CN115873852A - Recombinant nucleic acid sequence, genetic engineering bacteria and method for producing 1,5-pentanediamine - Google Patents

Recombinant nucleic acid sequence, genetic engineering bacteria and method for producing 1,5-pentanediamine Download PDF

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CN115873852A
CN115873852A CN202111153076.4A CN202111153076A CN115873852A CN 115873852 A CN115873852 A CN 115873852A CN 202111153076 A CN202111153076 A CN 202111153076A CN 115873852 A CN115873852 A CN 115873852A
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piclr
cada
gly
ala
promoter
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刘佳
雷云凤
刘修才
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Cathay Wusu Biomaterial Co ltd
Cathay R&D Center Co Ltd
CIBT America Inc
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Cathay Wusu Biomaterial Co ltd
Cathay R&D Center Co Ltd
CIBT America Inc
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention discloses a recombinant nucleic acid sequence, a recombinant nucleic acid combination, a genetic engineering bacterium and a method for producing 1,5-pentanediamine. Wherein the recombinant nucleic acid sequence comprises a tandem promoter sequence comprising a piclR promoter and a stationary phase specific promoter, and a lysine decarboxylase gene operably linked to the tandem promoter. According to the invention, the expression time and the expression quantity of the L-lysine decarboxylase are controlled by using the piclR-stationary phase specific promoters which are connected in series, so that the energy consumption in the 1,5-pentamethylene diamine tolerance process is reduced, the production capacity of the strain is promoted, and the yield of 1,5-pentamethylene diamine is further improved. Alternatively, the yield of 1,5-pentanediamine can be further improved by increasing the expression level of a gene which promotes 1,5-pentanediamine to be discharged out of cells, reducing the concentration of intracellular 1,5-pentanediamine and the inhibition of intracellular lysine decarboxylase activity, increasing the tolerance of the strain.

Description

Recombinant nucleic acid sequence, genetic engineering bacteria and method for producing 1,5-pentanediamine
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a recombinant nucleic acid sequence, a genetic engineering bacterium and a method for producing 1,5-pentamethylene diamine.
Background
1,5-pentanediamine has wide application and high economic value in industrial production, and can be used for synthesizing novel nylon by polymerization reaction with dibasic acid. Currently, biosynthesis of 1,5-pentanediamine is performed using mainly two strategies: fermentation production or in vitro enzyme catalysis. For fermentative production, 1,5-pentanediamine is produced by removing a carboxyl group from L-lysine by lysine decarboxylase (L-lysine decarboxylase, abbreviated as LDC, EC 4.1.1.18). Specifically, a lysine decarboxylase gene may be added to a lysine producing microorganism such as Corynebacterium glutamicum and Escherichia coli, thereby extending the lysine biosynthetic pathway to 1,5-pentanediamine biosynthetic pathway.
There are bacteria of the genus Corynebacterium and Escherichia having L-lysine-producing ability which have been modified by DNA recombination technology, and the efficiency thereof is improved by overexpressing genes related to the L-lysine synthesis pathway and genes related to desensitization of feedback inhibition, or enhancing the energy supply pathway from the start of glucose metabolism. Genes involved in desensitization to feedback inhibition, such as aspartokinase III (LysC), are specific key enzymes in the lysine synthesis pathway. However, since the cell body itself has a limited tolerance to 1,5-pentanediamine, if 1,5-pentanediamine generated by the conversion of lysine decarboxylase is expressed in the early stage of the fermentation system, it will poison the cell body, thereby inhibiting the growth of the cell body and the process for producing L-lysine from glucose (Qian, et al, biotechnol. Bioeng.2011; 108.
For example: WO2019006723A1, published as 2019, 1/19, AND named "HETEROLOGOUS EXPRESSION OF THERMOPHILIC LYSINE DEC-ARBOXYLASE AND USES THEREOF" discloses HETEROLOGOUS EXPRESSION OF THERMOPHILIC lysine decarboxylase AND use THEREOF. In the technique disclosed in this patent document, thermophilic lysine decarboxylase is used first, and the enzyme activity is controlled at a high temperature.
Another example is: chinese patent document with publication No. CN105368766A, publication No. 2016, 3 and 2, and entitled "a genetic engineering bacterium for producing pentamethylene diamine and a method for preparing pentamethylene diamine" discloses a pentamethylene diamine producing strain and a process for efficiently producing pentamethylene diamine by the same. In the technical scheme disclosed in the patent document, the lysine decarboxylase is expressed by using a temperature-controlled promoter, so that the stability problem is solved to a certain extent, but the catalysis by using high temperature additionally increases energy consumption and production cost.
Therefore, there is a need to develop a more economical, stable, and efficient production process of 1,5-pentanediamine. Also, superior cell physiology is one of the key factors in obtaining a highly efficient cell factory.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a recombinant nucleic acid sequence, a genetic engineering bacterium and a method for producing 1,5-pentamethylene diamine so as to realize stable, efficient and low-cost production of 1,5-pentamethylene diamine.
In order to achieve the above object, the present invention is achieved by the following aspects:
in a first aspect, the present invention provides a tandem promoter comprising a piclR promoter and a stationary phase specific promoter.
In some preferred embodiments, the piclR promoter is derived from any one or more of Escherichia coli (Escherichia coli), corynebacterium glutamicum (Corynebacterium glutamicum), and Hafnia alvei (Hafnia alvei); and/or, the stationary phase specific promoter is selected from one or more of the following: pcsiE, pbolA, posmY, pkatE, p21, p22, p23 and p24. The stationary phase specific promoter may be derived from a cell of a microorganism, animal or plant, including but not limited to Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus halodurans (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum) or Klebsiella oxytoca Bai Ganjun (Klebsiella oxytoca), and the like.
Preferably, the nucleotide sequence of the piclR promoter is shown in any one of SEQ ID NO 62-64, and the nucleotide sequences of pcsiE, pbolA, posmY, pkatE, p21, p22, p23 and p24 are shown in SEQ ID NO 1-8. Wherein the sequence of the pcsiE is shown as SEQ ID NO. 1; the sequence of pbolA is shown in SEQ ID NO. 2; the sequence of the posmY is shown as SEQ ID NO. 3; the sequence of pkatE is shown in SEQ ID NO 4; the sequence of p21 is shown as SEQ ID NO. 5; the sequence of p22 is shown as SEQ ID NO 6; the sequence of p23 is shown as SEQ ID NO. 7; the sequence of p24 is shown in SEQ ID NO 8.
More preferably, the tandem promoter is piclR-pcSiE, piclR-pbOLA, piclR-posmY, piclR-pkatE, piclR-p21, piclR-p22, piclR-p23 or piclR-p24.
The invention enhances the strength of the promoter by inserting the tandem promoter comprising the piclR promoter and the specificity promoter in the stationary phase and the lysine decarboxylase gene under the control of the tandem promoter into the chromosome of the bacteria in a recombination way, ensures the sufficient and stable expression of the L-lysine decarboxylase, and greatly reduces the energy consumption caused by the host cell tolerating 1,5-pentanediamine poison, thereby promoting the production of 1,5-pentanediamine without using resistance genes, induction elements and the like in the whole process. Optionally, genes that promote the discharge of 1,5-pentanediamine from cells, such as outer membrane porin genes (e.g., ompA, ompC, ompF, ompW, and ompX), may be further incorporated to further reduce intracellular 1,5-pentanediamine concentration, increasing the yield of 1,5-pentanediamine.
Herein, "under the control of the … … promoter" means that the promoter sequence and gene sequence are operably linked so as to ensure that transcription and expression of the gene is under the control of the promoter.
Thus, in a second aspect, the present invention provides a recombinant nucleic acid sequence comprising the tandem promoter sequence described above and a lysine decarboxylase gene operably linked to the tandem promoter.
The invention enhances the strength of the promoter by inserting the tandem promoter comprising the piclR promoter and the specificity promoter in the stationary phase and the lysine decarboxylase gene under the control of the tandem promoter into the chromosome of the bacteria in a recombination way, ensures the sufficient and stable expression of the L-lysine decarboxylase, and greatly reduces the energy consumption caused by the host cell tolerating 1,5-pentanediamine poison, thereby promoting the production of 1,5-pentanediamine without using resistance genes, induction elements and the like in the whole process.
Optionally, genes that promote the discharge of 1,5-pentanediamine from cells, such as outer membrane porin genes (e.g., ompA, ompC, ompF, ompW, and ompX), may be further incorporated to further reduce intracellular 1,5-pentanediamine concentration, increasing the yield of 1,5-pentanediamine.
In some embodiments, the gene for lysine decarboxylase (abbreviated as LDC, EC 4.1.1.18) may be derived from a cell of a microorganism, animal or plant, including, but not limited to, escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus halodurans (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum) or octocrytorum Bai Ganjun (Klebsiella oxytoca), and the like.
Preferably, the lysine decarboxylase gene is selected from the group consisting of: a cadA gene or an ldcC gene derived from any one of Escherichia coli, corynebacterium glutamicum and Hafnia alvei. The lysine decarboxylase may be derived from a strain obtained by subjecting the above-mentioned strain to mutagenesis or random mutagenesis, or a genetically engineered bacterium. Lysine decarboxylase may also be a mutant of lysine decarboxylase from the sources described above (including natural mutants and artificially recombinant mutants) or an active fragment (a truncated form of the protein fragment that retains lysine decarboxylase activity).
More preferably, the nucleotide sequence of the cadA gene is shown in SEQ ID NO 9.
In some embodiments, the recombinant nucleic acid sequence comprises piclR-pcSiE-cadA, piclR-pboloA-cadA, piclR-posmY-cadA, piclR-pkatE-cadA, piclR-p21-cadA, piclR-p22-cadA, piclR-p23-cadA, or piclR-p24-cadA.
It is understood by those skilled in the art that, in order to increase the expression of a gene of interest in a host cell, the coding sequence of the gene of interest may be optimized according to the codon preference of the host cell. For example, the rare codons of the gene of interest can be synonymously replaced to more closely approximate the codon usage pattern of the host cell. By this method, there have been many reports on the improvement of the expression level of a foreign gene in a host cell.
Herein, the gene promoting the excretion of 1,5-pentanediamine from cells means that its expression product promotes the excretion of 1,5-pentanediamine out of cells by microbial cells, thereby reducing intracellular 1,5-pentanediamine concentration and its inhibition of intracellular lysine decarboxylase activity, promoting the production of 1,5-pentanediamine. In some embodiments, the gene that promotes pentanediamine export comprises a gene for outer membrane porin. The outer membrane porins include OmpA, ompC, ompF, ompW, ompX, etc. More specifically, the protein promoting the excretion of pentamethylene diamine can also be a mutant (including a natural mutant and an artificial recombinant mutant) or an active fragment of the above protein.
Thus, in a third aspect, the present invention provides a recombinant nucleic acid combination comprising:
a first sequence comprising the tandem promoter described above or the recombinant nucleic acid sequence described above;
and a second sequence comprising a constitutive promoter or said constitutive promoter and operably linked thereto a gene encoding an outer membrane porin.
Preferably, the constitutive promoter is selected from any one or more of plac, trp, tac, trc and PL.
More preferably, the nucleotide sequence of the constitutive promoter is shown as SEQ ID NO. 61;
and/or, preferably, said outer membrane porin gene is selected from the group consisting of: any one or more of ompA, ompC, ompF, ompW and ompX.
In some embodiments, the nucleotide sequence of the gene encoding outer membrane porin is as set forth in any one of SEQ ID NOs 11-15.
In some embodiments, the ompA gene is represented by SEQ ID NO 11 and the OmpA protein is represented by SEQ ID NO 16;
the sequence of ompC gene is shown in SEQ ID NO. 12, and the sequence of ompC protein is shown in SEQ ID NO. 17;
the sequence of ompF gene is shown as SEQ ID NO. 13, and the sequence of OmpF is shown as SEQ ID NO. 18;
the sequence of ompW gene is shown as SEQ ID NO. 14, and the sequence of OmpW protein is shown as SEQ ID NO. 19;
the ompX gene has the sequence shown in SEQ ID NO. 15, and the ompX protein has the sequence shown in SEQ ID NO. 20.
In the recombinant nucleic acids described herein, the tandem promoter (which may be represented, for example, by element a) and lysine decarboxylase gene (which may be represented, for example, by element b) are operably linked, linking the resulting expression elements, which may be represented by expression elements a-b, such that transcription and expression of lysine decarboxylase is under the control of the pentanediamine-inducible promoter. Preferably, the linked expression elements a-b can achieve expression in a host cell under the control of the concentration of pentamethylenediamine, and the production of 1,5-pentamethylenediamine is improved by controlling the production of L-lysine decarboxylase by the tandem promoter.
In the recombinant nucleic acids described herein, a constitutive promoter (which may be represented, for example, as element c) and a gene that promotes the excretion of pentamethylenediamine (which may be represented, for example, as element d) are operably linked, linking the resulting expression element, which may be represented as expression elements c-d, such that transcription and expression of the gene that promotes the excretion of pentamethylenediamine is under the control of the constitutive promoter. Alternatively, the aforementioned attached a-b, c-d may be operably attached or may be independent.
More preferably, the first sequence is piclR-pcsiE-cadA, piclR-pbolA-cadA, piclR-posmY-cadA, piclR-pkatE-cadA, piclR-p21-cadA, piclR-p22-cadA, piclR-p23-cadA or piclR-p24-cadA; and/or the second sequence is plac-ompA, plac-ompC, plac-ompF or plac-ompW.
When the first sequence is operably linked to the second sequence, further embodiments of the invention are formed, i.e.piclR-p 24-cadA-plac-ompA, piclR-p24-cadA-plac-ompC, piclR-p24-cadA-plac-ompF, piclR-p24-cadA-plac-ompW or piclR-p24-cadA-plac-ompX.
Constitutive promoters used herein are well known to those skilled in the art and can express genes that promote the efflux of 1,5-pentanediamine from cells in host cells, for example, the plac, trp, tac, trc or PL promoters can be used. For example, the sequence comprising the plac promoter is shown in SEQ ID NO 61.
In a fourth aspect, the present invention also provides a biological material comprising a tandem promoter or a recombinant nucleic acid sequence as described above or a recombinant DNA nucleic acid as described above;
preferably, the biological material comprises an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.
More preferably, the backbone plasmids of the plasmid vector include pUC18, pUC19, pBR322, pACYC, pET, pSC101, and derivatives thereof. The backbone plasmid is, for example, pBR322, which is used to express the promoters and various elements of the genes described above. In addition, in the technical scheme of the invention, the Escherichia coli chromosome recombination is carried out by adopting pKD 46.
In a fifth aspect, the invention provides a genetically engineered bacterium for producing 1,5-pentamethylene diamine, which comprises the tandem promoter or the recombinant nucleic acid sequence or the recombinant nucleic acid combination.
Preferably, the chromosome of the genetically engineered bacterium contains the recombinant nucleic acid sequence or the recombinant nucleic acid combination.
More preferably, the genetically engineered bacterium is introduced into the chromosome by recombination of the tandem promoter or the recombinant nucleic acid sequence or the recombinant nucleic acid combination.
In some embodiments, the genetically engineered bacterium comprises a lysine decarboxylase gene in a chromosome under the control of the tandem promoter-stationary phase specific promoter.
In other embodiments, the genetically engineered bacterium has introduced by recombination into the chromosome a gene that facilitates the export of 1,5-pentanediamine from the cell, such as a tandem promoter or a recombinant nucleic acid sequence or a combination of recombinant nucleic acids as described above.
The gene for promoting 1,5-pentanediamine to be discharged from cells is under the control of the constitutive promoter, and the gene for promoting 1,5-pentanediamine to be discharged from cells can play a role in reducing the influence of 1,5-pentanediamine toxicity on cell growth.
In some preferred embodiments, the host bacterium of the genetically engineered bacterium is derived from a species of the genus Escherichia (Escherichia), corynebacterium (Corynebacterium), bacillus (Bacillus), thermus (Thermus), brevibacterium (Brevibacterium) or Hafnia (Hafnia).
Preferably from Escherichia coli, thermus, hafnia alvei, bacillus subtilis or Corynebacterium glutamicum.
Herein, the lysine decarboxylase gene may be contained in one plasmid.
Herein, the gene promoting the discharge of 1,5-pentanediamine from cells may be contained in the same plasmid as the lysine decarboxylase gene; alternatively, it may be contained in a different plasmid, expressed independently of the host chromosome in the host cell.
Herein, the genetically engineered bacterium comprises in its chromosome a lysine decarboxylase gene under the control of the tandem promoter. Therefore, the expression of L-lysine decarboxylase is regulated and controlled by using a piclR-stationary phase specific promoter in series with a promoter, so that the energy consumption in the 1,5-pentanediamine tolerance process is reduced, the production of L-lysine is promoted, and the yield of 1,5-pentanediamine is further improved. Furthermore, the genetically engineered bacteria can also comprise the gene which promotes 1,5-pentanediamine to be discharged from cells in a recombination manner in a chromosome, so that the concentration of the intracellular 1,5-pentanediamine and the inhibition of the intracellular lysine decarboxylase activity by the intracellular 1,5-pentanediamine are reduced by expressing the protein which promotes 1,5-pentanediamine to be discharged from the cells, and the yield of 1,5-pentanediamine is further improved.
As a starting strain, there can be used the L-lysine-producing Escherichia coli (Escherichia coli) M11A3 strain, which has been deposited in the China center for type culture Collection, at the address: wuhan, wuhan university, zip code 430072, preservation number CCTCC No: m2018456, date of deposit 2018, 7/6/month.
In a sixth aspect, the invention also provides a method for producing 1,5-pentamethylene diamine, which comprises culturing the genetically engineered bacteria containing the bacteria in a fermentation medium to produce 1,5-pentamethylene diamine.
In some embodiments, the culture temperature is 20-50 ℃.
In the method, the recombinant nucleic acid is constructed into engineering bacteria with the capacity of producing L-lysine, the recombinant bacteria are fermented and cultured, the lysine is accumulated, the fermentation culture temperature is controlled to be 20-50 ℃, the rapid growth of bacteria and the accumulation of the lysine are carried out, after the fermentation stabilization period, a large amount of lysine decarboxylase is expressed, and the 1,5-pentanediamine is transformed and produced.
As used herein, the term "about" when used to modify a value within a temperature range means that the value reasonably deviates from the value, e.g., within 1 ℃ or 2 ℃ below or above the value recited within the range is within the intended meaning of the value or range.
In some embodiments, the culturing is performed at a temperature of about 25 ℃ to about 45 ℃. In other embodiments, the culturing is performed at a temperature of about 30 ℃ to about 40 ℃. In a further embodiment, the culturing is carried out at a temperature of about 35 ℃ to about 39 ℃.
In a seventh aspect, the invention provides an application of the tandem promoter, the recombinant nucleic acid sequence, the recombinant nucleic acid composition, the biological material or the genetic engineering bacteria in the production of 1,5-pentamethylene diamine.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention enhances the strength of the promoter by inserting the tandem promoter comprising the piclR promoter and the specificity promoter in the stationary phase and the lysine decarboxylase gene under the control of the tandem promoter into the chromosome of the bacteria in a recombination way, ensures the sufficient and stable expression of the L-lysine decarboxylase, and greatly reduces the energy consumption caused by the host cell tolerating 1,5-pentanediamine poison, thereby promoting the production of 1,5-pentanediamine without using resistance genes, induction elements and the like in the whole process.
The invention uses a tandem promoter comprising a piclR promoter and a stationary phase specific promoter, and can start the sufficient expression of the lysine decarboxylase gene only after the thalli grow to a stationary phase. Compared with expression of thermophilic lysine decarboxylase or expression of lysine decarboxylase by a temperature-controlled promoter, the stable and sufficient expression of lysine decarboxylase can be realized by inserting the gene sequence of the tandem promoter-lysine decarboxylase into a chromosome, in addition, the strain removes the use of antibiotics, special environmental conditions or other inducers, and the whole fermentation culture process is self-regulated.
The application of the compound can obviously reduce the cell toxicity of 1,5-pentanediamine generated in the stages of cell growth and L-lysine production and improve the yield of L-lysine when being applied to the production of 1,5-pentanediamine; after the fermentation is finished, the L-lysine can be almost completely converted into 1,5-pentanediamine, so that the increase of the yield of 1,5-pentanediamine is realized. Meanwhile, the protein for promoting the discharge of the pentamethylene diamine is used, 1,5-pentamethylene diamine is output to the outside of cells while being converted, the yield of 1,5-pentamethylene diamine produced by the fermentation of the recombinant strain is comprehensively and remarkably improved, and the stable, efficient and low-cost production of 1,5-pentamethylene diamine is realized.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It should be understood that although a few embodiments of practicing the present invention have been illustrated herein, those skilled in the art will appreciate, in light of the present disclosure, that numerous modifications may be made without departing from the spirit and intended scope of the invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be defined only by the appended claims and equivalents thereof.
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, condition parameters and the like of PCR amplification, purification, plasmid extraction, enzyme digestion product ligation, transformation and the like, which are referred to in the following examples, are carried out according to the instructions of the purchased relevant enzymes and reagents. The DNA polymerase used for PCR amplification, the restriction enzyme used for enzyme digestion, and the ligase used for ligation of enzyme digestion products are all 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 (Wujiang) Co., ltd
Figure BDA0003287788300000102
Primers were purchased from INVITROGEN technologies (china) ltd.
The plasmid transformation methods referred to in the following examples are as follows: the ligation product was added to 100. Mu.l of E.coli BL21 (DE 3) competent cells, ice-cooled for 30min, and heat-shocked for 90s at 42 ℃. After incubation on ice for 5min 1ml of LB was added. Coating on the corresponding resistant plate.
In the present invention, the amounts of L-lysine and 1,5-pentamethylenediamine in the medium can be detected by a nuclear magnetic resonance method.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
The hosts, overexpressed proteins and plasmids used in the examples are summarized in Table 1 below.
TABLE 1
Figure BDA0003287788300000101
Figure BDA0003287788300000111
EXAMPLE 1 construction of the plasmid backbone of pBU
A genome of commercially available Escherichia coli K12 MG1655 is used as a template, a primer pair Upp-UF (shown in SEQ ID NO: 21) and Upp-UR (shown in SEQ ID NO: 22) are used for amplifying an Upp-U fragment (shown in SEQ ID NO: 23), a primer pair Upp-DF (shown in SEQ ID NO: 24) and an Upp-DR (shown in SEQ ID NO: 25) are used for amplifying an Upp-500bp-D fragment (shown in SEQ ID NO: 26), and a primer pair Upp-F (shown in SEQ ID NO: 27) and an Upp-R (shown in SEQ ID NO: 28) are used for amplifying a Pupp-uppp-D fragment (shown in SEQ ID NO: 29). A fragment P1P2-tetA (sequence shown as SEQ ID NO: 32) containing a P1P2 promoter and a tetA sequence (namely with a resistance marker) on the plasmid is amplified by taking a commercial plasmid pBR322 as a template and a primer pair P1P2-tetA-F (sequence shown as SEQ ID NO: 30) and P1P2-tetA-R (sequence shown as SEQ ID NO: 31). After the PCR products of the Upp-U, upp-500bp-D, P P2-tetA and Pupp-Upp-D fragments are cut and recovered, the PCR products are connected with a pBR322 vector after EcoRI and NaeI double enzyme digestion, and the gene fragment and the vector are subjected to recombinant connection by using a multi-fragment one-step cloning kit. The recombinant ligated mixture was transformed into E.coli JM109 (purchased from Takara Bio Inc.) competent cells and screened on LB plates containing ampicillin to obtain a plurality of single colonies. After the correctness is verified by colony PCR and sequencing, the plasmid is extracted to obtain a plasmid containing four fragments of Upp-U, upp-500bp-D, P P2-tetA and Pupp-Upp-D, and the plasmid is named as pBU vector.
EXAMPLE 2 cloning of the lysine decarboxylase CadA Gene
A cadA gene fragment (shown as SEQ ID NO: 9) is amplified by using a genome of commercial Escherichia coli K12 MG1655 as a template and using a primer pair cadA-F (shown as SEQ ID NO: 33) and cadA-R (shown as SEQ ID NO: 34), the cadA gene fragment and the pBU vector obtained in example 1 after EcoRI single enzyme digestion are subjected to gel cutting, recovered and purified, and the gene fragment and the vector are recombined by using a multi-fragment one-step cloning kit to generate a plasmid with the name of pBU-cadA.
A piclR promoter is amplified by using a genome of commercial escherichia coli K12 MG1655 as a template and a primer pair piclR-F (shown as SEQ ID NO: 35) and piclR (shown as SEQ ID NO: 36), and the sequence is shown as SEQ ID NO: 62. The piclR promoter fragment and the pBU-cadA vector after SacI single enzyme digestion are subjected to gel cutting, recovered and purified, and the gene fragment and the vector are subjected to recombinant connection by using a multi-fragment one-step cloning kit. The recombinant ligated mixture was transformed into E.coli JM109 (purchased from Takara Bio Inc.) competent cells, and screened on an ampicillin-containing LB plate to obtain a plurality of single colonies. After the correctness is verified by colony PCR and sequencing, the plasmid is extracted to obtain pBU-piclR-cadA plasmid, and the plasmid contains cadA gene under the control of piclR promoter.
Example 3 construction of pBU-piclR-stationary phase specific promoter-cadA and pBU-stationary phase specific promoter-cadA plasmids
The genome of commercial Escherichia coli K12 MG1655 is taken as a template, and primers pcSiE-F (shown in SEQ ID NO: 37) and pcSiE-R (shown in SEQ ID NO: 38) are respectively utilized, pbolA-F (shown in SEQ ID NO: 39) and pbolA-R (shown in SEQ ID NO: 40) are utilized, posmY-F (shown in SEQ ID NO: 41) and posmY-R (shown in SEQ ID NO: 42) are utilized, pkatE-F (shown in SEQ ID NO: 43) and pkatE-R (shown in SEQ ID NO: 44) are utilized, pcSiE-F2 (shown in SEQ ID NO: 45) and pcSiE-R (shown in SEQ ID NO: 38) are utilized, pbolA-F2 (shown in SEQ ID NO: 46) and pbolA-R (shown in SEQ ID NO: 40) are utilized, pbolA-F (shown in SEQ ID NO: 47) and pbolA-R (shown in SEQ ID NO: 48) are utilized; amplifying a stationary phase specific promoter pcsiE (shown as a sequence in SEQ ID NO: 1), pbolA (shown as a sequence in SEQ ID NO: 2), posmY (shown as a sequence in SEQ ID NO: 3) and pkatE (shown as a sequence in SEQ ID NO: 4), cutting and recovering PCR products, and respectively connecting the PCR products with pBU-piclR-cadA and pBU-cadA plasmids obtained in SacI-cut example 2 to obtain plasmids containing 4 tandem promoters: pBU-piclR-pcsiE-cadA, pBU-piclR-pbolA-cadA, pBU-piclR-posmY-cadA, pBU-piclR-pkatE-cadA; and plasmids containing only 4 stationary phase specific promoters: pBU-pcsiE-cadA, pBU-pbolA-cadA, pBU-posmY-cadA, and pBU-pkatE-cadA.
Double-stranded DNA sequences (5 '-3') of p21, p22, p23, and p24 promoters were synthesized using gene sequence synthesis methods commonly used in the art, and then ligated into SacI-digested plasmids pBU-picrcadA and pBU-cadA, respectively, obtained in example 2. Obtaining plasmids pBU-piclR-p21-cadA, pBU-piclR-p22-cadA, pBU-piclR-p23-cadA, pBU-piclR-p24-cadA containing 4 tandem promoters; and plasmids containing only 4 stationary phase specific promoters: pBU-p21-cadA, pBU-p22-cadA, pBU-p23-cadA, pBU-p24-cadA.
Example 4 construction of 1,5-Pentanediamine producing Strain and examination of 1,5-Pentanediamine production
The starting strain of the invention adopts Escherichia coli (Escherichia coli) M11A3 strain which can produce L-lysine, and the strain is preserved in China Center for Type Culture Collection (CCTCC) at the address: wuhan, wuhan university, post code 430072, preservation number CCTCC No: m2018456, date of deposit 2018, 7/6.
Firstly, preparing electrotransformation competence, transforming a commercial pKD46 plasmid to M11-A3 strain competence by a heat shock method, selecting and culturing a single colony in an LB resistance plate containing 100 mu g/mL ampicillin in a 5mL LB liquid culture medium at 30 ℃ and 200rpm for 8h; inoculating the mixture into 50mL of LB liquid culture medium with the inoculation amount of 1% to culture until the OD600 is about 0.15, adding 1mL of 2mM L-arabinose solution, and continuously culturing until the OD600 is 0.4-0.5; then transferring the mixture into a 50mL centrifuge tube for ice bath for 20min to stop growing; centrifuging at 4 ℃ and 4000rpm for 10min, and collecting thalli; then, 40mL of precooled sterile water is used for washing the thalli, and cells are collected by centrifugation; repeating the above steps; the cells were washed with 20mL of pre-cooled 10% glycerol and collected by centrifugation; finally, the cells were resuspended in 500. Mu.L of pre-cooled 10% glycerol and the resulting M11-A3/pKD46 competent cells were aliquoted for future use.
Using 16 plasmids (shown in Table 2 below) constructed in example 3 as templates, fragments usable for homologous recombination on chromosomes were obtained by amplification using the primer pair upp-UF/upp-R, respectively, and recovered by cutting the gel. Each fragment was transferred into the recipient strain M11A3/pKD 46. The selection was carried out by plating on LB-resistant plates containing 10. Mu.g/ml tetracycline. For each plasmid transformation, 3 single colonies were picked up in 600. Mu.l LB medium supplemented with ampicillin, cultured at 37 ℃ for 8h, 1. Mu.l of the cells were used as template for PCR validation to screen out the correct recombinant strains, and glycerol was used for conservation.
Inoculating 16 strains of glycerol-preserved strains into LB liquid culture medium containing 0.1ug/mL 5-FU (5-fluorouracil), and culturing at 37 ℃ and 200rpm for 8h; and respectively streaking the bacterial liquid to LB culture medium plates containing and not containing 5-fluorouracil, and culturing overnight. Plate analysis showed that strains failed to grow on plates containing 5-fluorouracil, 6 single clones were randomly selected for each strain from the corresponding plate without 5-fluorouracil, 1. Mu.l of the thallus was taken as a template for PCR validation again, and if P1P2-tetA and Pupp-Upp fragments were removed at the same time, indicating that the correct recombinant strain was obtained, the correct recombinant strain was glycerol-deposited.
3 transformants of each of the above recombinant strains were selected, and the resulting transformants were individually plated with an antibiotic-free seed medium (containing 4% glucose, 0.1% KH) together with the original strain M11A3 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract; the percent is qualityVolume percent, same below), incubated overnight at 37 ℃. Then, 3 single clones were picked up and used in 5ml of seed medium (containing 4% glucose, 0.1% KH) 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH4) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract) was cultured at 37 ℃ overnight at 225 rpm. Each strain was then transferred to 50ml fresh fermentation medium (30 g/L glucose, 0.7% Ca (HCO) 3 ) 2 ，0.1％KH 2 PO4，0.1％MgSO4,1.6％(NH4) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract medium) was further cultured at 37 ℃ and 170rpm for 48 hours, and the content of 1,5-pentanediamine in each medium was calculated by nuclear magnetic resonance detection (table 2).
TABLE 2 Nuclear magnetic assay of 1,5-pentanediamine yield and OD of recombinant compared with original strain 600
Figure BDA0003287788300000151
As can be seen from Table 2, the recombinant strains pcsiE-cadA/M11A3, pbolA-cadA/M11A3, posmY-cadA/M11A3, pkatE-cadA/M11A3, p21-cadA/M11A3, p22-cadA/M11A3, p23-cadA/M11A3, p24-cadA/M11A3, which directly express cadA using a stationary phase-specific promoter, detected 0.88 to 2.23g/kg of L-lysine and 1.14 to 2.12g/kg of 1,5-pentanediamine after 48h of fermentation, indicating that the lysine decarboxylase expression level is low and only a part of L-lysine is converted into 1,5-pentanediamine.
In contrast, the recombinant strains piclR-pcSiE-cadA/M11A3, piclR-pbolA-cadA/M11A3, piclR-posmY-cadA/M11A3, piclR-pkatE-cadA/M11A3, piclR-p21-cadA/M11A3, piclR-p22-cadA/M11A3, piclR-p23-cadA/M11A3, piclR-p24-cadA/M11A3 expressing cadA using the tandem promoters were tested to show that at 48h fermentation, the production of 1,5-pentanediamine was further increased, while the L-3252 was converted to total amounts of L-3252-pentanediamine, with a minimum residual production of 3532, and the strains had accumulated as little as much as L-lysine at 3532 g/g, 3532 g-pentanediamine, and no residual amounts of L-pxfT-pentanediamine at the final strain 3532 kg, 3432.
EXAMPLE 5 construction of pBU-piclR-p24-plac-ompA, pBU-piclR-p24-cadA-plac-ompC, pBU-piclR-p24-cadA-plac-ompF, pBU-piclR-p24-plac-ompW, pBU-piclR-p24-cadA-plac-ompX plasmids
The genome of commercial Escherichia coli K12 MG1655 is used as a template, ompA gene (sequence shown as SEQ ID NO: 11) is amplified by a primer pair ompA-F (sequence shown as SEQ ID NO: 49) and ompA-R (sequence shown as SEQ ID NO: 50), and ompC gene (sequence shown as SEQ ID NO: 12) is amplified by a primer pair ompC-F (sequence shown as SEQ ID NO: 51) and ompC-R (sequence shown as SEQ ID NO: 52). The ompF gene (sequence shown as SEQ ID NO: 13) was amplified by the primer pair ompF-F (sequence shown as SEQ ID NO: 53) and ompF-R (sequence shown as SEQ ID NO: 54). The ompW gene (shown as SEQ ID NO: 14) and ompW-R (shown as SEQ ID NO: 56) are amplified by a primer pair ompW-F (shown as SEQ ID NO: 14. The ompX-F (shown as SEQ ID NO: 57) and ompX-R (shown as SEQ ID NO: 58) to amplify the ompX gene (shown as SEQ ID NO: 15), a commercial pUC18 plasmid DNA is used as a template, a plac-F (shown as SEQ ID NO: 60) and a plac-R (shown as SEQ ID NO: 59) are amplified by a primer pair plac-F (shown as SEQ ID NO: 61), a plac promoter fragment, an ompA gene fragment and an XbaI single-digested 5754-picR-p 24-cacA dA and 3252-zxft 3252-p 24-cac plasmid are subjected to gel cutting purification, and a multi-cloning kit is used for generating a recombinant plasmid-35-25-3532 and a recombinant vector-34zxcaf-3532.
The plac promoter fragment, the ompC gene fragment and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids after XbaI single enzyme digestion are subjected to gel cutting, recovered and purified, the gene fragment and the vector are subjected to recombinant ligation by using a multi-fragment one-step cloning kit, and the generated plasmids are named as pBU-piclR-p24-cadA-plac-ompC and pBU-p24-cadA-plac-ompC.
The plac promoter fragment, ompF gene fragment and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids after XbaI single enzyme digestion are subjected to gel cutting, recovered and purified, the gene fragment and the vector are subjected to recombinant ligation by using a multi-fragment one-step cloning kit, and the generated plasmids are named as pBU-piclR-p24-cadA-plac-ompF and pBU-p24-cadA-plac-ompF.
The plac promoter fragment, the ompW gene fragment and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids which are subjected to single enzyme digestion by XbaI are subjected to gel cutting, recovered and purified, and the gene fragment and the vector are subjected to recombinant ligation by using a multi-fragment one-step cloning kit to generate plasmids with the names of pBU-piclR-p24-cadA-plac-ompW and pBU-p24-cadA-plac-ompW.
The plac promoter fragment, the ompX gene fragment and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids which are subjected to single enzyme digestion by XbaI are subjected to gel cutting, recovered and purified, and the gene fragment and the vector are subjected to recombinant ligation by using a multi-fragment one-step cloning kit to generate plasmids with the names of pBU-piclR-p24-cadA-plac-ompX and pBU-p24-cadA-plac-ompX.
Example 6 construction of 1,5-Pentanediamine producing Strain
Fragments usable for homologous recombination on chromosomes were obtained by amplifying 5 pairs of plasmids constructed in example 5 as templates with the primer pair upp-UF/upp-R, respectively, and then excised and recovered. Each fragment was transferred into the recipient strain M11-A3/pKD46, respectively. The selection was carried out by plating on LB-resistant plates containing 10. Mu.g/ml tetracycline. After 12 single colonies were picked up in 600. Mu.l LB medium supplemented with ampicillin and cultured at 37 ℃ for 8 hours, 1. Mu.l of the cells were used as a template for PCR verification to select correct recombinant strains, and glycerol was used for stock preservation.
Inoculating the strain protected by glycerol into an LB liquid culture medium containing 0.1ug/mL 5-FU (5-fluorouracil), and culturing at 37 ℃ and 200rpm for 8h; and respectively streaking the bacterial liquid to LB culture medium plates containing and not containing 5-fluorouracil, and culturing overnight. Plate analysis showed that strains that could not grow on plates containing 5-fluorouracil, 6 monoclonals were selected from the corresponding plates without 5-fluorouracil, 1 μ l of the thallus was taken as template for PCR validation again, if both P1P2-tetA and Pupp-Upp fragments had been removed at the same time, indicating that the correct recombinant strain was obtained, the correct recombinant strain was glycerol-preserved.
Example 7 detection of Strain 1,5-Pentanediamine production
3 transformants were selected from each of the recombinant strains obtained in example 6, and the starting strains M11A3, piclR-p24-cadA/M11A3 and p24-cadA/M11A3, which were used as control strains, were each plated with a seed medium (containing 4% glucose, 0.1% KH) containing no antibiotic 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4 ,0.001％FeSO 4 0.001% MnSO4,0.2% yeast extract), and incubated at 37 ℃ overnight. Then, 3 single clones were picked up and used for 5ml of seed medium (containing 4% glucose, 0.1% KH) 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract) was cultured at 37 ℃ overnight at 225 rpm. Then each strain was transferred to 50ml of fresh fermentation medium (30 g/L glucose, 0.7% Ca (HCO) 3 ) 2 ，0.1％KH 2 PO 4 ，0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4, 0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract medium, in addition to 30g/kg of pentanediamine added to one set of shake flasks) at 37 ℃ at 170rpm for an additional 48h, and samples were taken to determine the OD of each strain with or without pentanediamine addition 600 . The amount of pentamethylene diamine in each medium was determined and calculated by nuclear magnetic resonance for a group of samples to which pentamethylene diamine was not added (Table 3).
As shown in Table 3, the OD600 of the strain was determined after 20-fold dilution by adding a shake flask with pentamethylenediamine, and compared with 10 new strains of M11-A3, pcilR-p24-cadA/M11A3p24-cadA/M11A3 recombinant strains expressing the 1,5-pentamethylenediamine excreting protein OmpA, ompC, ompF, ompW, or OmpX, the OD600 was significantly reduced (i.e., the strain OD600 decreased), wherein the PicR-p21-cadA-plac-ompA/M11A3 strain OD600 was the highest, indicating that it had the highest ability to tolerate pentamethylenediamine.
As shown in Table 4, the strains piclR-p24-cadA-plac-ompA/M11A3, piclR-p24-cadA-plac-ompC/M11A3, piclR-p24-cadA-plac-ompF/M11A3, piclR-p24-cadA-plac-ompW/M11A3, piclR-p24-cadA-plac-ompX/M11A3, compared with the recombinant strain piclR-p24-cadA/M11A3, produced 1,5-pentanediamine with almost all L-lysine converted to 1,5-pentanediamine, wherein piclR-p 24-cadA-plac-323, had almost no residual lysine at 3745 kg/32 g/L3732, and finally accumulated as much more as L-lysine at 32 kg.
TABLE 3 OD measurement of the strains with and without addition of Pentanediamine 600
Figure BDA0003287788300000191
TABLE 4 Nuclear magnetic assay of Pentanediamine production of recombinant versus original strains
Figure BDA0003287788300000192
Figure BDA0003287788300000201
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Therefore, it is intended that all such modifications and improvements be made without departing from the spirit of the invention.
SEQUENCE LISTING
<110> Shanghai Kaiser Biotech Ltd
CIBT American corporation (CIBT America Inc.)
Kaisai (Wusu) Biomaterials Co., Ltd.
<120> recombinant nucleic acid sequence, genetically engineered bacterium and method for producing 1,5-pentamethylene diamine
<130> P21015799C
<160> 64
<170> PatentIn version 3.5
<210> 1
<211> 235
<212> DNA
<213> Escherichia coli
<400> 1
tgctttttcc gatcgtcacg gcgatgttta tcgcgaacag atggtggact ttatccttag 60
cgcgttgaat ccgcagaact aacccatgat cgctagcacg ataatcattc acaaaaccac 120
cttaagacat gctaatccac tggtcagaac agtttaagat gagaaaaatt ctgtgacgct 180
tgccaacatt tctgatgatt agcattccct tcgccatttc cttgagcaaa cttta 235
<210> 2
<211> 238
<212> DNA
<213> Escherichia coli
<400> 2
tgtttggtaa aaattcccgc catcataaca ttgccaacgg cgaggggaag tgggtaaggc 60
atgtaaattc atcatgttga cgaaataatc gcccctggta aaagaaacac tgatgcgagg 120
cctgtgtttc aatctttaaa tcagtaaact tcatacgctt gacggaaaaa ccaggacgaa 180
acctaaatat ttgttgttaa gctgcaatgg aaacggtaaa agcggctagt atttaaag 238
<210> 3
<211> 233
<212> DNA
<213> Escherichia coli
<400> 3
ctcgcttaca tcgctaccag catggtcaac ctgcgcctgg cacaggaacg ttatccggac 60
gttcagttcc accagacccg cgagcattaa ttcttgcctc cagggcgcgg tagccgctgc 120
gccctgtcaa tttcccttcc ttattagccg cttacggaat gttcttaaaa cattcacttt 180
tgcttatgtt ttcgctgata tcccgagcgg tttcaaaatt gtgatctata ttt 233
<210> 4
<211> 237
<212> DNA
<213> Escherichia coli
<400> 4
gcagaaatga ctctcccatc agtacaaacg caacatattt gccacgcagc atccagacat 60
cacgaaacga atccatcttt atcgcatgtt ctggcggcgc gggttccgtg cgtgggacat 120
agctaataat ctggcggttt tgctggcgga gcggtttctt cattactggc ttcactaaac 180
gcatattaaa aatcagaaaa actgtagttt agccgattta gcccctgtac gtcccgc 237
<210> 5
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> p21
<400> 5
cactcccgcc tttaggggtc aaaattgttc tatactgtat tg 42
<210> 6
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> p22
<400> 6
tcccgccaaa ttcccaattt tgttctatac tgtattg 37
<210> 7
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> p23
<400> 7
tcccgccttt aggggtgaat tgttctatac tgaattg 37
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> p24
<400> 8
tcccgccttt aggggctaat tgttctatac tgaaatg 37
<210> 9
<211> 2148
<212> DNA
<213> Artificial Sequence
<220>
<223> cadA Gene sequence
<400> 9
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> 10
<211> 715
<212> PRT
<213> Artificial Sequence
<220>
<223> cadA protein sequence
<400> 10
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> 11
<211> 1041
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sequence of ompA
<400> 11
atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60
gccgctccga aagataacac ctggtacact ggtgctaaac tgggctggtc ccagtaccat 120
gacactggtt tcatcaacaa caatggcccg acccatgaaa accaactggg cgctggtgct 180
tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggttacga ctggttaggt 240
cgtatgccgt acaaaggcag cgttgaaaac ggtgcataca aagctcaggg cgttcaactg 300
accgctaaac tgggttaccc aatcactgac gacctggaca tctacactcg tctgggtggc 360
atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga caccggcgtt 420
tctccggtct tcgctggcgg tgttgagtac gcgatcactc ctgaaatcgc tacccgtctg 480
gaataccagt ggaccaacaa catcggtgac gcacacacca tcggcactcg tccggacaac 540
ggcatgctga gcctgggtgt ttcctaccgt ttcggtcagg gcgaagcagc tccagtagtt 600
gctccggctc cagctccggc accggaagta cagaccaagc acttcactct gaagtctgac 660
gttctgttca acttcaacaa agcaaccctg aaaccggaag gtcaggctgc tctggatcag 720
ctgtacagcc agctgagcaa cctggatccg aaagacggtt ccgtagttgt tctgggttac 780
accgaccgca tcggttctga cgcttacaac cagggtctgt ccgagcgccg tgctcagtct 840
gttgttgatt acctgatctc caaaggtatc ccggcagaca agatctccgc acgtggtatg 900
ggcgaatcca acccggttac tggcaacacc tgtgacaacg tgaaacagcg tgctgcactg 960
atcgactgcc tggctccgga tcgtcgcgta gagatcgaag ttaaaggtat caaagacgtt 1020
gtaactcagc cgcaggctta a 1041
<210> 12
<211> 1104
<212> DNA
<213> Artificial Sequence
<220>
<223> ompC Gene sequence
<400> 12
atgaaagtta aagtactgtc cctcctggtc ccagctctgc tggtagcagg cgcagcaaac 60
gctgctgaag tttacaacaa agacggcaac aaattagatc tgtacggtaa agtagacggc 120
ctgcactatt tctctgacaa caaagatgta gatggcgacc agacctacat gcgtcttggc 180
ttcaaaggtg aaactcaggt tactgaccag ctgaccggtt acggccagtg ggaatatcag 240
atccagggca acagcgctga aaacgaaaac aactcctgga cccgtgtggc attcgcaggt 300
ctgaaattcc aggatgtggg ttctttcgac tacggtcgta actacggcgt tgtttatgac 360
gtaacttcct ggaccgacgt actgccagaa ttcggtggtg acacctacgg ttctgacaac 420
ttcatgcagc agcgtggtaa cggcttcgcg acctaccgta acactgactt cttcggtctg 480
gttgacggcc tgaactttgc tgttcagtac cagggtaaaa acggcaaccc atctggtgaa 540
ggctttacta gtggcgtaac taacaacggt cgtgacgcac tgcgtcaaaa cggcgacggc 600
gtcggcggtt ctatcactta tgattacgaa ggtttcggta tcggtggtgc gatctccagc 660
tccaaacgta ctgatgctca gaacaccgct gcttacatcg gtaacggcga ccgtgctgaa 720
acctacactg gtggtctgaa atacgacgct aacaacatct acctggctgc tcagtacacc 780
cagacctaca acgcaactcg cgtaggttcc ctgggttggg cgaacaaagc acagaacttc 840
gaagctgttg ctcagtacca gttcgacttc ggtctgcgtc cgtccctggc ttacctgcag 900
tctaaaggta aaaacctggg tcgtggctac gacgacgaag atatcctgaa atatgttgat 960
gttggtgcta cctactactt caacaaaaac atgtccacct acgttgacta caaaatcaac 1020
ctgctggacg acaaccagtt cactcgtgac gctggcatca acactgataa catcgtagct 1080
ctgggtctgg tttaccagtt ctaa 1104
<210> 13
<211> 1089
<212> DNA
<213> Artificial Sequence
<220>
<223> ompF Gene sequence
<400> 13
atgatgaagc gcaatattct ggcagtgatc gtccctgctc tgttagtagc aggtactgca 60
aacgctgcag aaatctataa caaagatggc aacaaagtag atctgtacgg taaagctgtt 120
ggtctgcatt atttttccaa gggtaacggt gaaaacagtt acggtggcaa tggcgacatg 180
acctatgccc gtcttggttt taaaggggaa actcaaatca attccgatct gaccggttat 240
ggtcagtggg aatataactt ccagggtaac aactctgaag gcgctgacgc tcaaactggt 300
aacaaaacgc gtctggcatt cgcgggtctt aaatacgctg acgttggttc tttcgattac 360
ggccgtaact acggtgtggt ttatgatgca ctgggttaca ccgatatgct gccagaattt 420
ggtggtgata ctgcatacag cgatgacttc ttcgttggtc gtgttggcgg cgttgctacc 480
tatcgtaact ccaacttctt tggtctggtt gatggcctga acttcgctgt tcagtacctg 540
ggtaaaaacg agcgtgacac tgcacgccgt tctaacggcg acggtgttgg cggttctatc 600
agctacgaat acgaaggctt tggtatcgtt ggtgcttatg gtgcagctga ccgtaccaac 660
ctgcaagaag ctcaacctct tggcaacggt aaaaaagctg aacagtgggc tactggtctg 720
aagtacgacg cgaacaacat ctacctggca gcgaactacg gtgaaacccg taacgctacg 780
ccgatcacta ataaatttac aaacaccagc ggcttcgcca acaaaacgca agacgttctg 840
ttagttgcgc aataccagtt cgatttcggt ctgcgtccgt ccatcgctta caccaaatct 900
aaagcgaaag acgtagaagg tatcggtgat gttgatctgg tgaactactt tgaagtgggc 960
gcaacctact acttcaacaa aaacatgtcc acctatgttg actacatcat caaccagatc 1020
gattctgaca acaaactggg cgtaggttca gacgacaccg ttgctgtggg tatcgtttac 1080
cagttctaa 1089
<210> 14
<211> 639
<212> DNA
<213> Artificial Sequence
<220>
<223> gene sequence of ompW
<400> 14
atgaaaaagt taacagtggc ggctttggca gtaacaactc ttctctctgg cagtgccttt 60
gcgcatgaag caggcgaatt ttttatgcgt gcaggttctg caaccgtacg tccaacagaa 120
ggtgctggtg gtacgttagg aagtctgggt ggattcagcg tgaccaataa cacgcaactg 180
ggccttacgt ttacttatat ggcgaccgac aacattggtg tggaattact ggcagcgacg 240
ccgttccgcc ataaaatcgg cacccgggcg accggcgata ttgcaaccgt tcatcatctg 300
ccaccaacac tgatggcgca gtggtatttt ggtgatgcca gcagcaaatt ccgtccttac 360
gttggggcag gtattaacta caccaccttc tttgataatg gatttaacga tcatggcaaa 420
gaggcagggc tttccgatct cagtctgaaa gattcctggg gagctgccgg gcaggtgggg 480
gttgattatc tgattaaccg tgactggttg gttaacatgt cagtgtggta catggatatc 540
gataccaccg ccaattataa gctgggcggt gcacagcaac acgatagcgt acgcctcgat 600
ccgtgggtgt ttatgttctc agcaggatat cgtttttaa 639
<210> 15
<211> 516
<212> DNA
<213> Artificial Sequence
<220>
<223> ompX Gene sequence
<400> 15
atgaaaaaaa ttgcatgtct ttcagcactg gccgcagttc tggctttcac cgcaggtact 60
tccgtagctg cgacttctac tgtaactggc ggttacgcac agagcgacgc tcagggccaa 120
atgaacaaaa tgggcggttt caacctgaaa taccgctatg aagaagacaa cagcccgctg 180
ggtgtgatcg gttctttcac ttacaccgag aaaagccgta ctgcaagctc tggtgactac 240
aacaaaaacc agtactacgg catcactgct ggtccggctt accgcattaa cgactgggca 300
agcatctacg gtgtagtggg tgtgggttat ggtaaattcc agaccactga atacccgacc 360
tacaaacacg acaccagcga ctacggtttc tcctacggtg cgggtctgca gttcaacccg 420
atggaaaacg ttgctctgga cttctcttac gagcagagcc gtattcgtag cgttgacgta 480
ggcacctgga ttgccggtgt tggttaccgc ttctaa 516
<210> 16
<211> 346
<212> PRT
<213> Artificial Sequence
<220>
<223> OmpA
<400> 16
Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala
1 5 10 15
Thr Val Ala Gln Ala Ala Pro Lys Asp Asn Thr Trp Tyr Thr Gly Ala
20 25 30
Lys Leu Gly Trp Ser Gln Tyr His Asp Thr Gly Phe Ile Asn Asn Asn
35 40 45
Gly Pro Thr His Glu Asn Gln Leu Gly Ala Gly Ala Phe Gly Gly Tyr
50 55 60
Gln Val Asn Pro Tyr Val Gly Phe Glu Met Gly Tyr Asp Trp Leu Gly
65 70 75 80
Arg Met Pro Tyr Lys Gly Ser Val Glu Asn Gly Ala Tyr Lys Ala Gln
85 90 95
Gly Val Gln Leu Thr Ala Lys Leu Gly Tyr Pro Ile Thr Asp Asp Leu
100 105 110
Asp Ile Tyr Thr Arg Leu Gly Gly Met Val Trp Arg Ala Asp Thr Lys
115 120 125
Ser Asn Val Tyr Gly Lys Asn His Asp Thr Gly Val Ser Pro Val Phe
130 135 140
Ala Gly Gly Val Glu Tyr Ala Ile Thr Pro Glu Ile Ala Thr Arg Leu
145 150 155 160
Glu Tyr Gln Trp Thr Asn Asn Ile Gly Asp Ala His Thr Ile Gly Thr
165 170 175
Arg Pro Asp Asn Gly Met Leu Ser Leu Gly Val Ser Tyr Arg Phe Gly
180 185 190
Gln Gly Glu Ala Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala Pro
195 200 205
Glu Val Gln Thr Lys His Phe Thr Leu Lys Ser Asp Val Leu Phe Asn
210 215 220
Phe Asn Lys Ala Thr Leu Lys Pro Glu Gly Gln Ala Ala Leu Asp Gln
225 230 235 240
Leu Tyr Ser Gln Leu Ser Asn Leu Asp Pro Lys Asp Gly Ser Val Val
245 250 255
Val Leu Gly Tyr Thr Asp Arg Ile Gly Ser Asp Ala Tyr Asn Gln Gly
260 265 270
Leu Ser Glu Arg Arg Ala Gln Ser Val Val Asp Tyr Leu Ile Ser Lys
275 280 285
Gly Ile Pro Ala Asp Lys Ile Ser Ala Arg Gly Met Gly Glu Ser Asn
290 295 300
Pro Val Thr Gly Asn Thr Cys Asp Asn Val Lys Gln Arg Ala Ala Leu
305 310 315 320
Ile Asp Cys Leu Ala Pro Asp Arg Arg Val Glu Ile Glu Val Lys Gly
325 330 335
Ile Lys Asp Val Val Thr Gln Pro Gln Ala
340 345
<210> 17
<211> 367
<212> PRT
<213> Artificial Sequence
<220>
<223> OmpC
<400> 17
Met Lys Val Lys Val Leu Ser Leu Leu Val Pro Ala Leu Leu Val Ala
1 5 10 15
Gly Ala Ala Asn Ala Ala Glu Val Tyr Asn Lys Asp Gly Asn Lys Leu
20 25 30
Asp Leu Tyr Gly Lys Val Asp Gly Leu His Tyr Phe Ser Asp Asn Lys
35 40 45
Asp Val Asp Gly Asp Gln Thr Tyr Met Arg Leu Gly Phe Lys Gly Glu
50 55 60
Thr Gln Val Thr Asp Gln Leu Thr Gly Tyr Gly Gln Trp Glu Tyr Gln
65 70 75 80
Ile Gln Gly Asn Ser Ala Glu Asn Glu Asn Asn Ser Trp Thr Arg Val
85 90 95
Ala Phe Ala Gly Leu Lys Phe Gln Asp Val Gly Ser Phe Asp Tyr Gly
100 105 110
Arg Asn Tyr Gly Val Val Tyr Asp Val Thr Ser Trp Thr Asp Val Leu
115 120 125
Pro Glu Phe Gly Gly Asp Thr Tyr Gly Ser Asp Asn Phe Met Gln Gln
130 135 140
Arg Gly Asn Gly Phe Ala Thr Tyr Arg Asn Thr Asp Phe Phe Gly Leu
145 150 155 160
Val Asp Gly Leu Asn Phe Ala Val Gln Tyr Gln Gly Lys Asn Gly Asn
165 170 175
Pro Ser Gly Glu Gly Phe Thr Ser Gly Val Thr Asn Asn Gly Arg Asp
180 185 190
Ala Leu Arg Gln Asn Gly Asp Gly Val Gly Gly Ser Ile Thr Tyr Asp
195 200 205
Tyr Glu Gly Phe Gly Ile Gly Gly Ala Ile Ser Ser Ser Lys Arg Thr
210 215 220
Asp Ala Gln Asn Thr Ala Ala Tyr Ile Gly Asn Gly Asp Arg Ala Glu
225 230 235 240
Thr Tyr Thr Gly Gly Leu Lys Tyr Asp Ala Asn Asn Ile Tyr Leu Ala
245 250 255
Ala Gln Tyr Thr Gln Thr Tyr Asn Ala Thr Arg Val Gly Ser Leu Gly
260 265 270
Trp Ala Asn Lys Ala Gln Asn Phe Glu Ala Val Ala Gln Tyr Gln Phe
275 280 285
Asp Phe Gly Leu Arg Pro Ser Leu Ala Tyr Leu Gln Ser Lys Gly Lys
290 295 300
Asn Leu Gly Arg Gly Tyr Asp Asp Glu Asp Ile Leu Lys Tyr Val Asp
305 310 315 320
Val Gly Ala Thr Tyr Tyr Phe Asn Lys Asn Met Ser Thr Tyr Val Asp
325 330 335
Tyr Lys Ile Asn Leu Leu Asp Asp Asn Gln Phe Thr Arg Asp Ala Gly
340 345 350
Ile Asn Thr Asp Asn Ile Val Ala Leu Gly Leu Val Tyr Gln Phe
355 360 365
<210> 18
<211> 362
<212> PRT
<213> Artificial Sequence
<220>
<223> OmpF
<400> 18
Met Met Lys Arg Asn Ile Leu Ala Val Ile Val Pro Ala Leu Leu Val
1 5 10 15
Ala Gly Thr Ala Asn Ala Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys
20 25 30
Val Asp Leu Tyr Gly Lys Ala Val Gly Leu His Tyr Phe Ser Lys Gly
35 40 45
Asn Gly Glu Asn Ser Tyr Gly Gly Asn Gly Asp Met Thr Tyr Ala Arg
50 55 60
Leu Gly Phe Lys Gly Glu Thr Gln Ile Asn Ser Asp Leu Thr Gly Tyr
65 70 75 80
Gly Gln Trp Glu Tyr Asn Phe Gln Gly Asn Asn Ser Glu Gly Ala Asp
85 90 95
Ala Gln Thr Gly Asn Lys Thr Arg Leu Ala Phe Ala Gly Leu Lys Tyr
100 105 110
Ala Asp Val Gly Ser Phe Asp Tyr Gly Arg Asn Tyr Gly Val Val Tyr
115 120 125
Asp Ala Leu Gly Tyr Thr Asp Met Leu Pro Glu Phe Gly Gly Asp Thr
130 135 140
Ala Tyr Ser Asp Asp Phe Phe Val Gly Arg Val Gly Gly Val Ala Thr
145 150 155 160
Tyr Arg Asn Ser Asn Phe Phe Gly Leu Val Asp Gly Leu Asn Phe Ala
165 170 175
Val Gln Tyr Leu Gly Lys Asn Glu Arg Asp Thr Ala Arg Arg Ser Asn
180 185 190
Gly Asp Gly Val Gly Gly Ser Ile Ser Tyr Glu Tyr Glu Gly Phe Gly
195 200 205
Ile Val Gly Ala Tyr Gly Ala Ala Asp Arg Thr Asn Leu Gln Glu Ala
210 215 220
Gln Pro Leu Gly Asn Gly Lys Lys Ala Glu Gln Trp Ala Thr Gly Leu
225 230 235 240
Lys Tyr Asp Ala Asn Asn Ile Tyr Leu Ala Ala Asn Tyr Gly Glu Thr
245 250 255
Arg Asn Ala Thr Pro Ile Thr Asn Lys Phe Thr Asn Thr Ser Gly Phe
260 265 270
Ala Asn Lys Thr Gln Asp Val Leu Leu Val Ala Gln Tyr Gln Phe Asp
275 280 285
Phe Gly Leu Arg Pro Ser Ile Ala Tyr Thr Lys Ser Lys Ala Lys Asp
290 295 300
Val Glu Gly Ile Gly Asp Val Asp Leu Val Asn Tyr Phe Glu Val Gly
305 310 315 320
Ala Thr Tyr Tyr Phe Asn Lys Asn Met Ser Thr Tyr Val Asp Tyr Ile
325 330 335
Ile Asn Gln Ile Asp Ser Asp Asn Lys Leu Gly Val Gly Ser Asp Asp
340 345 350
Thr Val Ala Val Gly Ile Val Tyr Gln Phe
355 360
<210> 19
<211> 212
<212> PRT
<213> Artificial Sequence
<220>
<223> OmpW
<400> 19
Met Lys Lys Leu Thr Val Ala Ala Leu Ala Val Thr Thr Leu Leu Ser
1 5 10 15
Gly Ser Ala Phe Ala His Glu Ala Gly Glu Phe Phe Met Arg Ala Gly
20 25 30
Ser Ala Thr Val Arg Pro Thr Glu Gly Ala Gly Gly Thr Leu Gly Ser
35 40 45
Leu Gly Gly Phe Ser Val Thr Asn Asn Thr Gln Leu Gly Leu Thr Phe
50 55 60
Thr Tyr Met Ala Thr Asp Asn Ile Gly Val Glu Leu Leu Ala Ala Thr
65 70 75 80
Pro Phe Arg His Lys Ile Gly Thr Arg Ala Thr Gly Asp Ile Ala Thr
85 90 95
Val His His Leu Pro Pro Thr Leu Met Ala Gln Trp Tyr Phe Gly Asp
100 105 110
Ala Ser Ser Lys Phe Arg Pro Tyr Val Gly Ala Gly Ile Asn Tyr Thr
115 120 125
Thr Phe Phe Asp Asn Gly Phe Asn Asp His Gly Lys Glu Ala Gly Leu
130 135 140
Ser Asp Leu Ser Leu Lys Asp Ser Trp Gly Ala Ala Gly Gln Val Gly
145 150 155 160
Val Asp Tyr Leu Ile Asn Arg Asp Trp Leu Val Asn Met Ser Val Trp
165 170 175
Tyr Met Asp Ile Asp Thr Thr Ala Asn Tyr Lys Leu Gly Gly Ala Gln
180 185 190
Gln His Asp Ser Val Arg Leu Asp Pro Trp Val Phe Met Phe Ser Ala
195 200 205
Gly Tyr Arg Phe
210
<210> 20
<211> 171
<212> PRT
<213> Artificial Sequence
<220>
<223> OmpX
<400> 20
Met Lys Lys Ile Ala Cys Leu Ser Ala Leu Ala Ala Val Leu Ala Phe
1 5 10 15
Thr Ala Gly Thr Ser Val Ala Ala Thr Ser Thr Val Thr Gly Gly Tyr
20 25 30
Ala Gln Ser Asp Ala Gln Gly Gln Met Asn Lys Met Gly Gly Phe Asn
35 40 45
Leu Lys Tyr Arg Tyr Glu Glu Asp Asn Ser Pro Leu Gly Val Ile Gly
50 55 60
Ser Phe Thr Tyr Thr Glu Lys Ser Arg Thr Ala Ser Ser Gly Asp Tyr
65 70 75 80
Asn Lys Asn Gln Tyr Tyr Gly Ile Thr Ala Gly Pro Ala Tyr Arg Ile
85 90 95
Asn Asp Trp Ala Ser Ile Tyr Gly Val Val Gly Val Gly Tyr Gly Lys
100 105 110
Phe Gln Thr Thr Glu Tyr Pro Thr Tyr Lys His Asp Thr Ser Asp Tyr
115 120 125
Gly Phe Ser Tyr Gly Ala Gly Leu Gln Phe Asn Pro Met Glu Asn Val
130 135 140
Ala Leu Asp Phe Ser Tyr Glu Gln Ser Arg Ile Arg Ser Val Asp Val
145 150 155 160
Gly Thr Trp Ile Ala Gly Val Gly Tyr Arg Phe
165 170
<210> 21
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-UF
<400> 21
aggcgtatca cgaggccctt tcgtcttcaa aaacccgcga catcgtaatc 50
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-UR
<400> 22
ggattatacc tcctttcttc aaggcg 26
<210> 23
<211> 790
<212> DNA
<213> Artificial Sequence
<220>
<223> Upp-U
<400> 23
aggcgtatca cgaggccctt tcgtcttcaa aaacccgcga catcgtaatc ctcaccgtga 60
tacatccccg gcatttctgc cgtttcgcca cccaccagtg aacagcctga ttgcagacaa 120
ccttccgcaa tgccgctgat caccgctgaa gcggtatcaa catccagttt tccggttgcg 180
taatagtcga ggaaaaacag cggctctgca ccttgcacca ccaggtcatt aacgcacatg 240
gcgaccagat caataccaat ggtgtcgtga cgttttaagt ccattgccag acgcagcttg 300
gtacctacgc cgtcagtgcc agaaaccagc acgggttcac gatatttttg cggcaatgca 360
cacagcgcac cgaagccgcc cagaccgccc atcacttccg gacgacgcgt tttcttcact 420
acgcctttga ttcttccaac cagagcatta cccgcgtcaa tatcaacacc ggcatctttg 480
tagctaagag aggttttatc ggtcactgct tgggtcccca cgcgttactt gcggtagaaa 540
aataaaattc ggcgcaattc taacagggaa agcaaacgtt tgcgagactg ctttacacaa 600
cctttttgca cgtcttttcc ccaggcgcgc ggcgaaagaa gacttgtgcc agggtaaagg 660
ttagttttcg gatggaataa tcttctttca taaccatctg aatataaaat aactttatct 720
caaaccgtta tcattttgac taaagtcaac gaaaagaata ttgccgcctt gaagaaagga 780
ggtataatcc 790
<210> 24
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-DF
<400> 24
tgccgccttg aagaaaggag gtataatccg aattcagtcg gctttttttt gagtaaagcg 60
<210> 25
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-DR
<400> 25
ccgcattaaa gcttatcgat gataagctgt caaacatgac cgggagtaaa cccgccata 59
<210> 26
<211> 574
<212> DNA
<213> Artificial Sequence
<220>
<223> Upp-500bp-D
<400> 26
tgccgccttg aagaaaggag gtataatccg aattcagtcg gctttttttt gagtaaagcg 60
cctataacac ataatacaga ggataatact atgacgcgcc gtgctatcgg ggtgagtgaa 120
agaccgccac ttttacagac aatcccgctt agtttgcaac atttgttcgc catgtttggt 180
gcaaccgtcc tggtgcccgt cttatttcat attaacccgg cgactgtact gttatttaac 240
ggtattggaa cgctgctgta tctcttcatc tgtaaaggga aaattccggc ttatcttggt 300
tccagctttg cctttatttc accggtattg ttactgttgc cgttagggta tgaagtcgcg 360
ctgggcggct ttattatgtg cggcgtgctg ttctgcctgg tttcttttat cgtgaagaaa 420
gcggggaccg gctggctgga cgtgctgttt ccacctgcgg caatgggcgc aatcgttgcc 480
gtcatcggtc tggagctggc gggcgtagct gccggtatgg cgggtttact cccggtcatg 540
tttgacagct tatcatcgat aagctttaat gcgg 574
<210> 27
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-F
<400> 27
tggagccggg ccacctcgac ctgaatggaa gccggcgtcg attttttttg tggctgccc 59
<210> 28
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> upp-R
<400> 28
tggagtggtg aatccgttag cgaggtgccg ctttgttgta atccactttc g 51
<210> 29
<211> 1905
<212> DNA
<213> Artificial Sequence
<220>
<223> Pupp-UPP-D
<400> 29
tggagccggg ccacctcgac ctgaatggaa gccggcgtcg attttttttg tggctgcccc 60
tcaaaggaga aagagtatga agatcgtgga agtcaaacac ccactcgtca aacacaagct 120
gggactgatg cgtgagcaag atatcagcac caagcgcttt cgcgaactcg cttccgaagt 180
gggtagcctg ctgacttacg aagcgaccgc cgacctcgaa acggaaaaag taactatcga 240
aggctggaac ggcccggtag aaatcgacca gatcaaaggt aagaaaatta ccgttgtgcc 300
aattctgcgt gcgggtcttg gtatgatgga cggtgtgctg gaaaacgttc cgagcgcgcg 360
catcagcgtt gtcggtatgt accgtaatga agaaacgctg gagccggtac cgtacttcca 420
gaaactggtt tctaacatcg atgagcgtat ggcgctgatc gttgacccaa tgctggcaac 480
cggtggttcc gttatcgcga ccatcgacct gctgaaaaaa gcgggctgca gcagcatcaa 540
agttctggtg ctggtagctg cgccagaagg tatcgctgcg ctggaaaaag cgcacccgga 600
cgtcgaactg tataccgcat cgattgatca gggactgaac gagcacggat acattattcc 660
gggcctcggc gatgccggtg acaaaatctt tggtacgaaa taaagaataa aaataattaa 720
agccgacttt aagagtcggc ttttttttga gtaaagcgcc tataacacat aatacagagg 780
ataatactat gacgcgccgt gctatcgggg tgagtgaaag accgccactt ttacagacaa 840
tcccgcttag tttgcaacat ttgttcgcca tgtttggtgc aaccgtcctg gtgcccgtct 900
tatttcatat taacccggcg actgtactgt tatttaacgg tattggaacg ctgctgtatc 960
tcttcatctg taaagggaaa attccggctt atcttggttc cagctttgcc tttatttcac 1020
cggtattgtt actgttgccg ttagggtatg aagtcgcgct gggcggcttt attatgtgcg 1080
gcgtgctgtt ctgcctggtt tcttttatcg tgaagaaagc ggggaccggc tggctggacg 1140
tgctgtttcc acctgcggca atgggcgcaa tcgttgccgt catcggtctg gagctggcgg 1200
gcgtagctgc cggtatggcg ggtttactcc cggctgaagg gcaaacgcca gactccaaaa 1260
ccatcatcat ctctattacc accctggcgg tcacggtttt aggttccgtg ctgtttcgtg 1320
gtttcctggc aattatcccg attttaattg gcgtgctggt ggggtacgcg ctctctttcg 1380
caatgggaat tgtcgatacc acgccgatta ttaatgctca ctggtttgcg ctgccaaccc 1440
tctatacgcc gcgcttcgag tggtttgcca ttctgactat tctgccagcg gcgttagtgg 1500
ttattgccga acacgtaggg cacctggtag taacggctaa tatcgtcaaa aaagatctgc 1560
tgcgcgatcc aggtctgcac cgttcgatgt ttgctaatgg cttgtcgacc gtgatttccg 1620
gcttctttgg ctctacgcca aatactactt acggagaaaa cattggcgtg atggcgatca 1680
cccgtgttta cagtacctgg gttatcggcg gggcggcgat tttcgctatc ctgctttcct 1740
gcgtcggtaa actggctgcc gctatccaga tgatcccatt gccggtgatg ggcggcgttt 1800
cgctgctgct ttatggtgtc atcggtgctt ccggtattcg tgttttgatc gaatcgaaag 1860
tggattacaa caaagcggca cctcgctaac ggattcacca ctcca 1905
<210> 30
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> P1P2-tetA-F
<400> 30
tcatgtttga cagcttatca tcgataagc 29
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> P1P2-tetA-R
<400> 31
gccggcttcc attcaggtcg 20
<210> 32
<211> 1285
<212> DNA
<213> Artificial Sequence
<220>
<223> P1P2-tetA
<400> 32
tcatgtttga cagcttatca tcgataagct ttaatgcggt agtttatcac agttaaattg 60
ctaacgcagt caggcaccgt gtatgaaatc taacaatgcg ctcatcgtca tcctcggcac 120
cgtcaccctg gatgctgtag gcataggctt ggttatgccg gtactgccgg gcctcttgcg 180
ggatatcgtc cattccgaca gcatcgccag tcactatggc gtgctgctag cgctatatgc 240
gttgatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgct ttggccgccg 300
cccagtcctg ctcgcttcgc tacttggagc cactatcgac tacgcgatca tggcgaccac 360
acccgtcctg tggatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg 420
tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt 480
cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact 540
gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa 600
cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gaccgatgcc 660
cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc 720
cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg 780
ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc 840
ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg 900
tttcggcgag aagcaggcca ttatcgccgg catggcggcc gacgcgctgg gctacgtctt 960
gctggcgttc gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg 1020
cggcatcggg atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca 1080
gggacagctt caaggatcgc tcgcggctct taccagccta acttcgatca ctggaccgct 1140
gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt 1200
aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc 1260
cacctcgacc tgaatggaag ccggc 1285
<210> 33
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> cadA-F
<400> 33
cgccttgaag aaaggaggta taatccgagc tcatgaacgt tattgcaata ttgaatcaca 60
tg 62
<210> 34
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> cadA-R
<400> 34
ggcgctttac tcaaaaaaaa gccgacttct agaccacttc ccttgtacga gctaa 55
<210> 35
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> piclR-F
<400> 35
ccttgaagaa aggaggtata atccatttgt tcaacattaa ctcatcggat 50
<210> 36
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> piclR-R
<400> 36
attcaatatt gcaataacgt tcatgagctc ctgaacctgt ccagtcgctg 50
<210> 37
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> pcsiE-F
<400> 37
cgccttgaag aaaggaggta taatccgagc tctgcttttt ccgatcgtca cg 52
<210> 38
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> pcsiE-R
<400> 38
aatattgcaa taacgttcat gagctctaaa gtttgctcaa ggaaatggc 49
<210> 39
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> pbolA-F
<400> 39
cgccttgaag aaaggaggta taatccgagc tctgtttggt aaaaattccc g 51
<210> 40
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> pbolA-R
<400> 40
aatattgcaa taacgttcat gagctccttt aaatactagc cgcttttac 49
<210> 41
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> posmY-F
<400> 41
cgccttgaag aaaggaggta taatccgagc tcctcgctta catcgctacc agc 53
<210> 42
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> posmY-R
<400> 42
aatattgcaa taacgttcat gagctcaaat atagatcaca attttgaa 48
<210> 43
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> pkatE-F
<400> 43
cgccttgaag aaaggaggta taatccgagc tcgcagaaat gactctccca tc 52
<210> 44
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> pkatE-R
<400> 44
aatattgcaa taacgttcat gagctcgcgg gacgtacagg ggc 43
<210> 45
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> pcsiE-F2
<400> 45
cgcaccagcg actggacagg ttcagtgctt tttccgatcg tcacg 45
<210> 46
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> pbolA-F2
<400> 46
cgcaccagcg actggacagg ttcagtgttt ggtaaaaatt cccg 44
<210> 47
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> posmY-F2
<400> 47
cgcaccagcg actggacagg ttcagctcgc ttacatcgct accagc 46
<210> 48
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> pkatE-F2
<400> 48
cgcaccagcg actggacagg ttcaggcaga aatgactctc ccatc 45
<210> 49
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> ompA-F
<400> 49
cggataacaa tttcacacag gaggagctca tgaaaaagac agctatcgcg 50
<210> 50
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> ompA-R
<400> 50
gctttactca aaaaaaagcc gacttctaga ttaagcctgc ggctgagtta 50
<210> 51
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> ompC-F
<400> 51
cggataacaa tttcacacag gaggagctca tgaaagttaa agtactgtcc ctcc 54
<210> 52
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> ompC-R
<400> 52
gctttactca aaaaaaagcc gacttctaga ttagaactgg taaaccagac cc 52
<210> 53
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> ompF-F
<400> 53
cggataacaa tttcacacag gaggagctca tgatgaagcg caatattctg g 51
<210> 54
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> ompF-R
<400> 54
gctttactca aaaaaaagcc gacttctaga ttagaactgg taaacgatac cca 53
<210> 55
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> ompW-F
<400> 55
cggataacaa tttcacacag gaggagctca tgaaaaagtt aacagtggcg g 51
<210> 56
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> ompW-R
<400> 56
gctttactca aaaaaaagcc gacttctaga ttaaaaacga tatcctgctg agaac 55
<210> 57
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> ompX-F
<400> 57
cggataacaa tttcacacag gaggagctca tgaaaaaaat tgcatgtctt tcagc 55
<210> 58
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> ompX-R
<400> 58
gctttactca aaaaaaagcc gacttctaga ttagaagcgg taaccaacac c 51
<210> 59
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> plac-F
<400> 59
gagctcctcc tgtgtgaaat tgttatc 27
<210> 60
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> plac-R
<400> 60
aaaaataatt agctcgtaca agggaagtgg ggataaccgt attaccgcct ttgag 55
<210> 61
<211> 308
<212> DNA
<213> Artificial Sequence
<220>
<223> sequence comprising a plac promoter
<400> 61
ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 60
gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 120
cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 180
cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 240
ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 300
aggagctc 308
<210> 62
<211> 175
<212> DNA
<213> Artificial Sequence
<220>
<223> piclR promoter sequence
<400> 62
atttgttcaa cattaactca tcggatcagt tcagtaacta ttgcattagc taacaataaa 60
aatgaaaatg atttccacga tacagaaaaa agagactgtc atggtcgcac ccattcccgc 120
gaaacgcggc agaaaacccg ccgttgccac cgcaccagcg actggacagg ttcag 175
<210> 63
<211> 113
<212> DNA
<213> Artificial Sequence
<220>
<223> Corynebacterium glutamicum (Corynebacterium glutamicum) of origin
Glutamicum) piclR promoter piclR-Cg
<400> 63
acagtatagc tattaagagg cgtaaatgtc acctcccgcc caaaatcttc ttataccccc 60
acacagtgaa tcccttcacc acgtctcatt gggtgaaatg ctaaattcaa ggt 113
<210> 64
<211> 71
<212> DNA
<213> Artificial Sequence
<220>
<223> piclR promoter piclR-Ha derived from Hafnia alvei (Hafnia alvei)
<400> 64
gagcagttaa aaaccacgat cgcatccata atgtcgaaaa cgaaaataga ttccattttt 60
ataacatttt t 71

Claims (10)

1. A tandem promoter, comprising a piclR promoter and a stationary phase specific promoter.
2. The tandem promoter according to claim 1, wherein the piclR promoter is derived from any one or more of Escherichia coli (Escherichia coli), corynebacterium glutamicum (Corynebacterium glutamicum), and Hafnia alvei (Hafnia alvei); and/or, the stationary phase specific promoter is selected from one or more of the following: pcsiE, pbolA, posmY, pkatE, p21, p22, p23, and p24;
preferably, the nucleotide sequence of the piclR promoter is shown in any one of SEQ ID NO 62-64, and the nucleotide sequences of pcsiE, pbolA, posmY, pkatE, p21, p22, p23 and p24 are shown in SEQ ID NO 1-8;
more preferably, the tandem promoter is piclR-pcSiE, piclR-pbOLA, piclR-posmY, piclR-pkatE, piclR-p21, piclR-p22, piclR-p23 or piclR-p24.
3. A recombinant nucleic acid sequence comprising the tandem promoter sequence of claim 1 or 2 and a lysine decarboxylase gene operably linked to the tandem promoter.
4. The recombinant nucleic acid sequence according to claim 3, wherein said lysine decarboxylase gene is selected from the group consisting of: a cadA gene or an ldcC gene derived from any one of Escherichia coli, corynebacterium glutamicum, and Hafnia alvei;
preferably, the nucleotide sequence of the cadA gene is shown as SEQ ID NO. 9;
more preferably, the recombinant nucleic acid sequence comprises piclR-pcSiE-cadA, piclR-pbOLA-cadA, piclR-posmY-cadA, piclR-pkatE-cadA, piclR-p21-cadA, piclR-p22-cadA, piclR-p23-cadA or piclR-p24-cadA.
5. A combination of recombinant nucleic acids, wherein said combination of recombinant nucleic acids comprises:
a first sequence comprising the tandem promoter of claim 1 or 2 or the recombinant nucleic acid sequence of claim 3 or 4; and a second sequence comprising a constitutive promoter or said constitutive promoter and operably linked thereto a gene encoding an outer membrane porin;
preferably, the constitutive promoter is selected from any one or more of plac, trp, tac, trc and PL; and/or, the gene encoding outer membrane porin is selected from the group consisting of: any one or more of ompA, ompC, ompF, ompW and ompX;
more preferably, the nucleotide sequence of the constitutive promoter is shown as SEQ ID NO. 61; and/or the nucleotide sequence of the gene for coding the outer membrane porin is shown in any one of SEQ ID NO 11-15;
even more preferably, the first sequence is piclR-pcsiE-cadA, piclR-pbolA-cadA, piclR-posmY-cadA, piclR-pkatE-cadA, piclR-p21-cadA, piclR-p22-cadA, piclR-p23-cadA or piclR-p24-cadA; and/or the second sequence is plac-ompA, plac-ompC, plac-ompF or plac-ompW;
most preferably, when the first sequence is operably linked to the second sequence, the recombinant nucleic acid combination comprises piclR-p24-cadA-plac-ompA, piclR-p24-cadA-plac-ompC, piclR-p24-cadA-plac-ompF, piclR-p24-cadA-plac-ompW or piclR-p24-cadA-plac-ompX.
6. A biomaterial comprising a tandem promoter according to claim 1 or 2 or a recombinant nucleic acid sequence according to claim 3 or 4 or a combination of recombinant nucleic acids according to claim 5;
preferably, the biological material comprises an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium;
more preferably, the backbone plasmids of the plasmid vector include pUC18, pUC19, pBR322, pACYC, pET, pSC101, pKD46, and derivatives thereof.
7. A genetically engineered bacterium comprising the tandem promoter of claim 1 or 2 or the recombinant nucleic acid sequence of claim 3 or 4 or the recombinant nucleic acid combination of claim 5;
preferably, the chromosome of the genetically engineered bacterium comprises the recombinant nucleic acid sequence of claim 3 or 4 or the recombinant nucleic acid combination of claim 5;
more preferably, the genetically engineered bacterium is introduced recombinantly in the chromosome with the tandem promoter according to claim 1 or 2 or the recombinant nucleic acid sequence according to claim 3 or 4 or the recombinant nucleic acid combination according to claim 5.
8. The genetically engineered bacterium of claim 7, wherein the host bacterium of the genetically engineered bacterium is derived from a species of the genus Escherichia (Escherichia), corynebacterium (Corynebacterium), bacillus (Bacillus), thermus (Thermus), brevibacterium (Brevibacterium) or Hafnia (Hafnia);
preferably, the host bacterium of the genetically engineered bacterium is derived from Escherichia coli (Escherichia coli), thermus thermophilus (Thermus thermophilus), hafnia alvei (Hafnia alvei), bacillus subtilis (Bacillus subtilis) or Corynebacterium glutamicum (Corynebacterium glutamicum).
9. A method for producing 1,5-pentanediamine, which comprises culturing the genetically engineered bacterium of claim 7 or 8 in a fermentation medium to produce 1,5-pentanediamine.
10. Use of the tandem promoter according to claim 1 or 2 or the recombinant nucleic acid sequence according to claim 3 or 4 or the recombinant nucleic acid combination according to claim 5 or the biological material according to claim 6 or the genetically engineered bacterium according to claim 7 or 8 for the production of 1,5-pentanediamine.
CN202111153076.4A 2021-09-29 2021-09-29 Recombinant nucleic acid sequence, genetic engineering bacteria and method for producing 1,5-pentanediamine Pending CN115873852A (en)

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CN115873852A true CN115873852A (en) 2023-03-31

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