Recombinant microorganism and method for producing 1, 5-pentanediamine
阅读说明：本技术 用于生产1,5-戊二胺的重组微生物及方法 (Recombinant microorganism and method for producing 1, 5-pentanediamine ) 是由 张嘉修 吴意珣 黄士芳 林宏益 李昇峰 蔡珈纬 黄智郁 丁婉雯 于 2020-08-24 设计创作，主要内容包括：本发明涉及用于生产1,5-戊二胺的重组微生物及方法,具体而言,本发明提供一种表达载体,其包括编码赖氨酸脱羧酶CadA的核苷酸序列以及控制该核苷酸序列表达的组成型启动子序列。本发明还提供一种包含该表达载体的重组微生物及使用该微生物生产1,5-戊二胺的方法。(The invention relates to a recombinant microorganism and a method for producing 1, 5-pentanediamine, and particularly provides an expression vector, which comprises a nucleotide sequence for coding lysine decarboxylase CadA and a constitutive promoter sequence for controlling the expression of the nucleotide sequence. The invention also provides a recombinant microorganism containing the expression vector and a method for producing 1, 5-pentanediamine by using the microorganism.)
1. An expression vector comprising a nucleotide sequence encoding lysine decarboxylase CadA and a constitutive promoter sequence controlling expression of said nucleotide sequence.
2. The expression vector of claim 1, wherein the nucleotide sequence encoding lysine decarboxylase CadA has a sequence identical to SEQ ID NO: 1 has at least 80% similarity to SEQ ID NO: 1 with the same activity.
3. The expression vector of claim 1, wherein the nucleotide sequence encoding lysine decarboxylase CadA is SEQ ID NO: 1.
4. the expression vector of claim, wherein the constitutive promoter is one of J series constitutive promoters.
5. The expression vector of claim 4, wherein the constitutive promoter is J23100, J23109 or J23114.
6. A microorganism producing 1, 5-pentanediamine comprising the expression vector according to any one of claims 1 to 5.
7. The microorganism according to claim 6, which belongs to the genus Escherichia, Klebsiella, Erwinia, Serratia, providencia, Corynebacterium or Brevibacterium.
8. The microorganism according to claim 6, which is a recombinant Escherichia coli (Escherichia coli) K-12W3110 strain.
9. The microorganism according to claim 6, which is Escherichia coli (Escherichia coli) W3110-JcadA deposited at the German Collection of microorganisms and cell cultures (DSMZ) under deposit number DSM 33576.
10. A method of producing 1, 5-pentanediamine, comprising:
mixing a microorganism according to any one of claims 6 to 9 with lysine in solution to convert the lysine to 1, 5-pentanediamine; and
separating 1, 5-pentanediamine from the solution.
11. The method of claim 10, further comprising culturing the microorganism in a high density fermentation process prior to mixing with the lysine in the solution.
12. The method of claim 10, further comprising adding a cofactor to the solution.
13. The method according to claim 12, wherein the cofactor is pyridoxal phosphate.
Technical Field
The present invention relates to a microorganism producing 1, 5-pentanediamine, and more particularly, to a microorganism genetically recombined and a method of producing 1, 5-pentanediamine using the same.
Background
1, 5-pentanediamine is an important monomer for synthesizing polymers such as polyamide (i.e., nylon). The existing approaches for producing 1, 5-pentanediamine by microorganisms can be roughly divided into three types: (1) the mode of the microbial metabolic pathway (in vivo); (2) co-cultivation, i.e. co-cultivation of two microorganisms, with the product of one microorganism acting as substrate for the other; and (3) whole cell catalysis (in vitro), which uses intact biological organisms as catalysts for chemical transformation, i.e., microorganisms are cultured to a certain cell size and then substrates are added for catalytic transformation into products.
Microorganisms that can be used to produce 1, 5-pentanediamine include E.coli, which can express lysine decarboxylase (CatA) and catalyze the conversion of lysine to pentanediamine. The studies on the production of pentanediamine by means of microbial metabolic pathways include: using glucose as carbon source, and after rejecting product metabolic gene, using low copy number carrier p15A containing Tac promoter to express CadA, its maximum yield is only 9.6 g/L1; and the highest yield of CadA expressed by galactose as carbon source and pETDuet as high copy number vector is only 8.8g/L [2 ]. From the two examples mentioned above, it is not possible to obtain high yields of pentanediamine by regulating the metabolic pathways of E.coli, probably because the cells produce both substrate and product at the same time, thus resulting in a slow rate of production of the product.
In 2018, Wang et al, by co-culture, produced substrates and products separately from different strains using different carbon sources (glucose for the former and glycerol for the latter), without competing with each other, and finally reached a yield of 28.5g/L after 50 hours of fermentation [3 ]. The co-cultivation does allow for improved yields, but yields remain low in terms of time efficiency.
The whole-cell catalysis method provides another alternative to increase bacterial load and accumulate enzyme load by high-density fermentation, and then catalyze the conversion of lysine into pentanediamine (1, 5-diamminepentane, DAP; also known as cadaverine). In 2014, the team of Weichao Ma et al expressed CatA and lysine/pentanediamine antiporter (CadB) simultaneously with pETDuet's expression system, and after 16 hours of catalysis with 8g/L cell mass, the highest yield can reach 221g/L [4 ]; in addition, in 2015, the group of Kim et al expressed CatA as pET24m, which had an enzymatic activity of 30.27 mmol/dry cell weight (mg)/min, and a final yield of 142.8g/L [5] after 2 hours of catalysis.
Further, Chinese patent publication No. 105316270 discloses that CadA gene and RBS-containing gene22The CadB gene of (3) was inserted into pET28a (+) vector, and E.coli B strain was used as a host. Chinese patent publication No. 104498519 discloses expression of CadA and CadB with pETDuet as vector, wherein periplasmic secretion signal is fused at 5' end of CadBPeptide (peptide) leader sequence). Furthermore, European patent publication No. 1482055 discloses that CadA is constructed in pUC18 vector and Escherichia coli K-12JM109 strain is used as a host.
However, the existing whole-cell catalytic production method of 1, 5-pentanediamine, including the whole-cell catalytic method, is mostly operated by using an escherichia coli T7 expression system, an expensive inducer, such as isopropyl β -D-thiogalactopyranoside (IPTG), must be added during the culture, the induction time and the required concentration must be precisely controlled, and the method is very disadvantageous for producing whole-cell enzyme in high quantity. In addition, the E.coli host BL21(DE3) strain using the T7 system has poor tolerance to 1, 5-pentanediamine, which is a product produced by itself, and thus limits the production of 1, 5-pentanediamine.
Accordingly, there is a need to provide a method for effectively increasing the productivity of producing 1, 5-pentanediamine, so as to solve the problems of the prior art.
Disclosure of Invention
In order to solve the above problems, the present invention provides an expression vector comprising a nucleotide sequence encoding lysine decarboxylase CadA and a constitutive promoter sequence controlling the expression of the nucleotide sequence.
In one embodiment, the nucleotide sequence encoding the lysine decarboxylase CadA has a sequence identical to SEQ ID NO: 1 has at least 80% similarity to SEQ ID NO: 1, for example, a protein having lysine decarboxylase activity can be expressed. In another embodiment, the lysine decarboxylase CadA has the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to SEQ ID NO: 2 with conservative variation.
In one embodiment, the constitutive promoter is one of the J series constitutive promoters. In another embodiment, the J-series constitutive promoter includes promoters J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23108, J23109, J23110, J23111, J23112, J23113, J23114, J23115, J23116, J23117, J23118 and J23119. In yet another embodiment, the constitutive promoter is J23100, J23109, or J23114.
In one embodiment, the constitutive promoter has a sequence identical to SEQ ID NO: 3 has at least 80% similarity to SEQ ID NO: 3, e.g. a sequence which is also a constitutive promoter.
In one embodiment, the expression vector has a sequence identical to SEQ ID NO: 4 and has at least 80% similarity to SEQ ID NO: 4 sequences with the same activity.
The invention also provides a recombinant microorganism which comprises the expression vector, and the recombinant microorganism can be used for producing the 1, 5-pentanediamine.
In one embodiment, the microorganism is of the genus Escherichia (Escherichia), Klebsiella (Klebsiella), Erwinia (Erwinia), Serratia (Serratia), Providence (Providecia), Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium). In another embodiment, the microorganism is Escherichia coli (Escherichia coli). In yet another embodiment, the microorganism is Escherichia coli K-12W3110 strain.
In one embodiment, the recombinant microorganism provided by the present invention is Escherichia coli (Escherichia coli) W3110-JcadA, deposited at the institute for food industry development of the financial group Farmland institute for food industry (New bamboo food road 331) at 12.19.2019 under the accession number BCRC940690, and deposited at the German Collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, Germany, Nonrorex, D-38124, Indonesia 7B) at 7.15.2020.
The present invention also provides a method for producing 1, 5-pentanediamine, comprising: mixing the microorganism with lysine in a solution to convert the lysine to 1, 5-pentanediamine; and separating the 1, 5-pentanediamine from the solution.
In one embodiment, the method further comprises culturing the above microorganism in a culture medium. In another embodiment, the culturing of the microorganism is performed as a high density fermentation process. In yet another embodiment, the culturing of the microorganism is performed before the microorganism is mixed with lysine in a solution.
In one embodiment, the microorganism has an absorbance (OD) at a wavelength of 600nm600) Mixed with lysine in solution at a concentration of 1 to 6. In another embodiment, the concentration of the lysine in the solution is 1M to 2M. In yet another embodiment, the concentration of the lysine in the solution is 1M, 1.2M, 1.4M, 1.5M, 1.6M, 1.8M, or 2M.
In one embodiment, the pH of the solution is 4 to 8. In another embodiment, the pH of the solution is 4, 4.5, 5, 5.5, 6, 6.5, 6.8, 7, 7.5, or 8.
In one embodiment, the method further comprises adding a cofactor to the solution, wherein the concentration of the cofactor in the solution is 0.01mM to 0.05 mM. In another embodiment, the concentration of the cofactor in the solution is 0.01mM, 0.02mM, 0.03mM, 0.04mM, or 0.05 mM. In yet another embodiment, the cofactor is pyridoxal phosphate (PLP).
The invention uses a non-inducible expression system to express the lysine decarboxylase CadA in a microbial host to be used as a whole-cell catalyst, has the effects of high protein expression quantity, high enzyme activity, slow degradation rate and the like, and can obviously improve the catalytic efficiency of the pentanediamine of the microbe. In addition, when the whole-cell catalyst is used for producing the 1, 5-pentanediamine, no additional inducer is needed to be added, the production cost of the 1, 5-pentanediamine can be reduced, the production procedure is simplified, the yield and the production rate of the pentanediamine are further improved, and the large-scale production of the 1, 5-pentanediamine is realized.
[ biological Material Collection ]
1. The center for storing and researching biological resources of the institute for developing and researching food industry of the financial group legal people is 12 months and 19 months in 2019, and the storage number is BCRC 940690;
2. the German Collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), year 2020, 7 and 15 days, deposit number DSM 33576.
Drawings
FIG. 1A is a schematic diagram of the gene map of constitutively expressed plasmid pSU-J23100-CadA. PJ23100: the J23100 promoter; b0034 RBS: a ribosome binding site; CadA: lysine decarboxylase CadA gene; pUC: a fragment of the replication origin sequence of the pSU plasmid; CmR: a chloramphenicol acetyltransferase (chloramphenicol) gene; HindIII, BglII: restriction enzyme cutting site.
FIG. 1B is a DNA electrophoresis of plasmid pSU-J23100-CadA showing that the plasmid is about 4000bp in length, consistent with that shown in FIG. 1A. Molecular weight markers 3k, 4k and 5k represent 3000, 4000 and 5000 base pairs (bp), respectively.
FIG. 2 shows the tolerance of three E.coli strains to pentanediamine. BL21, W3110, MG 1655: three Escherichia coli strains are BL21, K12W 3110 and MG1655 respectively; "+": adding pentanediamine into the culture medium; "-": no pentamethylene diamine was added to the medium.
FIG. 3 shows the protein expression level of the transfected strain after 12 hours of culture. WT: wild-type W3110; JcadA: the transformant JcadA/W3110. The unit of the molecular weight marker is kDa.
FIG. 4 shows the results of whole cell catalytic production of the transformant JcadA/W3110. 0 hours is the starting point for the addition of 1M substrate (i.e.lysine) and 0.05mM cofactor (PLP). W3110: wild type W3110.
FIG. 5 shows the number of bacteria grown and the activity status of the constitutive strain and the inducible strain within 30 hours, wherein the constitutive strain is JcadA/W3110, and the inducible strain is Escherichia coli T7cadA/BL21(DE 3). 0 h is the initiation point of induction of E.coli BL21(DE3) -T7cadA with IPTG addition. DCW: dry cell weight (dry cell weight).
FIGS. 6A and 6B show the plasmid copy number and protein expression level (indicated by arrows) of the transformant JcadA/W3110 of different generations in 12 hours of culture, respectively. The unit of the molecular weight marker is kDa.
FIGS. 6C and 6D show the plasmid copy number and protein expression level of the transformant JcadA/W3110 cultured in different resistant environments for 12 hours, respectively. PCN: plasmid copy number (plasmid copy number).
FIG. 7 shows the yield and activity status of whole-cell catalysts produced by different culture modes.
FIG. 8 shows the days of cryopreservation of whole cell catalysts at-80 ℃ and their activity status.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and those skilled in the art can easily understand the advantages and effects of the present invention from the description of the present specification. The invention is capable of other and different embodiments and its several details are capable of modification in various other respects, all without departing from the spirit and scope of the present invention. Moreover, all ranges and values herein are inclusive and combinable. Any value or point within the ranges set forth herein, such as any integer, may be treated as the minimum or maximum value to derive a range or the like for the lower rank.
As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
As used in the specification and the appended claims, the term "or" includes "and/or" is used in its sense unless the context clearly dictates otherwise.
The invention provides an expression vector, which comprises a nucleotide sequence for coding lysine decarboxylase CadA and a constitutive promoter sequence for controlling the expression of a nucleic acid molecule of the lysine decarboxylase. The invention also provides a recombinant microorganism containing the expression vector and a method for producing 1, 5-pentanediamine by using the microorganism.
As used herein, the term "lysine decarboxylase" refers to an enzyme involved in a reaction in which an organism produces 1, 5-pentanediamine, and includes two lysine decarboxylases, lysine decarboxylase 1 (cadA) and lysine decarboxylase 2(lysine decarboxylase 2, LdcC), wherein cadA is an inducible enzyme induced by hypoxia, excessive lysine supply and pH change, and LdcC is a constitutive enzyme not affected by an external pH change [6 ].
According to a particular embodiment of the invention, the nucleotide sequence encoding the lysine decarboxylase CadA has a sequence identical to SEQ ID NO: 1 has at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100%) similarity to SEQ ID NO: 1, for example, a protein having lysine decarboxylase activity can be expressed. In another embodiment, the lysine decarboxylase CadA has the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to SEQ ID NO: 2 with conservative variation.
As used herein, the term "percent sequence similarity" refers to the percentage of amino acid or nucleotide residues of a candidate protein or nucleic acid fragment that are identical to the amino acid or nucleotide residues of a reference protein or nucleic acid fragment. In the above alignment, the candidate protein or nucleic acid fragment and the protein or nucleic acid fragment may be aligned and, if necessary, gaps may be introduced to maximize the sequence similarity between the two sequences. Amino acid residues that are conservatively varied are considered different residues when calculating similarity; nucleotide residues of degenerate codons are also considered to be different residues, e.g. between the codons AAU and AAC, which also encode asparagine, a different residue U or C.
It is understood that amino acid or nucleotide sequences of candidate proteins or nucleic acid fragments in which at least a portion of the sequence is modified (e.g., deleted, substituted, or added) as compared to the amino acid or nucleotide sequence of a reference protein or nucleic acid fragment of the present invention are also within the scope of the present invention, so long as the resulting candidate protein or nucleic acid fragment has substantially the same biological activity as the amino acid or nucleotide sequence of the reference protein or nucleic acid fragment due to codon degeneracy. For example, in the nucleotide sequences encoding a CadA of the invention, various modifications can be made in the coding region, provided that it does not alter the activity of the polypeptide expressed from the coding region. Thus, the nucleotide sequence of the CadA encoded by the invention can be a nucleotide sequence having the sequence of SEQ ID NO: 1 or a nucleotide sequence identical to SEQ ID NO: 1, provided that the protein encoded by the nucleotide sequence is capable of exhibiting CadA activity. Similarly, a CadA of the invention can be a polypeptide having the sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to SEQ ID NO: 2, provided that the protein is capable of exhibiting substantially the activity of CadA.
As used herein, the term "constitutive promoter" refers to a promoter that maintains sustained activity in most or all tissues, relative to an inducible promoter, which must be controlled by an external signal or inducer, and which can sustainably express a particular gene.
Constitutive promoters suitable for use in the present invention include those belonging to the J series of constitutive 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 one embodiment, the constitutive promoter used in the present invention may be J23100, J23109 or J23114. In another embodiment, the constitutive promoter has the sequence of SEQ ID NO: 3 or a sequence identical to SEQ ID NO: 3 is at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100%) similar to SEQ ID NO: 3, e.g. a sequence which is also a constitutive promoter.
According to a specific embodiment of the present invention, the expression vector of the present invention further comprises at least one selected from the group consisting of: marker gene sequences, reporter gene sequences, antibiotic resistance gene sequences, restriction enzyme cleavage site sequences, polyadenylation site sequences, enhancer sequences, terminator sequences and regulator sequences. In another embodiment, the expression vector has the sequence of SEQ ID NO: 4 or a sequence identical to SEQ ID NO: 4 is at least 80% (e.g., at least 82%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100%) similar to SEQ ID NO: 4 sequences with the same activity.
As used herein, the term "recombinant" refers to the artificial combination of two sequence segments that are separated from each other. In general, the term "recombinant" refers to a nucleic acid, protein, or microorganism that contains, or is encoded by, genetic material derived from a plurality of different sources, such as from two or more different strains or species of organisms.
As used herein, the term "microorganism" belongs to a microscopic organism, including bacteria, archaea, viruses, or fungi, and the like. As used herein, a reference to "a microorganism" is understood to encompass "a bacterium.
Microbial hosts suitable for use as expression vectors in the present invention include, but are not limited to, microorganisms belonging to the genera Escherichia (Escherichia), Klebsiella (Klebsiella), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providecia), Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium). In one embodiment, the microbial host used in the present invention is capable of expressing the lysine decarboxylase CadA in vivo. In another embodiment, the microbial host used in the present invention is tolerant to pentanediamine in addition to being capable of expressing the lysine decarboxylase CadA in vivo.
According to a specific embodiment of the present invention, the method for producing 1, 5-pentanediamine of the present invention comprises culturing the above-described microbial host in a culture medium under conditions sufficient to produce a lysine decarboxylase CadA. In one embodiment, the medium may be LB medium. In another embodiment, the method comprises culturing the microorganism in a high density fermentation process.
According to a specific embodiment of the present invention, the method further comprises adjusting the OD of the cultured microorganism600After a concentration of 1 to 6, the microorganism is mixed with lysine in solution.
According to an embodiment of the invention, the concentration of lysine in the solution is between 1M and 2M. According to another embodiment of the invention, the pH of the solution may be 4 to 8.
According to an embodiment of the invention, the solution may further comprise a cofactor at a concentration of 0.01mM to 0.05 mM.
As used herein, the term "cofactor" comprises non-protein compounds required for having normal enzymatic activity. Such compounds may be organic or inorganic, for example, cofactors suitable for use in the present invention include, but are not limited to, pyridoxal phosphate (PLP).
The following examples further illustrate the features and effects of the present invention, but are not intended to limit the scope of the invention.
Examples
Chemical preparation:
sodium chloride was purchased from Sigma Aldrich Co. Yeast extracts were purchased from Oxoid (taiwan, china). Trypsin was purchased from Cyrusbioscience (Taiwan, China). Agar (agar) was purchased from BD Difco dehydration medium (France). Agarose (agarose) was purchased from GeneDireX (taiwan). Pyridoxal phosphate (PLP), diethyl ethoxymethylenemalonate (deem), and sodium acetate were purchased from Sigma Aldrich Co. L-lysine hydrochloride was purchased from Cyrusbioscience (Taiwan, China). D (+) -glucose was purchased from Comieco (Italian). Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Showa Chemical Industry Co. (Japan). Acetonitrile for HPLC analysis was purchased from Spectrum Chemical Manufacturing Corp. PCR reagents Ex-Tag were purchased from Takara Bio Inc. (USA). Restriction enzymes were purchased from New England Biolabs (USA). T4 DNA ligase was purchased from Leadgene co., ltd. (korea). Primers were synthesized by Integrated DNA Technologies (USA).
Example 1: construction of recombinant expression vectors
Primers HindIII-CadA-F (5'-GCA AGC TTA TGA ACG TTA TTG CAATAT TGA ATC AC-3' (SEQ ID NO: 5)) and BglII-CadA-R (5'-GCA GAT CTT CAT TTT TTG CTT TCT TCT TTC AAT ACC TTA ACG GTA TAG CGG CC-3' (SEQ ID NO: 6)) were designed using the genome of E.coli K-12MG1655 as a template, and Polymerase Chain Reaction (PCR) was performed.
The PCR reaction is used for amplifying a specific DNA sequence segment, and the required materials comprise a DNA template, a 5 'end primer, a 3' end primer, deoxynucleotide triphosphate (dNTP), 10x polymerase buffer solution and polymerase, wherein the polymerase used in the embodiment comprises Ex-Taq. And analyzing the PCR product by DNA electrophoresis and tapping and recovering to obtain an amplified lysine decarboxylase CadA fragment.
The amplified CadA fragment was digested with HindIII and BglII, and inserted into pSU-J23100 vector to construct pSU-J23100-CadA plasmid. As shown in FIG. 1A, the plasmid contains a constitutive promoter J23100 (Access: LP934757), a ribosome binding site B0034RBS (Biobrick No. BBa B0034), and a CadA gene expressed under the control of the promoter J23100.
For transformation, a tube of commercially available DH5a competent cells was added to pSU-J23100-CadA plasmid at about 10. mu.L, followed by 30 minutes on ice, followed by heating in a 42 ℃ water bath for 1.5 minutes, followed by 5 to 10 minutes on ice, followed by 400. mu.L of LB liquid medium or SOC (super optimal broth with lipids repression) medium, shaking in a 37 ℃ incubator for about 60 to 90 minutes, followed by 3 minutes of centrifugation at 4000rpm, removing 300. mu.L of supernatant, plating the total number of competent cells containing recombinant DNA on antibiotic-containing solid medium, and incubating overnight at 37 ℃.
Several colonies formed on the solid medium were inoculated into a liquid tube medium containing an antibiotic and cultured overnight in an incubator at 37 ℃. The next day, 2mL of the suspension from the preculture was taken for plasmid extraction and verified by restriction enzyme cleavage. The total length of pSU-J23100-CadA plasmid was 4267bp, which is consistent with the result of DNA electrophoresis shown in FIG. 1B (i.e., the fragment shown by the arrow), and the CadA gene was successfully constructed in the pSU vector.
Example 2: expression host assay
To select a microorganism with better tolerance to pentanediamine as an expression host, three common strains of E.coli were selected: BL21, K-12W3110 and MG 1655.
First, the three strains were individually cultured for 2 hours, and then 0.2M pentamethylenediamine was added to the culture solution of the three strains. As shown in FIG. 2, W3110 and MG1655 grew faster than BL21 without addition of pentamethylenediamine; after the addition of the pentanediamine, the growth rates of the three strains are delayed and the bacterial loads are reduced, but the growth rate of the W3110 strain is fastest and the reduction amplitude is minimum in the period of 4 to 12 hours, so that the strain has higher pentanediamine tolerance, and the Escherichia coli K-12W3110 strain is selected as an expression host.
Example 3: preparation of recombinant microorganisms
Plasmid pSU-J23100-CadA plasmids prepared in example 1 and stored in the selection host E.coli DH5a were extracted and transfected into E.coli K-12W3110 strain, and the protein expression level was analyzed after 12 hours of culture and as a control wild type W3110.
As shown in FIG. 3, the transfected JCadA (also referred to herein as JCadA/W3110 or W3110-JCadA) exhibited higher CadA expression levels (i.e., the fragment indicated by the arrow) compared to wild-type W3110.
The E.coli transformant W3110-JCadA has been deposited under the Budapest treaty at the Germany Collection of microorganisms and cell cultures (Germany, Delelix, D-38124, Inhoffentr.7B, D-38124 Braunschweig, Germany) on day 7, month 15 in 2020 and has been obtained under the accession number DSM 33576 from the International depository DSMZ. This biological material has been subjected to and passed viability tests.
Example 4: activity assay of recombinant microorganisms
After the transformant JcadA/W3110 of example 3 was cultured, it was centrifuged at 10,000rpm for 10 minutes, and the cell pellet (pellet) was suspended in deionized water, and the absorbance (OD) of the cell pellet at 600nm was adjusted600) To 6 (OD)6006), which was then added to a solution containing 1M lysine as a substrate, and 0.05mM pyridoxal phosphate (PLP) as a cofactor, and placed in an incubator for reaction with shaking (35 ℃, 200 rpm). During the reaction, the content of lysine remaining in the solution and the content of pentamethylenediamine produced were confirmed.
As shown in FIG. 4, when the transformant JcadA/W3110 was used as a whole-cell catalyst, the content of lysine decreased and the content of pentanediamine increased with the increase of the catalytic reaction time, which indicates that the transformant JcadA/W3110 catalyzes the conversion of lysine into pentanediamine; in contrast to the wild type W3110, the lysine residue was only slightly reduced, and no pentamethylenediamine was produced. It can be seen that, using a constitutive expression system as a whole cell catalyst, the activity of CadA was indeed more enhanced than that of wild-type W3110, and 1, 5-pentanediamine could be produced rapidly by in vitro means.
Example 5: comparison of yield and Activity of inducible and constitutive microbial systems
To compare the CadA activities of the strains of the constitutive expression system and the inducible expression system, JcadA/W3110 (constitutive strain) and T7cadA/BL21(DE3) (inducible strain) were cultured in a 5L fermentor (FB-6S, FIRSTEK, Taiwan) under the same conditions as LB medium, and the number of bacterial growth and activity status within 33 hours of culture were recorded. The conditions of the fermentation tank are as follows: dissolved Oxygen (DO) 10-30%, air flow rate 1.5vvm, pH 6.8, 32 ℃, 100rpm, and IPTG inducer (added at 0.00167g/L) was additionally added to the culture medium of T7cadA/BL21(DE 3). The number of bacteria was measured by OD at 600nm in a spectrophotometer, and the activity of lysine decarboxylase CadA was measured by BP assay.
The procedure for the BP assay is briefly as follows:
the pellet was first quantified as OD600The sample was then subjected to high pressure disruption to obtain a soluble protein sample containing lysine decarboxylase. The amount of pentanediamine produced via the lysine decarboxylase catalyzed reaction is increased. The BP color former can be detected at the wavelength of 595 nm. The lysine decarboxylase activity was measured by the difference of 595nm in wavelength (. DELTA.OD) using the reaction conditions listed in Table 1 below595) The enzyme activity was calculated by converting the assay curve of pentamethylenediamine quantified by HPLC.
TABLE 1 reaction conditions of the lysine decarboxylase Activity determination method
Article item
Concentration of
Lysine
40mM
Pyridoxal phosphate
0.2mM
BP color former
1/20
Sample to be tested
1/20
Sodium acetate buffer, pH 6
Total volume
500μL
As shown in FIG. 5, JcadA/W3110 produced CadA without addition of an inducer during the culture, and therefore had a biomass higher than that of the inducible strain at the same time, and the CadA activity was measured by the BP assay, showing that JcadA/W3110 still retained 150g of CadA after 30 hours of fermentationDAP/gDCWThe activity of CadA (i.e.the activity ratio) above/. sup.h was even 2-fold higher than that of the inducible strain.
Example 6: plastid stability testing
In order to test the plastid stability, the transformant JcadA/W3110 is subjected to subculture, wherein the subculture mode is that a bacterial bank at the temperature of-80 ℃ is inoculated into a culture solution for culturing, and after the culture is carried out for 12 hours, the subculture strain is diluted and coated on a plate culture medium for activation and is used as the 0 th generation; then, selecting a single colony from the activated plate culture medium, inoculating the single colony into a culture solution for culture, diluting and coating the single colony on the plate culture medium after culturing for 12 hours, and taking the single colony as a 1 st generation; thereafter, a single colony was selected from the 0 th generation of plate medium every 7 days and inoculated in the culture solution for 1 month.
The cells cultured for 12 hours were washed twice with sterilized water, concentrated to a certain OD, and then lysed by heating in a 100 ℃ water bath for 10 minutes, followed by centrifugation at the highest rotation speed for 10 minutes to separate the cell pellet from the material lysed in the cells. Plasmid copy number determination was performed using qpcr (quantitative pcr).
FIG. 6A shows that colonies activated for 2 weeks can reach maximum copy number; after 2 weeks, the copy number showed a decreasing trend; after 1 month of activation, the copy number remained slightly higher than the original copy number. In addition, figure 6B and table 2 shows that the activation of 2 weeks after protein expression level has a tendency to decrease, the results show that, although the plasmid copy number is reduced, but still can maintain stable expression.
TABLE 2 quantitative analysis of protein expression
Time (sky)
Relative amount of
0
1.00
7
0.32
14
0.43
28
0.77
In addition, the transformant JcadA/W3110 was cultured in medium supplemented with antibiotics and tested for the ability of this plasmid to maintain its stability at various resistance concentrations. The mode of resistance culture is as follows: the cells were inoculated in a culture medium from a-80 ℃ cell bank for culture, and used as preculture, 1% preculture cell suspension was inoculated into a 4mL cell culture tube, chloramphenicol (chloramphenicol) was added in different concentrations (0, 5, 10, 25ppm) in order, and after 12 hours of culture in a 37 ℃ incubator, 1 to 2mL of cell suspension was collected and analyzed.
As shown in FIGS. 6C and 6D and Table 3 below, the plasmid copy number of the strains grown at different chloramphenicol concentrations was maintained at a constant value, and the protein expression was consistent, even in an environment without resistance, indicating that the plasmid was stably present in JcadA/W3110.
TABLE 3 quantitative analysis of protein expression
Example 7: comparison of yields in scale-up production:
in this example, the whole cell catalyst JcadA/W3110 was produced under three culture strategy conditions of Erlenmeyer flask, fermenter and high density fermenter, and the amount and activity of the catalyst was measured. The number of bacteria was measured by OD at a wavelength of 600nm in a spectrophotometer, and the lysine decarboxylase activity was measured by BP assay. The composition of the medium and the culture conditions are shown in tables 4 and 5 below, respectively:
TABLE 4 composition of test media
TABLE 5 culture conditions
FIG. 7 shows the yield and activity status of whole-cell catalysts produced by the three culture modes, and the quantitative data thereof are further shown in Table 6 below. From these results, it was found that the culture in a high-density fermenter can increase the activity per unit cell amount as well as the cell amount as compared with the flask and the fermenter. More specifically, the bacterial quantity can be greatly increased by utilizing a high-density fermentation method, and lysine decarboxylase with 30 g/L and the activity of 170U/mg/h is generated and can be used for preparing the 1, 5-pentanediamine whole-cell catalyst with high lysine decarboxylase activity.
TABLE 6 Whole cell catalyst yields from different culture regimes
Example 8: cryopreservation activity degradation rate test
The whole cell catalyst JcadA/W3110 produced in the fermenter was stored at-80 ℃ in a frozen state, taken out on the 20 th and 130 th days of storage, cultured, and tested for its activity state. When thawing, the pellet was subjected to OD quantitative disruption, and its activity was measured by BP assay.
FIG. 8 shows that JcadA/W3110 still had the same CadA activity after 20 days of cryopreservation as when it was not frozen, and still had about half of the enzyme activity after 130 days of cryopreservation, with the remaining activity remaining at 95% of that of the inducible strain. From this, it was found that the lysine decarboxylase expressed in W3110 by the non-inducible expression system indeed had the effects of high protein expression, high enzyme activity, and slow degradation rate.
The above embodiments are merely illustrative, and not restrictive, of the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention is defined by the appended claims, and is intended to be covered by the technical disclosure unless it does not affect the effect and the practical purpose of the present invention.
Reference to the literature
[1]Z.G.Qian,X.X.Xia,S.Y.Lee(2011)Metabolic engineering of Escherichia coli for the production of cadaverine:a five carbon diamine.Biotechnology and Bioengineering,108(1),93-103.
[2]D.H.Kwak,H.G.Lim,J.Yang,S.W.Seo,G.Y.Jung(2017)Synthetic redesign of Escherichia coli for cadaverine production from galactose.Biotechnology for Biofuels,10(1),20.
[3]J.Wang,X.Lu,H.Ying,W.Ma,S.Xu,X.Wang,K.Chen,P.Ouyang(2018)A novel process for cadaverine bio-production using a consortium of two engineered Escherichia coli.Frontiers in Microbiology,9,1312.
[4]W.C.Ma,W.J.Cao,H.Zhang,K.Q.Chen,Y.Li,P.K.Ouyang(2015)Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB.Biotechnol.Lett.,37,799-806.
[5]H.J.Kim,Y.H.Kim,J.H.Shin,S.K.Bhatia,G.Sathiyanarayanan,H.M.Seo,K.Y.Choi,Y.H.Yang,K.Park(2015)Optimization of direct lysine decarboxylase biotransformation for cadaverine production with whole-cell biocatalysts at high lysine concentration,J.Microbiol.Biotechnol.,25,1108-1113.
[6]W.C.Ma,K.Q.Chen,Y.Li,N.Hao,X.Wang,P.K.Ouyang(2017)Advances in cadaverine bacterial production and its applications,Engineering,3,308-317.
Sequence listing
<110> China petrochemical Industrial development Ltd
<120> recombinant microorganism and method for producing 1, 5-pentanediamine
<130> 115196
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1932
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 1
atgaacgaga acctgccgtt gtacgcgttc gctaatacgt attccactct cgatgtaagc 60
ctgaatgacc tgcgtttaca gattagcttc tttgaatatg cgctgggtgc tgctgaagat 120
attgctaata agatcaagca gaccactgac gaatatatca acactattct gcctccgctg 180
actaaagcac tgtttaaata tgttcgtgaa ggtaaatata ctttctgtac tcctggtcac 240
atgggcggta ctgcattcca gaaaagcccg gtaggtagcc tgttctatga tttctttggt 300
ccgaatacca tgaaatctga tatttccatt tcagtatctg aactgggttc tctgctggat 360
cacagtggtc cacacaaaga agcagaacag tatatcgctc gcgtctttaa cgcagaccgc 420
agctacatgg tgaccaacgg tacttccact gcgaacaaaa ttgttggtat gtactctgct 480
ccagcaggcg gcaccattct gattgaccgt aactgccaca aatcgctgac ccacctgatg 540
atgatgagcg atgttacgcc aatctatttc cgcccgaccc gtaacgctta cggtattctt 600
ggtggtatcc cacagagtga attccagcac gctaccattg ctaagcgcgt gaaagaaaca 660
ccaaacgcaa cctggccggt acatgctgta attaccaact ctacctatga tggtctgctg 720
tacaacaccg acttcatcaa gaaaacactg gatgtgaaat ccatccactt tgactccgcg 780
tgggtgcctt acaccaactt ctcaccgatt tacgaaggta aatgcggtat gagcggtggc 840
cgtgtagaag ggaaagtgat ttacgaaacc cagtccactc acaaactgct ggcggcgttc 900
tctcaggctt ccatgatcca cgttaaaggt gacgtaaacg aagaaacctt taacgaagcc 960
tacatgatgc acaccaccac ttctccgcac tacggtatcg tggcgtccac tgaaaccgct 1020
gcggcgatga tgaaaggcaa tgcaggtaag cgtctgatta acggttctat tgaacgtgcg 1080
atcaaattcc gtaaagagat caaacgtctg agaacggaat ctgatggctg gttctttgat 1140
gtatggcagc cggatcatat cgatacgact gaatgctggc cgctgcgtcc tgacagcacc 1200
tggcacggct tcaaaaacat cgataacgag cacatgtatc ttgacccgat caaagtcacc 1260
ctgctgactc cggggatgga aaaagacggc accatgagcg actttggtat tccggccagc 1320
atcgtggcga aatacctcga cgaacatggc atcgttgttg agaaaaccgg tccgtataac 1380
ctgctgttcc tgttcagcat cggtatcgat aagaccaaag cactgagcct gctgcgtgct 1440
ctgactgact ttaaacgtgc gttcgacctg aacctgcgtg tgaaaaacat gctgccgtct 1500
ctgtatcgtg aagatcctga attctatgaa aacatgcgta ttcaggaact ggctcagaat 1560
atccacaaac tgattgttca ccacaatctg ccggatctga tgtatcgcgc atttgaagtg 1620
ctgccgacga tggtaatgac tccgtatgct gcattccaga aagagctgca cggtatgacc 1680
ggagaagttt acctcgacga aatggtaggt cgtattaacg ccaatatgat ccttccgtac 1740
ccgccgggag ttcctctggt aatgccgggt gaaatgatca ccgaagaaag ccgtccggtt 1800
ctggagttcc tgcagatgct gtgtgaaatc ggcgctcact atccgggctt tgaaaccgat 1860
attcacggtg cataccgtca ggctgatggc cgctataccg ttaaggtatt gaaagaagaa 1920
agcaaaaaat ga 1932
<210> 2
<211> 715
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 2
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> 3
<211> 35
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<223> promoter J23100
<400> 3
ttgacggcta gctcagtcct aggtacagtg ctagc 35
<210> 4
<211> 4239
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<223> pSU-J23100-CadA
<400> 4
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 1200
aaggtgacgt aaacgaagaa acctttaacg aagcctacat gatgcacacc accacttctc 1260
cgcactacgg tatcgtggcg tccactgaaa ccgctgcggc gatgatgaaa ggcaatgcag 1320
gtaagcgtct gattaacggt tctattgaac gtgcgatcaa attccgtaaa gagatcaaac 1380
gtctgagaac ggaatctgat ggctggttct ttgatgtatg gcagccggat catatcgata 1440
cgactgaatg ctggccgctg cgtcctgaca gcacctggca cggcttcaaa aacatcgata 1500
acgagcacat gtatcttgac ccgatcaaag tcaccctgct gactccgggg atggaaaaag 1560
acggcaccat gagcgacttt ggtattccgg ccagcatcgt ggcgaaatac ctcgacgaac 1620
atggcatcgt tgttgagaaa accggtccgt ataacctgct gttcctgttc agcatcggta 1680
tcgataagac caaagcactg agcctgctgc gtgctctgac tgactttaaa cgtgcgttcg 1740
acctgaacct gcgtgtgaaa aacatgctgc cgtctctgta tcgtgaagat cctgaattct 1800
atgaaaacat gcgtattcag gaactggctc agaatatcca caaactgatt gttcaccaca 1860
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 tctcattaat 2220
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 2280
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 2340
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 2400
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 2460
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 2520
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 2580
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 2640
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 2700
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 2760
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 2820
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 2880
ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 2940
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3000
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3060
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3120
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3180
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3240
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 3300
tacgggaggg cttaccatct gtcgacaaat tacgccccgc cctgccactc atcgcagtac 3360
tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc 3420
tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa 3480
acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc 3540
cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg 3600
ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg 3660
tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa 3720
gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg aaattccgga 3780
tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt 3840
ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat 3900
tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg 3960
gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat 4020
aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt 4080
acgtgcccga tcaactcgag tgccacctga cgtctaagaa accattatta tcatgacatt 4140
aacctataaa aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt 4200
cgctaaggat gatttctgga attcgcggcc gcttctaga 4239
<210> 5
<211> 35
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<223> HindIII-CadA-F
<400> 5
gcaagcttat gaacgttatt gcaatattga atcac 35
<210> 6
<211> 53
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<223> BglII-CadA-R
<400> 6
gcagatcttc attttttgct ttcttctttc aataccttaa cggtatagcg gcc 53