Recombinant nucleic acid sequences, genetically engineered bacteria, and methods for producing 1,5-pentanediamine

Abstract

The application discloses a recombinant nucleic acid sequence, a recombinant nucleic acid combination, a genetically engineered bacterium and a method for producing 1,5-pentanediamine. The recombinant nucleic acid sequence comprises a tandem promoter sequence and a lysine decarboxylase gene operably connected with the tandem promoter, and the tandem promoter comprises a piclR promoter and a stationary phase-specific promoter. The application controls the expression time and expression amount of L-lysine decarboxylase by using the piclR-stationary phase-specific promoter in series, reduces the energy consumption in the tolerance process of 1,5-pentanediamine, promotes the production capacity of the strain, and further improves the yield of 1,5-pentanediamine. Alternatively, the yield of 1,5-pentanediamine can be further improved by increasing the expression amount of the gene for promoting the discharge of 1,5-pentanediamine out of the cell, reducing the intracellular 1,5-pentanediamine concentration and the inhibition of the intracellular lysine decarboxylase activity, and increasing the tolerance capacity of the strain.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, specifically, it relates to recombinant nucleic acid sequences, genetically engineered bacteria, and methods for producing 1,5-pentanediamine. Background Technology

[0002] 1,5-Pentanediamine has a wide range of applications and high economic value in industrial production; for example, it can be polymerized with diacids to synthesize novel nylons. Currently, the biosynthesis of 1,5-pentanediamine mainly utilizes two strategies: fermentation production or in vitro enzymatic catalysis. For fermentation production, 1,5-pentanediamine is generated by removing a carboxyl group from L-lysine using lysine decarboxylase (LDC, EC 4.1.1.18). Specifically, the lysine decarboxylase gene can be added to lysine-producing microorganisms such as Corynebacterium glutamicum and Escherichia coli, thereby extending the lysine biosynthesis pathway to the 1,5-pentanediamine biosynthesis pathway.

[0003] Currently, bacteria of the genera *Corynebacterium* and *Escherichia* have been modified using DNA recombination technology to produce L-lysine. The increased efficiency is achieved by overexpressing genes related to the L-lysine synthesis pathway and genes related to feedback inhibition desensitization, or by enhancing the energy supply pathway starting from glucose metabolism. Genes related to feedback inhibition desensitization, such as aspartate kinase III (LysC), are specific key enzymes in the lysine synthesis pathway. However, because the bacterial cells have a limited tolerance to 1,5-pentanediamine concentrations, excessive expression of 1,5-pentanediamine generated from lysine decarboxylase in the early stages of fermentation can be toxic to the cells, inhibiting cell growth and the process of L-lysine production from glucose (Qian, et al., Biotechnol. Bioeng. 2011; 108:93–103).

[0004] For example, patent document No. WO2019006723A1, published on January 19, 2019, entitled "HETEROLOGOUSEXPRESSION OF THERMOPHILIC LYSINE DEC-ARBOXYLASE AND USES THEREOF", discloses a heterologous expression of thermophilic lysine decarboxylase and its uses. In the technical solution disclosed in this patent document, thermophilic lysine decarboxylase is first used, and the enzyme activity is controlled by high temperature.

[0005] For example, Chinese patent document CN105368766A, published on March 2, 2016, entitled "A genetically engineered bacterium for producing pentanediamine and a method for preparing pentanediamine therefrom," discloses a pentanediamine-producing strain and a process for efficiently producing pentanediamine. In the technical solution disclosed in this patent document, the stability problem is solved to some extent by using a temperature-controlled promoter to express lysine decarboxylase; however, using high temperature for catalysis further increases energy consumption and production costs.

[0006] Therefore, there is a need to develop a more economical, stable, and efficient production process for 1,5-pentanediamine. Furthermore, superior cellular physiological properties are one of the key factors for obtaining highly efficient cell factories. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the present invention aims to provide a recombinant nucleic acid sequence, genetically engineered bacteria and a method for producing 1,5-pentanediamine, so as to achieve stable, efficient and low-cost production of 1,5-pentanediamine.

[0008] To achieve the above objectives, the present invention is accomplished through the following aspects:

[0009] In a first aspect, the present invention provides a tandem promoter comprising a piclR promoter and a stationary phase-specific promoter.

[0010] In some preferred embodiments, the piclR promoter is derived from any one or more of *Escherichia coli*, *Corynebacterium glutamicum*, and *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 can originate from cells of microorganisms, animals, or plants, including but not limited to *Escherichia coli*, *Bacillus subtilis*, *Bacillus halodurans*, *Streptomyces coelicolor*, *Hafnia alvei*, *Corynebacterium glutamicum*, or *Klebsiella oxytoca*.

[0011] Preferably, the nucleotide sequences of the pclR promoter are 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, respectively. Specifically, the sequence of pcsiE is shown in SEQ ID NO:1; the sequence of pbolA is shown in SEQ ID NO:2; the sequence of posmY is shown in SEQ ID NO:3; the sequence of pkatE is shown in SEQ ID NO:4; the sequence of p21 is shown in SEQ ID NO:5; the sequence of p22 is shown in SEQ ID NO:6; the sequence of p23 is shown in SEQ ID NO:7; and the sequence of p24 is shown in SEQ ID NO:8.

[0012] More preferably, the tandem promoter is piclR-pcsiE, piclR-pbolA, piclR-posmY, piclR-pkatE, piclR-p21, piclR-p22, piclR-p23, or piclR-p24.

[0013] This invention enhances promoter strength by recombinantly inserting a tandem promoter, including a pclR promoter and a stationary-phase-specific promoter, and the lysine decarboxylase gene under its control into the bacterial chromosome. This ensures sufficient and stable expression of L-lysine decarboxylase, significantly reducing energy consumption caused by host cell tolerance to 1,5-pentanediamine toxicity, thereby promoting 1,5-pentanediamine production. The entire process requires no resistance genes, inducing elements, etc. Optionally, genes promoting 1,5-pentanediamine excretion from the cell, such as outer membrane porin genes (e.g., ompA, ompC, ompF, ompW, and ompX), can be further incorporated to further reduce intracellular 1,5-pentanediamine concentration and increase 1,5-pentanediamine production.

[0014] In this document, "under the control of the promoter" means that the promoter sequence and the gene sequence are operatively linked to ensure that the transcription and expression of the gene are controlled by the promoter.

[0015] Therefore, in a second aspect, the present invention provides a recombinant nucleic acid sequence comprising the aforementioned tandem promoter sequence and a lysine decarboxylase gene operably linked to the tandem promoter.

[0016] This invention enhances promoter strength and ensures sufficient and stable expression of L-lysine decarboxylase by recombinantly inserting a tandem promoter including the pclR promoter and a stationary phase-specific promoter, along with the lysine decarboxylase gene under its control, into the bacterial chromosome. This significantly reduces the energy consumption caused by host cell tolerance to 1,5-pentanediamine toxicity, thereby promoting the production of 1,5-pentanediamine. The entire process does not require the use of resistance genes, inducing elements, etc.

[0017] Optionally, genes that promote the expulsion of 1,5-pentanediamine from the cell, such as outer membrane porin genes (e.g., ompA, ompC, ompF, ompW, and ompX), can be further combined to further reduce the intracellular concentration of 1,5-pentanediamine and increase the production of 1,5-pentanediamine.

[0018] In some embodiments, the gene for the lysine decarboxylase (LDC, EC 4.1.1.18) may be derived from microbial, animal, or plant cells, including but not limited to Escherichia coli, Bacillus subtilis, Bacillus halodurans, Streptomyces coelicolor, Hafnia alvei, Corynebacterium glutamicum, or Klebsiella oxytoca.

[0019] Preferably, the lysine decarboxylase gene is selected from the cadA or ldcC gene of any one of *Escherichia coli*, *Corynebacterium glutamicum*, and *Haffniella vesicae*. Alternatively, the lysine decarboxylase can also originate from strains that have undergone mutagenesis or random mutation, or from genetically engineered bacteria. The lysine decarboxylase can also be a mutant (including natural mutants and artificial recombinant mutants) or an active fragment (a truncated protein fragment retaining lysine decarboxylase activity) of the aforementioned sources.

[0020] More preferably, the nucleotide sequence of the cadA gene is shown in SEQ ID NO:9.

[0021] In some implementations, the recombinant nucleic acid sequence includes 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.

[0022] Those skilled in the art will understand that, in order to improve the expression of a target gene in host cells, the coding sequence of the target gene can be optimized according to the codon preferences of the host cell. For example, rare codons of the target gene can be synonymously replaced to make them more similar to the codon usage patterns of the host cell. Numerous reports have been published on improving the expression levels of exogenous genes in host cells using this method.

[0023] In this document, a gene that promotes the expulsion of 1,5-pentanediamine from the cell refers to a gene whose expression product promotes the expulsion of 1,5-pentanediamine from the microbial cell, thereby reducing the intracellular concentration of 1,5-pentanediamine and inhibiting the activity of intracellular lysine decarboxylase, thus promoting the production of 1,5-pentanediamine. In some embodiments, the gene promoting pentanediamine expulsion includes genes for outer membrane porins. Outer membrane porins include OmpA, OmpC, OmpF, OmpW, OmpX, etc. More specifically, the protein promoting pentanediamine expulsion can also be a mutant (including natural mutants and artificial recombinant mutants) or an active fragment of the above proteins.

[0024] Therefore, in a third aspect, the present invention provides a recombinant nucleic acid combination, the recombinant nucleic acid combination comprising:

[0025] The first sequence includes the aforementioned tandem promoter or the aforementioned recombinant nucleic acid sequence;

[0026] And a second sequence comprising a constitutive promoter or the constitutive promoter and a gene encoding an outer membrane pore protein operatively linked thereto.

[0027] Preferably, the constitutive promoter is selected from any one or more of plac, trp, tac, trc and PL.

[0028] More preferably, the nucleotide sequence of the constitutive promoter is shown in SEQ ID NO:61;

[0029] And / or, preferably, the outer membrane pore protein gene is selected from any one or more of ompA, ompC, ompF, ompW and ompX.

[0030] In some embodiments, the nucleotide sequence of the gene encoding the outer membrane porin is shown in any one of SEQ ID NO:11-15.

[0031] In some implementations, the sequence of the ompA gene is shown in SEQ ID NO:11, and the sequence of the OmpA protein is shown in SEQ ID NO:16;

[0032] The sequence of the ompC gene is shown in SEQ ID NO:12, and the sequence of the OmpC protein is shown in SEQ ID NO:17.

[0033] The sequence of the ompF gene is shown in SEQ ID NO:13, and the sequence of OmpF is shown in SEQ ID NO:18;

[0034] The sequence of the ompW gene is shown in SEQ ID NO:14, and the sequence of the OmpW protein is shown in SEQ ID NO:19.

[0035] The sequence of the ompX gene is shown in SEQ ID NO:15, and the sequence of the OmpX protein is shown in SEQ ID NO:20.

[0036] In the recombinant nucleic acid described herein, the tandem promoter (e.g., element a) and the lysine decarboxylase gene (e.g., element b) are operatively linked, resulting in an expression element, denoted as expression element ab, such that the transcription and expression of the lysine decarboxylase are controlled by a pentanediamine-inducible promoter. Preferably, the linked expression element ab enables expression in host cells regulated by pentanediamine concentration, thereby controlling the production of L-lysine decarboxylase through the tandem promoter and increasing the yield of 1,5-pentanediamine.

[0037] In the recombinant nucleic acid described herein, a constitutive promoter (e.g., element c) and a gene promoting pentanediamine efflux (e.g., element d) are operatively linked, resulting in an expression element, which can be represented as expression element cd, such that the transcription and expression of the gene promoting pentanediamine efflux are under the control of the constitutive promoter. Optionally, the aforementioned linked ab and cd can be operatively linked or exist independently.

[0038] 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.

[0039] When the first sequence and the second sequence are operatively connected, other embodiments of the present invention are formed, namely 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.

[0040] The constitutive promoters used herein are well known to those skilled in the art and can enable the expression of genes that promote the excretion of 1,5-pentanediamine from cells in host cells, such as the plac, trp, tac, trc, or PL promoters. For example, sequences including the plac promoter are shown in SEQ ID NO:61.

[0041] Fourthly, the present invention also provides a biological material comprising the above-mentioned tandem promoter or recombinant nucleic acid sequence or the above-mentioned recombinant DNA nucleic acid;

[0042] Preferably, the biological material includes expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0043] More preferably, the backbone plasmid of the plasmid vector includes pUC18, pUC19, pBR322, pACYC, pET, pSC101, and their derivative plasmids. The backbone plasmid, for example, is pBR322, which is used to express the aforementioned promoter and multiple elements of the gene. Furthermore, in the technical solution described in this invention, pKD46 is used for E. coli chromosome recombination.

[0044] Fifthly, the present invention provides a genetically engineered bacterium for producing 1,5-pentanediamine, comprising the aforementioned tandem promoter or recombinant nucleic acid sequence or the aforementioned combination of recombinant nucleic acids.

[0045] Preferably, the chromosome of the genetically engineered bacteria contains the recombinant nucleic acid sequence described above or the recombinant nucleic acid combination described above.

[0046] More preferably, the genetically engineered bacteria are introduced into the chromosome via recombination using the aforementioned tandem promoter, the aforementioned recombinant nucleic acid sequence, or the aforementioned recombinant nucleic acid combination.

[0047] In some embodiments, the chromosome of the genetically engineered bacteria contains a lysine decarboxylase gene under the control of the tandem promoter-stationary phase-specific promoter.

[0048] In other embodiments, the genetically engineered bacteria incorporate a gene in the chromosome that promotes the expulsion of 1,5-pentanediamine from the cell via recombination, such as the aforementioned tandem promoter or recombinant nucleic acid sequence or combination of recombinant nucleic acids.

[0049] The gene that promotes the expulsion of 1,5-pentanediamine from the cell is under the control of the constitutive promoter described above. This gene can reduce the toxic effects of 1,5-pentanediamine on cell growth.

[0050] In some preferred embodiments, the host bacteria of the genetically engineered bacteria are derived from species of Escherichia, Corynebacterium, Bacillus, Thermus, Brevibacterium, or Hafnia.

[0051] Preferably, the source is Escherichia coli, Thermus thermophilus, Hafnia alvei, Bacillus subtilis, or Corynebacterium glutamicum.

[0052] In this paper, the lysine decarboxylase gene can be contained in a plasmid.

[0053] In this paper, the gene that promotes the expulsion of 1,5-pentanediamine from the cell may be contained in the same plasmid as the lysine decarboxylase gene; or it may be contained in a different plasmid and expressed in the host cell independently of the host chromosome.

[0054] In this study, the chromosome of the genetically engineered bacteria contains a lysine decarboxylase gene controlled by the tandem promoter. Thus, by regulating the expression of L-lysine decarboxylase using the piclR-stationary-phase-specific promoter tandem promoter, energy consumption during 1,5-pentanediamine tolerance is reduced, L-lysine production is promoted, and consequently, 1,5-pentanediamine production is increased. Furthermore, the genetically engineered bacteria can also include the gene promoting 1,5-pentanediamine excretion from the cell in the chromosome through recombination, thereby reducing intracellular 1,5-pentanediamine concentration and inhibiting intracellular lysine decarboxylase activity by expressing the protein promoting 1,5-pentanediamine excretion from the cell, thereby increasing 1,5-pentanediamine production.

[0055] As the starting strain, the L-lysine-producing Escherichia coli strain M11A3 can be used. This strain is now deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, China, accession number CCTCC No: M2018456, deposit date July 6, 2018.

[0056] In a sixth aspect, the present invention also provides a method for producing 1,5-pentanediamine, comprising culturing genetically engineered bacteria containing the above-mentioned material in a fermentation medium to produce 1,5-pentanediamine.

[0057] In some implementations, the culture temperature is 20-50°C.

[0058] In this paper, the recombinant nucleic acid was constructed into an engineered bacterium capable of producing L-lysine. The recombinant bacterium was fermented and cultured to accumulate lysine. The fermentation temperature was controlled at 20-50℃ to allow for rapid cell growth and lysine accumulation. After entering the stable fermentation phase, lysine decarboxylase was expressed in large quantities to produce 1,5-pentanediamine.

[0059] As used herein, the term “about” when used to modify a value within a temperature range indicates a reasonable deviation from that value, for example, within 1°C or 2°C below or above the value stated in the range, within the intended meaning of the value or range.

[0060] In some embodiments, the culture is carried out at a temperature of about 25°C to about 45°C. In other embodiments, the culture is carried out at a temperature of about 30°C to about 40°C. In a further embodiment, the culture is carried out at a temperature of about 35°C to about 39°C.

[0061] In a seventh aspect, the present invention provides the application of the above-mentioned tandem promoter or recombinant nucleic acid sequence or recombinant nucleic acid combination or biological material or genetically engineered bacteria in the production of 1,5-pentanediamine.

[0062] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0063] The reagents and raw materials used in this invention are all commercially available.

[0064] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0065] This invention enhances promoter strength and ensures sufficient and stable expression of L-lysine decarboxylase by recombinantly inserting a tandem promoter including the pclR promoter and a stationary phase-specific promoter, along with the lysine decarboxylase gene under its control, into the bacterial chromosome. This significantly reduces the energy consumption caused by host cell tolerance to 1,5-pentanediamine toxicity, thereby promoting the production of 1,5-pentanediamine. The entire process does not require the use of resistance genes, inducing elements, etc.

[0066] This invention utilizes a tandem promoter comprising the piclR promoter and a stationary-phase-specific promoter, ensuring sufficient expression of the lysine decarboxylase gene only after the bacterial cells reach the stationary phase. Compared to expressing thermophilic lysine decarboxylase or using temperature-controlled promoters, this invention achieves stable and sufficient expression of lysine decarboxylase by inserting the aforementioned tandem promoter-lysine decarboxylase gene sequence into the chromosome. Furthermore, this strain eliminates the need for antibiotics, special environmental conditions, or other inducers, and the entire fermentation process is self-regulated.

[0067] Applying this technology to 1,5-pentanediamine production significantly reduces the cytotoxicity of 1,5-pentanediamine produced during cell growth and L-lysine production, thereby increasing L-lysine yield. After fermentation, almost all L-lysine is converted to 1,5-pentanediamine, thus increasing the yield of 1,5-pentanediamine. Simultaneously, a protein promoting pentanediamine excretion is used, facilitating the extracellular export of 1,5-pentanediamine during conversion. Overall, this significantly improves the yield of 1,5-pentanediamine produced by the recombinant strain during fermentation, achieving stable, efficient, and low-cost production of 1,5-pentanediamine. Detailed Implementation

[0068] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0069] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. It should be understood that although some embodiments of the invention are illustrated herein, those skilled in the art will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention. It should also be understood that the terminology used herein is used only to describe particular embodiments and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims and their equivalents.

[0070] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer’s instructions.

[0071] In the following examples, the specific steps and conditions for PCR amplification, purification, plasmid extraction, enzyme digestion, ligation of digestion products, and transformation were all performed according to the instructions of the purchased enzymes and reagents. The DNA polymerase used for PCR amplification, the restriction endonucleases used for enzyme digestion, and the ligases used for ligation of digestion products were all purchased from Takara Bio Engineering (Dalian) Co., Ltd. The plasmid extraction kit, DNA gel recovery kit, and PCR purification kit were all purchased from Corning Life Sciences (Wujiang) Co., Ltd. All primers were purchased from Thermo Fisher Scientific (China) Co., Ltd. (INVITROGEN).

[0072] The plasmid transformation method involved in the following examples is as follows: The ligation product was added to 100 μl of E. coli BL21(DE3) competent cells, incubated on ice for 30 min, and then heat-shocked at 42°C for 90 s. After incubation on ice for 5 min, 1 ml of LB solution was added. The mixture was then plated onto the corresponding antibiotic plates.

[0073] In this invention, the amounts of L-lysine and 1,5-pentanediamine in the culture medium can be detected by nuclear magnetic resonance.

[0074] Unless otherwise specified, the percentage sign "%" used in this invention refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0075] The host, overexpressed protein, and plasmid used in the examples are summarized in Table 1 below.

[0076] Table 1

[0077]

[0078]

[0079] Example 1: Construction of the pBU plasmid backbone

[0080] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, primer pairs upp-UF (sequence shown in SEQ ID NO:21) and upp-UR (sequence shown in SEQ ID NO:22) amplified the Upp-U fragment (sequence shown in SEQ ID NO:23), primer pairs upp-DF (sequence shown in SEQ ID NO:24) and upp-DR (sequence shown in SEQ ID NO:25) amplified the Upp-500bp-D fragment (sequence shown in SEQ ID NO:26), and primer pairs upp-F (sequence shown in SEQ ID NO:27) and upp-R (sequence shown in SEQ ID NO:28) amplified the Pupp-Upp-D fragment (sequence shown in SEQ ID NO:29). Using the commercially available plasmid pBR322 as a template, primer pairs P1P2-tetA-F (sequence shown in SEQ ID NO:30) and P1P2-tetA-R (sequence shown in SEQ ID NO:31) amplified the fragment P1P2-tetA (sequence shown in SEQ ID NO:32), which contains the P1P2 promoter and the tetA sequence (i.e., with an antibiotic resistance marker). The PCR products of Upp-U, Upp-500bp-D, P1P2-tetA, and Pupp-Upp-D fragments were recovered from the gel and ligated into the pBR322 vector, which had been double-digested with EcoRI and NaeI. The gene fragments and vector were then recombinated using a multi-fragment one-step cloning kit. The recombinant ligation mixture was transformed into E. coli JM109 competent cells (purchased from Takara Bio Engineering (Dalian) Co., Ltd.), and multiple single colonies were obtained by screening on LB agar plates containing ampicillin. After verification by colony PCR and sequencing, plasmid was extracted to obtain a plasmid containing four fragments: Upp-U, Upp-500bp-D, P1P2-tetA, and Pupp-Upp-D. This plasmid was named the pBU vector.

[0081] Example 2 Cloning of the Lysine Decarboxylase CadA Gene

[0082] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, the cadA gene fragment (sequence shown in SEQ ID NO:33) was amplified using primer pairs cadA-F (sequence shown in SEQ ID NO:34) and cadA-R (sequence shown in SEQ ID NO:9). The cadA gene fragment and the pBU vector obtained in Example 1 after single digestion with EcoRI were purified by gel extraction. The gene fragment and the vector were recombined using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-cadA.

[0083] Using the genome of commercially available *E. coli* K12 MG1655 as a template, the piclR promoter was amplified using primer pairs piclR-F (sequence shown in SEQ ID NO:35) and piclR-R (sequence shown in SEQ ID NO:36), with the sequence shown in SEQ ID NO:62. The piclR promoter fragment and the SacI-digested pBU-cadA vector were purified by gel extraction. The gene fragment and vector were recombinantly ligated using a multi-fragment one-step cloning kit. The recombinant mixture was transformed into *E. coli* JM109 competent cells (purchased from Takara Bio Engineering (Dalian) Co., Ltd.), and multiple single colonies were obtained by screening on LB agar plates containing ampicillin. After verification by colony PCR and sequencing, the plasmid pBU-piclR-cadA was extracted, containing the cadA gene controlled by the piclR promoter.

[0084] Example 3: Construction of pBU-piclR-stability-specific promoter-cadA and pBU-stability-specific promoter-cadA plasmids

[0085] Using the genome of commercially available *Escherichia coli* K12 MG1655 as a template, primers pcsiE-F (sequence shown in SEQ ID NO:37) / pcsiE-R (sequence shown in SEQ ID NO:38), pbolA-F (sequence shown in SEQ ID NO:39) / pbolA-R (sequence shown in SEQ ID NO:40), posmY-F (sequence shown in SEQ ID NO:41) / posmY-R (sequence shown in SEQ ID NO:42), pkatE-F (sequence shown in SEQ ID NO:43) / pkatE-R (sequence shown in SEQ ID NO:44), pcsiE-F2 (sequence shown in SEQ ID NO:45) / pcsiE-R (sequence shown in SEQ ID NO:38), pbolA-F2 (sequence shown in SEQ ID NO:46) / pbolA-R (sequence shown in SEQ ID NO:40), and posmY-F2 (sequence shown in SEQ ID NO:45) / pcsiE-R (sequence shown in SEQ ID NO:38), pbolA-F2 (sequence shown in SEQ ID NO:46) / pbolA-R (sequence shown in SEQ ID NO:40), and posmY-F2 (sequence shown in SEQ ID NO:45) were used to conduct experiments. The sequence is shown in SEQ ID NO:47) / posmY-R (sequence shown in SEQ ID NO:42), pkatE-F2 (sequence shown in SEQ ID NO:48) / pkatE-R (sequence shown in SEQ ID NO:44); amplifying the stationary phase specific promoters pcsiE (sequence shown in SEQ ID NO:1), pbolA (sequence shown in SEQ ID NO:2), posmY (sequence shown in SEQ ID NO:3), pkatE (sequence shown in SEQ ID NO:44). As shown in NO:4, after gel extraction and recovery of the PCR products, they were ligated with the pBU-piclR-cadA and pBU-cadA plasmids obtained in Example 2 by SacI digestion, respectively, to obtain plasmids containing four tandem promoters: pBU-piclR-pcsiE-cadA, pBU-piclR-pbolA-cadA, pBU-piclR-posmY-cadA, and pBU-piclR-pkatE-cadA; and plasmids containing only four stationary phase-specific promoters: pBU-pcsiE-cadA, pBU-pbolA-cadA, pBU-posmY-cadA, and pBU-pkatE-cadA.

[0086] Double-stranded DNA sequences (5'-3') of promoters p21, p22, p23, and p24 were synthesized using commonly used gene sequence synthesis methods in the art, and then ligated into the pBU-piclR-cadA and pBU-cadA plasmids obtained in Example 2 by SacI digestion, respectively. This yielded plasmids pBU-piclR-p21-cadA, pBU-piclR-p22-cadA, pBU-piclR-p23-cadA, and pBU-piclR-p24-cadA containing four tandem promoters; and plasmids containing only four stationary-phase specific promoters: pBU-p21-cadA, pBU-p22-cadA, pBU-p23-cadA, and pBU-p24-cadA.

[0087] Example 4: Construction of a 1,5-pentanediamine-producing strain and detection of 1,5-pentanediamine yield.

[0088] The starting strain used in this invention is Escherichia coli M11A3, which is capable of producing L-lysine. This strain is now deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: M2018456 and deposit date July 6, 2018.

[0089] First, electroporation competent cells were prepared. The commercially available pKD46 plasmid was transformed into competent cells of strain M11-A3 via heat shock. After screening and culturing in LB resistant plates containing 100 μg / ml ampicillin, single colonies were picked and placed in 5 mL of LB liquid medium and incubated at 30°C and 200 rpm for 8 h. Then, 1% inoculum was transferred to 50 mL of LB liquid medium containing ampicillin and cultured until the OD600 reached approximately 0.15. Finally, 1 mL of ampicillin was added... Incubate the cells in a 2 mM L-arabinose solution until the OD600 reaches 0.4–0.5; then transfer the cells to 50 mL centrifuge tubes and incubate on ice for 20 min to stop growth; centrifuge at 4000 rpm for 10 min at 4 °C and collect the cells; wash the cells with 40 mL of pre-chilled sterile water and centrifuge to collect the cells; repeat the above steps; wash the cells with 20 mL of pre-chilled 10% glycerol and centrifuge to collect the cells; finally, resuspend the cells in 500 μL of pre-chilled 10% glycerol and dispense the resulting M11-A3 / pKD46 competent cells for use.

[0090] Using the 16 plasmids constructed in Example 3 (as shown in Table 2 below) as templates, fragments suitable for homologous recombination on chromosomes were amplified using primer pairs upp-UF / upp-R and then recovered via gel extraction. Each fragment was transformed into the recipient strain M11A3 / pKD46. The fragments were screened on LB agar plates containing 10 μg / ml tetracycline. For each plasmid transformation, three single colonies were picked and incubated in 600 μl LB medium supplemented with ampicillin at 37°C for 8 h. One μl of bacterial cells was then used as a template for PCR verification to screen for the correct recombinant strains, and the culture was preserved with glycerol.

[0091] Sixteen glycerol-preserved bacterial strains were inoculated into LB liquid medium containing 0.1 μg / mL 5-FU (5-fluorouracil) and incubated at 37°C and 200 rpm for 8 h. Bacterial cultures were then streaked onto LB agar plates containing and without 5-fluorouracil and incubated overnight. Plate analysis revealed strains that could not grow on plates containing 5-fluorouracil. For each strain, six single clones were randomly selected from the corresponding plate without 5-fluorouracil, and 1 μl of bacterial cells were used as templates for PCR verification. If both the P1P2-tetA and Pupp-Upp fragments were simultaneously removed, it indicated the correct recombinant strain was obtained. These correct recombinant strains were then used for glycerol preservation.

[0092] Three transformants from each of the above recombinant strains were selected and, together with the starting strain M11A3, were separately coated onto antibiotic-free seed culture medium (containing 4% glucose, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4, 0.001% FeSO4, 0.001% MnSO4, and 0.2% yeast extract; the percentages are by weight and volume, the same below), and cultured overnight at 37°C. Then, three single clones from each were picked and cultured in 5 ml of seed culture medium (containing 4% glucose, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4, 0.001% FeSO4, 0.001% MnSO4, and 0.2% yeast extract) overnight at 37°C and 225 rpm. Each strain was then transferred to 50 ml of fresh fermentation medium (30 g / L glucose, 0.7% Ca(HCO3)2, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4, 0.001% FeSO4, 0.001% MnSO4, 0.2% yeast extract) and cultured at 37°C and 170 rpm for 48 h. The content of 1,5-pentanediamine in each medium was detected and calculated using NMR (Table 2).

[0093] Table 2. 1,5-Pentanediamine yield and OD of the recombinant strain compared to the original strain, as determined by NMR. 600

[0094]

[0095] As shown in 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, and p24-cadA / M11A3, which directly express CadA using stationary-phase specific promoters, showed L-lysine levels of 0.88–2.23 g / kg and 1.14–2.12 g / kg of 1,5-pentanediamine after 48 h of fermentation. This indicates that the expression level of lysine decarboxylase was low, and only a portion of L-lysine was converted to 1,5-pentanediamine.

[0096] In contrast, recombinant strains expressing cadA using tandem promoters, namely 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, and piclR-p24-cadA, showed significant improvement. Through testing, it was found that at 48 hours of fermentation, the recombinant strains using the tandem promoter to express cadA showed a further increase in the production of 1,5-pentanediamine compared to the eight recombinant strains using the stationary-phase specific promoter to directly express cadA. Simultaneously, almost all L-lysine was converted to 1,5-pentanediamine. The pclR-p24-cadA / M11A3 strain had the highest 1,5-pentanediamine production, ultimately accumulating over 2.93 g / kg of 1,5-pentanediamine with almost no L-lysine residue.

[0097] 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, and pBU-piclR-p24-cadA-plac-ompX plasmids

[0098] Using the genome of commercially available *Escherichia coli* K12 MG1655 as a template, primer pairs ompA-F (sequence shown in SEQ ID NO:49) and ompA-R (sequence shown in SEQ ID NO:50) amplified the ompA gene (sequence shown in SEQ ID NO:11), primer pairs ompC-F (sequence shown in SEQ ID NO:51) and ompC-R (sequence shown in SEQ ID NO:52) amplified the ompC gene (sequence shown in SEQ ID NO:12), and primer pairs ompF-F (sequence shown in SEQ ID NO:53) and ompF-R (sequence shown in SEQ ID NO:54) amplified the ompF gene (sequence shown in SEQ ID NO:13). Primer pairs ompW-F (sequence shown in SEQ ID NO:55) and ompW-R (sequence shown in SEQ ID NO:56) amplified the ompW gene (sequence shown in SEQ ID NO:14). Primer pairs ompX-F (sequence shown in SEQ ID NO:57) and ompX-R (sequence shown in SEQ ID NO:58) amplified the ompX gene (sequence shown in SEQ ID NO:15). Using commercially available pUC18 plasmid DNA as a template, primer pairs plac-F (sequence shown in SEQ ID NO:60) and plac-R (sequence shown in SEQ ID NO:59) amplified the plac promoter (SEQ ID NO:60). NO:61); The plac promoter fragment, ompA gene fragment, and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids digested with XbaI were gel-cleaved and purified. The gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmids were named pBU-piclR-p24-cadA-plac-ompA and pBU-p24-cadA-plac-ompA.

[0099] The plac promoter fragment, ompC gene fragment, and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids digested with XbaI were gel-cleaved and purified. The gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmids were named pBU-piclR-p24-cadA-plac-ompC and pBU-p24-cadA-plac-ompC.

[0100] The plac promoter fragment, the ompF gene fragment, and the XbaI-digested pBU-piclR-p24-cadA and pBU-p24-cadA plasmids were purified by gel extraction. The gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmids were named pBU-piclR-p24-cadA-plac-ompF and pBU-p24-cadA-plac-ompF.

[0101] The plac promoter fragment, the ompW gene fragment, and the XbaI-digested pBU-piclR-p24-cadA and pBU-p24-cadA plasmids were purified by gel extraction. The gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmids were named pBU-piclR-p24-cadA-plac-ompW and pBU-p24-cadA-plac-ompW.

[0102] The plac promoter fragment, ompX gene fragment, and pBU-piclR-p24-cadA and pBU-p24-cadA plasmids digested with XbaI were gel-cleaved and purified. The gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmids were named pBU-piclR-p24-cadA-plac-ompX and pBU-p24-cadA-plac-ompX.

[0103] Example 6: Construction of a 1,5-pentanediamine-producing strain

[0104] Using the five plasmid pairs constructed in Example 5 as templates, fragments suitable for homologous recombination on chromosomes were amplified using primer pairs upp-UF / upp-R and then recovered via gel extraction. Each fragment was transformed into the recipient strain M11-A3 / pKD46. The fragments were plated on LB agar plates containing 10 μg / ml tetracycline for screening. Twelve single colonies were picked and incubated in 600 μl LB medium supplemented with ampicillin at 37°C for 8 h. One μl of bacterial cells was then used as a template for PCR verification of the selected recombinant strains, and the culture was preserved with glycerol.

[0105] The glycerol-preserved strain was inoculated into LB liquid medium containing 0.1 μg / mL 5-FU (5-fluorouracil) and incubated at 37°C and 200 rpm for 8 h. Bacterial culture was then streaked onto LB agar plates containing and without 5-fluorouracil, and incubated overnight. Plate analysis revealed that strains that could not grow on plates containing 5-fluorouracil were analyzed. Six single clones were selected from the corresponding plates without 5-fluorouracil, and 1 μl of bacterial cells were used as templates for PCR verification. If both the P1P2-tetA and Pupp-Upp fragments were simultaneously removed, the correct recombinant strain was obtained. The correct recombinant strain was then used for glycerol preservation.

[0106] Example 7: Detection of 1,5-pentanediamine production by strain

[0107] Three transformants were selected from each of the recombinant strains obtained in Example 6. The starting strains M11A3, piclR-p24--cadA / M11A3, and p24-cadA / M11A3 were used as control strains. These were spread onto antibiotic-free seed culture medium (containing 4% glucose, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4, 0.001% FeSO4, 0.001% MnSO4, and 0.2% yeast extract) and cultured overnight at 37°C. Then, three single clones from each strain were picked and cultured overnight at 37°C and 225 rpm using 5 ml of seed culture medium (containing 4% glucose, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4, 0.001% FeSO4, 0.001% MnSO4, and 0.2% yeast extract). Each strain was then transferred separately to 50 ml of fresh fermentation medium (30 g / L glucose, 0.7% Ca(HCO3)2, 0.1% KH2PO4, 0.1% MgSO4, 1.6% (NH4)2SO4). 4, A culture medium containing 0.001% FeSO4, 0.001% MnSO4, and 0.2% yeast extract was used, with an additional set of shake flasks containing 30 g / kg pentanediamine. The cultures were then incubated at 37°C and 170 rpm for 48 h. Samples were taken to determine the OD values ​​of each strain under both pentanediamine-added and pentanediamine-free conditions. 600 The content of pentamethylenediamine in each culture medium was detected and calculated using NMR for a group of samples without added pentamethylenediamine (Table 3).

[0108] As shown in Table 3, the OD600 of the strains after dilution 20-fold in shake flasks containing added pentanediamine was measured. Compared with the recombinant strains M11-A3 and PcilR-p24-cadA / M11A3p24-cadA / M11A3, the OD600 of the 10 new strains expressing the proteins OmpA, OmpC, OmpF, OmpW, or OmpX that promote the excretion of 1,5-pentanediamine was significantly reduced (i.e., the OD600 of the strains decreased less). Among them, the PicR-p21-cadA-plac-ompA / M11A3 strain had the highest OD600, indicating that it had the strongest tolerance to pentanediamine.

[0109] As shown in Table 4, the strains expressing the protein that promotes the excretion of 1,5-pentanediamine are: piclR-p24-cadA-plac-ompA / M11A3, piclR-p24-cadA-plac-ompC / M11A3, piclR-p24-cadA-plac-ompF / M11A3, piclR-p24-cadA-plac-ompW / M11A3, and piclR-p24-cadA-plac-ompW / M11A3. Compared with the recombinant strain piclR-p24-cadA / M11A3, ac-ompX / M11A3 produced a further increased yield of 1,5-pentanediamine, and almost all L-lysine was converted into 1,5-pentanediamine. Among them, the piclR-p24-cadA-plac-ompA / M11A3 strain had the highest yield of 1,5-pentanediamine, and finally accumulated more than 3.71 g / kg of 1,5-pentanediamine, with almost no L-lysine residue.

[0110] Table 3. OD values ​​of strains measured under conditions with and without pentamethylenediamine addition. 600

[0111]

[0112] Table 4. Pentanediamine yield of recombinant strain compared to the original strain, as determined by NMR.

[0113]

[0114]

[0115] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection of the present invention. SEQUENCE LISTING <110> Shanghai Kaisai Biotechnology Co., Ltd. CIBT America Inc. Kaisai (Urumqi) Biomaterials Co., Ltd. <120> Recombinant nucleic acid sequences, genetically engineered bacteria, and methods for producing 1,5-pentanediamine <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 ggtattgaa gagaaagca aaaataa 2148 <210> 10 <211> 715 <212> PRT <213> Artificial Sequence <220> <223> cadA flowers <400> 10 Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu 1 5 10 15 Glu Pro With 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 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 Leu Glu Cys Glu Glu Ile Ser Lys Met Asn Leu Glu 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 ctgtacagcc agctgagcaa cctggatccg aaagacggtt ccgtagttgt tctgggttac 780 accgaccgca tcggttctga cgcttacaac cagggtctgt ccgagcgccg tgctcagtct gttgttgatt acctgatctc caaaggtatc ccggcagaca agatctccgc acgtggtatg ggcgaatcca acccggttac tggcaacacc tgtgacaacg tgaacagcg tgctgcactg atcgactgcc tggctccgga tcgtcgcgta gagatcgaag ttaaaggtat caaagacgtt gtaactcagc cgcaggctta a <210> 12 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> ompC snowflakes <400> 12 atgaaagtta aagtactgtc cctcctggtc ccagctctgc tggtagcagg cgcagcaaac gctgctgaag tttacaacaa agacggcaac aaattagatc tgtacggtaa agtagcggc 180. ctgcactatt tctctgacaa caaagatgta gatggcgacc agacctacat gcgtcttggc ttcaaaggtg aaactcaggt tactgaccag ctgaccggtt acggccagtg ggaatatcag atccagggca acagcgctga aaacgaaac aactcctgga cccgtgtggc attcgcaggt 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 acctacactg gtggtctga acctacacgct aacaacatct acctggctgc tcagtacacc 840. cgacctaca acgcaactcg cgtaggttcc ctgggttggg cgaacaaagc acagaacttc gaagctgttg ctcagtacca gttcgacttc ggtctgcgtc cgtccctggc ttacctgcag 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> Gene sequence of ompF <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> Gene sequence of ompX <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> up‐UF <400> 21 aggcgtatca cgaggccctt tcgtcttcaa aaacccgcga catcgtaatc 50 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> up‐UR <400> 22 ggatatacc tccttctttc aaggcg 26 <210> 23 <211> 790 <212> DNA <213> Artificial Sequence <220> <223> Up‐U <400> 23 aggcgtatca cgaggccctt tcgtcttcaa aaacccgcga catcgtaatc ctcaccgtga 60 tacatccccg gcatttctgc cgtttcgcca cccaccagtg aacagcctga ttgcagacaa 120 ccttccgcaa tgccgctgat caccgctgaa gcggtatcaa catccagttt tccggttgcg 180 tatagtcga 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 acgccttga 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 aataaaat aactttatct 720 caaaccgtta tcattttgac taaagtcaac gaaaagaata ttgccgcctt gaagaaagga 780 ggtataatcc 790 <210> 24 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> up‐DF <400> 24 tgccgccttg aagaaaggag gtataatccg aattcagtcg gcttttttt gagtaaagcg 60 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> up‐DR <400> 25 ccgcattaaa gcttatcgat gataagctgt caaacatgac cgggagtaaa cccgccata 59 <210> 26 <211> 574 <212> DNA <213> Artificial Sequence <220> <223> Up‐500bp‐D <400> 26 tgccgccttg aagaaaggag gtataatccg aattcagtcg gcttttttt 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 cctttattc 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> up‐F <400> 27 tggagccggg ccacctcgac ctgaatggaa gccggcgtcg atttttttg tggctgccc 59 <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> up‐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 aggctgggaac 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 caccgttct 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 gtcgcatag 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 ccgctcggc gagcacatgg aacgggttgg catggattgt 1200 aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc 1260 cacctcgacc tgaatggaag ccggc <210> 33 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> cadA‐F <400> 33 cgccttgaag aaaggaggta taatccgagc tcatgaacgt tattgcaata ttgaatcaca tg 62 <210> 34 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> cadA‐R <400> 34 55. ggcgctttac tcaaaaaaaa gccgacttct agaccacttc ccttgtacga gctaa <210> 35 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> piclR‐F <400> 35 ccttgaagaa aggaggtata atccatttgt tcaacattaa ctcatcggat <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 <210> 54 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ompF‐R <400> 54 gctttactca aaaaaaagcc gacttctaga ttagaactgg taaacgatac cca <210> 55 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ompW‐F <400> 55 cggataacaa tttcacacag gaggagctca tgaaaaagtt aacagtggcg g <210> 56 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> ompW‐R <400> 56 gctttactca aaaaaaagcc gacttctga ttaaaaacga tatcctgctg agaac <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 containing the 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 3​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<220> <223> Source: Corynebacterium glutamate glutamicum) pclR promoter pclR-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> The piclR promoter piclR-Ha from Hafnia alvei <400> 64 gagcagttaa aaaccacgat cgcatccata atgtcgaaaa cgaaaataga ttccattttt 60 ataacatttt t 71

Claims

1. A tandem promoter, characterized in that, which comprises a piclR promoter and a stationary phase-specific promoter; the stationary phase-specific promoter is selected from one of the following: pcsiE, pbolA, posmY, pkatE, p21, p22, p23 and p24; the nucleotide sequence of the piclR promoter is shown as SEQ ID NO: 62, and the nucleotide sequences of the pcsiE, pbolA, posmY, pkatE, p21, p22, p23 and p24 are shown as SEQ ID NOs: 1-8, respectively; the tandem promoter is piclR-pcsiE, piclR-pbolA, piclR-posmY, piclR-pkatE, piclR-p21, piclR-p22, piclR-p23 or piclR-p24.

2. A recombinant nucleic acid, characterized in that, The recombinant nucleic acid comprises the tandem promoter as claimed in claim 1 and a lysine decarboxylase gene operably linked to the tandem promoter; the lysine decarboxylase gene is a cadA gene from Escherichia coli.

3. The recombinant nucleic acid of claim 2, wherein, The nucleotide sequence of the cadA gene is shown as SEQ ID NO:

9.

4. The recombinant nucleic acid of claim 3, wherein, The recombinant nucleic acid 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 recombinant nucleic acid combination, comprising, The recombinant nucleic acid combination comprises: a first nucleic acid comprising the tandem promoter as claimed in claim 1 or the recombinant nucleic acid as claimed in any one of claims 2-4; and a second nucleic acid comprising a constitutive promoter and a gene encoding an outer membrane porin operably linked thereto; the gene encoding the outer membrane porin is selected from any one of the following: ompA, ompC, ompF, ompW and ompX; the constitutive promoter is plac.

6. The recombinant nucleic acid combination of claim 5, wherein, The nucleotide sequence of the constitutive promoter is shown as SEQ ID NO: 61; and / or the nucleotide sequence of the gene encoding the outer membrane porin is shown as any one of SEQ ID NOs: 11-15.

7. The recombinant nucleic acid combination of claim 6, wherein, The first nucleic acid 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 nucleic acid is plac-ompA, plac-ompC, plac-ompF, plac-ompW or plac-ompX.

8. The recombinant nucleic acid combination of claim 7, wherein, 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 when the first nucleic acid is operably linked to the second nucleic acid.

9. A biomaterial, characterized by, The biological material comprises the tandem promoter of claim 1 or the recombinant nucleic acid of any one of claims 2-4 or the recombinant nucleic acid combination of any one of claims 5-8.

10. The biomaterial of claim 9, wherein The biological material comprises an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

11. The biomaterial of claim 10, wherein The backbone plasmid of the plasmid vector comprises pUC18, pUC19, pBR322, pACYC, pET, pSC101, pKD46 and their derivative plasmids.

12. A genetically engineered bacterium, characterized in that, The engineered bacterium comprises the tandem promoter of claim 1 or the recombinant nucleic acid of any one of claims 2-4 or the recombinant nucleic acid combination of any one of claims 5-8.

13. The genetically engineered bacteria as described in claim 12, characterized in that, The engineered bacterium comprises the recombinant nucleic acid of any one of claims 2-4 or the recombinant nucleic acid combination of any one of claims 5-8 in its chromosome.

14. The genetically engineered bacterium of claim 13, wherein, The engineered bacterium comprises the recombinant nucleic acid of any one of claims 2-4 recombined in its chromosome.

15. The genetically engineered bacteria as described in claim 13, characterized in that, The engineered bacterium comprises the recombinant nucleic acid combination of any one of claims 5-8 recombined in its chromosome.

16. The genetically engineered bacterium of any one of claims 12-15, wherein, The host bacterium of the engineered bacterium is derived from Escherichia coli, Hafnia alvei or Corynebacterium glutamicum.

17. Process for the production of 1,5-pentanediamine, characterized in that, The fermentation medium comprises the engineered bacterium of any one of claims 12-16 for producing 1,5-pentanediamine.

18. Use of the tandem promoter of claim 1 or the recombinant nucleic acid of any one of claims 2-4 or the recombinant nucleic acid combination of any one of claims 5-8 or the biological material of any one of claims 9-11 or the engineered bacterium of any one of claims 12-16 for producing 1,5-pentanediamine.

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