Recombinant microorganism and method for producing 1,5-pentanediamine

Abstract

The present disclosure provides a recombinant microorganism, which comprises an expression vector, the expression vector comprising a nucleotide sequence encoding a pyridoxal phosphate gene and a continuous promoter sequence controlling the expression of the nucleotide sequence. The present disclosure also provides a method for producing 1,5-pentanediamine using the microorganism.

Description

Technical Field

[0001] The present disclosure relates to a microorganism for producing 1,5-pentanediamine, and in particular to a genetically recombinant microorganism and a method for producing 1,5-pentanediamine using the microorganism. Background Art

[0002] 1,5-Pentanediamine is an important raw material for synthesizing polymers in the chemical industry. The improvement and efficiency enhancement of its production and preparation methods have always been the main topics in this field.

[0003] The in vitro whole-cell biotransformation method is one of the existing methods for producing 1,5-pentanediamine by microorganisms. It uses a complete biological organism as a catalyst for chemical transformation, that is, after the microorganisms are cultured to a certain cell mass, a substrate is added for catalysis and then converted into products. For example, genetically modified microorganisms are used to express lysine decarboxylase (CadA), and the bacterial mass is increased through high-density fermentation to increase the expression of lysine decarboxylase, thereby improving its catalytic conversion efficiency of lysine to produce pentamethylenediamine. However, when producing 1,5-pentanediamine by the whole-cell biotransformation method, expensive pyridoxal 5'-phosphate (PLP) must be added to the reaction system to activate lysine decarboxylase (CadA) to further improve the efficiency of the whole-cell biotransformation method.

[0004] Pyridoxal phosphate (PLP) is the active form of vitamin B6. Escherichia coli cells have two self-generated pathways for pyridoxal phosphate, one of which is the de novo synthesis method, which uses glucose to produce pyridoxal phosphate, and the other is called the super rescue pathway, which uses vitamin B6 precursors such as pyridoxal (PL) and pyridoxine (PN) to catalyze the production of pyridoxal phosphate.

[0005] In the prior art, a method of adding a vitamin B6 precursor to replace pyridoxal phosphate in the catalytic process has been disclosed, such as patent publication number CN109536542A, which discloses adding pyridoxal, pyridoxal phosphate, pyridoxine, pyridoxamine and other coenzymes at a concentration of 0.05 to 0.5 mM in the reaction system; in addition, patent publication number CN109097408A discloses adding lysine decarboxylase and vitamin B6 coenzyme in a continuous enzyme reactor; and patent publication number CN108997141A also discloses adding pyridoxamine, pyridoxine, pyridoxal, pyridoxal phosphate and other vitamin B6 coenzymes in a mixed system of lysine and lysine decarboxylase. However, the above-mentioned direct addition of pyridoxal phosphate precursor is not effective for the production of 1,5-pentanediamine, and other whole-cell catalytic methods for improving the production capacity of 1,5-pentanediamine are still needed.

[0006] In 2015, a research team from Nanjing University of Technology (Ma, W., Cao, W., Zhang, B., Chen, K., Liu, Q., Li, Y., & Ouyang, P. (2015). Engineering a pyridoxal 5'-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis. Scientific Reports, 5(1), 1-10) attempted to increase the production of pentamethylenediamine by expressing genes in the de novo synthesis pathway, while a team from South Korea led by Kim et al. (Kim, JH, Kim, J., Kim, HJ, Sathiyanarayanan, G., Bhatia, SK, Song, HS & Yang, YH (2017). Biotransformation of pyridoxal 5'-phosphate from pyridoxal by pyridoxal kinase (pdxY) to support cadaverine production in Escherichia coli. Enzyme and Microbial Technology, 104, 9-15) expressed PdxY and added PL as a precursor to obtain a maximum of 33 g / L of pentamethylenediamine with a productivity of 5.5 g / L / h. However, the production capacity and efficiency of these methods do not meet the requirements, so there is still a need for a method to further increase the production of 1,5-pentanediamine.

[0007] In view of this, there is still a need in the technical field to which the present disclosure belongs for a method for effectively increasing the production capacity of 1,5-pentanediamine to solve the problems existing in the known technology. Summary of the invention

[0008] To solve the above problems, the present disclosure provides a recombinant microorganism for producing 1,5-pentanediamine, which comprises at least two genes, and at least one of the at least two genes is located on an expression vector, wherein the at least two genes comprise a gene encoding a lysine decarboxylase and a gene encoding an enzyme for producing pyridoxal phosphate. In at least one specific embodiment of the present disclosure, the gene expression of the lysine decarboxylase and the enzyme for producing pyridoxal phosphate included in the recombinant microorganism is enhanced.

[0009] In at least one specific embodiment of the present disclosure, the recombinant microorganism producing 1,5-pentanediamine comprises at least two exogenous genes, wherein the exogenous genes are a gene encoding lysine decarboxylase (CadA) and a gene encoding an enzyme producing pyridoxal phosphate (PLP).

[0010] In at least one specific embodiment of the present disclosure, the gene encoding the enzyme that produces pyridoxal phosphate is pdxH, pdxY, pdK or any combination thereof.

[0011] In at least one specific embodiment of the present disclosure, the recombinant microorganism comprises an expression vector, wherein the expression vector comprises at least one nucleotide sequence encoding lysine decarboxylase (CadA), encoding PdxH, PdxY or PdxK for producing pyridoxal phosphate, and a continuous promoter sequence controlling the expression of the nucleotide sequence.

[0012] In at least one specific embodiment of the present disclosure, in the expression vector contained in the recombinant microorganism, the nucleotide sequence encoding lysine decarboxylase (CadA) is a sequence having at least 80% sequence identity with SEQ ID NO: 11 and having the same activity as SEQ ID NO: 11, the nucleotide sequence encoding PdxH is a sequence having at least 80% sequence identity with SEQ ID NO: 1 and having the same activity as SEQ ID NO: 1, the nucleotide sequence encoding PdxY is a sequence having at least 80% sequence identity with SEQ ID NO: 3 and having the same activity as SEQ ID NO: 3, and the nucleotide sequence encoding PdxK is a sequence having at least 80% similarity with SEQ ID NO: 5 and having the same activity as SEQ ID NO: 5.

[0013] In at least one embodiment of the present disclosure, the persistent promoter is one of the J series persistent promoters. In some embodiments of the present disclosure, the J series persistent promoters include J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23108, J23109, J23110, J23111, J23112, J23113, J23114, J23115, J23116, J23117, J23118 and J23119. In some embodiments of the present disclosure, the persistent promoter is J23100, J23109 or J23114.

[0014] In some embodiments of the present disclosure, the persistent promoter is a PlacI persistent promoter.

[0015] In at least one embodiment of the present disclosure, the microorganism belongs to the genus Escherichia, Klebsiella, Erwinia, Serratia, Providencia, Corynebacterium or Brevibacterium. In some embodiments of the present disclosure, the microorganism is Escherichia coli K-12W3110 strain or BL21 strain.

[0016] In at least one specific embodiment of the present disclosure, the recombinant microorganism provided by the present disclosure is Escherichia coli JcadA-Wmut, which was deposited with the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, D-38124, Inhofenstrasse 7B, Brunswick, Germany) on January 11, 2021 with the deposit number DSM 33754.

[0017] The present disclosure also provides a method for producing 1,5-pentanediamine, comprising: mixing the above-mentioned microorganism with lysine in a first solution to convert the lysine into 1,5-pentanediamine; and separating 1,5-pentanediamine from the first solution.

[0018] In some embodiments of the present disclosure, the method further includes adding a cofactor to the first solution. In some embodiments of the present disclosure, the cofactor is pyridoxal-5'-phosphate (PLP) or a precursor thereof, such as pyridoxal (PL) or pyridoxine (PN). In at least one embodiment of the present disclosure, the concentration of lysine in the first solution is 0.1M to 2M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2M.

[0019] In at least one specific embodiment of the present disclosure, the method further comprises: after separating and obtaining 1,5-pentanediamine, recovering the microorganisms in the solution; adding the recovered microorganisms to a second solution containing lysine; adding the aforementioned microorganisms to the second solution; and separating and obtaining 1,5-pentanediamine from the second solution.

[0020] In at least one embodiment of the present disclosure, the concentration of lysine in the second solution is 0.1M to 2M, for example 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2M.

[0021] In at least one embodiment of the present disclosure, the amount of the microorganism added to the second solution is OD 600 The value is counted from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and is not limited thereto.

[0022] The present invention uses a non-inducible expression system to simultaneously express lysine decarboxylase (CadA) and generate pyridoxal phosphate (PLP) in a microbial host as a whole-cell catalyst, thereby eliminating the step and cost of adding PLP as a coenzyme, and further improving the catalytic efficiency and yield of pentamethylenediamine of the microorganism. The whole-cell catalyst can also be recovered and recycled, further reducing the production cost of 1,5-pentamethylenediamine. When the whole-cell catalyst is used to produce 1,5-pentamethylenediamine, it is not necessary to add additional pyridoxal phosphate (PLP) coenzyme, thereby reducing the production cost of 1,5-pentamethylenediamine and simplifying the production process, thereby increasing the yield and production rate of pentamethylenediamine, and enabling large-scale production of 1,5-pentamethylenediamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The figure shows that different precursors such as pyridoxal (PL) or pyridoxine (PN) were added to W3110 and BL21 strains to drive protein expression of pdxH, pdxY or pdxK genes using a continuous plasmid containing the spontaneous promoter pSU-J23100 or pSU-PlacI. The arrows in the figure indicate the positions of PdxH (25.5 KDa), PdxY (31.7 KDa) and PdxK (31.2 KDa) in protein electrophoresis. JH: pSU-J23100 promoter drives the expression of pdxH gene; PH: pSU-PlacI promoter drives the expression of pdxH gene; JY: pSU-J23100 promoter drives the expression of pdxY gene; PY: pSU-PlacI promoter drives the expression of pdxY gene; JK: pSU-J23100 promoter drives the expression of pdxK gene; PK: pSU-PlacI promoter drives the expression of pdxK gene.

[0024] Figure 2 The figure shows the amount of PLP produced by driving pdxH, pdxY or pdxK genes with a continuous plasmid containing the spontaneous promoter pSU-J23100 or pSU-PlacI in W3110 and BL21 strains under the addition of pyridoxal (PL) or pyridoxine (PN). WT: original strain without any plasmid transformation; JH: driving the expression of pdxH gene with the pSU-J23100 promoter; PH: driving the expression of pdxH gene with the pSU-PlacI promoter; JY: driving the expression of pdxY gene with the pSU-J23100 promoter; PY: driving the expression of pdxY gene with the pSU-PlacI promoter; JK: driving the expression of pdxK gene with the pSU-J23100 promoter; PK: driving the expression of pdxK gene with the pSU-PlacI promoter. DCW: dry cell weight. *P<0.05.

[0025] FIG. 3A to FIG. 3C The production of pentamethylenediamine by the two-plasmid BL21 strain containing the CadA plasmid and either the PdxY or PdxK plasmid is shown. Figure 3A Show the growth status of cells; Figure 3B The yield of pentamethylenediamine is shown; Figure 3C Shows the amount of self-generated intracellular PLP. DCW: dry cell weight. *P<0.05.

[0026] Figure 4 Shown are the pentamethylenediamine production and lysine consumption rate of the dual-plasmid BL21 strain co-expressing CadA and PdxY or PdxK in different lysine concentrations.

[0027] Figure 5 The activity analysis of lysine decarboxylase (CadA) of the PLP self-producing strain after cloning with the pSU-J23100-CadA plasmid (JcadA-Wmut strain) is shown. The white columns in the bar graph represent the addition of pyridoxal phosphate (PLP) as a cofactor, the light gray columns represent the addition of pyridoxine (PN) as a cofactor, and the dark gray columns represent the addition of pyridoxal (PL) as a cofactor. DAP: 1,5-diaminopentane

[0028] Figure 6 The results show that the PLP self-producing strain cloned with the pSU-J23100-CadA plasmid (JcadA-Wmut strain) catalyzes the production of 1,5-diaminopentane (DAP) from lysine (1.2 M) using PN or PL as a precursor.

[0029] Figure 7 The figure shows the lysine conversion rate after recycling of the PLP self-producing strain after cloning with the pSU-J23100-CadA plasmid (JcadA-Wmut strain). DETAILED DESCRIPTION

[0030] The following is an explanation of the embodiments of the present disclosure by specific specific examples, and those skilled in the art can easily understand the advantages and effects of the present disclosure from the contents described in this specification. The present disclosure can also be implemented or applied through other different embodiments, and the details in this specification can also be given different modifications and changes based on different viewpoints and applications without departing from the scope described in this disclosure. In addition, all ranges and values ​​herein are inclusive and combinable. Any value or point falling within the range described herein, such as any integer, can be used as a minimum or maximum value to derive a lower range.

[0031] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural individuals unless the context dictates otherwise.

[0032] As used in this disclosure and the appended claims, the term "or" includes the meaning of "and / or" unless the context dictates otherwise.

[0033] The present disclosure provides an expression vector, which includes a gene encoding an enzyme for producing pyridoxal phosphate, including nucleotide sequences of pdxH, pdxY and pdxK, and a continuous promoter sequence for controlling the expression of the nucleic acid molecule of the lysine decarboxylase. The present disclosure also provides a recombinant microorganism comprising the expression vector, and a method for producing 1,5-pentanediamine using the microorganism.

[0034] As used herein, the term "pyridoxal phosphate" (PLP) refers to a coenzyme (or auxiliary factor) of lysine decarboxylase that catalyzes the reaction of organisms to produce 1,5-pentanediamine, and pyridoxal phosphate is the active form of vitamin B6. As mentioned above, there are two self-generated pathways for PLP in Escherichia coli cells, one is the de novo synthesis method, that is, the production of PLP using glucose, and the other is the super rescue pathway, that is, the use of vitamin B6 precursors, including pyridoxal (PL) and pyridoxine (PN), etc., to catalyze the production of PLP.

[0035] According to at least one embodiment of the present disclosure, the nucleotide sequence encoding the pdxH gene that produces pyridoxal phosphate is a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 1 and having the same activity as SEQ ID NO: 1, for example, a protein having the activity of producing PdxH of pyridoxal phosphate. In some embodiments of the present disclosure, the PdxH that produces pyridoxal phosphate has the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having conservative variations with SEQ ID NO: 2.

[0036] According to at least one embodiment of the present disclosure, the nucleotide sequence encoding the pdxY gene that produces pyridoxal phosphate is a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 3 and having the same activity as SEQ ID NO: 3, for example, a protein having the activity of producing PdxY for producing pyridoxal phosphate. In some embodiments of the present disclosure, the PdxY gene that produces pyridoxal phosphate has the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that has conservative variations with SEQ ID NO: 4.

[0037] According to at least one embodiment of the present disclosure, the nucleotide sequence encoding the pdxK gene that produces pyridoxal phosphate is a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 5 and having the same activity as SEQ ID NO: 5, for example, a protein having the activity of producing PdxK for producing pyridoxal phosphate. In some embodiments of the present disclosure, the PdxK gene that produces pyridoxal phosphate has the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having conservative variations with SEQ ID NO: 6.

[0038] As used herein, the term "sequence identity percentage" refers to the percentage of amino acid or nucleotide residues of a candidate protein or nucleic acid fragment that are exactly the same as the amino acid or nucleotide residues of a reference protein or nucleic acid fragment. When performing the above comparison, the candidate protein or nucleic acid fragment can be first aligned with the protein or nucleic acid fragment, and gaps can be introduced if necessary to allow the two sequences to form the highest sequence identity. When calculating sequence identity, conservatively varied amino acid residues will be considered different residues; nucleotide residues of degenerate codons will also be considered different residues, for example, between the codons AAU and AAC that also encode asparagine, it will be considered that there is a different residue U or C.

[0039] It should be understood that the amino acid or nucleotide sequence of a candidate protein or nucleic acid fragment in which at least a portion of the sequence is modified (e.g., deleted, substituted, or added) compared to the amino acid or nucleotide sequence of the reference protein or nucleic acid fragment in the present disclosure is also within the scope of the present disclosure, as long as the resulting candidate protein or nucleic acid fragment has substantially the same biological activity as the amino acid or nucleotide of the reference protein or nucleic acid fragment, due to the degeneracy of codons. For example, in the nucleotide sequence of the enzyme encoding pyridoxal phosphate producing enzyme disclosed in the present disclosure, various modifications can be made in the coding region, provided that it does not change the activity of the polypeptide expressed from the coding region. Therefore, the nucleotide sequence of the pdxH gene encoding pyridoxal phosphate producing enzyme disclosed in the present disclosure can be a nucleotide sequence having SEQ ID NO: 1 or any nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1, as long as the protein encoded by the nucleotide sequence can exhibit the activity of PdxH producing pyridoxal phosphate. Similarly, the PdxH that generates pyridoxal phosphate in the present disclosure may be any protein having an amino acid sequence of SEQ ID NO: 2 or a protein homologous to SEQ ID NO: 2, as long as the protein can substantially exhibit the activity of PdxH that generates pyridoxal phosphate.

[0040] As used herein, the term "constitutive promoter" refers to a promoter that maintains continuous activity in most or all tissues. Compared with an inducible promoter that must be regulated by external signals or inducers, a constitutive promoter can continuously express a specific gene.

[0041] The persistent promoters suitable for the present disclosure include members belonging to the J series persistent promoters, such as: J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23108, J23109, J23110, J23111, J23112, J23113, J23114, J23115, J23116, J23117, J23118 and J23119. In at least one specific embodiment, the persistent promoter used in the present disclosure may be J23100, J23109 or J23114. In some specific embodiments, the persistent promoter used in the present disclosure has the sequence of SEQ ID NO: 7 or a sequence that has at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 7 and has the same activity as SEQ ID NO: 7, for example, a sequence that can also serve as a persistent promoter.

[0042] The persistent promoters suitable for the present disclosure further include PlacI persistent promoters. In some specific embodiments, the persistent promoter has the sequence of SEQ ID NO: 8 or a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 8 and having the same activity as SEQ ID NO: 8, such as a sequence that can also serve as a persistent promoter.

[0043] According to at least one specific embodiment of the present disclosure, the expression vector of the present disclosure further includes at least one selected from the following group: a marker gene sequence, a reporter gene sequence, an antibiotic resistance gene sequence, a restriction enzyme cleavage site sequence, a polyadenylation site sequence, a enhancer sequence, a terminator sequence and a regulator sequence.

[0044] In some embodiments, the expression vector of the present disclosure has the sequence of SEQ ID NO: 9, or a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) sequence identity to SEQ ID NO: 9 and having the same activity as SEQ ID NO: 9. In some embodiments, the expression vector of the present disclosure has the sequence of SEQ ID NO: 10, or a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) sequence identity to SEQ ID NO: 10 and having the same activity as SEQ ID NO: 10.

[0045] As used herein, the term "lysine decarboxylase" refers to an enzyme involved in the reaction of generating 1,5-pentanediamine in an organism, including two lysine decarboxylases, namely lysine decarboxylase 1 (CadA) and lysine decarboxylase 2 (LdcC), wherein CadA is an inducible enzyme induced by hypoxia, excess lysine supply and pH change, while LdcC is a continuous enzyme that is not affected by external pH changes.

[0046] According to at least one specific embodiment of the present disclosure, the nucleotide sequence encoding lysine decarboxylase CadA is a sequence having at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 11 and having the same activity as SEQ ID NO: 11, for example, a protein having lysine decarboxylase activity can be exhibited. In some specific embodiments, the lysine decarboxylase CadA has an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a conservative variation with SEQ ID NO: 12. An expression vector provided in at least one specific embodiment of the present disclosure comprises a nucleotide sequence encoding lysine decarboxylase CadA and a continuous promoter sequence for controlling the expression of the nucleic acid molecule of the lysine decarboxylase. In some specific embodiments of the present disclosure, a recombinant microorganism comprising the lysine decarboxylase CadA expression vector and a method for producing 1,5-pentanediamine using the microorganism are provided. In some embodiments of the present disclosure, the expression vector has the sequence of SEQ ID NO: 13 or a sequence that has at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98% or 100%) sequence identity with SEQ ID NO: 13 and has the same activity as SEQ ID NO: 13.

[0047] According to at least one specific embodiment of the present disclosure, the present disclosure provides a microorganism comprising two or more different expression vectors, which includes an expression vector expressing lysine decarboxylase CadA and a recombinant microorganism expressing PdxH, PdxY or PdxK that produces pyridoxal phosphate. In some specific embodiments, the recombinant microorganism provided by the present disclosure can simultaneously express lysine decarboxylase CadA and produce pyridoxal phosphate, and the enzyme that produces the pyridoxal phosphate is PdxH, PdxY or PdxK that produces pyridoxal phosphate encoded by the pdxH, pdxY or pdxK gene. In some specific embodiments, the present disclosure provides a recombinant microorganism that can produce pyridoxal phosphate by itself.

[0048] As used herein, the term "recombinant" refers to the artificial combination of two separate sequence fragments. In general, the term "recombinant" refers to a nucleic acid, protein or microorganism containing genetic material derived from multiple different sources, or encoded by genetic material derived from multiple different sources, such as organisms derived from two or more different strains or species.

[0049] As used herein, the term "exogenous gene" refers to genetic material encoding derived from one or more different sources, such as organisms derived from two or more different strains or species, and the exogenous gene can be expressed in a selected host using known genetic recombination techniques, for example, using an expression vector to express the exogenous gene in the host.

[0050] As used herein, the term "microorganism" pertains to microscopic organisms, including bacteria, archaea, viruses or fungi, etc. As used herein, references to "microorganisms" should be understood to encompass "bacteria".

[0051] Microbial hosts suitable for use as expression vectors of the present disclosure include, but are not limited to, microorganisms belonging to the genus Escherichia, Klebsiella, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium. In some embodiments, the microbial host used in the present disclosure is capable of expressing lysine decarboxylase CadA and pyridoxal phosphate generating enzymes encoded by pdxK, pdxH, or pdxY in vivo. In some embodiments, the microbial host used in the present disclosure is not only capable of expressing lysine decarboxylase CadA and pyridoxal phosphate generating enzymes encoded by pdxK, pdxH, or pdxY in vivo, but also has tolerance to pentamethylenediamine.

[0052] As used herein, the term "coenzyme" or "cofactor" includes non-protein compounds required for normal enzyme activity. Such compounds can be organic or inorganic. For example, coenzymes or cofactors suitable for the present disclosure include, but are not limited to, precursors of vitamin B6 such as pyridoxal phosphate (PLP), pyridoxal (PL) and pyridoxine (PN).

[0053] The features and effects of the present disclosure are further illustrated below through specific embodiments, but are not intended to limit the scope of the present disclosure.

[0054] Example

[0055] Example 1: Construction of expression vector

[0056] Using the genome of E. coli K-12MG1655 as a template, primers with HindIII and SpeI cutting sites were designed to construct a constitutive promoter to express the super rescue genes pdxH, pdxY and pdxK of PLP. The primers used are shown in Table 1 below.

[0057] Table 1. Primers used to construct expression vectors

[0058]

[0059]

[0060] The MG1655 genome was used as a template and the above primers were used to perform polymerase chain reaction (PCR) to obtain specific DNA sequence amplification fragments of the relevant genes. The materials required for the PCR reaction for amplifying specific DNA sequence fragments include DNA template, 5' end primer, 3' end primer, triphosphate deoxynucleotide (dNTP), 10x polymerase buffer and polymerase, wherein the polymerase used in this embodiment includes Ex-Taq. The PCR products were analyzed by DNA electrophoresis and recovered by gel tapping to obtain amplified PLP super rescue gene pdxH, pdxY and pdxK fragments.

[0061] The amplified gene fragment was digested with HindIII and SpeI, and then the amplified PLP super rescue gene fragment was inserted into the pSU-J23100 or pSU-PlacI vector to construct pSU-J23100-pdxH (indicated by JH, i.e., the pSU-J23100 promoter drives the expression of pdxH gene), pSU-PlacI-pdxH (indicated by PH, i.e., the pSU-PlacI promoter drives the expression of pdxH gene), pSU-J23100-pdxY (indicated by JY, i.e., the pSU-J23100 promoter drives the expression of pdxY gene), pSU-PlacI-pdxY (indicated by PY, i.e., the pSU-PlacI promoter drives the expression of pdxY gene), pSU-J23100-pdxK (indicated by JK, i.e., the pSU-J23100 promoter drives the expression of pdxK gene), And plasmids such as pSU-PlacI-pdxK (indicated by PK, that is, the pSU-PlacI promoter drives the expression of the pdxK gene), and then the plasmids are transformed into the DH5α strain.

[0062] When performing thermal transformation, take a tube of commercially available DH5α competent cells, add about 10μL of the plasmid obtained above, place it on ice for 30 minutes, then heat it in a 42℃ water bath for 1.5 minutes, then place it on ice for 5 to 10 minutes, then add 400μL of LB (Luria Bertani) liquid medium or SOC (super optimal broth with catabolitesrepression) medium, place it in a 37℃ incubator and shake culture for about 60 to 90 minutes, then centrifuge it at 4,000rpm for 3 minutes, remove 300μL of supernatant, and spread all the competent cells containing recombinant DNA on a solid medium containing antibiotics, and culture it at 37℃ overnight. Select a few colonies generated on the solid medium and inoculate them in a liquid medium containing antibiotics, and culture them in a 37℃ incubator overnight to obtain DH5α bacterial liquid containing various plasmids.

[0063] Example 2: Preparation of a continuous self-producing PLP strain and its PLP expression level

[0064] The plasmids pSU-J23100-pdxH, pSU-PlacI-pdxH, pSU-J23100-pdxY, pSU-PlacI-pdxY, pSU-J23100-pdxK and pSU-PlacI-pdxK prepared in Example 1 and stored in DH5α cells were extracted and introduced into Escherichia coli BL21 or W3110 strains through thermal transformation. Competent cells of BL21 or W3110 strains containing the above plasmids were spread on a solid culture medium containing antibiotics and cultured overnight at 37°C. Several cells were then inoculated into a liquid culture medium containing antibiotics and cultured overnight in a 37°C incubator to obtain BL21 or W3110 strains containing the plasmids, which were strains that continuously self-produced PLP.

[0065] The strains that continuously self-produce PLP prepared above were cultured in liquid culture medium, to which 0.2 mM pyridoxine (PN) or pyridoxal (PL) was added for culture, and then the protein expression level of each strain was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the wild-type BL21 or W3110 was used as a control.

[0066] Here we further describe the steps of SDS-PAGE protein analysis. First, cells were collected and adjusted to OD 6004, washed twice with deionized water, and then the cell protein was extracted. Take 30μL of protein sample and mix it with 10μL of protein dye, heat it at 100℃ for 10 minutes, add 15μL of sample solution to the SDS film sample slot, and then perform SDS-PAGE electrophoresis analysis. The setting program of SDS-PAGE is to first perform it at 80V for 30 minutes, then at 120V for 60 minutes, stop when the blue indicator line runs out of the bottom of the film, and then stain with Coomassie Brilliant Blue for 40 minutes to 60 minutes, and decolorize for about 40 minutes. Change the decolorizing solution according to the actual staining situation. After the background blue is completely removed, replace it with deionized water, and scan it after the background color is clear. The results are as follows Figure 1 As shown, the protein expression levels of various plasmids in different host strains and after adding different precursors such as PN or PL were different. For example, when the PL precursor was added, the BL21 strain containing JY, PY and PK plasmids had a higher PLP protein expression.

[0067] The intracellular PLP of the strain was further quantified. The overnight culture cells were quantified to OD 600 The cells were washed three times with sterile water, centrifuged at 12,000 rpm for 3 minutes, and then dissolved in 1 mL of sterile water. The cells were disrupted with a high pressure disruptor at 30 KPsi to obtain intracellular metabolites, 10% trichloroacetic acid was added, and the cells were shaken vigorously and placed on ice for 10 minutes. The proteins were removed by centrifugation at 14,000 g for 10 minutes, and the supernatant was used to quantify the PLP production in the continuous self-producing PLP strain by high performance liquid chromatography (HPLC).

[0068] The HPLC method and analysis conditions for quantitative analysis of PLP include filtering the sample to be analyzed through a 0.2 μm filter membrane, using a reverse phase chromatography C18 column (YMC-C18 column, 4.6×250 mm, particle size 5 μm), eluting with 0.1 M potassium dihydrogen phosphate (pH 6.6) as the elution buffer, eluting for 0 to 20 minutes, and setting the flow rate at 0.8 mL / min. The injection volume is 50 μL. The column temperature is maintained at 35°C, and PLP is measured at a wavelength of 340 nm. Using different concentrations of standard PLP as a control, the PLP concentration in the sample can be measured.

[0069] Figure 2The results of PLP quantification driven by different plasmids in BL21 or W3110 strains are shown, which show that the expression level of pSU-J23100-pdxH (JH) plasmid in the W3110 strain host is significantly different from that of the original strain without any plasmid transformation, while the expression levels of pSU-J23100-pdxH (JH), pSU-PlacI-pdxH (PH), pSU-J23100-pdxY (JY) and pSU-PlacI-pdxK (PK) plasmids in the BL21 strain host are significantly different from that of the original strain without any plasmid transformation.

[0070] Example 3: Preparation of dual-plasmid strains and testing of activity and yield

[0071] The pSIT-CadA expression vector was constructed by amplifying the lysine decarboxylase gene from the genomic DNA of Escherichia coli MG1655 (GenBank: NC_000913), cutting the sticky ends with restriction endonucleases BamHI and NotI, and then ligating into the pSIT vector (pSIT vector purchased from Addgene, vector trade name: pSKDuet16, number: #41684) with ligase to generate pSIT-CadA (SEQ ID NO.20). pSIT-CadA and pSU-J23100-pdxY or pSU-PlacI-pdxK were simultaneously introduced into BL21 cells and selected in a dual resistance medium containing kanamycin and chloramphenicol to construct a dual plasmid strain.

[0072] Next, the biofermentation method was performed, that is, the above dual plasmid system was cultured in a liquid culture medium at 37°C and shaken at 200 rpm. 600 When the pH reached 0.6, 0.01 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), 0.2 mM PN and 50 g / L lysine were added, and the cells were changed to 30°C and cultured with shaking at 200 rpm. Samples were taken at regular intervals to measure cell growth, lysine consumption and pentamethylenediamine production.

[0073] like Figure 3AAs shown in the results, after 10 hours, the biomass of the dual-plasmid system containing pSIT-CadA and pSU-J23100-pdxY (AJY) and the dual-plasmid system containing pSIT-CadA and pSU-PlacI-pdxK (APK) was higher than that of the strain containing only pSIT-CadA (A), indicating that their growth was better. Figure 3B It was shown that AJY could promote the production of pentamethylenediamine in a short time, while APK could achieve the maximum conversion. Figure 3C It was also shown that the dual-plasmid system has a high ability to self-generate PLP.

[0074] The whole-cell transformation was performed using a dual-plasmid system, which included collecting cells cultured overnight by centrifugation at 8,000g for 5 minutes, washing them twice with pure water, and then placing them in lysine solutions of different concentrations. The whole-cell catalytic reaction was performed at 37°C and 200 rpm. After a certain period of time, the reaction solution was boiled at 100°C for 10 minutes, and then the supernatant was obtained by high-speed centrifugation, and the lysine consumption rate and pentamethylenediamine production rate were quantitatively analyzed by HPLC.

[0075] The HPLC quantitative analysis of lysine and pentamethylenediamine derivatization system consisted of 340 μL of 0.05 M boric acid buffer (pH 9), 240 μL of 100% methanol, 6 μL of sample and 12 μL of 200 mM diethylethoxymethylenemalonate (DEEMM). The above sample was heated at 70°C for 2 hours to completely degrade excess DEEMM and derivatization byproducts. After derivatization with DEEMM, HPLC was used for analysis. The C18 column (YMC-C18 column, 4.6×250 mm, particle size 5 μm) was maintained at 35°C. Mobile phase A consisted of 100% acetonitrile, and mobile phase B consisted of 25 mM sodium acetate aqueous solution (pH 4.8). A flow rate of 0.8 mL / min was used with the following gradient program: 0–2 min, 20–25% A; 2–32 min, 25–60% A; 32–40 min, 60–20% A. Detection was performed at 284 nm.

[0076] Figure 4 The catalytic effect of the dual-plasmid system at different concentrations of lysine was shown. The results showed that the APK system had the best catalytic ability and the highest pentamethylenediamine yield and lysine consumption rate.

[0077] Example 4: Comparison of the activity of self-generated PLP domesticated strains

[0078] A continuous self-producing PLP strain was obtained by domestication, including culturing the W3110 strain containing pSU-J23100-CadA in an LB liquid medium containing a high concentration of pyridoxal (PL), wherein the concentration of pyridoxal (PL) was 10 to 150 μM, and inoculating the strain into a fresh medium containing a high concentration of pyridoxal (PL) after overnight culture, and repeating the subculture 10 times. The above strain was evenly spread on an LB agar medium (pH 5.0) containing 20 g / L lysine and 100 μM pyridoxal (PL), and the obtained single colonies were selected for CadA enzyme activity test, and the strain with the highest enzyme activity was screened out, thereby obtaining a self-producing PLP strain that simultaneously expresses CadA and PLP-related genes (hereinafter referred to as JcadA-Wmut to represent this domesticated strain).

[0079] The cells cultured overnight were collected by centrifugation at 8000 x g for 5 minutes, washed twice with pure water, and then put into 1.2 M lysine solution. Different precursors such as PLP, PN or PL were added. The whole-cell catalytic reaction was carried out at 37°C and 200 rpm shaking. The CadA activity was determined by bromophenol purple test (BP test) according to the literature (Ting, WW, Huang, CY, Wu, PY, Huang, SF, Lin, HY, Li, SF, Chang, JS&Ng, IS (2021). Whole-cell biocatalyst for cadaverine production usingstable, constitutive and high expression of L-lysine decarboxylase inrecombinant Escherichia coli W3110. Enzyme and Microbial Technology, 109811).

[0080] The BP analysis steps for determining lysine decarboxylase (CadA) activity are as follows: first, obtain OD 600The bacterial cells obtained after the liquid culture with a quantitative value of 5 were subjected to high pressure sterilization (30Kpsi) and centrifuged at 12,000 rpm for 5 minutes to obtain a soluble protein sample containing lysine decarboxylase. The relationship between different concentrations of lysine and the maximum reaction rate was initially tested, and it was found that the best correlation between pentamethylenediamine and HPLC results was obtained when the initial lysine concentration was 40mM. The BP analysis method will appear purple in alkaline conditions and yellow in acidic conditions. After the reaction of lysine decarboxylase, pentamethylenediamine can be increased and the pH can be increased. Therefore, the BP analysis method can detect the color at a wavelength of 595nm to determine the activity of lysine decarboxylase (CadA).

[0081] The activity of lysine decarboxylase was measured using the reaction conditions in Table 2 below. Finally, 3% trichloroacetic acid (TCA) was added to terminate the reaction of lysine decarboxylase. The change in wavelength at 595 nm (△OD 595 ) A calibration curve was obtained by quantification of pentamethylenediamine via HPLC.

[0082] Table 2. Reaction conditions of lysine decarboxylase activity measurement method

[0083] project concentration Volume (μL) Lysine 40mM 40 PLP 0.01mM 10* BP Analysis 0.2g / L 25 <![CDATA[Specimen (OD 600 )]]> 5 25 Sodium acetate buffer, pH = 6 0.5M 400 Total volume 500μL

[0084] *PLP can be omitted when testing the CadA enzyme activity of mutant strains

[0085] like Figure 5 As shown, the CadA activity of JcadA-Wmut was highest when PL was added as a cofactor, and was even higher than when PLP was added as a cofactor.

[0086] In addition, if Figure 6 As shown, when PL is used as a precursor and JcadA-Wmut is used as a biocatalyst, 1.2 M lysine can be completely converted into pentamethylenediamine without adding the auxiliary factor PLP, and ultimately a pentamethylenediamine conversion rate of more than 95% and a pentamethylenediamine yield of 124 g / L can be achieved.

[0087] Example 5: Use of JcadA-Wmut as a recycling catalyst

[0088] The cells in the 2M lysine whole cell catalyst solution were centrifuged at 12,000 rpm for 1 minute, recovered, and then placed in a 2M lysine solution. New JcadA-Wmut strain cells were added. The amount of addition was measured by OD 600 The values ​​were 1, 2, 3 and 5 respectively, and the process was repeated twice. The catalytic time was 4 hours each time. The concentrations of lysine and pentamethylenediamine in the catalyzed solution were quantified by HPLC.

[0089] The results are as follows Figure 7 As shown, JcadA-Wmut increased at each feeding OD 600 When the value is 5, it can be recycled at least twice and still maintain a lysine conversion rate of more than 95%.

[0090] The above embodiments are merely illustrative and are not intended to limit the present disclosure. Any person skilled in the art may modify and change the above embodiments without departing from the scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be defined by the appended claims, and any claims that do not affect the effects and implementation purposes of the present disclosure shall be included in the content of the present disclosure.

[0091] Deposit of biological materials

[0092] The recombinant microorganism provided in the present disclosure is Escherichia coli JcadA-Wmut, which was deposited with the German Collection of Microorganisms (Deutsche Sammlungvon Mikroorganismen und Zellkulturen, DSMZ, D-38124, Inhofenstrasse 7B, Brunswick, Germany) on January 11, 2021 under the deposit number DSM 33754. Sequence Listing <110> China Petrochemical Industry Development Co., Ltd., Taiwan <120> Recombinant microorganism and method for producing 1,5-pentanediamine <130> 115589 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 657 <212> DNA <213> Escherichia coli <400> 1 atgtctgata acgacgaatt gcagcaaatc gcgcatctgc gctgtgaata caccaaaggc 60 gggttacgcc gccgcgatct tcccgccgat ccattaaccc tttttgaacg ttggctctct 120 caggcttgtg aagccaaact ggcggaccct accgcgatgg tggtcgctac cgtggatgaa 180 catggtcagc cttatcagcg catcgtttta ctcaaacatt acgacgaaaa aggcatggtg 240 ttttacacca acctcggcag ccgtaaagca catcaaatcg aaaataatcc gcgcgttagc 300 ctgctgttcc cgtggcatac ccttgagcgc caggtgatgg tgatcggtaa agcagaacga 360 ctttcgactc tcgaagtgat gaaatatttt catagccgcc cgcgtgatag ccagattggt 420 gcatgggttt cgaagcagtc cagtcgcatt tctgcccgcg gtatccttga aagtaaattc 480 ctggagctga agcagaagtt tcaacagggc gaagtgccat tgccgagctt ttggggcggt 540 tttcgcgtca gccttgaaca gattgagttc tggcagggtg gtgagcatcg cctgcatgac 600 cgctttttgt accagcgtga aaatgatgcg tggaagattg atcgtcttgc accctga 657 <210> 2 <211> 218 <212> PRT <213> Escherichia coli <400> 2 Met Ser Asp Asn Asp Glu Leu Gln Gln Ile Ala His Leu Arg Cys Glu 1 5 10 15 Tyr Thr Lys Gly Gly Leu Arg Arg Arg Asp Leu Pro Ala Asp Pro Leu 20 25 30 Thr Leu Phe Glu Arg Trp Leu Ser Gln Ala Cys Glu Ala Lys Leu Ala 35 40 45 Asp Pro Thr Ala Met Val Val Ala Thr Val Asp Glu His Gly Gln Pro 50 55 60 Tyr Gln Arg Ile Val Leu Leu Lys His Tyr Asp Glu Lys Gly Met Val 65 70 75 80 Phe Tyr Thr Asn Leu Gly Ser Arg Lys Ala His Gln Ile Glu Asn Asn 85 90 95 Pro Arg Val Ser Leu Leu Phe Pro Trp His Thr Leu Glu Arg Gln Val 100 105 110 Met Val Ile Gly Lys Ala Glu Arg Leu Ser Thr Leu Glu Val Met Lys 115 120 125 Tyr Phe His Ser Arg Pro Arg Asp Ser Gln Ile Gly Ala Trp Val Ser 130 135 140 Lys Gln Ser Ser Arg Ile Ser Ala Arg Gly Ile Leu Glu Ser Lys Phe 145 150 155 160 Leu Glu Leu Lys Gln Lys Phe Gln Gln Gly Glu Val Pro Leu Pro Ser 165 170 175 Phe Trp Gly Gly Phe Arg Val Ser Leu Glu Gln Ile Glu Phe Trp Gln 180 185 190 Gly Gly Glu His Arg Leu His Asp Arg Phe Leu Tyr Gln Arg Glu Asn 195 200 205 Asp Ala Trp Lys Ile Asp Arg Leu Ala Pro 210 215 <210> 3 <211> 864 <212> DNA <213> Escherichia coli <400> 3 atgatgaaaa atattctcgc tatccagtct cacgttgttt atggtcatgc gggtaacagt 60 gcggcagagt ttccgatgcg ccgcctgggc gcgaacgtct ggccgctgaa caccgttcaa 120 ttttctaatc acacccaata cggcaaatgg actggctgcg tgatgccgcc cagccattta 180 accgaaattg tgcaaggcat tgccgccatt gataaattac acacctgtga tgccgtatta 240 agtggctatc tgggatcggc ggagcagggt gaacatatcc tcggtatcgt ccgtcaggtg 300 aaagccgcga atccgcaggc gaaatatttt tgcgatccgg taatgggtca tccggaaaaa 360 ggctgtatcg ttgcaccggg tgtcgcagag tttcatgtgc ggcacggttt gcctgccagc 420 gatatcattg cgccaaatct ggttgagctg gaaatactct gtgagcatgc ggtaaataac 480 gtcgaagaag cggttctggc agcgcgcgaa ctcattgcgc aagggccaca aattgtgttg 540 gttaaacacc tggcgcgagc tggctacagc cgtgaccgtt ttgaaatgct gctggtcacc 600 gccgatgaag cctggcatat cagccgtccg ctggtggatt ttggtatgcg ccagccggta 660 ggtgttggtg atgtgacgag cggtttactg ctggtgaaac cgcttcaggg ggcaacgctg 720 caggaggcgc tggaacatgt gaccgctgca gtctacgaaa tcatggtgac caccaaagca 780 atgcaggaat atgagctgca agtggtggct gctcaggatc gtattgccaa accagaacat 840 tacttcagcg caacaaagct ctga 864 <210> 4 <211> 287 <212> PRT <213> Escherichia coli <400> 4 Met Met Lys Asn Ile Leu Ala Ile Gln Ser His Val Val Tyr Gly His 1 5 10 15 Ala Gly Asn Ser Ala Ala Glu Phe Pro Met Arg Arg Leu Gly Ala Asn 20 25 30 Val Trp Pro Leu Asn Thr Val Gln Phe Ser Asn His Thr Gln Tyr Gly 35 40 45 Lys Trp Thr Gly Cys Val Met Pro Pro Ser His Leu Thr Glu Ile Val 50 55 60 Gln Gly Ile Ala Ala Ile Asp Lys Leu His Thr Cys Asp Ala Val Leu 65 70 75 80 Ser Gly Tyr Leu Gly Ser Ala Glu Gln Gly Glu His Ile Leu Gly Ile 85 90 95 Val Arg Gln Val Lys Ala Ala Asn Pro Gln Ala Lys Tyr Phe Cys Asp 100 105 110 Pro Val Met Gly His Pro Glu Lys Gly Cys Ile Val Ala Pro Gly Val 115 120 125 Ala Glu Phe His Val Arg His Gly Leu Pro Ala Ser Asp Ile Ile Ala 130 135 140 Pro Asn Leu Val Glu Leu Glu Ile Leu Cys Glu His Ala Val Asn Asn 145 150 155 160 Val Glu Glu Ala Val Leu Ala Ala Arg Glu Leu Ile Ala Gln Gly Pro 165 170 175 Gln Ile Val Leu Val Lys His Leu Ala Arg Ala Gly Tyr Ser Arg Asp 180 185 190 Arg Phe Glu Met Leu Leu Val Thr Ala Asp Glu Ala Trp His Ile Ser 195 200 205 Arg Pro Leu Val Asp Phe Gly Met Arg Gln Pro Val Gly Val Gly Asp 210 215 220 Val Thr Ser Gly Leu Leu Leu Val Lys Pro Leu Gln Gly Ala Thr Leu 225 230 235 240 Gln Glu Ala Leu Glu His Val Thr Ala Ala Val Tyr Glu Ile Met Val 245 250 255 Thr Thr Lys Ala Met Gln Glu Tyr Glu Leu Gln Val Val Ala Ala Gln 260 265 270 Asp Arg Ile Ala Lys Pro Glu His Tyr Phe Ser Ala Thr Lys Leu 275 280 285 <210> 5 <211> 852 <212> DNA <213> Escherichia coli <400> 5 atgagtagtt tgttgttgtt taacgataag agtagggcac tgcaggcgga tatcgtcgcc 60 gtgcagtcgc aggtggttta cggcagcgtg ggcaacagca ttgccgtgcc tgctatcaaa 120 cagaacggcc tgaatgtctt tgccgtgccg acggtattgc tgagcaatac gccgcattat 180 gacactttct acggtggtgc gattccggac gaatggttta gcggctattt gcgtgcgctt 240 caggagcgtg atgcgctgcg ccaacttcgt gctgtaacca cgggctatat gggaacggca 300 tcgcaaatca aaatccttgc cgagtggctg actgcgctac gcaaagacca tcctgaccta 360 ttgatcatgg tcgatccggt gattggcgat attgatagcg gaatttatgt caaacctgac 420 cttcccgaag cgtatcgaca atatttactg ccgctggcgc agggaattac ccccaatatc 480 tttgagttgg aaatcctgac cggtaaaaat tgccgcgatc tcgacagtgc cattgctgcc 540 gcaaaaagtc tgctttcaga cacattaaaa tgggtggtgg ttaccagcgc ctccggtaat 600 gaagaaaatc aggagatgca ggttgtggtg gtcactgccg acagcgtgaa tgtcatttcc 660 cattcacggg taaaaaccga cctgaaaggg actggcgacc tgttttgtgc tcagctcatc 720 agtggcttgc tgaaagggaa ggcgttaacc gatgcagtgc accgagcggg gttgcgcgta 780 ctggaagtga tgcgctacac ccagcagcat gagagcgatg aattgatttt gccgccgctg 840 gcggaagcat aa 852 <210> 6 <211> 283 <212> PRT <213> Escherichia coli <400> 6 Met Ser Ser Leu Leu Leu Phe Asn Asp Lys Ser Arg Ala Leu Gln Ala 1 5 10 15 Asp Ile Val Ala Val Gln Ser Gln Val Val Tyr Gly Ser Val Gly Asn 20 25 30 Ser Ile Ala Val Pro Ala Ile Lys Gln Asn Gly Leu Asn Val Phe Ala 35 40 45 Val Pro Thr Val Leu Leu Ser Asn Thr Pro His Tyr Asp Thr Phe Tyr 50 55 60 Gly Gly Ala Ile Pro Asp Glu Trp Phe Ser Gly Tyr Leu Arg Ala Leu 65 70 75 80 Gln Glu Arg Asp Ala Leu Arg Gln Leu Arg Ala Val Thr Thr Gly Tyr 85 90 95 Met Gly Thr Ala Ser Gln Ile Lys Ile Leu Ala Glu Trp Leu Thr Ala 100 105 110 Leu Arg Lys Asp His Pro Asp Leu Leu Ile Met Val Asp Pro Val Ile 115 120 125 Gly Asp Ile Asp Ser Gly Ile Tyr Val Lys Pro Asp Leu Pro Glu Ala 130 135 140 Tyr Arg Gln Tyr Leu Leu Pro Leu Ala Gln Gly Ile Thr Pro Asn Ile 145 150 155 160 Phe Glu Leu Glu Ile Leu Thr Gly Lys Asn Cys Arg Asp Leu Asp Ser 165 170 175 Ala Ile Ala Ala Ala Lys Ser Leu Leu Ser Asp Thr Leu Lys Trp Val 180 185 190 Val Val Thr Ser Ala Ser Gly Asn Glu Glu Asn Gln Glu Met Gln Val 195 200 205 Val Val Val Thr Ala Asp Ser Val Asn Val Ile Ser His Ser Arg Val 210 215 220 Lys Thr Asp Leu Lys Gly Thr Gly Asp Leu Phe Cys Ala Gln Leu Ile 225 230 235 240 Ser Gly Leu Leu Lys Gly Lys Ala Leu Thr Asp Ala Val His Arg Ala 245 250 255 Gly Leu Arg Val Leu Glu Val Met Arg Tyr Thr Gln Gln His Glu Ser 260 265 270 Asp Glu Leu Ile Leu Pro Pro Leu Ala Glu Ala 275 280 <210> 7 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Promoter sequence <400> 7 ttgacggcta gctcagtcct aggtacagtg ctagc 35 <210> 8 <211> 200 <212> DNA <213> Artificial sequence <220> <223> Promoter sequence <400> 8 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 60 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 120 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 180 gcggataaca atttcacaca 200 <210> 9 <211> 3077 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence <400> 9 ttgacggcta gctcagtcct aggtacagtg ctagcggatc caaagaggag aaaaagctta 60 tgatgaaaaa tattctcgct atccagtctc acgttgttta tggtcatgcg ggtaacagtg 120 cggcagagtt tccgatgcgc cgcctgggcg cgaacgtctg gccgctgaac accgttcaat 180 tttctaatca cacccaatac ggcaaatgga ctggctgcgt gatgccgccc agccatttaa 240 ccgaaattgt gcaaggcatt gccgccattg ataaattaca cacctgtgat gccgtattaa 300 gtggctatct gggatcggcg gagcagggtg aacatatcct cggtatcgtc cgtcaggtga 360 aagccgcgaa tccgcaggcg aaatattttt gcgatccggt aatgggtcat ccggaaaaag gctgtatcgt tgcaccgggt gtcgcagagt ttcatgtgcg gcacggtttg cctgccagcg 480 atatcattgc gccaaatctg gttgagctgg aaatactctg tgagcatgcg gtaaataacg tcgaagagc ggttctggca gcgcgcgac tcattgcgca agggccaca attgtgttgg ttaacacct ggcgcgagct ggctacagcc gtgaccgttt tgaaatgctg ctggtcaccg 660 ccgatgaagc ctggcatatc agccgtccgc tggtggattt tggtatgcgc cagccggtag 720 gtgttggtga tgtgacgagc ggtttactgc tggtgaaact gcttcagggg gcaacgctgc 780 aggaggcgct ggaacatgtg accgctgcag tctacgaaat catggtgacc accaaagcaa tgcaggata tgagctgcaa gtggtggctg ctcaggatcg tattgccaaa ccagaacatt acttcagcgc aacaaagctc tgaactgta gcggccgctg cagtccggca aaaaaagggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag attacttcgc gttatgcaga tctattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 1080 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 1140 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 1200 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 1260 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 1320 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 1380 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 1440 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 1500 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 1560 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 1620 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 1680 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 1740 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 1800 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 1860 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 1920 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 1980 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 2040 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 2100 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagagt 2160 cgacaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc 2220 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca 2280 ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca 2340 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa 2400 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat 2460 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg 2520 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca 2580 ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa 2640 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg 2700 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct 2760 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt 2820 tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta 2880 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgcccgatc aactcgagtg 2940 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 3000 acgaggcaga atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctggaat 3060 tcgcggccgc ttctaga 3077 <210> 10 <211> 3230 <212> DNA <213> Artificial Sequence <220> <223> Plasmid Sequence <400> 10 60. accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 240. gcggataaca atttcacaca ggatccaaag aggagaaaaa gcttatgagt agtttgttgt tgtttaacga taagagtagg gcactgcagg cggatatcgt cgccgtgcag tcgcaggtgg tttacggcag cgtgggcaac agcattgccg tgcctgctat caaacagaac ggcctgaatg tctttgccgt gccgacggta ttgctgagca atacgccgca ttatgacact ttctacggtg 420 gtgcgattcc ggacgaatgg tttagcggct atttgcgtgc gcttcaggag cgtgatgcgc 480 540. tgcgccaact tcgtgctgta accacgggct atatgggaac ggcatcgcaa atcaaaatcc ttgccgagtg gctgactgcg ctacgcaaag accatcctga cctattgatc atggtcgatc 600 cggtgattgg cgatattgat agcggaattt atgtcaaacc tgaccttccc gaagcgtatc 660 gacaattt actgccgctg gcgcaggga ttacccccaa tatctttgag ttggaaatcc tgaccggtaa aaattgccgc gatctcgaca gtgccattgc tgccgcaaaa agtctgcttt 780 cagacacatt aaaatgggtg gtggttacca gcgcctccgg taatgaagaa aatcaggaga 840 tgcaggttgt ggtggtcact gccgacagcg tgaatgtcat ttcccattca cgggtaaaaa 900 ccgacctgaa agggactggc gacctgtttt gtgctcagct catcagtggc ttgctgaaag 960 ggaaggcgtt aaccgatgca gtgcaccgag cggggttgcg cgtactggaa gtgatgcgct 1020 acacccagca gcatgagagc gatgaattga ttttgccgcc gctggcggaa gcataaacta 1080 gtagcggccg ctgcagtccg gcaaaaaagg gcaaggtgtc accaccctgc cctttttctt 1140 taaaaccgaa aagattactt cgcgttatgc agatctatta atgaatcggc caacgcgcgg 1200 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1260 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1320 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1380 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1440 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1500 cgtttcccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1560 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 1620 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1680 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1740 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1800 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 1860 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 1920 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 1980 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2040 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 2100 tcctttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 2160 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 2220 catccatagt tgcctgactc cccgtcgtgt agataactac gatacggggag ggcttaccat 2280 ctggccccag tgctgcaatg ataccgcgag agtcgacaaa ttacgccccg ccctgccact 2340 catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg 2400 catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta tatattgc 2460 ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg 2520 tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga 2580 aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc 2640 ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa 2700 cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac 2760 gaaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact 2820 tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt 2880 tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg 2940 atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg 3000 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt 3060 tggaacctct tacgtgcccg atcaactcga gtgccacctg acgtctaaga aaccattatt 3120 atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag ataaaaaaaa 3180 tccttagctt tcgctaagga tgatttctgg aattcgcggc cgcttctaga 3230 <210> 11 <211> 2148 <212> DNA <213> Escherichia coli <400> 11 atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60 gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120 gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180 aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240 gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300 agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360 actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420 cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480 agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540 tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600 gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660 tccactgcga acaaaattgt tggtatgtac tctgctccag caggcggcac 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 tgattaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320 cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380 acgactgaat gctggccgct gcgtcctgac 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 atgaccggag 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 aaaaatga 2148 <210> 12 <211> 715 <212> PRT <213> Escherichia coli <400> 12 Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu 1 5 10 15 Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln 20 25 30 Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn 35 40 45 Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu 50 55 60 Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu 85 90 95 Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp 100 105 110 Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile 115 120 125 Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys 145 150 155 160 Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met 165 170 175 Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe 195 200 205 Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn 210 215 220 Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Gly 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 Pro 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 Gly Glu Val Tyr Leu Asp Glu Met 625 630 635 640 Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val 660 665 670 Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr 690 695 700 Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys 705 710 715 <210> 13 <211> 4267 <212> DNA <213> Artificial sequence <220> <223> Plasmid sequence <400> 13 ttgacggcta gctcagtcct aggtacagtg ctagcggatc caaagaggag aaaaagctta 60 tgaacgttat tgcaatattg aatcacatgg gggtttattt taaagaagaa cccatccgtg 120 aacttcatcg cgcgcttgaa cgtctgaact tccagattgt ttacccgaac gaccgtgacg 180 acttattaaa actgatcgaa aacaatgcgc gtctgtgcgg cgttattttt gactgggata 240 aatataatct cgagctgtgc gaagaaatta gcaaaatgaa cgagaacctg ccgttgtacg 300 cgttcgctaa tacgtattcc actctcgatg taagcctgaa tgacctgcgt ttacagatta 360 gcttctttga atatgcgctg ggtgctgctg aagatattgc taataagatc aagcagacca 420 ctgacgaata tatcaacact attctgcctc cgctgactaa agcactgttt aaatatgttc 480 gtgaaggtaa atatactttc tgtactcctg gtcacatggg cggtactgca ttccagaaaa 540 gcccggtagg tagcctgttc tatgatttct ttggtccgaa taccatgaaa tctgatattt 600 ccatttcagt atctgaactg ggttctctgc tggatcacag tggtccacac aaagaagcag 660 aacagtatat cgctcgcgtc tttaacgcag accgcagcta catggtgacc aacggtactt 720 ccactgcgaa caaaattgtt ggtatgtact ctgctccagc aggcggcacc attctgattg 780 accgtaactg ccacaaatcg ctgacccacc tgatgatgat gagcgatgtt acgccaatct 840 atttccgccc gacccgtaac gcttacggta ttcttggtgg tatcccacag agtgaattcc 900 agcacgctac cattgctaag cgcgtgaaag aaacaccaaa cgcaacctgg ccggtacatg 960 ctgtaattac caactctacc tatgatggtc tgctgtacaa caccgacttc atcaagaaaa 1020 cactggatgt gaaatccatc cactttgact ccgcgtgggt gccttacacc aacttctcac 1080 cgatttacga aggtaaatgc ggtatgagcg gtggccgtgt agaagggaaa gtgatttacg 1140 aaacccagtc cactcacaaa ctgctggcgg cgttctctca ggcttccatg atccacgtta aaggtgacgt aaacgaaga acctttaacg aagcctacat gatgcacacc accacttctc cgcactacgg tatcgtggcg tccactgaaa ccgctgcggc gatgatgaaa ggcaatgcag gtaagcgtct gattaacggt tctattgaac gtgcgatcaa attccgtaaa gagatcaaac gtctgagaac ggaatctgat ggctggttct ttgatgtatg gcagccggat catatcgata cgactgaatg ctggccgctg cgtcctgaca gcacctggca cggcttcaaa aacatcgata acgagcacat gtatcttgac ccgatcaaag tcaccctgct gactccgggg atggaaaaag acggcaccat gagcgacttt ggtattccgg ccagcatcgt ggcgaatac ctcgacgaac atggcatcgt tgttgagaaa accggtccgt atacctgct gttcctgttc agcatcggta 1680. tcgataagac caaagcactg agcctgctgc gtgctctgac tgactttaaa cgtgcgttcg acctgaacct gcgtgtgaaa aacatgctgc cgtctctgta tcgtgaagat cctgaattct atgaaaacat gcgtattcag gaactggctc agaatatcca caaactgatt gttcaccaca atctgccgga tctgatgtat cgcgcatttg aagtgctgcc gacgatggta atgactccgt 1920 atgctgcatt ccagaaagag ctgcacggta tgaccggaga agtttacctc gacgaaatgg 1980 taggtcgtat taacgccaat atgatccttc cgtacccgcc gggagttcct ctggtaatgc 2040 cgggtgaaat gatcaccgaa gaaagccgtc cggttctgga gttcctgcag atgctgtgtg 2100 aaatcggcgc tcactatccg ggctttgaaa ccgatattca cggtgcatac cgtcaggctg 2160 atggccgcta taccgttaag gtattgaaag aagaaagcaa aaaatgaaga tctattaatg 2220 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 2280 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 2340 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 2400 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 2460 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2520 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 2580 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 2640 tagctcacgc ttaggttatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 2700 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 2760 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 2820 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 2880 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaagagt 2940 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 3000 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 3060 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 3120 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 3180 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 3240 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 3300 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagagt cgacaaatta 3360 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 3420 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 3480 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac 3540 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 3600 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 3660 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 3720 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 3780 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat 3840 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 3900 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 3960 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt 4020 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatctttat 4080 ttcattatgg tgaaagttgg aacctcttac gtgcccgatc aactcgagtg ccacctgacg 4140 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggcaga 4200 atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctggaat tcgcggccgc 4260 ttctaga 4267 <210> 14 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 14 aaaagcttat gtctgataac gacgaattgc 30 <210> 15 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 cctactagttc agggtgcaag acgatcaatc 30 <210> 16 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 16 aagcttatga tgaaaaatat tctcgctatc c 31 <210> 17 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 cctactagttc agagctttgt tgcgctgaag t 31 <210> 18 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 aagcttatga gtagtttgtt gttgtttaac g 31 <210> 19 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 19 actagtttat gcttccgcca gcggcggcaa 30 <210> 20 <211> 5926 <212> DNA <213> Artificial sequence <220> <223> Plasmid sequence <400> 20 tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg aattgtgagc 60 ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga tataccatgt 120 ctgaattcga cggatccatg aacgttattg caatattgaa tcacatgggg gtttatttta 180 aagaagaacc catccgtgaa cttcatcgcg cgcttgaacg tctgaacttc cagattgttt 240 acccgaacga ccgtgacgac ttattaaaac tgatcgaaaa caatgcgcgt ctgtgcggcg 300 ttatttttga ctgggataaa tataatctcg agctgtgcga agaaattagc aaaatgaacg 360 agaacctgcc gttgtacgcg ttcgctaata cgtattccac tctcgatgta agcctgaatg 420 acctgcgttt acagattagc ttctttgaat atgcgctggg tgctgctgaa gatattgcta 480 ataagatcaa gcagaccact gacgaatata tcaacactat tctgcctccg ctgactaaag 540 cactgtttaa atatgttcgt gaaggtaaat atactttctg tactcctggt cacatgggcg 600 gtactgcatt ccagaaaagc ccggtaggta gcctgttcta tgatttcttt ggtccgaata 660 ccatgaaatc tgatatttcc atttcagtat ctgaactggg ttctctgctg gatcacagtg 720 gtccacacaa agaagcagaa cagtatatcg ctcgcgtctt taacgcagac cgcagctaca 780 tggtgaccaa cggtacttcc actgcgaaca aaattgttgg tatgtactct gctccagcag 840 gcggcaccat tctgattgac cgtaactgcc acaaatcgct gacccacctg atgatgatga 900 gcgatgttac gccaatctat ttccgcccga cccgtaacgc ttacggtatt cttggtggta 960 tcccacagag tgaattccag cacgctacca ttgctaagcg cgtgaaagaa acaccaaacg 1020 caacctggcc ggtacatgct gtaattacca actctaccta tgatggtctg ctgtacaaca 1080 ccgacttcat caagaaaaca ctggatgtga aatccatcca ctttgactcc gcgtgggtgc 1140 cttacaccaa cttctcaccg atttacgaag gtaaatgcgg tatgagcggt ggccgtgtag 1200 aagggaaagt gatttacgaa acccagtcca ctcacaaact gctggcggcg ttctctcagg 1260 cttccatgat ccacgttaa ggtgacgtaa acgaagaaac ctttaacgaa gcctacatga 1320 tgcacaccac cacttctccg cactacggta tcgtggcgtc cactgaaacc gctgcggcga 1380 tgatgaaagg caatgcaggt aagcgtctga ttaacggttc tattgaacgt gcgatcaaat 1440 tccgtaaaga gatcaaacgt ctgagaacgg aatctgatgg ctggttcttt gatgtatggc 1500 agccggatca tatcgatacg actgaatgct ggccgctgcg tcctgacagc acctggcacg 1560 gcttcaaaaa catcgataac gagcacatgt atcttgaccc gatcaaagtc accctgctga 1620 ctccggggat ggaaaaagac ggcaccatga gcgactttgg tattccggcc agcatcgtgg 1680 cgaaatacct cgacgaacat ggcatcgttg ttgagaaaac cggtccgtat aacctgctgt 1740 tcctgttcag catcggtatc gataagacca aagcactgag cctgctgcgt gctctgactg 1800 actttaaacg tgcgttcgac ctgaacctgc gtgtgaaaaa catgctgccg tctctgtatc 1860 gtgaagatcc tgaattctat gaaaacatgc gtattcagga actggctcag aatatccaca 1920 aactgattgt tcaccacaat ctgccggatc tgatgtatcg cgcatttgaa gtgctgccga 1980 cgatggtaat gactccgtat gctgcattcc agaaagagct gcacggtatg accggagaag 2040 tttacctcga cgaaatggta ggtcgtatta acgccaatat gatccttccg tacccgccgg 2100 gagttcctct ggtaatgccg ggtgaaatga tcaccgaaga aagccgtccg gttctggagt 2160 tcctgcagat gctgtgtgaa atcggcgctc actatccggg ctttgaaacc gatattcacg 2220 gtgcataccg tcaggctgat ggccgctata ccgttaaggt attgaaagaa gaaagcaaaa 2280 aaaagcttca tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg 2340 gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 2400 ccatcttagt attack ggagatatac attack tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaacc tcaggcattt gagagcaca cggtcacact gcttccggta gtcaataac cggtaaacca gcaatagaca tagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaatga actgcaattt atcaatca ggattatca taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc attaggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc 3180 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt 3240 gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa acaggaatcg 3300 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat 3360 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat 3420 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt 3480 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa 3540 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga 3600 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg 3660 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt 3720 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 3780 ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg ctacgctcgg 3840 tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg ttatccacag 3900 aatcaggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg gaagaagcca 3960 acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4020 tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4080 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4140 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct cagttcggtg 4200 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4260 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4320 gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca cctgttaagg 4380 ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac cttggttcaa 4440 agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg tttttcttta 4500 tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc cgttactcta 4560 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa 4620 gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag 4680 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt 4740 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4800 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt 4860 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag 4920 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg 4980 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc 5040 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc 5100 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc 5160 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag 5220 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa 5280 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc 5340 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc 5400 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt 5460 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc 5520 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga 5580 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc 5640 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 5700 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt 5760 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt 5820 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc 5880 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctc 5926

Claims

1. A recombinant microorganism for producing 1,5-pentanediamine, comprising at least two genes, and at least one of the at least two genes is located on an expression vector, wherein: The at least two genes include a gene encoding lysine decarboxylase and a gene encoding an enzyme producing pyridoxal phosphate, and wherein the recombinant microorganism is deposited in the German Collection of Microorganisms under the deposit number DSM 33754.

2. A method for producing 1,5-pentanediamine, comprising: mixing the recombinant microorganism according to claim 1 with lysine in a first solution to convert the lysine into 1,5-pentanediamine; as well as 1,5-Pentanediamine is separated from the first solution.

3. The method of claim 2, further comprising adding a cofactor to the first solution.

4. The method according to claim 3, wherein: The cofactor is pyridoxal phosphate, pyridoxal, pyridoxine or any combination thereof.

5. The method according to claim 2, further comprising: After separating and obtaining 1,5-pentanediamine, recovering the microorganisms in the solution; adding the recovered microorganism to a second solution containing lysine; adding the recombinant microorganism according to claim 1 to the second solution; as well as 1,5-Pentanediamine is separated from the second solution.

6. The method according to claim 5, characterized in that The amount of the microorganism added to the second solution is expressed as OD 600 The values ​​are counted from 1 to 10.

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