CN111662903B - Logarithmic phase specific promoter and application thereof - Google Patents

Abstract

The invention provides a logarithmic phase specific promoter (shown as SEQ ID NO: 1-10) and application thereof. The log-phase specific induction promoter provided by the invention mainly focuses on the log-phase starting recombinant expression of thallus growth, which has important significance for producing lysine decarboxylase by fermentation. The invention provides powerful technical support for expressing lysine decarboxylase by using microorganisms and producing 1, 5-pentanediamine by using the lysine decarboxylase as in vitro enzyme catalysis L-lysine.

Description

Logarithmic phase specific promoter and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a log-phase specific promoter and application thereof.

Background

Lysine decarboxylase (LDC, code number EC 4.1.1.18) is widely present in microorganisms, animals and higher plants, and can remove one carboxyl group from L-lysine to produce 1, 5-pentanediamine (cadaverine) and CO ₂ . The 1, 5-pentanediamine has wide application, for example, the 1, 5-pentanediamine can be polymerized with dibasic acid to synthesize novel polyamide, and has high application value in industrial production. At present, the microbial method for producing the pentamethylene diamine mainly adopts microbial fermentation production and microbial in-vitro enzyme catalysis production for producing the pentamethylene diamine.

When the microbial in-vitro enzyme catalysis is used for producing the 1, 5-pentanediamine, a lysine decarboxylase gene expression cassette is mostly constructed according to a lactose operon, the lac promoter can be expressed constitutively by utilizing the lactose operon with lacI function deletion, and the mass expression of heterologous genes along with the growth of thalli is realized without depending on the addition of an inducer.

In addition to the lac promoter, which is dependent on the lactose operon, it is desirable to have new promoters that induce transcription of downstream genes starting at the log phase of bacterial growth, and induction of these promoters does not require addition of an inducer.

Disclosure of Invention

The invention aims to provide a log-phase specific promoter and application thereof.

Another object of the present invention is to provide a method for producing 1, 5-pentanediamine by whole-cell catalysis.

In order to achieve the purpose of the invention, the log-phase induced promoter provided by the inventor can use microorganisms to express a large amount of heterologous proteins in the log phase, and provides technical support for using the microorganisms to express proteins which generate toxicity on the growth of thalli. Further, in order to realize the whole-cell catalytic production of 1, 5-pentanediamine, a gene encoding lysine decarboxylase is constructed after a log-phase specific promoter and transformed into a bacterial strain, and the obtained recombinant strain can be used for the whole-cell catalytic production of 1, 5-pentanediamine.

In a first aspect, the log-phase specific promoter provided by the invention has a size of 37-42bp, and at least comprises a-10 region and RNA polymerase sigma ^s A specific recognition site and-35 region;

recording the position of a 3' terminal base of the promoter sequence corresponding to a transcription start site of a downstream gene as-1 position, positioning the-10 region at-12 to-7 positions, and forming a base composition into 5' -TATACT-3';

the-13 base of the promoter sequence is C;

the RNA polymerase Be ^s The specific recognition site is positioned at-18 to-14, and the base composition is 5'-TTGTT-3';

the base composition of the-35 region is 5'-T (C or T) G (C or T or A) (T or C) -3', except 5'-TCCCGCC-3', and the number of bases of the interval between the-35 region and the-10 region is 15-20bp.

Further, the sequence of the log phase specific promoter is:

i) 1-10 of the nucleotide sequence shown in SEQ ID NO; or

ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and/or added to the nucleotide sequence shown in any one of SEQ ID NOs 1 to 10 and which exhibits promoter activity in a log-phase specific manner; or

iii) A nucleotide sequence which hybridizes with a sequence shown in any one of SEQ ID NOs 1 to 10 under stringent conditions which hybridize at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, and which shows promoter activity in a log-phase specific manner, and washing a membrane with the solution; or

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and shows promoter activity in a log phase-specific manner.

In a second aspect, the present invention provides biological materials containing the log phase promoter, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors or engineered bacteria.

In a third aspect, the invention provides any one of the following uses of the log phase promoter:

1) The application of the promoter as a log-phase specific promoter of prokaryotes;

2) The application in constructing recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, virus vectors or engineering bacteria.

The prokaryotes are bacteria, such as bacteria in the genera Escherichia (Escherichia), corynebacterium (Corynebacterium), brevibacterium (Brevibacterium), streptomyces (Streptomyces), hafnia (Hafnia); preferably, the bacteria are selected from escherichia coli (e.coli), bacillus subtilis (b. Subtilis), streptomyces coelicolor (s.coelicolor), hafnia alvei (h.alvei), corynebacterium glutamicum (c. Glutamicum), or strains or genetically engineered bacteria after mutagenesis or random mutagenesis.

Further, the invention provides application of the log phase promoter as a log phase specific promoter for regulating and controlling expression of a lysine decarboxylase gene in fermentation production of lysine decarboxylase by using escherichia coli (e.coli), bacillus subtilis (b.subtilis), streptomyces coelicolor (s.coelicolor), hafnia alvei (h.alvei) or corynebacterium glutamicum (c.glutamicum).

In a fourth aspect, the present invention provides a recombinant DNA, which is formed by operably linking the promoter and a downstream target gene.

In the present invention, the target gene is selected from the group consisting of a nucleic acid encoding a protein, a nucleic acid encoding a ribozyme, and a nucleic acid encoding an antisense RNA; preferably, the protein is an enzyme, hormone, antibody or growth factor; more preferably, the enzyme is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Further, the lyase is a decarboxylase; the decarboxylase is an amino acid decarboxylase, such as lysine decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine decarboxylase, or glutamic acid decarboxylase. Still further, the lysine decarboxylase is derived from Escherichia coli (Escherichia coli), bacillus subtilis, bacillus alkalophilus (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum) or Klebsiella oxytoca (Klebsiella oxytoca). Further, the lysine decarboxylase is encoded by a cadA gene, an ldcC gene, a haldc gene, a fragment of a cadA gene, a fragment of an ldcC gene, or a fragment of a haldc gene. Further preferred is lysine decarboxylase encoded by cadA gene.

In a fifth aspect, the present invention provides an expression vector comprising said recombinant DNA.

In some embodiments, the expression vector carries an expression cassette comprising a lysine decarboxylase gene, and the expression cassette drives expression of the lysine decarboxylase gene by the log phase specific promoter.

In some embodiments, the expression vector further comprises a backbone plasmid capable of autonomous replication in a host cell; preferably, the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101, and derivatives thereof.

In a sixth aspect, the invention provides a transformant, wherein the transformant is a host bacterium carrying the expression vector.

The host bacteria are selected from the strains in the genera Escherichia (Escherichia), corynebacterium (Corynebacterium), brevibacterium (Brevibacterium), streptomyces (Streptomyces) and Hafnia (Hafnia); preferably, the host bacterium is selected from escherichia coli (e.coli), bacillus subtilis (b.subtilis), streptomyces coelicolor (s.coelicolor), hafnia alvei (h.alvei), corynebacterium glutamicum (c.glutamicum), or a strain or genetically engineered bacterium after mutagenesis or random mutation; more preferably, the host bacterium is escherichia coli (e.coli) or hafnia alvei (h.alvei).

In a seventh aspect, the invention provides the use of said transformant for the fermentative production of an amino acid, a polypeptide or a protein.

In an eighth aspect, the invention provides an engineering bacterium for producing lysine decarboxylase, wherein the starting strain of the engineering bacterium is escherichia coli (e.coli) or hafnia alvei (h.alvei), and the engineering bacterium carries a plasmid containing a lysine decarboxylase gene expression cassette, and the lysine decarboxylase gene expression cassette drives the expression of the lysine decarboxylase gene by the log-phase specific promoter.

Preferably, the lysine decarboxylase gene is an endogenous cadA gene from E.coli (SEQ ID NO: 11) encoding an E.coli inducible lysine decarboxylase cadA (SEQ ID NO: 12).

Preferably, the log phase specific promoter is a promoter shown in any one of SEQ ID NO 1, 2, 3, 4 and 6.

Further, the engineering bacterium for producing lysine decarboxylase provided by the invention contains the log-phase specific promoter or carries a plasmid containing a lysine decarboxylase gene expression cassette, and the lysine decarboxylase gene expression cassette drives the expression of the lysine decarboxylase gene by the log-phase specific promoter.

In a ninth aspect, the invention provides a method for producing lysine decarboxylase by fermentation, which comprises culturing the engineering bacteria producing lysine decarboxylase in a fermentation medium.

In a tenth aspect, the invention provides a method for producing 1, 5-pentanediamine by fermentation, which comprises the steps of producing lysine decarboxylase by fermentation of the engineering bacteria obtained in the ninth aspect, and catalyzing L-lysine to convert into 1, 5-pentanediamine by using the obtained lysine decarboxylase.

Preferably, the engineering bacteria are fermented to obtain enzyme fermentation liquor, and the enzyme fermentation liquor is mixed with L-lysine, an L-lysine salt solution or an L-lysine fermentation liquor to catalyze the conversion of the L-lysine into the 1, 5-pentanediamine.

Preferably, the L-lysine fermentation liquid is selected from L-lysine fermentation stock solution, concentrated solution or diluted solution of the L-lysine fermentation stock solution, sterilized L-lysine fermentation liquid obtained by removing thalli from the L-lysine fermentation stock solution and concentrated solution or diluted solution of the sterilized L-lysine fermentation liquid. The L-lysine fermentation liquor can be prepared by the prior art, for example, the L-lysine fermentation liquor can be obtained by culturing engineering bacteria for producing L-lysine in a fermentation culture medium, see CN104762336A, or the L-lysine fermentation liquor can be obtained by the market.

Preferably, the method further comprises adding a coenzyme selected from one or more of pyridoxal, pyridoxal phosphate, pyridoxine, pyridoxamine, more preferably pyridoxal 5' -phosphate, to the L-lysine fermentation broth.

Preferably, the temperature of the reaction system for catalyzing the conversion of L-lysine into 1, 5-pentanediamine is 25 ℃ to 55 ℃, more preferably 28 ℃ to 40 ℃.

By means of the technical scheme, the invention at least has the following advantages and beneficial effects:

compared with a constitutive Plac promoter, the log-phase promoter protected by the invention can start to induce the expression of the lysine decarboxylase gene after the growth of the strain is over-stagnating, so that the pressure of the bacteria on adapting to the fermentation environment caused by the start of expressing the gene by the bacteria in the stagnating phase is avoided, and more lysine decarboxylase can be expressed in the same fermentation time. The invention provides powerful technical support for producing 1, 5-pentanediamine by expressing lysine decarboxylase by using microorganisms and catalyzing L-lysine by using the lysine decarboxylase.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

The specific steps of PCR amplification, plasmid extraction, digestion, ligation of digested products, transformation, and the like, and the condition parameters, etc. described in the following examples were performed according to the conditions suggested by the specifications of the relevant enzymes and reagents purchased. Wherein, DNA polymerase used for PCR amplification, restriction enzyme used for enzyme digestion, ligase used for enzyme digestion product connection and escherichia coli E.coli BL21 competent cells are purchased from Takara Bio-engineering (Dalian) Co., ltd. The plasmid extraction kit, the DNA gel recovery kit and the PCR purification kit are all purchased from Kangning Life sciences (Wu Jiang) Co., ltd., trademark Axygen, and the primers are purchased from Saimer Feishale science, ltd., trademark INVITROGEN.

Hafnia alvei Am607 (Hafnia alvei Am 607) described in the following examples has now been deposited at the China center for type culture Collection, at the address: wuhan, wuhan university, post code 430072, preservation number M2018737, preservation date 2018, 11 months and 1 days.

The following examples illustrate the conversion of L-lysine-1, 5-pentanediamine, i.e., the percentage of mole ratio of 1, 5-pentanediamine to the initial L-lysine in the reaction system, wherein the detection of L-lysine and 1, 5-pentanediamine is determined using nuclear magnetic resonance (BRUKER ULTRASHIEDDT M400PLUS, beckman NMR).

The plasmid transformation methods described in the following examples are as follows: the ligation product was added to 100. Mu.l of E.coli BL21 (DE 3) competent cells, heat-shocked for 90s at 42 ℃ after ice-bath for 20 min. After 5min incubation on ice 1ml of LB was added. Coating on the corresponding resistant plate.

The primers used in the following examples are as follows:

example 1 promoter sequences for log phase expression

The invention designs and synthesizes 10 promoter sequences, the length of the promoter is 37-42bp, which are respectively shown as follows (5 ' -3', the position of the last base at the 3' end in the promoter sequence corresponding to the transcription initiation site of the downstream gene is-1 bit), wherein double underlines are marked as-10 region (-12 to-7), and the base composition of the 10 region is 5' -TATACT-3'; the extended-10 region is a conserved base C at the (-13 th position); the-18 to-14 base has the composition of 5'-TTGTT-3', and the region is RNA polymerase sigma ^s Is required for specific recognition; the single underline marks a possible-35 region, and the base sequence of the promoter can be 5'-T (C/T) (C/T) (C/T) G (C/T/A) (T/C) -3' for regulating the promoter initiation time, but the sequence of the promoter can not be 5'-TCCCGCC-3', and the number of the spacer bases between the-35 region and the-10 region of the promoter can be 15-20bp.

1.CAGTCTTGTCAAATTTGTAAAATTGTTC

GTATTG(SEQ ID NO:1)；

2.CCATTCCTGACAAATTTTTAATATTGTTC

GTATTG(SEQ ID NO:2)；

3.AATCTCGCCAAATTTCAATTTGTTC

GTATTG(SEQ ID NO:3)；

4.TAAGTCTTGCCAAATCCTTATTGTTC

GTATTG(SEQ ID NO:4)；

5.TCCTGACAAATTTACAATATTGTTC

GTATTG(SEQ ID NO:5)；

6.TCCTGCCAAATCCGTAAATTTTGTTC

GTATTG(SEQ ID NO:6)；

7.CACTTTCGTCAAATTTGTAATTATTGTTC

GTATTG(SEQ ID NO:7)；

8.TCTTGTTAAATTTGTAATTATTGTTC

GTATTG(SEQ ID NO:8)；

9.TTTTGCTAAATTTGTAAATTATTGTTC

GTATTG(SEQ ID NO:9)；

10.CGGAGTCTTGACAATTGTAAATTATTGTTC

GTATTG(SEQ ID NO:10)。

Example 2 construction of Red fluorescent protein expression plasmids containing different promoters

The coding gene of the mCherry red fluorescent protein is synthesized by a gene assembly method. The amino acid sequence of the red fluorescent protein mCherry is SEQ ID NO:14, and the nucleotide sequence of the mCherry is obtained by codon optimization and has the nucleotide sequence of SEQ ID NO:15. codon optimization and manipulation of its DNA assembly reference is made to Hoover DM & Lubkowski J, nucleic Acids Research 30, 2002.

PCR amplification is carried out on the mCherry gene after codon optimization by using primers mCherry-F (SEQ ID NO: 16) and mCherry-R (SEQ ID NO: 17), and a ribose binding site (RBS, SEQ ID NO: 18) is introduced at the upstream of the gene initiation codon; cutting the gel and recovering a DNA fragment with a target size, adding A for reaction, then connecting with a pMD18-T (purchased from Takara Bio-engineering Co., ltd.), converting a connecting product into an E.coli BL21 competent cell, screening in a plate containing ampicillin resistance with a final concentration of 100 mug/ml, culturing overnight at 37 ℃ (16 h, the same below), respectively selecting a plurality of cloning shake bacteria, then sending the cloning shake bacteria for sequencing, selecting a cloning extraction plasmid with a correct sequencing sequence and mCherry gene reversely connected into a lactose promoter plac, and obtaining the plasmid T-mCherry containing the mCherry gene. During the sequencing process, it was found that mCherry gene sequencing verified that the correct and forward inserted clone after the plac could appear red on the plate. Therefore, the cloned mCherry gene can be smoothly expressed in Escherichia coli, and translation products are functional proteins. And the cloning of the mCherry gene after being reversely inserted into the plac has no red color although the sequencing verification is correct, which indicates that the plasmid T-mCherry can be used for the subsequent verification of the starting time of the promoter.

Plasmid T-mCherry is used as a template, a primer T-mC-F1 (SEQ ID NO: 19) and a primer T-mC-R1 (SEQ ID NO: 20) are used for carrying out plasmid amplification, a PCR product is purified by a PCR purification kit, the template is digested by restriction enzyme DpnI and then is transformed into E.coli BL21 cells, screening is carried out on a resistance plate containing ampicillin with the final concentration of 100 mu g/ml, and the cells are cultured overnight at 37 ℃. Three single clones were randomly picked for sequencing identification. Selecting a clone with correct sequencing to extract a plasmid, taking the plasmid as a template, carrying out PCR amplification on the plasmid by using a primer T-mC-F2 (SEQ ID NO: 21) and a primer T-mC-R2 (SEQ ID NO: 22), purifying a PCR product by using a PCR purification kit, digesting the plasmid template by using a restriction enzyme DpnI, transforming the plasmid template into an escherichia coli E.coli BL21 competent cell, screening on a resistance plate containing ampicillin with the final concentration of 100 mu g/ml, and culturing at 37 ℃ for overnight. Three single clones were randomly picked for sequencing identification. Selecting a clone with correct sequencing to extract a plasmid, and obtaining the plasmid T-Psyn-mCherry with an RBS sequence and a multiple cloning site inserted in front of the mCherry gene.

Double-stranded DNA sequences containing 10 promoters listed in example 1 were synthesized by a gene sequence synthesis method commonly used in the art, and ligated with cleavage sites KpnI and ClaI at the 5 'and 3' ends of the sequences, respectively, and after double cleavage with KpnI and ClaI, ligated into KpnI and ClaI double-cleaved plasmids T-Psyn-mCherry, respectively. Plasmids containing different promoters were obtained. Coli BL21 competent cells were transformed with the 10 plasmids, respectively, to obtain strains containing the 10 plasmids, respectively, and the strains were named BL21-1 to BL21-10, respectively.

Example 3 verification of promoter Start time Using Red fluorescent protein

After 4 clones growing from 10 plasmid-transformed plates were picked up in a 96-well deep-well plate in 600. Mu.l each of LB liquid medium containing ampicillin at a final concentration of 100. Mu.g/ml, incubated overnight in a shaker at 37 ℃ and 250rpm, 20. Mu.l of each of the bacterial solutions was transferred to 600. Mu.l each of LB liquid medium containing ampicillin at a final concentration of 100. Mu.g/ml, incubated for 2hrs in a shaker at 37 ℃ and 250rpm, and 8. Mu.l of each of the bacterial solutions was transferred to 200. Mu.l each of LB liquid medium containing ampicillin at a final concentration of 100. Mu.g/ml in a microplate (four replicates were set up) and placed in a microplate reader. And setting a bacterial liquid of the strain which is determined not to contain the red fluorescent protein as a negative control, wherein the negative control thallus normally grows but is not detected by fluorescence. Setting the temperature to be 37 ℃, and setting the working process: firstly, shake the bacteria for 15s, and determine the OD of the bacteria _600nm Then measuring the fluorescence value of the bacterial cells under the conditions of 575nm excitation light and 610nm emission light, and finally shaking the bacterial cells for 600s, wherein the process is circulated, and the total length time is 24hrs and the measurement is performed every 15 min. After the measurement, the growth curve of the cells and the fluorescence curve (the ratio of the fluorescence value of the cells at a certain time point to the OD measured at that time point) in OD unit were plotted, respectively. And obtaining the time point of the remarkable enhancement of the fluorescence of the thallus as which stage of the thallus growth according to the corresponding relation of the two curves. The promoter contained in the clone whose fluorescence value starts to increase significantly after the log phase of the growth of the cells was determined to be a log phase-specific inducible promoter.

As can be seen from Table 1, when the recombinant strain containing the promoter of the invention is cultured for 360-480 min, the OD of the thallus increases rapidly, from about 0.2 to about 0.4, and the fluorescence value/OD of the thallus also increases significantly, from about 80 units to about 120 units; as the culture time of the bacterial cells continues to be prolonged, the OD and the fluorescence value/OD of the bacterial cells are not obviously increased, which indicates that the promoters start to start the transcription of downstream genes at the logarithmic phase of the growth of the bacterial cells.

TABLE 1 OD value and fluorescence value/OD value of each strain at different culture times, measured by microplate reader

EXAMPLE 4 log phase promoter for in vitro enzymatic production of 1, 5-Pentanediamine by expression of lysine decarboxylase

1. Construction of plasmid containing logarithmic phase promoter and inducing expression of lysine decarboxylase and recombinant strain

Taking the genome of Escherichia coli MG 1655K 12 as a template, performing first round amplification by using primers cadA-F (SEQ ID NO: 23) and cadA-R (SEQ ID NO: 24), performing second round amplification by using primers cadA-F2 (SEQ ID NO: 25) and cadA-R (SEQ ID NO: 24) after gel cutting and recycling target fragments as the template, and obtaining cadA gene (SEQ ID NO: 13) with HindIII enzyme cutting sites and ribosome binding sites at the 5' end after gel cutting and recycling; the plasmid containing 10 promoters obtained in example 2 was extracted and ligated to the similarly double-digested cadA gene (SEQ ID NO: 13) by double digestion with HindIII and XbaI to obtain 10 cadA expression plasmids of T-1-cadA, T-2-cadA, T-3-cadA, T-4-cadA, T-5-cadA, T-6-cadA, T-7-cadA, T-8-cadA, T-9-cadA, and T-10-cadA, and these 10 plasmids were transformed into Hafnia alvei strain Am607 to obtain 10 recombinant strains, HA-1-cadA, HA-2-cadA, HA-3-cadA, HA-4-cadA, HA-5-cadA, HA-5-cadA, HA-6-cadA, HA-7-cadA, HA-8-cadA, and HA-9-cadA, respectively.

It is known that in the absence of expression of the LacI repressor, the plac promoter may be a constitutive promoter, and the downstream gene is expressed following growth of the bacterial cells, in order to compare the advantages and disadvantages of the plac and the log-phase inducible promoter of the present invention in the expression of heterologous proteins. Meanwhile, a pUC18 plasmid is used as a template, a plac constitutive promoter (SEQ ID NO: 28) is obtained by utilizing the amplification of primers plac-F (SEQ ID NO: 26) and plac-R (SEQ ID NO: 27), after the two enzyme digestion of KpnI and ClaI, the plasmid is connected with the plasmid T-1-cadA which is subjected to the same two enzyme digestion, and the plasmid T-plac-cadA is obtained. It was also transformed into the Hafnia alvei strain Am607 to give the HA-plac-cadA recombinant strain.

2. Fermentation culture recombinant strain

The recombinant strains are respectively selected to be three monoclonals to be added into a 5mL LB liquid culture medium containing 1% (w/v) of tryptone (tryptone), 0.5% (w/v) of yeast extract (yeast extract), 1% (w/v) of NaCl and 100 mu g/mL of ampicillin, and are cultured for 24 hours at 37 ℃ to obtain enzyme fermentation liquid, and OD of the enzyme fermentation liquid of each strain is respectively measured _560nm 。

3. Catalytic conversion of L-lysine to 1, 5-pentanediamine

Taking 500 mu L of enzyme fermentation liquid of each recombinant strain obtained in the step, adding 500 mu L of L-lysine hydrochloride aqueous solution (the concentration of L-lysine is 400 g/L), pyridoxal 5' -phosphate with the final concentration of 0.05mM, the pH is natural, the reaction temperature is 37 ℃, the rotating speed is 250rpm, reacting for 2h, centrifuging the reaction liquid (12000rpm, 3 min) after the reaction is finished, taking 500 mu L of the reaction liquid, detecting and calculating the conversion rate of L-lysine-1, 5-pentanediamine of each reaction system (Table 2).

Table 2 shows the levels of the remaining L-lysine and the converted 1, 5-pentanediamine in each reaction system

As can be seen from Table 2, by comparing the expression of lysine decarboxylase induced by 10 log phase promoters in the present invention with the constitutive plac promoter, the results show that the log phase promoters 1-10 screened in the present invention can catalyze the conversion of L-lysine into 1, 5-pentanediamine, and the conversions obtained by inducing the expression of lysine decarboxylase by log phase promoters 1, 2, 3, 4, 6 are more advantageous than the constitutive plac promoter, and the conversions are 78%, 75%, 71%, 68% and 62%, respectively.

EXAMPLE 5 logarithmic phase promoter for expression of lysine decarboxylase for in vitro enzymatic production of 1, 5-pentanediamine

The construction of a plasmid containing a log-phase promoter for inducible expression of lysine decarboxylase, a recombinant strain, and a method for fermentation culture of the recombinant strain are the same as in example 4, except that the plasmid catalyzes the conversion of L-lysine to 1, 5-pentanediamine: mixing enzyme fermentation liquor of the obtained recombinant strains HA-1-cadA, HA-2-cadA, HA-3-cadA, HA-4-cadA, HA-6-cadA and HA-plac-cadA with L-lysine fermentation liquor, and converting to generate 1, 5-pentanediamine, specifically comprising the following steps:

600. Mu.l of lysine fermentation stock (purchased from Kaiser (Jinxiang) biomaterials Co., ltd.) was taken, and the lysine content was 11%. Mu.l of the above enzyme fermentation broth and pyridoxal 5' -phosphate at a final concentration of 0.05mM were added to the lysine fermentation stock solution, respectively, the pH was natural, the reaction temperature was 38 ℃, the rotation speed was 250rpm, the reaction was carried out for 2 hours, the reaction solution was centrifuged (12000rpm, 3 min) after the reaction was completed, 500. Mu.l of the reaction solution was taken for detection, and the conversion of L-lysine-1, 5-pentanediamine of each reaction system was calculated (Table 3).

Table 3 shows the levels of the remaining L-lysine and the converted 1, 5-pentanediamine in each reaction system

As can be seen from Table 3, the lysine decarboxylase of the logarithmic phase promoters 1, 2, 3, 4 and 6, which is induced and expressed by the invention, also obtains higher conversion rate of the L-lysine-1, 5-pentanediamine when the lysine fermentation stock solution is subjected to catalytic conversion, and the logarithmic phase promoters which are screened by the invention promote the simplification of the process for catalytically converting the L-lysine into the 1, 5-pentanediamine.

Although the invention has been described in detail above with reference to a general description and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made on the basis of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shanghai Kaiser Biotechnology research and development center, inc. CIBT US Corp

<120> logarithmic phase-specific promoter and use thereof

<130> KHP181115191.8

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<170> SIPOSequenceListing 1.0

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ccattcctga caaattttta atattgttct atactgtatt g 41

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aatctcgcca aatttcaatt tgttctatac tgtattg 37

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

taagtcttgc caaatcctta ttgttctata ctgtattg 38

<210> 5

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<213> Artificial Sequence (Artificial Sequence)

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tcctgacaaa tttacaatat tgttctatac tgtattg 37

<210> 6

<211> 38

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<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcctgccaaa tccgtaaatt ttgttctata ctgtattg 38

<210> 7

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cactttcgtc aaatttgtaa ttattgttct atactgtatt g 41

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tcttgttaaa tttgtaatta ttgttctata ctgtattg 38

<210> 9

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ttttgctaaa tttgtaaatt attgttctat actgtattg 39

<210> 10

<211> 42

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<400> 10

cggagtcttg acaattgtaa attattgttc tatactgtat tg 42

<210> 11

<211> 2148

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 11

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 tctgctccag 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 aggcaatgca 1260

ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320

cgtctgagaa cggaatctga tggctggttc tttgatgtat 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 ctgactttaa acgtgcgttc 1680

gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740

tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800

aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860

tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920

gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980

ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040

gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100

gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148

<210> 12

<211> 715

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 12

Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu

1 5 10 15

Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln

20 25 30

Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

35 40 45

Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu

50 55 60

Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr

65 70 75 80

Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu

85 90 95

Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp

100 105 110

Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile

115 120 125

Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

145 150 155 160

Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met

165 170 175

Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp

180 185 190

His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe

195 200 205

Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu

260 265 270

Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg

275 280 285

Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr

290 295 300

Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

325 330 335

Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala

420 425 430

Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly

435 440 445

Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys

450 455 460

Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser

500 505 510

Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

515 520 525

Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr

530 535 540

Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn

580 585 590

Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg

595 600 605

Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met

625 630 635 640

Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val

645 650 655

Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val

660 665 670

Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

675 680 685

Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

690 695 700

Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys

705 710 715

<210> 13

<211> 1642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aagcgaacga acaacaagaa ggaaaacaag aacgagcaaa gaacacaggg ggaaaagaag 60

aacccaccgg aaccacgcgc gcgaacgcga acccagagac ccgaacgacc ggacgacaaa 120

aacgacgaaa acaagcgcgc ggcggcgaga cgggaaaaaa accgagcggc gaagaaaagc 180

aaaagaacga gaaccgccgg acgcgcgcaa acgaccaccc gagaagccga agaccgcgac 240

agaagccgaa agcgcggggc gcgaagaagc aaaagacaag cagaccacga cgaaaacaac 300

acacgccccg cgacaaagca cgaaaagcgg aaggaaaaac cgacccggca cagggcggac 360

gcaccagaaa agcccggagg agccgcagac ggccgaaacc agaaacgaac cacagacgaa 420

cgggccgcgg acacagggcc acacaaagaa gcagaacaga acgccgcgca acgcagaccg 480

cagcacaggg accaacggac ccacgcgaac aaaagggaga ccgcccagca ggcagcacca 540

cgagaccgaa cgccacaaac gcgacccacc gagagagagc gagacgccaa caccgcccga 600

cccgaacgca cggacgggga cccacagagg aaccagcacg caccagcaag cgcggaaaga 660

aacaccaaac gcaaccggcc ggacagcgaa accaaccacc agaggcgcga caacaccgac 720

cacaagaaaa cacggaggaa accaccacga cccgcggggg ccacaccaac ccaccgaacg 780

aaggaaagcg gagagcgggg ccggagaagg gaaaggaacg aaacccagcc accacaaacg 840

cggcggcgcc caggcccaga ccacgaaagg gacgaaacga agaaaccaac gaagccacag 900

agcacaccac caccccgcac acggacgggc gccacgaaac cgcgcggcga gagaaaggca 960

agcaggaagc gcgacaacgg cagaacggcg acaaaccgaa agagacaaac gcgagaacgg 1020

aacgaggcgg cgagaggcag ccggacaacg aacgacgaag cggccgcgcg cgacagcacc 1080

ggcacggcca aaaacacgaa acgagcacag acgacccgac aaagcacccg cgacccgggg 1140

aggaaaaaga cggcaccaga gcgacggacc ggccagcacg ggcgaaaacc cgacgaacag 1200

gcacgggaga aaaccggccg aaaccgcgcc gcagcacgga cgaaagacca aagcacgagc 1260

cgcgcggccg acgacaaacg gcgcgaccga accgcgggaa aaacagcgcc gccgacggaa 1320

gaccgaacag aaaacagcga caggaacggc cagaaaccac aaacgagcac cacaacgccg 1380

gacgagacgc gcagaaggcg ccgacgagga agacccgagc gcaccagaaa gagcgcacgg 1440

agaccgaaga agacccgacg aaaggaggcg aaacgccaaa gaccccgacc cgccgggagc 1500

ccggaagccg gggaaagaca ccgaagaaag ccgccggcgg agccgcagag cgggaaacgg 1560

cgccacaccg ggcgaaaccg aacacgggca accgcaggcg aggccgcaac cgaaggagaa 1620

agaagaaagc aaaaaaaaca ga 1642

<210> 14

<211> 236

<212> PRT

<213> Mushroom coral (mushroom coral)

<400> 14

Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

1 5 10 15

Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

100 105 110

Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 15

<211> 711

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60

gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120

cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180

tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240

ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300

gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360

ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420

atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480

gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540

gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600

aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660

cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaata a 711

<210> 16

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gctctagagt ttttttggga attcacacac aggaggagct gatggtgtct aaaggcgag 59

<210> 17

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gctctagatt atttatacag ttcatccatt ccgcccg 37

<210> 18

<211> 744

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gtttttttgg gaattcacac acaggaggag ctgatggtgt ctaaaggcga ggaagataat 60

atggcgatta tcaaagaatt tatgcgtttt aaagtgcata tggaaggcag cgtgaatggg 120

catgagtttg aaattgaagg cgaaggagaa ggccgtccgt atgaaggcac ccagaccgct 180

aaactgaaag tgaccaaagg cggaccactg ccgtttgcgt gggacattct gagcccgcag 240

tttatgtatg gcagcaaagc gtatgtgaaa catccggcgg atattccgga ttatctgaaa 300

ctgagctttc cggagggctt caaatgggaa cgtgtgatga attttgaaga tggcggcgtg 360

gtgaccgtga cccaggatag cagcctgcaa gacggcgaat tcatttacaa ggtgaagctg 420

cgtggcacca actttcccag cgatggcccg gtgatgcaga aaaagaccat gggctgggag 480

gcgagcagcg aacgtatgta cccggaggat ggcgcgctga agggcgaaat taagcagcgt 540

ctgaagttaa aagatggtgg gcactatgat gcggaagtga aaaccaccta taaagcgaaa 600

aaaccggtgc agttaccagg cgcttataat gtgaacatta agctggatat taccagccat 660

aatgaagatt ataccattgt ggaacagtat gagcgtgcgg agggacggca tagcacgggc 720

ggaatggatg aactgtataa ataa 744

<210> 19

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gatatcgaat tcttaacttt aagaaggaat atacatatgg tgtctaaagg cgaggaa 57

<210> 20

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aaagttaaga attcgatatc ggcactggcc gtcgttttac 40

<210> 21

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaattcgagc tcggtaccat cgataagctt gatatcgaat tc 42

<210> 22

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gatggtaccg agctcgaatt cggcactggc cgtcgtttta c 41

<210> 23

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gaattcttaa ctttaagaag gaatatacat atgaacgtta ttgcaatatt g 51

<210> 24

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gcctctagac cacttccctt gtacgagc 28

<210> 25

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gccaagcttg atatcgaatt cttaacttta agaag 35

<210> 26

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggggtaccga gtgagctgat accgctcgcc g 31

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ggatcgatag ctgtttcctg tgtgaaattg 30

<210> 28

<211> 286

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 60

gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 120

tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 180

gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 240

ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagct 286

Claims (15)

1. The log phase specific promoter is characterized in that the promoter is a nucleotide sequence shown by any one of SEQ ID NO 1-10.

2. A biological material comprising the promoter of claim 1, said biological material comprising a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

3. The promoter of claim 1, for any one of the following uses:

1) The application of the promoter as a log-phase specific promoter of prokaryotes;

2) The application in constructing recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineering bacteria.

4. A recombinant DNA comprising the promoter of claim 1 operably linked to a downstream gene of interest; the gene of interest is selected from the group consisting of a nucleic acid encoding a protein, a nucleic acid encoding a ribozyme, and a nucleic acid encoding an antisense RNA.

5. The recombinant DNA according to claim 4, wherein the protein is an enzyme, a hormone, an antibody or a growth factor.

6. The recombinant DNA according to claim 5, wherein the enzyme is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases.

7. An expression vector comprising the recombinant DNA of any one of claims 4 to 6.

8. A transformant characterized in that it is a host bacterium carrying the expression vector of claim 7.

9. The transformant according to claim 8, wherein the host bacterium is selected from the genera Escherichia (A), (B), (C), and (C)Escherichia) Corynebacterium (I) and (II)Corynebacterium) Brevibacterium (Brevibacterium) (II)Brevibacterium) Streptomyces (I), (II)Streptomyces) Hafnia genus (a)Hafnia) The strain of (1).

10. The transformant according to claim 8, wherein the host bacterium is selected from the group consisting of Escherichia coli (E.coli) ((E.coli))E. coli) Bacillus subtilis preparation (B)B. subtilis) Streptomyces coelicolor (I), (II)S. coelicolor) Hafnia alvei: (H. alvei) Corynebacterium glutamicum (C.glutamicum)C. glutamicum)。

11. The transformant according to claim 10, wherein the host bacterium is Escherichia coli (E.coli) ((E.coli))E. coli) Or Hafnia alvei (a)H. alvei)。

12. Use of a transformant according to any one of claims 8 to 11 for the fermentative production of an amino acid, a polypeptide or a protein.

13. An engineered bacterium producing lysine decarboxylase, wherein the engineered bacterium contains the promoter of claim 1, or the engineered bacterium carries a plasmid containing a lysine decarboxylase gene expression cassette, and the expression of the lysine decarboxylase gene is driven by the promoter of claim 1.

14. A method for producing 1, 5-pentanediamine by fermentation, which is characterized in that the method comprises the steps of utilizing the engineering bacteria of claim 13 to produce lysine decarboxylase by fermentation, and utilizing the obtained lysine decarboxylase to catalyze the conversion of L-lysine to produce 1, 5-pentanediamine.

15. The method as claimed in claim 14, wherein the engineering bacteria are fermented to obtain an enzyme fermentation broth, and the enzyme fermentation broth is mixed with L-lysine, an L-lysine salt solution or an L-lysine fermentation broth to catalyze the conversion of L-lysine into 1, 5-pentanediamine.