Logarithmic phase specific promoter and application thereof
阅读说明：本技术 对数期特异性启动子及其应用 (Logarithmic phase specific promoter and application thereof ) 是由 陈玲 周豪宏 刘修才 于 2019-03-25 设计创作，主要内容包括：本发明提供了对数期特异性启动子(序列如SEQ ID NO:1-10所示)及其应用。本发明提供的对数期特异性诱导的启动子主要集中在菌体生长的对数期启动重组表达,这对于发酵生产赖氨酸脱羧酶具有很重要的意义,本发明保护的对数期启动子可以在菌株的生长度过停滞期后开始诱导赖氨酸脱羧酶基因的表达,因此避免了菌体在停滞期就开始表达基因给菌体适应发酵环境带来的压力,可以在同样的发酵时间内表达更多的赖氨酸脱羧酶。本发明为利用微生物表达赖氨酸脱羧酶并利用赖氨酸脱羧酶作为体外酶催化L-赖氨酸生产1,5-戊二胺提供有力的技术支持。(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 producing 1, 5-pentanediamine by using the lysine decarboxylase expressed by microorganisms and using the lysine decarboxylase as in vitro enzyme to catalyze L-lysine.)
1. Log phase specific promoter, characterized in that the size of said promoter is 37-42bp, comprising at leastRegion-10, RNA polymerase бsA 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 бsThe 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 15-20 bp.
2. The promoter according to claim 1, wherein the 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 with which the membrane is washed, and shows promoter activity in a log-phase specific manner; 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.
3. Biological material comprising a promoter according to claim 1 or 2, said biological material comprising recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineered bacterium.
4. Use of the promoter of claim 1 or 2 for any one of the following applications:
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.
5. A recombinant DNA comprising the promoter of claim 1 or 2 operably linked to a downstream gene of interest; the target gene is selected from nucleic acid encoding protein, nucleic acid encoding ribozyme, and nucleic acid encoding 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.
6. An expression vector comprising the recombinant DNA of claim 5.
7. A transformant characterized by being a host bacterium carrying the expression vector of claim 6.
8. The transformant according to claim 7, wherein the host bacterium is selected from the group consisting of species of Escherichia (Escherichia), Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium), Streptomyces (Streptomyces), 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).
9. Use of the transformant of claim 7 or 8 for the fermentative production of an amino acid, a polypeptide or a protein.
10. An engineered bacterium producing lysine decarboxylase, wherein the engineered bacterium contains the promoter according to claim 1 or 2, 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 according to claim 1 or 2.
11. 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 10 to produce lysine decarboxylase by fermentation, and utilizing the obtained lysine decarboxylase to catalyze the conversion of L-lysine to produce 1, 5-pentanediamine.
12. The method as claimed in claim 11, 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.
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 CO2. 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 coding for lysine decarboxylase is constructed after a log-phase specific promoter is constructed, and is 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 invention provides a log phase specific promoter, wherein the promoter has a size of 37-42bp, and at least comprises a-10 region, RNA polymerase бsA 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 бsThe 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 15-20 bp.
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 with which the membrane is washed, and shows promoter activity in a log-phase specific manner; 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 prokaryote is a bacterium, such as a bacterium in the genus 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 the 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, L-lysine salt solution or L-lysine fermentation liquor to catalyze the conversion of the L-lysine to generate 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 the L-lysine producing engineering bacteria 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, when the L-lysine is catalyzed to convert into the 1, 5-pentanediamine, the temperature of the reaction system is 25-55 ℃, and more preferably 28-40 ℃.
By 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. The DNA polymerase used for PCR amplification, the restriction enzyme used for enzyme digestion, the ligase used for enzyme digestion product ligation, and the competent cells of Escherichia coli E.coli BL21 were purchased from Takara Bio Inc. 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 Am607) 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 detected using nuclear magnetic resonance (BRUKERULTRASHIEDTM400PLUS, 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(DE3) competent cells and heat-shocked for 90s at 42 ℃ after ice-bath for 20 min. After incubation on ice for 5min 1ml of LB was added. Coating on the corresponding resistant plate.
The primers used in the following examples are as follows:
