, the saltant type of natural promoter or Artificial promoters.
The promoter can inhibit to transcribe by the mutant of bacteriophage lambda aporepressor CI (abbreviation λ CI) or λ CI.λ CI are mutated
The λ CI albumen of the preferred thermally labile of body.The λ CI albumen of thermally labile can inhibit the transcription of promoter in lower temperature, and work as
Temperature then loses inhibitory activity when increasing.Thermally labile saltant type λ CI albumen of the present invention includes but not limited to CI857,
I21S, G53S, A62T, V73A, F141I/P153L, N207T, K224E.CI857 the 66th amino acids compared with wild type λ CI
Residue becomes threonine from alanine.In certain embodiments of the present invention, it is residual also to can further include other amino acid by CI857
The mutation in base site, the mutation of other acid residues sites are not influenced caused by its 66th amino acids residue mutations
Thermal instability.
The second aspect of the present invention is to propose a kind of second polynucleotide, second polynucleotide contain promoter and
The manipulation sequence of promoter, the manipulation sequence of the promoter include the first of bacteriophage lambda aporepressor CI and manipulate site (abbreviation λ
CI first manipulates site), the first manipulation site of the λ CI is the first polynucleotide as described above.
The promoter can be natural promoter, the saltant type of natural promoter or Artificial promoters.In an implementation
In example, second polynucleotide includes the saltant type G-38A/T-41C of bacteriophage lambda R promoters.Alternatively, second poly-nuclear
The complementary base sequences thereof of thuja acid sequence comprising the sequence as shown in SEQ ID NO 17 or as shown in SEQ ID NO 17.
The manipulation sequence of promoter of the present invention includes λ CI first of the sequence as shown in SEQ ID NO 1 and manipulates position
Point.Further, the manipulation sequence of the promoter can also include the manipulation position of 1,2,3,4 or 5 the 2nd λ CI
Point.The promoter manipulates 1,2,3, the 4 or 5 λ CI second also contained in sequence and manipulates site selected from λ phagocytosis
The saltant type of manipulation site OL1, OL2, OL3, OR1, OR2, OR3 and above-mentioned manipulation site in body genome.
The promoter can inhibit to transcribe by the mutant of bacteriophage lambda aporepressor CI and λ CI.
The promoter is heat shock promoter, chemically inducible promoter or radiation-induced promoter.
Second polynucleotide is expression cassette, plasmid vector, plasmid, phage genome, transposons either in place
Polynucleotide in key-gene group.
Further, second polynucleotide includes the polynucleotide of coding polypeptide, and the expression of the polypeptide is opened by described
Mover controls.
Further, the polypeptide includes enzyme and polypeptide drug.The enzyme includes oxidoreducing enzyme, transferase, water
Solve enzyme, lyase, at least one of isomerase and ligase.Still further, the enzyme is amino acid decarboxylases, is relied in this way
Propylhomoserin decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase.Polypeptide drug packet
Include hormone, antibody, growth factor etc..In one embodiment, the polypeptide of expression is proinsulin.
Further, the polynucleotide of the coding lysine decarboxylase is selected from following at least one：CadA genes, ldcC
Gene, haldc genes, the segment of cadA genes, the segment of ldcC genes, the segment of haldc genes；Alternatively, the coding relies
The polynucleotide of propylhomoserin decarboxylase is selected from following at least one：The sequence DNA as shown in SEQ ID NO 2, SEQ ID NO 3
Shown DNA, DNA of the sequence as shown in SEQ ID NO 4, the segment of DNA of the sequence as shown in SEQ ID NO 2, sequence is such as
The segment of DNA shown in SEQ ID NO 3, the segment of DNA of the sequence as shown in SEQ ID NO 4.
The third aspect of the present invention is to propose a kind of transformant, and the transformant contains the second poly-nuclear glycosides as described above
Acid.
A kind of transformant, the transformant include the second polynucleotide as described in any one technical solution above.
Preferably, the transformant includes the second polynucleotide described in technical solution above, institute in the technical solution
It states polypeptide and includes lysine decarboxylase.
On the basis of any of the above-described technical solution further, coding λ CI or its saltant type are contained in the transformant
Polynucleotide.Further, the coding λ CI or the polynucleotide of its saltant type are on plasmid；Further, the coding λ
The polynucleotide of CI or the polynucleotide of its saltant type and the coding polypeptide is on same plasmid.Alternatively, the coding λ CI
Or the polynucleotide of its saltant type is in host genome.
On the basis of any of the above-described technical solution further, the host of the transformant is prokaryote.
On the basis of any of the above-described technical solution further, the host of the transformant includes but not limited to Escherichia coli
(Escherichia.coli), hafnia alvei (Hafnia alvei), Corynebacterium glutamicum (Corynebacterium
Glutamicum), clostridium acetobutylicum (Clostridium acetobutylicum), bacillus subtilis (Bacillus
subtilis)。
The fourth aspect of the present invention purpose is to propose a kind of method of fermenting and producing polypeptide.
A kind of method of fermenting and producing polypeptide, which is characterized in that include the following steps：
A the transformant as above described in any one technical solution) is cultivated；
B polypeptide is obtained in the bacterium solution or thalline that) are obtained from step A.
Preferably, the method for a kind of fermenting and producing polypeptide is the method for fermenting and producing lysine decarboxylase, including
Following steps：
1) transformant (certainly, the technical solution described in polypeptide packet of the culture as described in any one technical solution above
Include lysine decarboxylase)；
2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1.
On the basis of any of the above-described technical solution further, the transformant starts recombinant expression under inductive condition,
Inductive condition is thermal induction, chemical induction or radiation-induced.
On the basis of any of the above-described technical solution further, inductive condition is thermal induction, and the inductive condition refers to temperature
It is 32 DEG C -48 DEG C.
The fifth aspect of the present invention purpose is to propose a kind of method of fermenting and producing 1,5- pentanediamines and a kind of biology base
1,5- pentanediamines.
A kind of fermenting and producing 1, the method for 5- pentanediamines include the following steps：
I) the step 1 production lysine decarboxylase of the method for fermenting and producing lysine decarboxylase as described above；
II it) is catalyzed with the bacterium solution or thalline obtained in step I or with the lysine decarboxylase in bacterium solution, thalline bad
Propylhomoserin decarboxylation generates 1,5- pentanediamines.
The present invention proposes a kind of new λ PRThe saltant type of promoter, simultaneously comprising published T-41C base mutations and
One new base mutation G-38A (λ PRG-38A/T-41C).With published λ PRUnlike T-41C saltant types, λ PR
Expression effects of the G-38A/T-41C under inducing temperature and wild type λ PRPromoter is essentially identical.The present invention is induction recombination table
Up to providing new selection.
Description of the drawings
Fig. 1 is the structure diagram of the pPR2-cadA recombinant expression plasmids described in embodiment 1；
Fig. 2 is λ PRAnd its sequence chart is manipulated, wherein λ CI manipulate site OR1, OR2, OR3 and are marked with box, transcription initiation
Point use+1 marks, and the base positions use -10, -20, -30 of upstream sequence marks, saltant type λ PRAt the two of G-38A/T-41C
Base mutation indicates below promoter；
Fig. 3 is recombinant strains JM109/pPRT41C-cadA (swimming lane 1-3), the JM109/ described in embodiment 1-2
PPRG38AT41C-cadA (swimming lane 4-6) mycoprotein electrophoretogram.
Specific embodiment
Below in conjunction with the accompanying drawings, the present invention will be described in detail.
The invention discloses a kind of new λ PRThe saltant type of promoter, simultaneously comprising T-41C and G-38A base mutations
(λPRG-38A/T-41C).New λ P disclosed in this inventionRTwo base mutations in the saltant type of promoter are all located at λ CI
It manipulates in the OR2 of site.Therefore present invention provides a new λ CI to manipulate site, this saltant type λ CI manipulate the sequence in site
As shown in SEQ ID NO 1.
In the present invention, inventor has found published phageλ PRThe saltant type λ P of promoterRT-41C is in induction item
Expression quantity under part is far below wild type λ PRPromoter.And a new point is introduced on the basis of this single base mutation type and is dashed forward
Become, the λ P of gainedRG-38A/T-41C expression effects and wild type λ PRPromoter is essentially identical.
The present invention relates to the promoter from bacteriophage lambda and manipulate sequence.
A kind of first polynucleotide of the present invention, base sequence is as shown in SEQ ID NO 1.The first poly-nuclear glycosides
The purposes of acid is used as the manipulation site of bacteriophage lambda aporepressor CI.
The manipulation sequence of promoter of the present invention includes the first polynucleotide as described above.First poly-nuclear
Thuja acid is located at the upstream of the promoter, in downstream or embedded promoter sequence.In one embodiment, first poly-nuclear
Thuja acid is located at -49 to -33 base positions of promoter (digital representation base is located at the position of promoter transcription starting point upstream).
Further, for the manipulation sequence of the promoter also comprising 1,2,3,4,5 λ CI manipulate site, the λ
CI manipulates site and is selected from OL1, OL2, OL3, OR1, OR2, OR3, polynucleotide of the sequence as shown in SEQ ID NO 1, bacteriophage lambda
The saltant type of manipulation site OL1, OL2, OL3, OR1, OR2, OR3 in genome and the saltant type composition of the first polynucleotide
Set.
The invention further relates to a kind of second polynucleotide, second polynucleotide includes promoter and opens as described above
Mover manipulates sequence.Alternatively, second polynucleotide includes the sequence or such as SEQ ID NO as shown in SEQ ID NO 17
The complementary base sequences thereof of sequence shown in 17.
The promoter can inhibit to transcribe by the mutant of bacteriophage lambda aporepressor CI and bacteriophage lambda aporepressor CI.
Promoter of the present invention can be natural promoter, the saltant type of natural promoter or artificial constructed open
Mover.In certain embodiments, the promoter is natural promoter, and the natural promoter is including but not limited to λ PL, λ
PR.In certain embodiments, the promoter is the saltant type of natural promoter, such as in natural promoter nucleotide sequence
There are one or more base replacement, insertion or deletions.In certain embodiments, the promoter is artificial constructed synthesis
Promoter.
Promoter of the present invention is heat shock promoter, chemically inducible promoter or radiation-induced promoter.
Second polynucleotide is plasmid.Certainly, second polynucleotide can also be phage genome, swivel base
Son or the nucleotide sequence in host genome.The plasmid is in any matter that can be replicated in host
It is built on the basis of grain carrier.Plasmid vector includes but not limited to pUC18, pUC19, pBR322, pACYC, pSC101 plasmid,
With their plasmid.Further, the plasmid includes the polynucleotide of coding polypeptide, and the expression of the polypeptide is opened by described
Mover controls.Preferably, the polypeptide includes enzyme and polypeptide drug.Signified enzyme includes oxidoreducing enzyme, transferase, hydrolysis
Enzyme, lyase, at least one of isomerase and ligase.Still further, the enzyme is amino acid decarboxylases, relies ammonia in this way
Acid decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase.Polypeptide drug includes
Hormone, antibody, growth factor etc..In one embodiment, the polypeptide of expression is proinsulin.In another embodiment, institute
It states polypeptide and includes lysine decarboxylase.Still further preferably, the polynucleotide of the coding lysine decarboxylase is selected from down
At least one of row：CadA genes, ldcC genes, haldc genes and cadA genes, ldcC genes, haldc genes segment.
Alternatively, the polynucleotide of the coding lysine decarboxylase is sequence such as SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO
The segment of the DNA of DNA or sequence as shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 shown in 4.
The invention further relates to a kind of transformant, the transformant includes the poly-nuclear glycosides as described in any technical solution above
Acid.Preferably, the transformant includes the as above polynucleotide described in any technical solution, polypeptide described in the technical solution
Including lysine decarboxylase.
On the basis of any of the above-described technical solution further, the transformant also expresses bacteriophage lambda aporepressor CI (letters
Claim λ CI) or its saltant type, preferably thermally labile type λ CI albumen.Thermally labile type λ CI albumen lower temperature can inhibit L or
The transcription of R promoters, and inhibitory activity is then lost when the temperature increases.The thermally labile type λ CI albumen includes but unlimited
In CI857, I21S, G53S, A62T, V73A, F141I/P153L, N207T, K224E.CI857 is compared with wild type at the 66th
Threonine is become from alanine on acid residues sites.In certain embodiments, it is residual to further include other amino acid by CI857
The mutation in base site, the mutation of other acid residues sites do not influence the mutation of its 66th amino acids Residue positions and are led
The thermally sensitive characteristic caused.
In one embodiment, the poly-nuclear containing coding bacteriophage lambda aporepressor CI or its saltant type in the transformant
Thuja acid.Further, the coding λ CI or the polynucleotide of its saltant type are on plasmid.Further, the coding λ CI
Or the polynucleotide of the polynucleotide of its saltant type and the coding polypeptide is on same plasmid.Alternatively, it is described coding λ CI or
The polynucleotide of its saltant type is in host genome.The host of the transformant is prokaryote.Further, described turn
The host of beggar includes Escherichia coli, hafnia alvei, Corynebacterium glutamicum, clostridium acetobutylicum, bacillus subtilis.
A kind of preferred method of fermenting and producing polypeptide of the present invention, includes the following steps：
A) transformant of the culture as described in any one technical solution above；
B polypeptide is obtained in the bacterium solution or thalline that) are obtained from step A.
Preferably, a kind of method of fermenting and producing polypeptide is the method for fermenting and producing lysine decarboxylase, including with
Lower step：
1) transformant (certainly, the technical solution described in polypeptide packet of the culture as described in any one technical solution above
Include lysine decarboxylase)；
2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1.
In a preferred embodiment, inductive condition has been used in fermentation process.The inductive condition is thermal induction, chemical
Induction is radiation-induced.Preferably, inductive condition is thermal induction, and it is 32 DEG C -48 DEG C that the inductive condition, which refers to temperature,.
A kind of fermenting and producing 1, the method for 5- pentanediamines include the following steps：
I) the step 1 production lysine decarboxylase of the method for fermenting and producing lysine decarboxylase as described above；
II it) is catalyzed with the bacterium solution or thalline obtained in step I or with the lysine decarboxylase in bacterium solution, thalline bad
Propylhomoserin decarboxylation generates 1,5- pentanediamines.
Embodiment 1.
The PCR amplification mentioned in the present embodiment, plasmid extraction, digestion, the specific steps of digestion products connection, conditional parameter
It is carried out Deng the condition for pressing the specification suggestion for purchasing relevant enzyme and reagent.
1.1 contain the wild type λ P regulated and controled by CI857RThe structure of the expression plasmid of promoter
With λ-HindIII digest DNA (purchased from precious bioengineering (Dalian) Co., Ltd) for template, with primer 1 and 2
(sequence of primer 1 is shown in SEQ ID NO：5, the sequence of primer 2 is shown in SEQ ID NO：6) sequence of the genes of cI857 containing part is expanded；
With primer 3 and 4, (sequence of primer 3 is shown in SEQ ID NO：7, the sequence of primer 4 is shown in SEQ ID NO：8) cI857 containing part is expanded
The sequence of gene and R promoters.Again with overlap PCR methods, using above-mentioned two PCR product as template, expanded with primer 1 and 4
Sequence containing cI857 genes and R promoters (it is limited to be purchased from precious bioengineering (Dalian) with restriction endonuclease BamHI and BglII
Company) the digestion overlap PCR products.With pPlac-cadA plasmids, (preparation method is shown in Chinese invention patent application
The embodiment 1 of CN201210177392.X, publication number CN102851307A, publication date 2013-01-02) it is template, with primer 5
(sequence of primer 5 is shown in SEQ ID NO with 6：9, the sequence of primer 6 is shown in SEQ ID NO：10) amplification is containing carrier framework and cadA bases
The sequence of cause equally uses BamHI and BglII digestions.Two digestion products are connected, conversion e. coli jm109 (is won purchased from Beijing
Mai De gene technology Co., Ltd), obtain plasmid pPR-cadA.
In order to remove the BglII restriction enzyme sites of cadA upstream region of gene, using pPR-cadA as template, with 7 and 8 (primer 7 of primer
Sequence see SEQ ID NO：11, the sequence of primer 8 is shown in SEQ ID NO：12) PCR duplications are carried out.It (is purchased from restriction endonuclease DpnI
Precious bioengineering (Dalian) Co., Ltd) and BglII processing PCR products.PCR product is converted into JM109.Extract transformant matter
Grain, with primer 9, (sequence of primer 9 is shown in SEQ ID NO：13) cadA gene upstream sequences are sequenced.Correct (removal is examined in sequencing
BglII sites) plasmid be named as pPR2-cadA (Fig. 1).
1.2 contain saltant type λ PRThe structure of the expression plasmid of T-41C and G-38A/T-41C promoters
Using pPR2-cadA as template, with primer 1 and 10, (sequence of primer 10 is shown in SEQ ID NO：14) amplification is containing cI857
Gene and part λ PRSequence, with primer 11 and 12, (sequence of primer 11 is shown in SEQ ID NO：15, the sequence of primer 12 is shown in SEQ
ID NO 16) amplification λ containing part PRThe sequence of sequence and part cadA genes.Overlap PCR methods are used again, with above-mentioned two
PCR product is template, is obtained with the amplification of primer 1 and 12 containing λ P after modificationRSequence (i.e. λ PRT-41C PCR product), uses BamHI
With XhoI enzymes the PCR product is cut (purchased from precious bioengineering (Dalian) Co., Ltd).By pPR2-cadA plasmids with BamHI and
XhoI digestions isolate and purify~DNA fragmentation (the AxyPrep DNA Gel Extraction of 4.5kb with agarose gel electrophoresis
Kit, purchased from healthy and free from worry life science (Wujiang) Co., Ltd), it is connect with the PCR product after above-mentioned digestion, converts JM109.With drawing
Object 12 finds in addition to obtaining expected λ P several transformant plasmid order-checkingsRT-41C saltant types also obtain a λ PR G-
38A/T-41C double-mutants.λ P will be containedRThe plasmid of T-41C saltant types is named as pPRT41C-cadA, contains λ PR G-38A/
The plasmid of T-41C double-mutants is named as pPRG38AT41C-cadA.
1.3 contain wild type and saltant type λ PRThe expression of the expression plasmid of promoter
JM109/pPRT41C-cadA and JM109/pPRG38AT41C-cadA single bacterium colonies are inoculated in LB/Amp liquid
(ampicillin concentration 100mg/L), 30 DEG C of shaking table cultures are stayed overnight.Bacterium solution is inoculated in 1%v/v in fresh LB/Amp,
30 DEG C of shaking table culture 3hr.Every part of bacterium solution is divided into 3 parts, the overnight incubation in 30 DEG C, 37 DEG C and 42 DEG C shaking tables, takes 1ml bacterium respectively
Liquid, thalline were collected by centrifugation.
SDS-PAGE separation gels are prepared, component includes：10%w/v acrylamides/methene acrylamide (29/1), 0.375M
Tris-HCl (pH8.8), 0.1%w/v SDS, 0.1%w/v ammonium persulfate, 0.04%v/v TEMED.By the thalline sample of collection
Product are respectively with the 600 sterile aqueous suspensions of μ l.15 μ l suspension are respectively taken, add 5 μ 4 × SDS-PAGE of l sample solutions (purchased from precious bioengineering
(Dalian) Co., Ltd) mixing, heats 5min in boiling water bath.By above-mentioned 20 μ l samples whole loading electrophoresis.Molecular weight of albumen is used
Protein Marker (width) instruction of precious bioengineering (Dalian) Co., Ltd.Running gel is dyed with coomassie brilliant blue R_250
Liquid dyes, and component includes：0.1%w/v coomassie brilliant blue R_250s, 25%v/v isopropanols, 10%v/v glacial acetic acid.
Electrophoretogram is shown in Fig. 3, and positions of the recombinant expression protein CadA in electrophoretogram is indicated with black arrow.Wherein swimming lane 1
It is that each recombinant bacterium inoculation cultivates 3hr, and continue the albumen of the collected thalline after 30 DEG C of overnight incubations at 30 DEG C later with 4；Swimming
Road 2 and 5 is that each recombinant bacterium inoculation cultivates 3hr at 30 DEG C later, and temperature is increased to 37 DEG C, continues collected bacterium after overnight incubation
The albumen of body；Swimming lane 3 and 6 is that each recombinant bacterium inoculation cultivates 3hr at 30 DEG C later, and temperature is increased to 42 DEG C, continues to cultivate
The albumen of collected thalline after night.
As seen from Figure 3, the CadA expression quantity of two kinds of plasmids all increases with temperature and increases (comparison swimming lane 1-3,4-6),
But contain λ PRThe plasmid pPRT41C-cadA expression quantity of promoter single base mutation T-41C is in the expression of inducing temperature (42 DEG C)
Measure the half (comparison swimming lane 3 and 6) less than pPRG38AT41C-cadA.
SEQUENCE LISTING
<110>Kai Sai biological industrys Co., Ltd of Shanghai Cathay Biological Technology Research Center Co., Ltd
<120>A kind of polynucleotide, transformant and its application
<130> 123456789
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> operator
<400> 1
taacaccgcg catgttg 17
<210> 2
<211> 2148
<212> DNA
<213> Escherichia coli
<400> 2
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccgg caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa gggtaatgct 1260
ggtaagcgtc tgatcaacgg ttccattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgttt ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgacttcaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct caaaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcgt tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtatccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 3
<211> 2142
<212> DNA
<213> Escherichia coli
<400> 3
atgaacatca ttgccattat gggaccgcat ggcgtctttt ataaagatga gcccatcaaa 60
gaactggagt cggcgctggt ggcgcaaggc tttcagatta tctggccaca aaacagcgtt 120
gatttgctga aatttatcga gcataaccct cgaatttgcg gcgtgatttt tgactgggat 180
gagtacagtc tcgatttatg tagcgatatc aatcagctta atgaatatct cccgctttat 240
gccttcatca acacccactc gacgatggat gtcagcgtgc aggatatgcg gatggcgctc 300
tggttttttg aatatgcgct ggggcaggcg gaagatatcg ccattcgtat gcgtcagtac 360
accgacgaat atcttgataa cattacaccg ccgttcacga aagccttgtt tacctacgtc 420
aaagagcgga agtacacctt ttgtacgccg gggcatatgg gcggcaccgc atatcaaaaa 480
agcccggttg gctgtctgtt ttatgatttt ttcggcggga atactcttaa ggctgatgtc 540
tctatttcgg tcaccgagct tggttcgttg ctcgaccaca ccgggccaca cctggaagcg 600
gaagagtaca tcgcgcggac ttttggcgcg gaacagagtt atatcgttac caacggaaca 660
tcgacgtcga acaaaattgt gggtatgtac gccgcgccat ccggcagtac gctgttgatc 720
gaccgcaatt gtcataaatc gctggcgcat ctgttgatga tgaacgatgt agtgccagtc 780
tggctgaaac cgacgcgtaa tgcgttgggg attcttggtg ggatcccgcg ccgtgaattt 840
actcgcgaca gcatcgaaga gaaagtcgct gctaccacgc aagcacaatg gccggttcat 900
gcggtgatca ccaactccac ctatgatggc ttgctctaca acaccgactg gatcaaacag 960
acgctggatg tcccgtcgat tcacttcgat tctgcctggg tgccgtacac ccattttcat 1020
ccgatctacc agggtaaaag tggtatgagc ggcgagcgtg ttgcgggaaa agtgatcttc 1080
gaaacgcaat cgacccacaa aatgctggcg gcgttatcgc aggcttcgct gatccacatt 1140
aaaggcgagt atgacgaaga ggcctttaac gaagccttta tgatgcatac caccacctcg 1200
cccagttatc ccattgttgc ttcggttgag acggcggcgg cgatgctgcg tggtaatccg 1260
ggcaaacggc tgattaaccg ttcagtagaa cgagctctgc attttcgcaa agaggtccag 1320
cggctgcggg aagagtctga cggttggttt ttcgatatct ggcaaccgcc gcaggtggat 1380
gaagccgaat gctggcccgt tgcgcctggc gaacagtggc acggctttaa cgatgcggat 1440
gccgatcata tgtttctcga tccggttaaa gtcactattt tgacaccggg gatggacgag 1500
cagggcaata tgagcgagga ggggatcccg gcggcgctgg tagcaaaatt cctcgacgaa 1560
cgtgggatcg tagtagagaa aaccggccct tataacctgc tgtttctctt tagtattggc 1620
atcgataaaa ccaaagcaat gggattattg cgtgggttga cggaattcaa acgctcttac 1680
gatctcaacc tgcggatcaa aaatatgcta cccgatctct atgcagaaga tcccgatttc 1740
taccgcaata tgcgtattca ggatctggca caagggatcc ataagctgat tcgtaaacac 1800
gatcttcccg gtttgatgtt gcgggcattc gatactttgc cggagatgat catgacgcca 1860
catcaggcat ggcaacgaca aattaaaggc gaagtagaaa ccattgcgct ggaacaactg 1920
gtcggtagag tatcggcaaa tatgatcctg ccttatccac cgggcgtacc gctgttgatg 1980
cctggagaaa tgctgaccaa agagagccgc acagtactcg attttctact gatgctttgt 2040
tccgtcgggc aacattaccc cggttttgaa acggatattc acggcgcgaa acaggacgaa 2100
gacggcgttt accgcgtacg agtcctaaaa atggcgggat aa 2142
<210> 4
<211> 2220
<212> DNA
<213> Hafnia alvei
<400> 4
atgaatatca ttgccatcat gaacgattta agcgcttatt ttaaggaaga acccctgcgc 60
gagctgcatc aagagttaga gaaggaaggc ttccgtattg cttatcccaa agaccgcaac 120
gatctgctga agctgattga aaacaactcc cgcctgtgtg gcgtcatttt cgactgggat 180
aaatataacc tcgaactcag cgctgaaatc agcgagctca acaaactgct gccaatttat 240
gccttcgcca atacctattc gacgcttgac gtcaacatga gcgacctgcg tcttaatgtt 300
cgcttctttg aatatgcatt aggcagcgcg caagacattg ccaccaagat ccgccaaagc 360
accgatcagt atattgatac cattctgcca ccgctgacca aggcgctgtt caaatacgtc 420
aaagaagaga aatacacagt ctgtacgccg gggcatatgg gcggaactgc gttcgataaa 480
agccctgtcg gtagcctgtt ctatgatttc ttcggtgaaa acaccatgcg ttcggatatc 540
tcgatctccg tatctgagct cggatcgctg ctcgatcata gcggcccaca ccgtgacgcc 600
gaagagtata tcgcgcgcac gttcaacgcc gatcgcagct atatcgtaac caacggaaca 660
tctacggcga ataaaattgt cggcatgtat tcatctcctg ccggtgccac tattctgata 720
gaccgtaact gccataaatc attgacccat ttgatgatga tgagcaacgt tgtccccgtc 780
tatctgcgcc caacccgtaa cgcctacggc attttaggcg ggataccgca aagcgagttc 840
acccgcgcca gcattgaaga gaaagtgaaa aatacgccca atgcgacatg gccggtgcat 900
gcggtagtca ccaactctac ctatgacggc ctgttctaca ataccgaata catcaaaaac 960
acgcttgatg ttaagtcgat tcacttcgat tcggcatggg tgccttacac caacttccat 1020
ccgatttacc aaggcaaagc agggatgagc ggtgaacgtg tgccggggaa aatcatctac 1080
gagactcagt ccacccacaa actgctggcg gcattctcgc aggcatcgat gatccacgtg 1140
aaaggtgaga tcaacgaaga aaccttcaat gaagcctata tgatgcatac ctcaacatca 1200
ccgcattacg ggatcgttgc gtcgacggaa accgcggcgg ctatgatgaa gggcaacgcc 1260
ggtaagcgtt taattaacgg ttcaattgaa cgagcgatcc gcttccgtaa agagatccgc 1320
cgcttacgta cagaatctga tggctggttc tttgacgtat ggcagccgga taacattgac 1380
gaggttgctt gctggccact caatccacgt aatgaatggc atggattccc gaacatcgac 1440
aacgatcata tgtatcttga tccgatcaaa gtcactctgc tgaccccagg tttaagcccc 1500
aatggcactc tggaagagga agggataccg gcgtcgatcg tgtcgaaata tctggatgag 1560
cacggcatca tcgtggaaaa aaccgggcca tataacctgc tcttcctgtt tagtatcggg 1620
atcgataaaa ccaaggcgtt gagcttgttg cgggcattaa ccgatttcaa acgcgtgtat 1680
gacctcaacc tgcgcgtgaa aaacgtgttg ccatcgctct ataacgaggc gcctgatttc 1740
tataaagaga tgcgaattca ggagttggct caggggattc atgctctggt gaaacaccac 1800
aatctaccag acctgatgta tcgtgcattt gaggtattac caaagctggt gatgacgccg 1860
catgatgcgt tccaagaaga agtgcgtggc aatattgagc catgtgcctt ggatgatatg 1920
ttagggaaag ttagcgccaa catgatcttg ccgtatcctc cgggtgttcc ggtggttatg 1980
ccgggagaaa tgctcactaa ggagagccgc cctgttctga gcttcttgca gatgctatgt 2040
gaaattggcg cacactatcc gggctttgaa acggatattc acggcgttca tcgtgatggt 2100
gcaacgggta aatacatggt cgtggtgctc aaacaaggcg cagatgaacc gggtgataaa 2160
ccgagtgata cggtgaagaa agcgccgggt aaaaaaccat cagcggcgaa gaagtcataa 2220
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
actgacggat cctcagccaa acgtctcttc ag 32
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
acaggctcca agccaagctt tcctgacgga atgttaattc 40
<210> 7
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 7
aagcttggct tggagcctgt tggtgcggtc atggaattac c 41
<210> 8
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 8
actgacagat ctacctcctt agtacatgca accattatca ccgccag 47
<210> 9
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 9
actgacagat ctatgaacgt tattgcaata ttgaatcac 39
<210> 10
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 10
actgacggat cccttcctcg ctcactgact cg 32
<210> 11
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 11
caatattgca ataacgttca tacaacctcc ttagtacatg caaccattat caccg 55
<210> 12
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 12
cggtgataat ggttgcatgt actaaggagg ttgtatgaac gttattgcaa tattg 55
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 13
caggcttaca tcgagagtgg 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 14
gcggtgttag atatttatcc 20
<210> 15
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 15
ggataaatat ctaacaccgc gcgtgttgac tattttacc 39
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 16
caggcttaca tcgagagtgg 20
<210> 17
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> promoter
<400> 17
taacaccgcg catgttgact attttacctc tggcggtgat aatggttgca 50
Claims (20)
1. a kind of first polynucleotide, which is characterized in that the sequence of first polynucleotide is as shown in SEQ ID NO 1
Sequence or as shown in SEQ ID NO 1 sequence complementary base sequences thereof.
2. a kind of purposes of first polynucleotide, which is characterized in that first polynucleotide is as described in claim 1, described
The purposes of first polynucleotide is used as the manipulation site of bacteriophage lambda aporepressor CI.
3. a kind of second polynucleotide, which is characterized in that second polynucleotide includes a promoter and the promoter
Sequence is manipulated, wherein, the manipulation sequence of the promoter includes the first of bacteriophage lambda aporepressor CI and manipulates site, and the λ bites
It is the first polynucleotide as described in claim 1 that the first of thalline aporepressor CI, which manipulates site,.
4. such as a kind of second polynucleotide of claim 3 any one of them, which is characterized in that the manipulation sequence of the promoter
Second also comprising 1,2,3,4 or 5 bacteriophage lambda aporepressor CI manipulates site；It is further preferred that the λ
The second manipulation site of phage repressor protein CI is selected from：Manipulation site OL1, OL2, OL3, OR1 in bacteriophage lambda genome,
OR2, OR3, polynucleotide sequence as described in claim 1, manipulation site OL1, OL2, OL3 in bacteriophage lambda genome,
The set of the saltant type composition of the saltant type of OR1, OR2, OR3 and polynucleotide sequence as described in claim 1.
5. such as the second polynucleotide of claim 3~4 any one of them, which is characterized in that the promoter is that thermal induction is opened
Mover, chemically inducible promoter or radiation-induced promoter.
6. such as the second polynucleotide of claim 3~5 any one of them, which is characterized in that second polynucleotide includes λ
The saltant type G-38A/T-41C of bacteriophage R promoters；Alternatively, second polynucleotide is included as shown in SEQ ID NO 17
Sequence or as shown in SEQ ID NO 17 sequence complementary base sequences thereof.
7. the second polynucleotide as described in claim 3~6, which is characterized in that second polynucleotide is expression cassette, matter
Grain carrier, the polynucleotide of plasmid, phage genome, transposons either in host genome.
8. the second polynucleotide as claimed in claim 7, which is characterized in that the plasmid includes the poly-nuclear glycosides of coding polypeptide
Acid, the expression of the polypeptide are controlled by the promoter.
9. the second polynucleotide as claimed in claim 8, which is characterized in that the polypeptide includes enzyme and polypeptide drug；Into
Preferably, the enzyme includes at least one in oxidoreducing enzyme, transferase, hydrolase, lyase, isomerase and ligase to one step
Kind；Further preferably, the lyase includes decarboxylase, still more preferably including lysine decarboxylase, tyrosine
Decarboxylase, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase.
10. the second polynucleotide as claimed in claim 9, which is characterized in that the poly-nuclear glycosides of the coding lysine decarboxylase
Acid is selected from following at least one：CadA genes, ldcC genes, haldc genes, the segment of cadA genes, the segment of ldcC genes,
The segment of haldc genes；Alternatively, the polynucleotide of the coding lysine decarboxylase is selected from following at least one：Sequence is such as
DNA shown in SEQ ID NO 2, DNA of the sequence as shown in SEQ ID NO 3, DNA of the sequence as shown in SEQ ID NO 4, sequence
Arrange the segment of the DNA as shown in SEQ ID NO 2, the segment of DNA of the sequence as shown in SEQ ID NO 3, sequence such as SEQ ID
The segment of DNA shown in NO 4.
11. a kind of transformant, which is characterized in that the transformant is included as claim 3~10 any one of them second is poly-
Nucleotide.
12. a kind of transformant, which is characterized in that the transformant is included as claim 8~10 any one of them second is poly-
Nucleotide.
13. a kind of transformant as claimed in claim 12, which is characterized in that the transformant is included as claim 10 is any
The second polynucleotide described in item technical solution.
14. the transformant as described in claim 11~13, which is characterized in that contain coding bacteriophage lambda resistance in the transformant
Hold back PROTEIN C I or the polynucleotide of its saltant type.
15. transformant as claimed in claim 14, which is characterized in that the coding bacteriophage lambda aporepressor CI or its mutation
The polynucleotide of type is on plasmid, it is further preferred that the coding bacteriophage lambda aporepressor CI or the poly-nuclear glycosides of its saltant type
The polynucleotide of sour and described coding polypeptide is on same plasmid；Alternatively, the coding bacteriophage lambda aporepressor CI or it is prominent
The polynucleotide of modification is in host genome.
16. such as claim 11~15 any one of them transformant, which is characterized in that the host of the transformant gives birth to for protokaryon
Object cell；It is further preferred that the host of the transformant include Escherichia coli, hafnia alvei, Corynebacterium glutamicum, third
Ketone Clostridium acetobutylicum or bacillus subtilis.
A kind of 17. method of fermenting and producing polypeptide, which is characterized in that include the following steps：
A) culture such as claim 12~16 any one of them transformant；
B polypeptide is obtained in the bacterium solution or thalline that) are obtained from step A.
18. a kind of method of fermenting and producing polypeptide as claimed in claim 17, which is characterized in that be that fermenting and producing lysine takes off
The method of carboxylic acid, includes the following steps：
1) culture such as claim 13~16 any one of them transformant；
2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1.
19. the method for the fermenting and producing polypeptide according to claim 17 or 18, which is characterized in that the transformant is inducing
Under the conditions of start recombinant expression, inductive condition for thermal induction, chemical induction or radiation-induced；It is further preferred that the induction
Condition is thermal induction, and it is 32 DEG C -48 DEG C that the inductive condition, which refers to temperature,.
20. a kind of fermenting and producing 1, the method for 5- pentanediamines, which is characterized in that include the following steps：
I) lysine decarboxylase is produced according to the step 1 of claim 18；
II) lysine is catalyzed with the bacterium solution or thalline obtained in step I or with the lysine decarboxylase in bacterium solution, thalline
Decarboxylation generates 1,5- pentanediamines.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2077586C1 (en) * | 1992-10-07 | 1997-04-20 | Государственный научно-исследовательский институт прикладной микробиологии | RECOMBINANT PLASMID DNA PTHY 315 ENCODING HYBRID PROTEIN TUMOR NECROSIS α-FACTOR - THYMOSIN-a1 |
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