Recombinant nucleic acid sequence, recombinant expression vector and genetically engineered bacterium

Abstract

The present invention discloses a recombinant nucleic acid sequence, a recombinant expression vector, and a genetically engineered bacterium. Among them, the recombinant nucleic acid sequence includes a tandem promoter, a lysine decarboxylase gene, and a gene for enhancing cell stress tolerance. The tandem promoter includes a piclR promoter and a stationary-phase specific promoter. The present invention controls the expression time and expression level of L-lysine decarboxylase by using the tandem piclR-stationary-phase specific promoter, reduces the energy consumption during the tolerance process of 1,5-pentanediamine, promotes the production capacity of the strain, and thereby increases the yield of 1,5-pentanediamine. And by increasing the expression level of the gene that promotes the excretion of 1,5-pentanediamine from the cell, the intracellular 1,5-pentanediamine concentration and its inhibition of the activity of intracellular lysine decarboxylase are reduced, the tolerance ability of the strain is increased, and the yield of 1,5-pentanediamine is further increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and specifically relates to recombinant DNA, strains for fermentative production of 1,5-pentanediamine, and their uses. Background Art

[0002] 1,5-Pentanediamine has quite extensive uses and high economic value in industrial production. For example, it can undergo a polymerization reaction with a dicarboxylic acid to synthesize a new type of nylon. Currently, the biosynthesis of 1,5-pentanediamine mainly uses two strategies: fermentative production or in vitro enzymatic catalysis. For fermentative production, 1,5-pentanediamine is generated by removing a carboxyl group from L-lysine through lysine decarboxylase (abbreviated as 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 biosynthetic pathway to the 1,5-pentanediamine biosynthetic pathway.

[0003] Currently, there are bacteria of the genus Corynebacterium and Escherichia coli with the ability to produce L-lysine that have been modified by DNA recombinant technology, and the improvement in efficiency is achieved by overexpressing genes related to the L-lysine synthesis pathway and genes related to feedback inhibition desensitization, or by strengthening the energy supply pathway starting from glucose metabolism. Genes related to feedback inhibition desensitization, such as aspartokinase III (LysC), are specific key enzymes in the lysine synthesis pathway. However, since the concentration of 1,5-pentanediamine tolerated by the bacterial cells themselves is limited, if too much 1,5-pentanediamine is generated by the expression of lysine decarboxylase in the early stage of the fermentation system, it will be toxic to the bacterial cells, thereby inhibiting the growth of the bacterial cells and the process of using glucose to produce L-lysine (Qian, et al., Biotechnol. Bioeng. 2011; 108: 93–103).

[0004] For example: The patent document with the publication number WO2019006723A1, publication date January 19, 2019, and title "HETEROLOGOUS EXPRESSION OF THERMOPHILIC LYSINE DEC-ARBOXYLASE AND USES THEREOF" discloses the heterologous expression of a thermophilic lysine decarboxylase and its uses. In the technical solution disclosed in this patent document, a thermophilic lysine decarboxylase is first used to control the enzyme activity through high temperature.

[0005] For another example, a Chinese patent document with the publication number CN105368766A, publication date of March 2, 2016, and title of "A genetically engineered bacterium for producing pentanediamine and a method for producing pentanediamine therefrom" discloses a pentanediamine-producing strain and a process for its efficient production. In the technical solution disclosed in this patent document, the use of a temperature-controlled promoter to express lysine decarboxylase solves the stability problem to a certain extent, but the use of high temperature for catalysis additionally increases energy consumption and production costs.

[0006] Therefore, there is a need to develop a more economical, stable, and efficient process for producing 1,5-pentanediamine. Summary of the Invention

[0007] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a recombinant nucleic acid sequence, a recombinant expression vector, and a genetically engineered bacterium to achieve the stable, efficient, and low-cost production of 1,5-pentanediamine.

[0008] To achieve the above object, the present invention is realized through the following aspects:

[0009] In a first aspect, the present invention provides a recombinant nucleic acid sequence, which comprises a tandem promoter, a lysine decarboxylase gene, and a gene for enhancing cell stress tolerance. Among them, the tandem promoter comprises a piclR promoter and a stationary-phase specific promoter, and the stationary-phase specific promoter is selected from one or more of the following: pcsiE, pbolA, posmY, pkatE, p21, p22, p23, and p24.

[0010] Osmotic stress tolerance is an important aspect of cell physiological performance. For bulk chemicals secreted extracellularly, the higher the production, the greater the osmotic stress, and cells need to have osmotic stress tolerance to achieve high yields.

[0011] On the one hand, the tandem promoter has the starting ability of a strong promoter. On the other hand, IclR is a local transcriptional regulatory inhibitor, and the IclR protein can inhibit the transcription of itself and the arcABC operon in the glyoxylate pathway. The activation of the piclR promoter is also affected by FadR.

[0012] In the present invention, by recombinantly inserting a tandem promoter comprising a piclR promoter and a stationary-phase specific promoter, and the lysine decarboxylase gene under its control, into the bacterial chromosome, the promoter strength is enhanced, ensuring the sufficient and stable expression of L-lysine decarboxylase, greatly reducing the energy consumption caused by the host cell's tolerance to the toxicity of 1,5-pentanediamine, thereby promoting the production of 1,5-pentanediamine, and the whole process does not require the use of resistance genes, induction elements, etc.

[0013] Those skilled in the art understand that in order to improve the expression of a target gene in a host cell, the coding sequence of the target gene can be optimized according to the codon preference of the host cell. For example, rare codons of the target gene can be replaced with synonymous codons to make it closer to the codon usage pattern of the host cell. There have been many reports on increasing the expression level of foreign genes in host cells by this method.

[0014] Preferably, the piclR promoter is derived from any one or more of Escherichia coli, Corynebacterium glutamicum, and Hafnia alvei;

[0015] Preferably, the nucleotide sequence of the piclR promoter is shown as any one of SEQ ID NO: 83-85; the nucleotide sequence of the stationary-phase specific promoter is shown as any one of SEQ ID NO: 1-8; and / or, the lysine decarboxylase gene is selected from the cadA gene or ldcC gene of any one of Escherichia coli, Corynebacterium glutamicum, and Hafnia alvei;

[0016] More preferably, the nucleotide sequence of the cadA gene is shown as SEQ ID NO: 9.

[0017] In some embodiments, the gene of the lysine decarboxylase (abbreviated as LDC, EC 4.1.1.18) can be 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, etc.

[0018] In some embodiments, the lysine decarboxylase gene is the cadA gene or the ldcC gene from Escherichia coli. For example, the sequence of the cadA gene can be SEQ ID NO:9, and the sequence of the CadA protein can be SEQ ID NO:10. Additionally, the lysine decarboxylase can also be from a strain obtained by mutagenesis or random mutation of the above-mentioned strain, or a genetically engineered bacterium. The lysine decarboxylase can also be a mutant (including natural mutants and artificial recombinant mutants) or an active fragment (a truncated protein fragment that retains the lysine decarboxylase activity) of the lysine decarboxylase from the above sources.

[0019] In some embodiments, the stationary-phase specific promoter in the tandem promoter is selected from any one or more of pcsiE, pbolA, posmY, pkatE, p21, p22, p23, and p24.

[0020] The stationary-phase specific promoter can be from the 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, etc.

[0021] The sequence of pcsiE can be SEQ ID NO:1; the sequence of pbolA can be SEQ ID NO:2; the sequence of posmY can be SEQ ID NO:3; the sequence of pkatE can be SEQ ID NO:4; the sequence of p21 can be SEQ ID NO:5; the sequence of p22 can be SEQ ID NO:6; the sequence of p23 can be SEQ ID NO:7; the sequence of p24 can be SEQ ID NO:8.

[0022] In this text, the gene that promotes the excretion of 1,5-pentanediamine from cells refers to a gene whose expression product promotes the excretion of 1,5-pentanediamine from microbial cells to the extracellular environment, thereby reducing the intracellular concentration of 1,5-pentanediamine and its inhibition of the activity of intracellular lysine decarboxylase, and promoting the production of 1,5-pentanediamine. In some embodiments, the gene that promotes the excretion of pentanediamine includes the gene of outer membrane porin or the gene that increases the cell's pressure resistance. More specifically, the outer membrane porin that promotes the excretion of pentanediamine or the protein that increases the cell's pressure resistance can also be a mutant (including natural mutants and artificially recombinant mutants) or an active fragment of the above proteins. Optionally, genes that increase the cell's tolerance to stress (such as rpoS, crp, puuR, pepA, and soxS) and / or outer membrane porin genes (such as ompA, ompC, ompF, ompW, and ompX) are combined to further reduce the intracellular concentration of 1,5-pentanediamine and increase the yield of 1,5-pentanediamine.

[0023] Preferably, the recombinant nucleic acid sequence further includes a constitutive promoter;

[0024] 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 as shown in SEQ ID NO:60.

[0025] The constitutive promoter used in this text is well-known to those skilled in the art, and it can enable the gene that promotes the excretion of 1,5-pentanediamine from cells or the gene that increases the cell's pressure resistance to be expressed in the host cell. For example, the plac, trp, tac, trc, or PL promoter can be used. For example, the sequence of plac can be SEQ ID NO:60.

[0026] Preferably, the gene that enhances the cell's tolerance to stress is operably linked to the constitutive promoter, and the gene that enhances the cell's tolerance to stress is selected from any one or more of rpoS, crp, puuR, pepA, and soxS;

[0027] Preferably, the nucleotide sequence of the gene enhancing cell stress tolerance is shown in any one of SEQ ID NO: 11-15. Among them, the sequence of the rpoS gene can be SEQ ID NO: 11, and its protein sequence is shown in SEQ ID NO: 16; the sequence of the crp gene can be SEQ ID NO: 12, and its protein sequence is shown in SEQ ID NO: 17; the sequence of the puuR gene can be the coding sequence of SEQ ID NO: 13, and its protein sequence is shown in SEQ ID NO: 18; the sequence of the pepA gene can be SEQ ID NO: 14, and its protein sequence is shown in SEQ ID NO: 19; the sequence of the soxS gene can be SEQ ID NO: 15, and its protein sequence is shown in SEQ ID NO: 20.

[0028] Preferably, the recombinant nucleic acid sequence further comprises a gene encoding an outer membrane porin.

[0029] Preferably, the gene encoding an outer membrane porin is operably linked to a constitutive promoter, and / or the gene encoding an outer membrane porin is selected from any one or more of ompA, ompC, ompF, ompW and ompX; more preferably, the nucleotide sequence of the gene encoding an outer membrane porin is shown in any one of SEQ ID NO: 62-66. Among them, the sequence of the ompA gene can be SEQ ID NO: 62, and its protein sequence is shown in SEQ ID NO: 67; the sequence of the ompC gene can be SEQ ID NO: 63, and its protein sequence is shown in SEQ ID NO: 68; the sequence of the ompF gene can be the coding sequence of SEQ ID NO: 64, and its protein sequence is shown in SEQ ID NO: 69; the sequence of the ompW gene can be SEQ ID NO: 65, and its protein sequence is shown in SEQ ID NO: 70; the sequence of the ompX gene can be SEQ ID NO: 66, and its protein sequence is shown in SEQ ID NO: 71.

[0030] In the recombinant DNA described herein, the tandem promoter (element a) and the lysine decarboxylase gene (element b) are operably linked (denoted as a-b), such that the transcription and expression of lysine decarboxylase are under the control of a cadaverine-inducible promoter. Preferably, the linked a-b can achieve the regulation of expression in the host cell by the concentration of cadaverine, control the production of L-lysine decarboxylase through the tandem promoter, and increase the yield of 1,5-cadaverine.

[0031] In the recombinant DNA described herein, a constitutive promoter (element c) and a gene promoting the excretion of cadaverine (element d) are operably linked (denoted as c-d) such that the transcription and expression of the gene promoting the excretion of cadaverine are under the control of the constitutive promoter; a constitutive promoter (element e) and a gene increasing the cell's pressure resistance (element f) are operably linked (denoted as e-f). Optionally, the aforementioned linked a-b, c-d, and e-f can be operably linked or exist independently.

[0032] Preferably, the recombinant nucleic acid sequence is selected from the following group:

[0033] (1) piclR-p24-cadA-plac-rpoS, piclR-p24-cadA-plac-crp, piclR-p24-cadA-plac-puuR, piclR-p24-cadA-plac-pepA or piclR-p24-cadA-plac-soxS; and,

[0034] (2) piclR-p24-cadA-plac-rpoS-plac-ompA, piclR-p24-cadA-plac-rpoS-plac-ompC, piclR-p24-cadA-plac-rpoS-plac-ompF, piclR-p24-cadA-plac-rpoS-plac-ompW or piclR-p24-cadA-plac-rpoS-plac-ompX.

[0035] In a second aspect, the present invention provides a recombinant expression vector, which comprises the above-mentioned recombinant nucleic acid sequence;

[0036] Preferably, the backbone plasmid of the recombinant expression vector includes pUC18, pUC19, pBR322, pACYC, pET, pSC101 and their derivative plasmids.

[0037] Preferably, in the technical solution of the present invention, pKD46 is used for the recombination of the Escherichia coli chromosome.

[0038] In this article, the lysine decarboxylase gene can be contained in a plasmid.

[0039] In this article, the gene promoting the excretion of 1,5-cadaverine from cells or the gene increasing the cell's pressure resistance can be contained in the same plasmid as the lysine decarboxylase gene; or, it can also be contained in a different plasmid and expressed in the host cell independently of the host chromosome.

[0040] In a third aspect, the present invention provides a genetically engineered bacterium for producing 1,5-pentanediamine, which genetically engineered bacterium comprises the above-described recombinant nucleic acid sequence.

[0041] Herein, the chromosome of the genetically engineered bacterium contains a lysine decarboxylase gene under the control of the tandem promoter. Thus, by using the piclR-stationary phase-specific promoter tandem promoter to regulate the expression of L-lysine decarboxylase, the energy consumption during the 1,5-pentanediamine tolerance process is reduced, the production of L-lysine is promoted, and thus the yield of 1,5-pentanediamine is increased. Further, the genetically engineered bacterium can also recombinantly contain the gene that promotes the excretion of 1,5-pentanediamine from the cell in the chromosome or contain the gene that promotes the excretion of 1,5-pentanediamine from the cell in the transformed expression plasmid. Thus, by expressing the protein that promotes the excretion of 1,5-pentanediamine from the cell, the intracellular concentration of 1,5-pentanediamine and its inhibition of the intracellular lysine decarboxylase activity are reduced, and thus the yield of 1,5-pentanediamine is increased.

[0042] As the starting strain, Escherichia coli M11A3 strain capable of producing L-lysine can be used. This strain has been deposited at the China Center for Type Culture Collection, address: Wuhan, China, Wuhan University, postal code 430072, deposit number CCTCC No: M2018456, deposit date July 6, 2018.

[0043] Preferably, the recombinant nucleic acid sequence is located in a free recombinant expression vector or integrated into the chromosome.

[0044] More preferably, the host bacterium of the genetically engineered bacterium is derived from a strain of the genus Escherichia, Corynebacterium, Bacillus, Thermus, Brevibacterium or Hafnia.

[0045] Even more preferably, the host bacterium of the genetically engineered bacterium is derived from Escherichia coli, Thermus thermophilus, Hafnia alvei, Bacillus subtilis or Corynebacterium glutamicum.

[0046] In a fourth aspect, the present invention provides a method for producing 1,5-pentanediamine, which method comprises the step of culturing the above-described genetically engineered bacterium in a fermentation medium to produce 1,5-pentanediamine.

[0047] In some embodiments, the culture temperature of the genetically engineered bacterium is 20 - 50 °C.

[0048] In this article, the recombinant DNA is constructed into an engineered bacterium capable of producing L-lysine. The recombinant bacterium is fermentatively cultured for lysine accumulation. The fermentation culture temperature is controlled at 20 - 50 °C for rapid growth of the bacteria and lysine accumulation. After entering the fermentation stationary phase, lysine decarboxylase is highly expressed to convert and produce 1,5-pentanediamine.

[0049] As used herein, the term "about", when used to modify a numerical value within a temperature range, means a reasonable deviation from that value, e.g., within 1 °C or 2 °C below or above the value within the range, which is within the intended meaning of the value or range.

[0050] 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 further embodiments, the culture is carried out at a temperature of about 35 °C to about 39 °C.

[0051] In a fifth aspect, the present invention provides a method for producing 1,5-pentanediamine, the method comprising the step of culturing the above-mentioned genetically engineered bacterium in a fermentation medium to produce 1,5-pentanediamine.

[0052] In a sixth aspect, the present invention provides the use of the above-mentioned recombinant nucleic acid sequence, recombinant expression vector or genetically engineered bacterium in the production of 1,5-pentanediamine.

[0053] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0054] The present invention uses a tandem promoter comprising a piclR promoter and a stationary-phase specific promoter, and sufficient expression of the lysine decarboxylase gene is initiated only when the bacteria grow to the stationary phase. Compared with expressing thermophilic lysine decarboxylase or expressing lysine decarboxylase with a temperature-controlled promoter, the present invention can achieve stable and sufficient expression of lysine decarboxylase by inserting the tandem promoter-lysine decarboxylase gene sequence into the chromosome. In addition, the use of antibiotics, special environmental conditions or other inducers is removed in this strain, and the whole fermentation culture process is self-regulated.

[0055] Applying it to the production of 1,5-pentanediamine can significantly reduce the cytotoxicity of 1,5-pentanediamine generated during the cell growth and L-lysine production stages, and increase the L-lysine yield; after fermentation, almost all of the L-lysine can be converted into 1,5-pentanediamine, thereby achieving an increase in the yield of 1,5-pentanediamine. Meanwhile, proteins that increase cell tolerance and / or proteins that promote the excretion of pentanediamine are used, and 1,5-pentanediamine is exported extracellularly during the conversion, comprehensively and significantly increasing the yield of recombinant strains in the fermentation production of 1,5-pentanediamine, and achieving stable, efficient, and low-cost production of 1,5-pentanediamine. Detailed implementation manners

[0056] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0057] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. It should be understood that although some embodiments of implementing the present invention are exemplified herein, those skilled in the art will recognize, based on the present disclosure, that numerous modifications can be made without departing from the spirit and intended scope of the present invention. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be restrictive, because the scope of the present invention will only be defined by the appended claims and their equivalents.

[0058] Unless otherwise specified, the examples are all carried out under conventional experimental conditions, such as the Molecular Cloning Laboratory Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0059] For the specific steps, condition parameters, etc. of PCR amplification, purification, plasmid extraction, enzyme digestion, ligation of enzyme digestion products, transformation, etc. involved in the following examples, they are all carried out according to the instructions of the relevant enzymes and reagents purchased. The DNA polymerase used for PCR amplification, the restriction endonucleases used for enzyme digestion, and the ligase used for ligation of enzyme digestion products are all purchased from Takara Bio Inc. (Dalian). The plasmid extraction kit, DNA gel extraction kit, and PCR purification kit are all purchased from Corning Life Sciences (Wujiang) Co., Ltd. The primers are all purchased from Thermo Fisher Scientific (China) Co., Ltd. (INVITROGEN).

[0060] The plasmid transformation method involved in the following examples is as follows: Add the ligation product to 100 μl of Escherichia coli E. coli BL21(DE3) competent cells, incubate on ice for 30 min, and then perform heat shock at 42 °C for 90 s. After incubating on ice for 5 min, add 1 ml of LB. Coat it onto the corresponding resistant plate.

[0061] In the present invention, the amounts of L-lysine and 1,5-pentanediamine in the culture medium can be detected by nuclear magnetic resonance method.

[0062] In the present invention, the percentage sign "%" refers to mass percentage unless otherwise specified; however, for the percentage of a solution, unless otherwise specified, it refers to the number of grams of solute contained in 100 mL of the solution.

[0063] The plasmids used in the examples are summarized in Table 1 below.

[0064] Table 1.

[0065]

[0066]

[0067] Construction of the pBU plasmid backbone in Example 1

[0068] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, the Upp-U fragment (SEQ ID NO: 23) was amplified with the primer pair upp-UF (SEQ ID NO: 21) and upp-UR (SEQ ID NO: 22), the Upp-500bp-D (SEQ ID NO: 26) fragment was amplified with the primer pair upp-DF (SEQ ID NO: 24) and upp-DR (SEQ ID NO: 25), and the Pupp-Upp-D fragment (SEQ ID NO: 29) was amplified with the primer pair upp-F (SEQ ID NO: 27) and upp-R (SEQ ID NO: 28).

[0069] Using the commercially available plasmid pBR322 as a template, the primer pair P1P2-tetA-F (SEQ ID NO:30) and P1P2-tetA-R (SEQ ID NO:31) were used to amplify the fragment P1P2-tetA (SEQ ID NO:32) on the plasmid containing the P1P2 promoter and the tetA sequence (i.e., with a resistance marker). After gel extraction and recovery of the PCR products of the upp-U, upp-500bp-D, P1P2-tetA, and Pupp-Upp-D fragments, they were ligated to the pBR322 vector digested with EcoRI and NaeI, and the gene fragments and the vector were recombinantly ligated using a multi-fragment one-step cloning kit. The recombinant ligation mixture was transformed into competent E. coli JM109 (purchased from Takara Bio Inc. (Dalian)) cells and screened on LB plates containing ampicillin to obtain multiple single colonies. After verification by colony PCR and sequencing, the plasmid was extracted to obtain a plasmid containing the four fragments Upp-U, Upp-500bp-D, P1P2-tetA, and Pupp-Upp-D, and this plasmid was named the pBU vector.

[0070] Example 2 Cloning of the lysine decarboxylase CadA gene

[0071] Using the genome of the commercially available Escherichia coli K12 MG1655 as a template, the primer pair cadA-F (the sequence is as shown in SEQ ID NO:33) and cadA-R (the sequence is as shown in SEQ ID NO:34) were used to amplify the cadA gene fragment (the sequence is as shown in SEQ ID NO:9). The cadA gene fragment and the pBU vector obtained in Example 1 digested with EcoRI were subjected to gel extraction and purification, and the gene fragment and the vector were recombined using a multi-fragment one-step cloning kit to produce a plasmid named pBU-cadA.

[0072] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, the piclR promoter was amplified with the primer pair piclR-F (sequence shown in SEQ ID NO:35) and piclR-R (sequence shown in SEQ ID NO:36), and the sequence was shown in SEQ ID NO:83. Of course, in some other embodiments, depending on the source, the pirclR sequence can also be derived from piclR-Cg shown in SEQ ID NO:84 of Corynebacterium glutamicum, or from piclR-Ha shown in SEQ ID NO:85 of Hafnia alvei. The piclR promoter fragment and the pBU-cadA vector digested with sacI alone were recovered and purified by gel cutting. The gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit. The recombinant ligation mixture was transformed into competent E. coli JM109 (purchased from Takara Bio Inc. (Dalian)) cells and screened on an LB plate containing ampicillin to obtain multiple single colonies. After verification by colony PCR and sequencing, the plasmid was extracted to obtain the pBU-piclR-cadA plasmid, which contains the cadA gene under the control of the piclR promoter.

[0073] Example 3 Construction of pBU-piclR-stationary-phase specific promoter-cadA and pBU-stationary-phase specific promoter-cadA plasmids

[0074] 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), posmY-F2 (sequence 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) were used respectively; to amplify 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: 4). After the PCR products were recovered by gel extraction, they were respectively ligated with the pBU-piclR-cadA and pBU-cadA plasmids obtained by SacI digestion in 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.

[0075] The double-stranded DNA sequences (5'-3') of the p21, p22, p23, and p24 promoters were respectively synthesized using common gene sequence synthesis methods in the art, and then ligated into the pBU-piclR-cadA and pBU-cadA plasmids obtained by SacI digestion in Example 2. Plasmids pBU-piclR-p21-cadA, pBU-piclR-p22-cadA, pBU-piclR-p23-cadA, and pBU-piclR-p24-cadA containing 4 tandem promoters were obtained; and plasmids pBU-p21-cadA, pBU-p22-cadA, pBU-p23-cadA, and pBU-p24-cadA containing only 4 stationary-phase specific promoters were obtained.

[0076] Example 4 Construction of 1,5-pentanediamine-producing strain and detection of 1,5-pentanediamine production

[0077] In the present invention, the starting strain is Escherichia coli M11-A3 strain capable of producing L-lysine. This strain has been deposited at the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC No: M2018456, deposit date July 6, 2018.

[0078] First, prepare electrocompetent cells. Transform the commercially available pKD46 plasmid into the electrocompetent cells of the M11-A3 strain by heat shock method. After screening and culturing on an LB resistance plate containing 100 μg / ml ampicillin, pick a single colony into 5 mL of LB liquid medium and culture at 30 °C and 200 rpm for 8 h; transfer it to 50 mL of LB liquid medium containing at an inoculation amount of 1% and culture until the OD600 is about 0.15, add 1 mL of 2 mM L-arabinose solution, and continue to culture until the OD600 is 0.4 - 0.5; immediately transfer it to a 50 mL centrifuge tube and ice-bath for 20 min to stop its growth; centrifuge at 4 °C and 4000 rpm for 10 min to collect the cells; then wash the cells with 40 mL of pre-cooled sterile water and centrifuge to collect the cells; repeat the above steps; wash the cells with 20 mL of pre-cooled 10% glycerol and centrifuge to collect the cells; finally, resuspend the cells with 500 μL of pre-cooled 10% glycerol, and the obtained M11-A3 / pKD46 electrocompetent cells are aliquoted and stored for use.

[0079] Using the 16 plasmids constructed in Example 3 (shown in Table 2 below) as templates, the fragments for homologous recombination on the chromosome were amplified respectively using the primer pair upp-UF / upp-R, and gel extraction was performed. Each fragment was separately transferred into the recipient bacterium M11A3 / pKD46 strain. It was spread on an LB resistant plate containing 10 μg / ml tetracycline for screening. For each plasmid transformation, 3 single colonies were picked into 600 μl of LB medium supplemented with ampicillin, and after culturing at 37 °C for 8 h, 1 μl of the bacterial cells was taken as a template for PCR verification to screen out the correct recombinant strains, and glycerol stocks were prepared.

[0080] The 16 strains with glycerol stocks were inoculated into an LB liquid medium containing 0.1 μg / mL 5-FU (5-fluorouracil), and cultured at 37 °C and 200 rpm for 8 h; the bacterial solutions were respectively streaked onto LB medium plates with and without 5-fluorouracil and cultured overnight. Plate analysis showed that for the strains that could not grow on the plate containing 5-fluorouracil, 6 monoclonal colonies were randomly selected from the corresponding plate without 5-fluorouracil, and 1 μl of the bacterial cells was taken as a template for PCR verification again. If both the P1P2-tetA and Pupp-Upp fragments had been removed simultaneously, it indicated that the correct recombinant strain was obtained, and the correct recombinant strain was prepared as a glycerol stock.

[0081] For each of the above recombinant strains, 3 transformants were selected and spread together with the starting strain M11A3 onto a seed medium without antibiotics (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2% yeast extract), and cultured overnight at 37 °C. Then, 3 single colonies were respectively picked and cultured using 5 ml of the seed medium (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2 wt% yeast extract) overnight at 37 °C and 225 rpm. Then each strain was further transferred to 50 ml of fresh fermentation medium (a medium containing 30 g / L glucose, 0.7 wt% Ca(HCO3)2, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2% yeast extract) and continued to be cultured at 37 °C and 170 rpm for 48 h, and the content of 1,5-pentanediamine in each medium was detected by NMR and calculated (Table 2).

[0082] Table 2 NMR detection of 1,5-pentanediamine production and OD600 of recombinant strains compared with the starting strain

[0083]

[0084] As can be seen from Table 2, for 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 that directly express CadA using stationary-phase specific promoters, after 48 h of fermentation, 0.88 - 2.23 g / kg of L-lysine and 1.14 - 2.12 g / kg of 1,5-pentanediamine were detected, indicating that the expression level of lysine decarboxylase was relatively low, and only part of the L-lysine was converted into 1,5-pentanediamine.

[0085] In contrast, for 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 that express cadA using tandem promoters, through testing, it was found that at 48 h of fermentation, compared with the 8 recombinant strains that directly express cadA using stationary-phase specific promoters, the yield of 1,5-pentanediamine produced by each recombinant strain using tandem promoters was further increased, and almost all of the L-lysine was converted into 1,5-pentanediamine. Among them, the 1,5-pentanediamine yield of the piclR-p24-cadA / M11A3 strain was the highest, and finally more than 2.93 g / kg of 1,5-pentanediamine was accumulated, and almost no L-lysine remained.

[0086] Example 5 Construction of plasmids pBU-piclR-p24-plac-rpoS, pBU-piclR-p24-cadA-plac-crp, pBU-piclR-p24-cadA-plac-puuR, pBU-piclR-p24-plac-pepA, pBU-piclR-p24-cadA-plac-soxS

[0087] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, the rpoS gene (sequence as shown in SEQ ID NO:11) was amplified with the primer pair rpoS-F (sequence as shown in SEQ ID NO:49) and rpoS-R (sequence as shown in SEQ ID NO:50), the crp gene (sequence as shown in SEQ ID NO:12) was amplified with the primer pair crp-F (sequence as shown in SEQ ID NO:51) and crp-R (sequence as shown in SEQ ID NO:52), the puuR gene (sequence as shown in SEQ ID NO:13) was amplified with the primer pair puuR-F (sequence as shown in SEQ ID NO:53) and puuR-R (sequence as shown in SEQ ID NO:54), the pepA gene (sequence as shown in SEQ ID NO:14) was amplified with the primer pair pepA-F (sequence as shown in SEQ ID NO:55) and pepA-R (sequence as shown in SEQ ID NO:56), and the soxS gene (sequence as shown in SEQ ID NO:15) was amplified with the primer pair soxS-F (sequence as shown in SEQ ID NO:57) and soxS-R (sequence as shown in SEQ ID NO:58). Using the commercially available pUC18 plasmid DNA as a template, the plac promoter (SEQ ID NO:61) was amplified with the primer pair plac-F (sequence as shown in SEQ ID NO:59) and plac-R (sequence as shown in SEQ ID NO:60);

[0088] The plac promoter fragment, rpos gene fragment, and the pBU-piclR-p24-cadA and pBU-p24-cadA plasmids digested with XbaI were subjected to gel extraction and purification, and the gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, generating plasmids named pBU-piclR-p24-cadA-plac-rpos and pBU-p24-cadA-plac-rpos.

[0089] The plac promoter fragment, crp gene fragment, and the pBU-piclR-p24-cadA and pBU-p24-cadA plasmids digested with XbaI were subjected to gel extraction and purification, and the gene fragments and vectors were recombinantly ligated using a multi-fragment one-step cloning kit, generating plasmids named pBU-piclR-p24-cadA-plac-crp and pBU-p24-cadA-plac-crp.

[0090] The plac promoter fragment, puuR gene fragment, and the plasmids pBU-piclR-p24-cadA and pBU-p24-cadA after single digestion with XbaI were subjected to gel extraction and purification. 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-puuR and pBU-p24-cadA-plac-puuR.

[0091] The plac promoter fragment, pepA gene fragment, and the plasmids pBU-piclR-p24-cadA and pBU-p24-cadA after single digestion with XbaI were subjected to gel extraction and purification. 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-pepA and pBU-p24-cadA-plac-pepA.

[0092] The plac promoter fragment, soxS gene fragment, and the plasmids pBU-piclR-p24-cadA and pBU-p24-cadA after single digestion with XbaI were subjected to gel extraction and purification. 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-soxS and pBU-p24-cadA-plac-soxS.

[0093] Example 6 Construction of 1,5-pentanediamine-producing strain

[0094] Using the 5 pairs of plasmids constructed in Example 5 as templates, the fragments for homologous recombination on the chromosome were amplified using the primer pair upp-UF / upp-R and subjected to gel extraction. Each fragment was separately transformed into the recipient strain M11-A3 / pKD46. It was spread on an LB resistance plate containing 10 μg / ml tetracycline for screening. Twelve single colonies were picked into 600 μl of LB medium supplemented with ampicillin and cultured at 37°C for 8 h. Then, 1 μl of the bacterial cells was taken as a template for PCR verification to screen the correct recombinant strains, which were stored in glycerol.

[0095] Inoculate the strain preserved with glycerol into an LB liquid medium containing 0.1 μg / mL 5-FU (5-fluorouracil), culture at 37 °C and 200 rpm for 8 h; streak the bacterial solution onto LB medium plates with and without 5-fluorouracil respectively, and culture overnight. Plate analysis shows that for the strains that cannot grow on the plates containing 5-fluorouracil, select 6 monoclonal colonies from the corresponding plates without 5-fluorouracil, take 1 μL of the bacterial cells as a template and perform PCR verification again. If the P1P2-tetA and Pupp-Upp fragments have been removed simultaneously, it indicates that the correct recombinant strain has been obtained, and preserve the correct recombinant strain with glycerol.

[0096] Example 7 Detection of the 1,5-pentanediamine production of the strain

[0097] Select 3 transformants from each of the recombinant strains obtained in Example 6, and use the starting strain M11A3, piclR-p24--cadA / M11A3 and p24-cadA / M11A3 as control strains. Spread them on a seed medium without antibiotics (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2% yeast extract), and culture overnight at 37 °C. Then pick 3 monoclonal colonies respectively, and culture them using 5 mL of the seed medium (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2 wt% yeast extract) overnight at 37 °C and 225 rpm. Then transfer each strain 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)2SO 4, 0.001% FeSO4, 0.001% MnSO4, 0.2% yeast extract medium. In addition, add 30 g / kg of pentanediamine to one group of shake flasks) and continue to culture at 37 °C and 170 rpm for 48 h. Sampling is carried out to measure the OD600 of each strain under the conditions of adding or not adding pentanediamine. For the group of samples without adding pentanediamine, use nuclear magnetic resonance to detect and calculate the content of pentanediamine in each medium (the results are shown in Table 3).

[0098] As shown in Table 3, for the shake flasks supplemented with cadaverine, after diluting 20-fold, the OD600 of the strains was measured. Compared with the recombinant strains M11-A3, pcilR-p24-cadA / M11A3, p24-cadA / M11A3, and M11A3, for 10 new strains expressing genes rpoS, crp, puuR, pepA, and soxS that enhance the cell's stress tolerance ability, the OD600 was significantly less affected (i.e., the decrease in the strain's OD600 was small). Among them, the picR-p24-cadA-plac-rpoS / M11A3 strain had the highest OD600, indicating the strongest ability to tolerate cadaverine.

[0099] As shown in Table 4, for the strains piclR-p24-cadA-plac-rpoS / M11A3, piclR-p24-cadA-plac-crp / M11A3, piclR-p24-cadA-plac-puuR / M11A3, piclR-p24-cadA-plac-pepA / M11A3, piclR-p24-cadA-plac-soxS / M11A3, which express proteins promoting the excretion of 1,5-cadaverine, compared with the recombinant strain piclR-p24-cadA / M11A3, the yield of 1,5-cadaverine was further increased. At the same time, almost all of the L-lysine was converted into 1,5-cadaverine. Among them, the piclR-p24-cadA-plac-rpoS / M11A3 strain had the highest yield of 1,5-cadaverine, and finally accumulated more than 3.68 g / kg of 1,5-cadaverine with almost no residual L-lysine.

[0100] Table 3 OD600 of strains measured under conditions with and without cadaverine addition

[0101] Strain OD600 - without adding cadaverine OD600 - with adding cadaverine M11A3 0.826±0.007 0.521±0.008 piclR - cadA / M11A3 0.842±0.005 0.507±0.003 piclR - p24 - cadA / M11A3 0.863±0.003 0.516±0.004 p24 - cadA / M11A3 0.856±0.014 0.497±0.001 piclR - p24 - cadA - plac - rpoS / M11A3 0.889±0.004 0.794±0.007 p24 - cadA - plac - rpoS / M11A3 0.878±0.002 0.743±0.006 piclR - p24 - cadA - plac - crp / M11A3 0.841±0.004 0.693±0.003 p24 - cadA - plac - crp / M11A3 0.854±0.009 0.634±0.005 piclR - p24 - cadA - plac - puuR / M11A3 0.839±0.005 0.675±0.005 p24 - cadA - plac - puuR11A3 0.849±0.006 0.615±0.006 piclR - p24 - cadA - plac - pepA / M11A3 0.875±0.009 0.733±0.008 p24 - cadA - plac - pepA / M11A3 0.859±0.003 0.671±0.003 piclR - p24 - cadA - plac - soxS / M11A3 0.847±0.002 0.690±0.007 p24 - cadA - plac - soxS / M11A3 0.818±0.004 0.686±0.006

[0102] Table 4 Cadaverine yield detected by NMR of recombinant strains compared with the starting strain

[0103]

[0104]

[0105] Example 8 Construction of plasmids pBU-piclR-p24-plac-rpoS-plac-ompA, pBU-piclR-p24-cadA-plac-rpoS-plac-ompC, pBU-piclR-p24-cadA-plac-rpoS-plac-ompF, pBU-piclR-p24-plac-rpoS-plac-ompW, pBU-piclR-p24-cadA-plac-rpoS-plac-ompX

[0106] Using the genome of commercially available Escherichia coli K12 MG1655 as a template, the ompA gene (sequence shown in SEQ ID NO:62) was amplified with the primer pair ompA-F (sequence shown in SEQ ID NO:73) and ompA-R (sequence shown in SEQ ID NO:74), the ompC gene (sequence shown in SEQ ID NO:63) was amplified with the primer pair ompC-F (sequence shown in SEQ ID NO:75) and ompC-R (sequence shown in SEQ ID NO:76), the ompF gene (sequence shown in SEQ ID NO:64) was amplified with the primer pair ompF-F (sequence shown in SEQ ID NO:77) and ompF-R (sequence shown in SEQ ID NO:78), and the ompW gene (sequence shown in SEQ ID NO:65) was amplified with the primer pair ompW-F (sequence shown in SEQ ID NO:79) and ompW-R (sequence shown in SEQ ID NO:80). The ompX gene (sequence shown in SEQ ID NO:66) was amplified with the primer pair ompX-F (sequence shown in SEQ ID NO:81) and ompX-R (sequence shown in SEQ ID NO:82). Using the commercially available pUC18 plasmid DNA as a template, the plac promoter (sequence shown in SEQ ID NO:61) was amplified with the primer pair plac-F2 (sequence shown in SEQ ID NO:72) and plac-R (sequence shown in SEQ ID NO:59); the plac promoter fragment, the ompA gene fragment, and the pBU-piclR-p24-cadA-plac-rpoS plasmid digested with XbaI were subjected to gel extraction and purification, and the gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-piclR-p24-cadA-plac-rpoS-plac-ompA.

[0107] The plac promoter fragment, the ompC gene fragment, and the pBU-piclR-p24-cadA-plac-rpoS plasmid digested with XbaI were subjected to gel extraction and purification, and the gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-piclR-p24-cadA-plac-rpoS-plac-ompC.

[0108] The plac promoter fragment, the ompF gene fragment, and the pBU-piclR-p24-cadA-plac-rpoS plasmid digested with XbaI alone were subjected to gel extraction and purification. The gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-piclR-p24-cadA-plac-rpoS-plac-ompF.

[0109] The plac promoter fragment, the ompW gene fragment, and the pBU-piclR-p24-cadA-plac-rpoS plasmid digested with XbaI alone were subjected to gel extraction and purification. The gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-piclR-p24-cadA-plac-rpoS-plac-ompW.

[0110] The plac promoter fragment, the ompX gene fragment, and the pBU-piclR-p24-cadA-plac-rpoS plasmid digested with XbaI alone were subjected to gel extraction and purification. The gene fragment and the vector were recombinantly ligated using a multi-fragment one-step cloning kit, and the resulting plasmid was named pBU-piclR-p24-cadA-plac-rpoS-plac-ompX.

[0111] Example 9 Construction of 1,5-pentanediamine-producing strain

[0112] Using the 5 plasmids constructed in Example 8 as templates, the fragments for homologous recombination on the chromosome were amplified respectively using the primer pair upp-UF / upp-R, and subjected to gel extraction. Each fragment was separately transformed into the recipient strain M11-A3 / pKD46. It was spread on an LB resistant plate containing 10 μg / ml tetracycline for screening. 12 single colonies were picked into 600 μl of LB medium supplemented with ampicillin and cultured at 37 °C for 8 h. Then, 1 μl of the bacterial cells was taken as a template for PCR verification to screen the correct recombinant strains, and they were stored in glycerol.

[0113] The strains stored in glycerol were inoculated into an LB liquid medium containing 0.1 μg / mL 5-FU (5-fluorouracil), and cultured at 37 °C and 200 rpm for 8 h; the bacterial liquid was streaked on LB medium plates with and without 5-fluorouracil respectively and cultured overnight. Plate analysis showed the strains that could not grow on the plate containing 5-fluorouracil. Six monoclonal colonies were selected from the corresponding plate without 5-fluorouracil. 1 μl of the bacterial cells was taken as a template for PCR verification again. If both the P1P2-tetA and Pupp-Upp fragments had been removed simultaneously, it indicated that the correct recombinant strains were obtained, and the correct recombinant strains were stored in glycerol.

[0114] Example 10 Detection of the Yield of 1,5-Pentanediamine by the Test Strain

[0115] Three transformants were selected from each of the recombinant strains obtained in Example 9, and the starting strain M11A3, piclR-p24--cadA / M11A3, and piclR-p24-cadA-plac-rpoS / M11A3 were used as control strains. They were respectively spread on a seed medium without antibiotics (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2 wt% yeast extract) and cultured overnight at 37 °C. Then, three single colonies were picked respectively and cultured using 5 ml of the seed medium (containing 4 wt% glucose, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2% yeast extract) overnight at 37 °C and 225 rpm. Then, each strain was transferred to 50 ml of fresh fermentation medium (30 g / L glucose, 0.7 wt% Ca(HCO3)2, 0.1 wt% KH2PO4, 0.1 wt% MgSO4, 1.6 wt% (NH4)2SO 4, 0.001 wt% FeSO4, 0.001 wt% MnSO4, 0.2 wt% yeast extract medium) and continued to be cultured at 37 °C and 170 rpm for 48 h. The content of pentanediamine in each medium was detected by NMR and calculated.

[0116] As shown in Table 5, compared with the recombinant strains piclR-p24-cadA-plac-rpoS-plac-ompA / M11A3, piclR-p24-cadA-plac-rpoS-plac-ompC / M11A3, piclR-p24-cadA-plac-rpoS-plac-ompF / M11A3, piclR-p24-cadA-plac-rpoS-plac-ompW / M11A3, piclR-p24-cadA-plac-rpoS-plac-ompX / M11A3 that express proteins promoting the excretion of 1,5-pentanediamine, the yield of 1,5-pentanediamine produced is further increased. At the same time, almost all L-lysine is converted into 1,5-pentanediamine. Among them, the strain piclR-p24-cadA-plac-rpoS-plac-ompA / M11A3 has the highest yield of 1,5-pentanediamine, and finally accumulates more than 4.18 g / kg of 1,5-pentanediamine with almost no residual L-lysine.

[0117] Table 5 Pentanediamine yield of recombinant strains detected by NMR compared with the starting strain

[0118]

[0119] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention. SEQUENCE LISTING <110> Shanghai Kaisai Biotechnology Co., Ltd. CIBT America Inc. Kaisai (Wusu) Biomaterials Co., Ltd. <120> Recombinant nucleic acid sequences, recombinant expression vectors and genetically engineered bacteria <130> P21015902C <160> 85 <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> 993 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of rpoS <400> 11 atgagtcaga atacgctgaa agttcatgat ttaaatgaag atgcggaatt tgatgagaac 60 ggagttgagg tttttgacga aaaggcctta gtagaacagg aacccagtga taacgatttg 120 gccgaagagg aactgttatc gcagggagcc acacagcgtg tgttggacgc gactcagctt 180 taccttggtg agattggtta ttcaccactg ttaacggccg aagaagaagt ttattttgcg 240 cgtcgcgcac tgcgtggaga tgtcgcctct cgccgccgga tgatcgagag taacttgcgt 300 ctggtggtaa aaattgcccg ccgttatggc aatcgtggtc tggcgttgct ggaccttatc 360 gaagagggca acctggggct gatccgcgcg gtagagaagt ttgacccgga acgtggtttc 420 cgcttctcaa catacgcaac ctggtggatt cgccagacga ttgaacgggc gattatgaac 480 caaacccgta ctattcgttt gccgattcac atcgtaaagg agctgaacgt ttacctgcga 540 accgcacgtg agttgtccca taagctggac catgaaccaa gtgcggaaga gatcgcagag 600 caactggata agccagttga tgacgtcagc cgtatgcttc gtcttaacga gcgcattacc 660 tcggtagaca ccccgctggg tggtgattcc gaaaaagcgt tgctggacat cctggccgat 720 gaaaaagaga acggtccgga agataccacg caagatgacg atatgaagca gagcatcgtc 780 aaatggctgt tcgagctgaa cgccaaacag cgtgaagtgc tggcacgtcg attcggtttg 840 ctggggtacg aagcggcaac actggaagat gtaggtcgtg aaattggcct cacccgtgaa 900 cgtgttcgcc agattcaggt tgaaggcctg cgccgtttgc gcgaaatcct gcaaacgcag 960 gggctgaata tcgaagcgct gttccgcgag taa 993 <210> 12 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of crp <400> 12 atggtgcttg gcaaaccgca aacagacccg actctcgaat ggttcttgtc tcattgccac 60 attcataagt acccatccaa gagcacgctt attcaccagg gtgaaaaagc ggaaacgctg 120 tactacatcg ttaaaggctc tgtggcagtg ctgatcaaag acgaagaggg taaagaaatg 180 atcctctcct atctgaatca gggtgatttt attggcgaac tgggcctgtt tgaagagggc 240 caggaacgta gcgcatgggt acgtgcgaaa accgcctgtg aagtggctga aatttcgtac 300 aaaaaatttc gccaattgat tcaggtaaac ccggacattc tgatgcgttt gtctgcacag 360 atggcgcgtc gtctgcaagt cacttcagag aaagtgggca acctggcgtt cctcgacgtg 420 acgggccgca ttgcacagac tctgctgaat ctggcaaaac aaccagacgc tatgactcac 480 ccggacggta tgcaaatcaa aattacccgt caggaaattg gtcagattgt cggctgttct 540 cgtgaaaccg tgggacgcat tctgaagatg ctggaagatc agaacctgat ctccgcacac 600 ggtaaaacca tcgtcgttta cggcactcgt taa 633 <210> 13 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of puuR <400> 13 atgagtgatg agggactggc gccaggaaaa cgcttgtcgg aaatccgcca gcagcagggg 60 ctttcacaac gtcgtgccgc cgaactctcc gggctgactc acagtgctat cagtacgata 120 gaacaagata aagtcagccc tgccatcagt acgctgcaaa agctgctgaa ggtgtatggt 180 ctgtcactct cggaattctt ttccgagccg gaaaaacctg atgagccgca ggtcgtcatt 240 aatcaggacg acttaattga gatgggtagt cagggtgtgt caatgaagct ggttcataac 300 ggtaacccga atcgcacgct ggcgatgatc tttgaaacgt accagccggg cacaaccact 360 ggggaaagaa ttaagcatca gggtgaggaa ataggcactg tactggaagg tgaaattgtt 420 ctgacgatta atggtcagga ttaccacctc gtcgcggggc aaagttatgc cattaatacc 480 ggcatcccgc acagtttcag taatacgtcg gcaggtattt gccgaattat cagcgcccat 540 acgcccacca cgttttaa 558 <210> 14 <211> 1512 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of pepA <400> 14 atggagttta gtgtaaaaag cggtagcccg gagaaacagc ggagtgcctg catcgtcgtg 60 ggcgtcttcg aaccacgtcg cctttctccg attgcagaac agctcgataa aatcagcgat 120 gggtacatca gcgccctgct acgtcggggc gaactggaag gaaaaccggg gcagacattg 180 ttgctgcacc atgttccgaa tgtactttcc gagcgaattc tccttattgg ttgcggcaaa 240 gaacgtgagc tggatgagcg tcagtacaag caggttattc agaaaaccat taatacgctg 300 aatgatactg gctcaatgga agcggtctgc tttctgactg agctgcacgt taaaggccgt 360 aacaactact ggaaagtgcg tcaggctgtc gagacggcaa aagagacgct ctacagtttc 420 gatcagctga aaacgaacaa gagcgaaccg cgtcgtccgc tgcgtaagat ggtgttcaac 480 gtgccgaccc gccgtgaact gaccagcggt gagcgcgcga tccagcacgg tctggcgatt 540 gccgccggga ttaaagcagc aaaagatctc ggcaatatgc cgccgaatat ctgtaacgcc 600 gcttacctcg cttcacaagc gcgccagctg gctgacagct acagcaagaa tgtcatcacc 660 cgcgttatcg gcgaacagca gatgaaagag ctggggatgc attcctatct ggcggtcggt 720 cagggttcgc aaaacgaatc gctgatgtcg gtgattgagt acaaaggcaa cgcgtcggaa 780 gatgcacgcc caatcgtgct ggtgggtaaa ggtttaacct tcgactccgg cggtatctcg 840 atcaagcctt cagaaggcat ggatgagatg aagtacgata tgtgcggtgc ggcagcggtt 900 tacggcgtga tgcggatggt cgcggagcta caactgccga ttaacgttat cggcgtgttg 960 gcaggctgcg aaaacatgcc tggcggacga gcctatcgtc cgggcgatgt gttaaccacc 1020 atgtccggtc aaaccgttga agtgctgaac accgacgctg aaggccgcct ggtactgtgc 1080 gacgtgttaa cttacgttga gcgttttgag ccggaagcgg tgattgacgt ggcgacgctg 1140 accggtgcct gcgtgatcgc gctgggtcat catattactg gtctgatggc gaaccataat 1200 ccgctggccc atgaactgat tgccgcgtct gaacaatccg gtgaccgcgc atggcgctta 1260 ccgctgggtg acgagtatca ggaacaactg gagtccaatt ttgccgatat ggcgaacatt 1320 ggcggtcgtc ctggtggggc gattaccgca ggttgcttcc tgtcacgctt tacccgtaag 1380 tacaactggg cgcacctgga tatcgccggt accgcctggc gttctggtaa agcaaaaggc 1440 gccaccggtc gtccggtagc gttgctggca cagttcctgt taaaccgcgc tgggtttaac 1500 ggcgaagagt aa 1512 <210> 15 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of soxS <400> 15 atgtcccatc agaaaattat tcaggatctt atcgcatgga ttgacgagca tattgaccag 60 ccgcttaaca ttgatgtagt cgcaaaaaaa tcaggctatt caaagtggta cttgcaacga 120 atgttccgca cggtgacgca tcagacgctt ggcgattaca ttcgccaacg ccgcctgtta 180 ctggccgccg ttgagttgcg caccaccgag cgtccgattt ttgatatcgc aatggacctg 240 ggttatgtct cgcagcagac cttctcccgc gttttccgtc ggcagtttga tcgcactccc 300 agcgattatc gccaccgcct gtaa 324 <210> 16 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> RpoS <400> 16 Met Ser Gln Asn Thr Leu Lys Val His Asp Leu Asn Glu Asp Ala Glu 1 5 10 15 Phe Asp Glu Asn Gly Val Glu Val Phe Asp Glu Lys Ala Leu Val Glu 20 25 30 Gln Glu Pro Ser Asp Asn Asp Leu Ala Glu Glu Glu Leu Leu Ser Gln 35 40 45 Gly Ala Thr Gln Arg Val Leu Asp Ala Thr Gln Leu Tyr Leu Gly Glu 50 55 60 Ile Gly Tyr Ser Pro Leu Leu Thr Ala Glu Glu Glu Val Tyr Phe Ala 65 70 75 80 Arg Arg Ala Leu Arg Gly Asp Val Ala Ser Arg Arg Arg Met Ile Glu 85 90 95 Ser Asn Leu Arg Leu Val Val Lys Ile Ala Arg Arg Tyr Gly Asn Arg 100 105 110 Gly Leu Ala Leu Leu Asp Leu Ile Glu Glu Gly Asn Leu Gly Leu Ile 115 120 125 Arg Ala Val Glu Lys Phe Asp Pro Glu Arg Gly Phe Arg Phe Ser Thr 130 135 140 Tyr Ala Thr Trp Trp Ile Arg Gln Thr Ile Glu Arg Ala Ile Met Asn 145 150 155 160 Gln Thr Arg Thr Ile Arg Leu Pro Ile His Ile Val Lys Glu Leu Asn 165 170 175 Val Tyr Leu Arg Thr Ala Arg Glu Leu Ser His Lys Leu Asp His Glu 180 185 190 Pro Ser Ala Glu Glu Ile Ala Glu Gln Leu Asp Lys Pro Val Asp Asp 195 200 205 Val Ser Arg Met Leu Arg Leu Asn Glu Arg Ile Thr Ser Val Asp Thr 210 215 220 Pro Leu Gly Gly Asp Ser Glu Lys Ala Leu Leu Asp Ile Leu Ala Asp 225 230 235 240 Glu Lys Glu Asn Gly Pro Glu Asp Thr Thr Gln Asp Asp Asp Met Lys 245 250 255 Gln Ser Ile Val Lys Trp Leu Phe Glu Leu Asn Ala Lys Gln Arg Glu 260 265 270 Val Leu Ala Arg Arg Phe Gly Leu Leu Gly Tyr Glu Ala Ala Thr Leu 275 280 285 Glu Asp Val Gly Arg Glu Ile Gly Leu Thr Arg Glu Arg Val Arg Gln 290 295 300 Ile Gln Val Glu Gly Leu Arg Arg Leu Arg Glu Ile Leu Gln Thr Gln 305 310 315 320 Gly Leu Asn Ile Glu Ala Leu Phe Arg Glu 325 330 <210> 17 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> CRP <400> 17 Met Val Leu Gly Lys Pro Gln Thr Asp Pro Thr Leu Glu Trp Phe Leu 1 5 10 15 Ser His Cys His Ile His Lys Tyr Pro Ser Lys Ser Thr Leu Ile His 20 25 30 Gln Gly Glu Lys Ala Glu Thr Leu Tyr Tyr Ile Val Lys Gly Ser Val 35 40 45 Ala Val Leu Ile Lys Asp Glu Glu Gly Lys Glu Met Ile Leu Ser Tyr 50 55 60 Leu Asn Gln Gly Asp Phe Ile Gly Glu Leu Gly Leu Phe Glu Glu Gly 65 70 75 80 Gln Glu Arg Ser Ala Trp Val Arg Ala Lys Thr Ala Cys Glu Val Ala 85 90 95 Glu Ile Ser Tyr Lys Lys Phe Arg Gln Leu Ile Gln Val Asn Pro Asp 100 105 110 Ile Leu Met Arg Leu Ser Ala Gln Met Ala Arg Arg Leu Gln Val Thr 115 120 125 Ser Glu Lys Val Gly Asn Leu Ala Phe Leu Asp Val Thr Gly Arg Ile 130 135 140 Ala Gln Thr Leu Leu Asn Leu Ala Lys Gln Pro Asp Ala Met Thr His 145 150 155 160 Pro Asp Gly Met Gln Ile Lys Ile Thr Arg Gln Glu Ile Gly Gln Ile 165 170 175 Val Gly Cys Ser Arg Glu Thr Val Gly Arg Ile Leu Lys Met Leu Glu 180 185 190 Asp Gln Asn Leu Ile Ser Ala His Gly Lys Thr Ile Val Val Tyr Gly 195 200 205 Thr Arg 210 <210> 18 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> PuuR <400> 18 Met Ser Asp Glu Gly Leu Ala Pro Gly Lys Arg Leu Ser Glu Ile Arg 1 5 10 15 Gln Gln Gln Gly Leu Ser Gln Arg Arg Ala Ala Glu Leu Ser Gly Leu 20 25 30 Thr His Ser Ala Ile Ser Thr Ile Glu Gln Asp Lys Val Ser Pro Ala 35 40 45 Ile Ser Thr Leu Gln Lys Leu Leu Lys Val Tyr Gly Leu Ser Leu Ser 50 55 60 Glu Phe Phe Ser Glu Pro Glu Lys Pro Asp Glu Pro Gln Val Val Ile 65 70 75 80 Asn Gln Asp Asp Leu Ile Glu Met Gly Ser Gln Gly Val Ser Met Lys 85 90 95 Leu Val His Asn Gly Asn Pro Asn Arg Thr Leu Ala Met Ile Phe Glu 100 105 110 Thr Tyr Gln Pro Gly Thr Thr Thr Gly Glu Arg Ile Lys His Gln Gly 115 120 125 Glu Glu Ile Gly Thr Val Leu Glu Gly Glu Ile Val Leu Thr Ile Asn 130 135 140 Gly Gln Asp Tyr His Leu Val Ala Gly Gln Ser Tyr Ala Ile Asn Thr 145 150 155 160 Gly Ile Pro His Ser Phe Ser Asn Thr Ser Ala Gly Ile Cys Arg Ile 165 170 175 Ile Ser Ala His Thr Pro Thr Thr Phe 180 185 <210> 19 <211> 503 <212> PRT <213> Artificial Sequence <220> <223> PepA <400> 19 Met Glu Phe Ser Val Lys Ser Gly Ser Pro Glu Lys Gln Arg Ser Ala 1 5 10 15 Cys Ile Val Val Gly Val Phe Glu Pro Arg Arg Leu Ser Pro Ile Ala 20 25 30 Glu Gln Leu Asp Lys Ile Ser Asp Gly Tyr Ile Ser Ala Leu Leu Arg 35 40 45 Arg Gly Glu Leu Glu Gly Lys Pro Gly Gln Thr Leu Leu Leu His His 50 55 60 Val Pro Asn Val Leu Ser Glu Arg Ile Leu Leu Ile Gly Cys Gly Lys 65 70 75 80 Glu Arg Glu Leu Asp Glu Arg Gln Tyr Lys Gln Val Ile Gln Lys Thr 85 90 95 Ile Asn Thr Leu Asn Asp Thr Gly Ser Met Glu Ala Val Cys Phe Leu 100 105 110 Thr Glu Leu His Val Lys Gly Arg Asn Asn Tyr Trp Lys Val Arg Gln 115 120 125 Ala Val Glu Thr Ala Lys Glu Thr Leu Tyr Ser Phe Asp Gln Leu Lys 130 135 140 Thr Asn Lys Ser Glu Pro Arg Arg Pro Leu Arg Lys Met Val Phe Asn 145 150 155 160 Val Pro Thr Arg Arg Glu Leu Thr Ser Gly Glu Arg Ala Ile Gln His 165 170 175 Gly Leu Ala Ile Ala Ala Gly Ile Lys Ala Ala Lys Asp Leu Gly Asn 180 185 190 Met Pro Pro Asn Ile Cys Asn Ala Ala Tyr Leu Ala Ser Gln Ala Arg 195 200 205 Gln Leu Ala Asp Ser Tyr Ser Lys Asn Val Ile Thr Arg Val Ile Gly 210 215 220 Glu Gln Gln Met Lys Glu Leu Gly Met His Ser Tyr Leu Ala Val Gly 225 230 235 240 Gln Gly Ser Gln Asn Glu Ser Leu Met Ser Val Ile Glu Tyr Lys Gly 245 250 255 Asn Ala Ser Glu Asp Ala Arg Pro Ile Val Leu Val Gly Lys Gly Leu 260 265 270 Thr Phe Asp Ser Gly Gly Ile Ser Ile Lys Pro Ser Glu Gly Met Asp 275 280 285 Glu Met Lys Tyr Asp Met Cys Gly Ala Ala Ala Val Tyr Gly Val Met 290 295 300 Arg Met Val Ala Glu Leu Gln Leu Pro Ile Asn Val Ile Gly Val Leu 305 310 315 320 Ala Gly Cys Glu Asn Met Pro Gly Gly Arg Ala Tyr Arg Pro Gly Asp 325 330 335 Val Leu Thr Thr Met Ser Gly Gln Thr Val Glu Val Leu Asn Thr Asp 340 345 350 Ala Glu Gly Arg Leu Val Leu Cys Asp Val Leu Thr Tyr Val Glu Arg 355 360 365 Phe Glu Pro Glu Ala Val Ile Asp Val Ala Thr Leu Thr Gly Ala Cys 370 375 380 Val Ile Ala Leu Gly His His Ile Thr Gly Leu Met Ala Asn His Asn 385 390 395 400 Pro Leu Ala His Glu Leu Ile Ala Ala Ser Glu Gln Ser Gly Asp Arg 405 410 415 Ala Trp Arg Leu Pro Leu Gly Asp Glu Tyr Gln Glu Gln Leu Glu Ser 420 425 430 Asn Phe Ala Asp Met Ala Asn Ile Gly Gly Arg Pro Gly Gly Ala Ile 435 440 445 Thr Ala Gly Cys Phe Leu Ser Arg Phe Thr Arg Lys Tyr Asn Trp Ala 450 455 460 His Leu Asp Ile Ala Gly Thr Ala Trp Arg Ser Gly Lys Ala Lys Gly 465 470 475 480 Ala Thr Gly Arg Pro Val Ala Leu Leu Ala Gln Phe Leu Leu Asn Arg 485 490 495 Ala Gly Phe Asn Gly Glu Glu 500 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> SoxS <400> 20 Met Ser His Gln Lys Ile Ile Gln Asp Leu Ile Ala Trp Ile Asp Glu 1 5 10 15 His Ile Asp Gln Pro Leu Asn Ile Asp Val Val Ala Lys Lys Ser Gly 20 25 30 Tyr Ser Lys Trp Tyr Leu Gln Arg Met Phe Arg Thr Val Thr His Gln 35 40 45 Thr Leu Gly Asp Tyr Ile Arg Gln Arg Arg Leu Leu Leu Ala Ala Val 50 55 60 Glu Leu Arg Thr Thr Glu Arg Pro Ile Phe Asp Ile Ala Met Asp Leu 65 70 75 80 Gly Tyr Val Ser Gln Gln Thr Phe Ser Arg Val Phe Arg Arg Gln Phe 85 90 95 Asp Arg Thr Pro Ser Asp Tyr Arg His Arg Leu 100 105 <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> 53 <212> DNA <213> Artificial Sequence <220> <223> rpoS-F <400> 49 cggataacaa tttcacacag gaggagctca tgagtcagaa tacgctgaaa gtt 53 <210> 50 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> rpoS-R <400> 50 gctttactca aaaaaaagcc gacttctaga ttactcgcgg aacagcgctt 50 <210> 51 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> crp-F <400> 51 cggataacaa tttcacacag gaggagctca tggtgcttgg caaaccgca 49 <210> 52 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> crp-R <400> 52 gctttactca aaaaaaagcc gacttctaga ttaacgagtg ccgtaaacga 50 <210> 53 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> puuR-F <400> 53 cggataacaa tttcacacag gaggagctca tgagtgatga gggactggc 49 <210> 54 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> puuR-R <400> 54 gctttactca aaaaaaagcc gacttctaga ttaaaacgtg gtgggcgtat 50 <210> 55 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> pepA-F <400> 55 cggataacaa tttcacacag gaggagctca tggagtttag tgtaaaaagc gg 52 <210> 56 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> pepA-R <400> 56 gctttactca aaaaaaagcc gacttctaga ttactcttcg ccgttaaacc ca 52 <210> 57 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> soxS-F <400> 57 cggataacaa tttcacacag gaggagctca tgtcccatca gaaaattatt cagg 54 <210> 58 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> soxS-R <400> 58 gctttactca aaaaaaagcc gacttctaga ttacaggcgg tggcgataat 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 308 <210> 62 <211> 1041 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of ompA <400> 62 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> 63 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of ompC <400> 63 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> 64 <211> 1089 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of ompF <400> 64 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> 65 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of ompW <400> 65 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> 66 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> Gene sequence of ompX <400> 66 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> 67 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> OmpA <400> 67 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> 68 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> OmpC <400> 68 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> 69 <211> 362 <212> PRT <213> Artificial Sequence <220> <223> OmpF <400> 69 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> 70 <211> 212 <212> PRT <213> Artificial Sequence <220> <223> OmpW <400> 70 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> 71 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> OmpX <400> 71 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> 72 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> plac-F2 <400> 72 ctgaatatcg aagcgctgtt ccgcgagtaa ggataaccgt attaccgcct ttgag 55 <210> 73 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ompA-F <400> 73 cggataacaa tttcacacag gaggagctca tgaaaaagac agctatcgcg 50 <210> 74 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ompA-R <400> 74 gctttactca aaaaaaagcc gacttctaga ttaagcctgc ggctgagtta 50 <210> 75 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> ompC-F <400> 75 cggataacaa tttcacacag gaggagctca tgaaagttaa agtactgtcc ctcc 54 <210> 76 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> ompC-R <400> 76 gctttactca aaaaaaagcc gacttctaga ttagaactgg taaaccagac cc 52 <210> 77 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ompF-F <400> 77 cggataacaa tttcacacag gaggagctca tgatgaagcg caatattctg g 51 <210> 78 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ompF-R <400> 78 gctttactca aaaaaaagcc gacttctaga ttagaactgg taaacgatac cca 53 <210> 79 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ompW-F <400> 79 cggataacaa tttcacacag gaggagctca tgaaaaagtt aacagtggcg g 51 <210> 80 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> ompW-R <400> 80 gctttactca aaaaaaagcc gacttctaga ttaaaaacga tatcctgctg agaac 55 <210> 81 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> ompX-F <400> 81 cggataacaa tttcacacag gaggagctca tgaaaaaaat tgcatgtctt tcagc 55 <210> 82 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ompX-R <400> 82 gctttactca aaaaaaagcc gacttctaga ttagaagcgg taaccaacac c 51 <210> 83 <211> 175 <212> DNA <213> Artificial Sequence <220> <223> piclR promoter sequence <400> 83 atttgttcaa cattaactca tcggatcagt tcagtaacta ttgcattagc taacaataaa 60 aatgaaaatg atttccacga tacagaaaaa agagactgtc atggtcgcac ccattcccgc 120 gaaacgcggc agaaaacccg ccgttgccac cgcaccagcg actggacagg ttcag 175 <210> 84 <211> 113 <212> DNA <213> Artificial Sequence <220> <223> Source: piclR promoter piclR-Cg from Corynebacterium glutamicum <400> 84 acagtatagc tattaagagg cgtaaatgtc acctcccgcc caaaatcttc ttataccccc 60 acacagtgaa tcccttcacc acgtctcatt gggtgaaatg ctaaattcaa ggt 113 <210> 85 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Source: piclR promoter piclR-Ha from Hafnia alvei <400> 85 gagcagttaa aaaccacgat cgcatccata atgtcgaaaa cgaaaataga ttccattttt 60 ataacatttt t 71

Claims

1. A recombinant nucleic acid sequence, characterized in that, The recombinant nucleic acid sequence includes a tandem promoter, a lysine decarboxylase gene, and a gene for enhancing cell stress tolerance, wherein the tandem promoter includes a piclR promoter and a stationary-phase specific promoter, and 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 as shown in SEQ ID NO: 83; the nucleotide sequence of the stationary-phase specific promoter is as shown in any one of SEQ ID NOs: 1-8; the lysine decarboxylase gene is the cadA gene from Escherichia coli; the gene for enhancing cell stress tolerance is selected from any one of rpoS, crp, puuR, pepA, and soxS from Escherichia coli.

2. The recombinant nucleic acid sequence according to claim 1, characterized in that, The nucleotide sequence of the cadA gene is as shown in SEQ ID NO:

9.

3. The recombinant nucleic acid sequence according to claim 1, wherein The recombinant nucleic acid sequence further includes a constitutive promoter; the gene for enhancing cell stress tolerance is operably linked to the constitutive promoter; the constitutive promoter is selected from any one or more of plac, trp, tac, trc, and PL.

4. The recombinant nucleic acid sequence according to claim 3, wherein The nucleotide sequence of the constitutive promoter is as shown in SEQ ID NO:

60.

5. The recombinant nucleic acid sequence according to claim 1, wherein The nucleotide sequence of the gene for enhancing cell stress tolerance is as shown in any one of SEQ ID NOs: 11-15.

6. The recombinant nucleic acid sequence according to claim 1, wherein The recombinant nucleic acid sequence further includes a gene encoding an outer membrane porin.

7. The recombinant nucleic acid sequence according to claim 6, wherein The gene encoding an outer membrane porin is operably linked to the constitutive promoter, and / or the gene encoding an outer membrane porin is selected from any one or more of ompA, ompC, ompF, ompW, and ompX.

8. The recombinant nucleic acid sequence according to claim 7, wherein The nucleotide sequence of the gene encoding an outer membrane porin is as shown in any one of SEQ ID NOs: 62-66.

9. The recombinant nucleic acid sequence according to any one of claims 1 to 8, characterized in that, The recombinant nucleic acid sequence is selected from the following groups: (1) piclR-p24-cadA-plac-rpoS, piclR-p24-cadA-plac-crp, piclR-p24-cadA-plac-puuR, piclR-p24-cadA-plac-pepA, or piclR-p24-cadA-plac-soxS; and, (2) piclR-p24-cadA-plac-rpoS-plac-ompA, piclR-p24-cadA-plac-rpoS-plac-ompC, piclR-p24-cadA-plac-rpoS-plac-ompF, piclR-p24-cadA-plac-rpoS-plac-ompW, or piclR-p24-cadA-plac-rpoS-plac-ompX.

10. A recombinant expression vector, characterized in that, The recombinant expression vector includes the recombinant nucleic acid sequence as described in any one of claims 1-9.

11. The recombinant expression vector according to claim 10, characterized in that, The backbone plasmids of the recombinant expression vector include pUC18, pUC19, pBR322, pACYC, pET, pSC101, and their derivative plasmids.

12. A genetically engineered bacterium for producing 1,5-pentanediamine, characterized in that, The genetically engineered bacterium contains the recombinant nucleic acid sequence described in any one of claims 1 to 9.

13. The genetically engineered bacterium according to claim 12, wherein, The host bacterium of the genetically engineered bacterium is derived from Escherichia coli.

14. The genetically engineered bacterium according to claim 12 or 13, characterized in that, The recombinant nucleic acid sequence is located on a free recombinant expression vector or integrated into the chromosome.

15. A method for producing 1,5-pentanediamine, characterized in that, The method includes the steps of culturing the genetically engineered bacterium containing the one described in any one of claims 12 to 14 in a fermentation medium to produce 1,5-pentanediamine.

16. Use of the recombinant nucleic acid sequence described in any one of claims 1 to 9, the recombinant expression vector described in claim 10 or 11, or the genetically engineered bacterium described in any one of claims 12 to 14 in the production of 1,5-pentanediamine.

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