Expression of polypeptides involved in lysine decarboxylation and methods and applications thereof

Abstract

The expression plasmid vector comprises a polynucleotide sequence encoding an Ldc2 polypeptide, a fragment thereof and / or a mutant thereof. The backbone plasmid is capable of autonomous replication in a host cell. The host cell is not a Pseudomonas aeruginosa cell. The transformant is transformed with the expression plasmid vector. The transformant is not Pseudomonas aeruginosa. The mutant host cell comprises a polynucleotide sequence encoding an Ldc2 polypeptide, a fragment thereof and / or a mutant that has been integrated into the host cell chromosome. The polypeptide, fragment and / or mutant comprises Ldc2. The non-naturally occurring polynucleotide and / or mutant encodes a polypeptide comprising Ldc2. The transformant is used to prepare bio-based cadaverine and the bio-based cadaverine prepared by the method. Polyamides and compositions formed using bio-based cadaverine.

Description

Background Art

[0001] Cadaverine is a platform compound involved in the production of a variety of products. Cadaverine can be synthesized by decarboxylation of lysine in microorganisms. Lysine decarboxylase is an enzyme that catalyzes the production of cadaverine by removing the carboxyl group from lysine. For example, in Escherichia coli (E. coli), cadaverine is biosynthesized directly from L-lysine by two lysine decarboxylase polypeptides, CadA and LdcC.

[0002] At present, the biosynthesis of cadaverine is carried out using two strategies: fermentation production or in vitro enzyme catalysis. In the fermentation production path of L-lysine, lysine decarboxylase, usually CadA or LdcC, is added to lysine-producing bacterial strains (e.g., Corynebacterium glutamicum and Escherichia coli) to extend the lysine biosynthetic pathway to the cadaverine biosynthetic pathway. Alternatively, for in vitro enzyme catalysis, bacteria can be engineered or induced to produce lysine decarboxylase, usually CadA or LdcC, which can then be used to convert lysine into cadaverine by decarboxylation.

[0003] However, the production of cadaverine is currently limited and results in low yields.Therefore, there is a need for methods of producing cadaverine in higher yields. SUMMARY OF THE INVENTION

[0005] One aspect of the present invention relates to a polypeptide comprising one or more mutants of Pseudomonas aeruginosa (P. aeruginosa) polypeptide Ldc2, consisting of one or more mutants of Pseudomonas aeruginosa (P. aeruginosa) polypeptide Ldc2, or consisting essentially of one or more mutants of Pseudomonas aeruginosa (P. aeruginosa) polypeptide Ldc2. As used herein, Pseudomonas aeruginosa polypeptide Ldc2 is referred to as "Pseudomonas aeruginosa Ldc2", "Ldc2" or "Ldc2 polypeptide", and has an amino acid sequence of SEQ ID NO: 4.

[0006] Another aspect of the present invention relates to a first polynucleotide encoding one or more first polypeptides, the first polypeptide comprising, consisting of or consisting essentially of one or more second polypeptides, the second polypeptides being selected from Ldc2, Ldc2 fragments and Ldc2 mutants, wherein the first polynucleotide comprises one or more second polynucleotides encoding one or more second polypeptides, respectively; when there are multiple first polypeptides, each first polypeptide may be the same or different; when there are multiple second polypeptides, each second polypeptide may be the same or different; when there are multiple second polynucleotides, each second polynucleotide may be the same or different; the one or more first polypeptides may be expressed alone or as a fusion protein; and when the second polypeptide is Ldc2, at least one corresponding second polynucleotide encoding the second polypeptide comprises, consists of or consists essentially of a mutant Pseudomonas aeruginosa ldc2 gene. As used herein, the Pseudomonas aeruginosa ldc2 gene is referred to as "Pseudomonas aeruginosa ldc2" or "ldc2" and has a polynucleotide sequence of SEQ ID NO: 3. An example of an ldc2 mutant encodes Ldc2, and may be a codon-optimized ldc2 (eg, ldc2-co1 (SEQ ID NO: 17)).

[0007] Another aspect of the present invention relates to a third polynucleotide encoding one or more third polypeptides, the third polypeptide comprising, consisting of or consisting essentially of one or more fourth polypeptides, the fourth polypeptide being selected from Ldc2 (SEQ ID NO: 4), a fragment of Ldc2 and a mutant of Ldc2; wherein the third polynucleotide comprises one or more fourth polynucleotides encoding one or more fourth polypeptides, respectively; when there are multiple third polypeptides, each third polypeptide may be the same or different; when there are multiple fourth polypeptides, each fourth polypeptide may be the same or different; when there are multiple fourth polynucleotides, each fourth polynucleotide may be the same or different; and the one or more third polypeptides may be expressed alone or as a fusion protein. In certain embodiments, the polynucleotide comprises, consists of or consists essentially of ldc2.

[0008] Another aspect of the present invention relates to a first expression plasmid vector, comprising, consisting of, or consisting essentially of a fifth polynucleotide encoding one or more fifth polypeptides, the fifth polypeptide comprising, consisting of, or consisting essentially of one or more sixth polypeptides, the sixth polypeptide being selected from Ldc2, a fragment of Ldc2, and a mutant of Ldc2; and a backbone plasmid capable of autonomous replication in a host cell. In certain embodiments, the host cell is a Pseudomonas aeruginosa cell.

[0009] Another aspect of the present invention relates to a second expression plasmid vector, comprising, consisting of, or substantially consisting of a seventh polynucleotide encoding one or more seventh polypeptides, the seventh polypeptide comprising, consisting of, or substantially consisting of one or more eighth polypeptides, the eighth polypeptide being selected from Ldc2, a fragment of Ldc2, and a mutant of Ldc2; and a backbone plasmid capable of autonomous replication in a host cell. In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell.

[0010] Another aspect of the present invention relates to a first transformant comprising the first expression plasmid vector in a host cell. In certain embodiments, the host cell is a Pseudomonas aeruginosa cell.

[0011] Another aspect of the present invention relates to a second transformant comprising the second expression plasmid vector in a host cell. In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell.

[0012] Another aspect of the present invention relates to a first mutant host cell comprising the first or third polynucleotide as disclosed herein, wherein the first or third polynucleotide has been integrated into the chromosome of the host cell. In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell. In certain embodiments, the host cell is a Pseudomonas aeruginosa cell.

[0013] Another aspect of the present invention relates to a method for preparing one or more ninth polypeptides, wherein the ninth polypeptide is selected from Ldc2, a fragment of Ldc2 and a mutant of Ldc2, the method comprising obtaining a first transformant, a second transformant or a first mutant host cell as described herein, culturing the first transformant, the second transformant or the first mutant host cell under conditions effective for expressing the one or more ninth polypeptides, and harvesting the one or more ninth polypeptides.

[0014] Another aspect of the present invention relates to a method for preparing cadaverine, the method comprising culturing the first transformant, the second transformant or the first mutant host cell as described herein, preparing cadaverine by decarboxylating lysine using a culture obtained from culturing the first transformant, the second transformant or the first mutant host cell, and extracting and purifying cadaverine.

[0015] Another aspect of the present invention relates to polyamides prepared from bio-based cadaverine prepared as disclosed herein and 1,5-diisocyanatopentane, and compositions and methods of preparation thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Protein tree generated from a sequence similarity search of the E. coli protein CadA (highlighted) using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST).

[0017] Figure 2 : Protein tree generated from a sequence similarity search of the E. coli protein LdcC (highlighted) using BLAST.

[0018] Figure 3 : Polymerase chain reaction (PCR) primer sequences used to construct a recombinant expression plasmid vector containing a Pseudomonas aeruginosa gene (hereinafter "ldc1") having a sequence of SEQ ID NO: 1 or ldc2 according to an embodiment of the present invention, and primer sequences (psyn-1 and psyn-2) used to construct a promoter sequence of SEQ ID NO: 5.

[0019] Figure 4 : Recombinant expression plasmid vector maps used to express the Pseudomonas aeruginosa protein having the sequence of SEQ ID NO: 2 (hereinafter "Ldc1") and the Ldc2 protein according to an embodiment of the present invention. A) Vector map of pCIB10; B) Vector map of pCIB45; C) Vector map of pCIB46; D) Vector map of pCIB47; and E) Vector map of pCIB48.

[0020] Figure 5 : Sequence alignment of a portion of lysine decarboxylase polypeptides from different species, including Escherichia coli LdcC, Shigella sonnei CadA, Salmonella enterica lysine decarboxylase, Escherichia coli CadA and Pseudomonas aeruginosa Ldc2, with a conserved serine (boxed region).

[0021] Figure 6: Sequence alignment of a portion of lysine decarboxylase polypeptides from different species, including Escherichia coli LdcC, Shigella sonnei CadA, Salmonella enterica lysine decarboxylase, Escherichia coli CadA, and Pseudomonas aeruginosa Ldc2, with conserved asparagine and serine (boxed region). DETAILED DESCRIPTION OF THE INVENTION

[0023] The following description provides specific details for fully understanding and implementing the embodiments of the present disclosure. However, those skilled in the art will appreciate that the present disclosure can be implemented without these details. In other cases, well-known structures and functions are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the present disclosure.

[0024] Pseudomonas aeruginosa Ldc2 (Accession: WP_014603046.1) was characterized as a β-elimination lyase polypeptide in a BLAST search. Pseudomonas aeruginosa Ldc2 has low sequence similarity to the known E. coli lysine decarboxylases CadA and LdcC (39.44% and 38.71% sequence identity, respectively). The BLAST search results did not indicate that Ldc2 is a lysine decarboxylase.

[0025] Functional heterologous expression of Pseudomonas proteins in E. coli is challenging, in part due to low gene expression and insoluble proteins (West, 1988). For example, heterologous expression of the previously characterized Pseudomonas aeruginosa lysine decarboxylase Ldc1 (SEQ ID NO: 2, accession: EME94559.1) in E. coli did not result in increased cadaverine production, although its BLAST results showed that it was a putative member of the lysine decarboxylase family (the corresponding Pseudomonas aeruginosa gene is ldc1 (SEQ ID NO: 1)).

[0026] As disclosed herein, it was unexpectedly found that heterologous expression of Pseudomonas aeruginosa Ldc2 resulted in unexpectedly high yields of cadaverine production, with E. coli expressing Ldc2 exhibiting higher cadaverine production than E. coli expressing the E. coli lysine decarboxylase CadA (see, e.g., Example 5). Expression of Pseudomonas aeruginosa Ldc2 in other host cells (e.g., H. alvei) also resulted in unexpectedly higher yields of cadaverine production compared to E. coli CadA expression (see, e.g., Example 5). In addition, it was unexpectedly found that expression of the Ldc2 mutant polypeptides disclosed herein resulted in high cadaverine production.

[0027] One aspect of the present invention relates to a polypeptide comprising, consisting of or consisting essentially of one or more mutants of Ldc2. The mutant of Ldc2 may include deletion, substitution, addition and / or insertion of one or more amino acids of the amino acid sequence of SEQ ID NO: 4, while the mutant of Ldc2 provides substantially the same function as Ldc2 (i.e., the mutant of Ldc2 has about 80% or more lysine decarboxylase activity compared to Ldc2; about 90% or more lysine decarboxylase activity compared to Ldc2; about 95% or more lysine decarboxylase activity compared to Ldc2; about 97% or more lysine decarboxylase activity compared to Ldc2; about 99% or more lysine decarboxylase activity compared to Ldc2; or about 100% or more lysine decarboxylase activity compared to Ldc2).

[0028] Examples of Ldc2 mutants include, but are not limited to, SEQ ID NO: 6 (Ldc2S111C), SEQ ID NO: 11 (Ldc2N262T), SEQ ID NO: 12 (Ldc2K265N), SEQ ID NO: 13 (Ldc2S111C / N262T), SEQ ID NO: 14 (Ldc2S111C / K265N), SEQ ID NO: 15 (Ldc2N262T / K265N), SEQ ID NO: 16 (Ldc2S111C / N262T), SEQ ID NO: 17 (Ldc2S111C / K265N), SEQ ID NO: 18 (Ldc2S111C / N262T), SEQ ID NO: 19 (Ldc2S111C / K265N), SEQ ID NO: 20 (Ldc2S111C / N262T), SEQ ID NO: 21 (Ldc2S111C / K265N), SEQ ID NO: NO:16 (Ldc2S111C / N262T / K265N), homologous polypeptides of Ldc2, homologous polypeptides of Ldc2S111C (e.g., Ldc2S111X), homologous polypeptides of Ldc2N262T (e.g., Ldc2N262X'), homologous polypeptides of Ldc2K265N (e.g., Ldc2K265X'), homologous polypeptides of Ldc2S111C / N262T (e.g., Ldc2S111X / N262X'), Homologous polypeptides of K265N (e.g., Ldc2S111X / K265X"), homologous polypeptides of Ldc2N262T / K265N (e.g., Ldc2N262X' / K265X"), and homologous polypeptides of Ldc2S111C / N262T / K265N (e.g., Ldc2S111X / N262X' / K265X"). X is any amino acid that is not serine, X' is any amino acid that is not asparagine, and X" is any amino acid that is not lysine. As used herein, homologous polypeptides are at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% homologous to the polypeptide. When the Ldc2 mutant has multiple mutations, each mutation can be the same or different.

[0029] As used herein, a polypeptide comprising a specific polypeptide sequence may include fragments and / or mutants of the specific polypeptide sequence while still providing substantially the same function as the intact original unmutated specific polypeptide sequence. A fragment of a polypeptide means a portion of the polypeptide that provides substantially the same function as the intact polypeptide. Examples of mutants of a specific polypeptide sequence include deletions, substitutions, additions and / or insertions of one or more amino acids of the specific polypeptide sequence. For example, a fragment or mutant of Ldc2 has substantially the same function (e.g., lysine decarboxylase activity) of a Ldc2 polypeptide.

[0030] Another aspect of the present invention relates to a first polynucleotide encoding one or more first polypeptides, the first polypeptide comprising, consisting of or consisting essentially of one or more second polypeptides, the second polypeptides being selected from Ldc2, fragments of Ldc2 and mutants of Ldc2; wherein the first polynucleotide comprises one or more second polynucleotides encoding one or more second polypeptides, respectively; when there are multiple first polypeptides, each first polypeptide may be the same or different; when there are multiple second polypeptides, each second polypeptide may be the same or different; when there are multiple second polynucleotides, each second polynucleotide may be the same or different; the one or more first polypeptides may be expressed alone or as a fusion protein; and when the second polypeptide is Ldc2, at least one corresponding second polynucleotide encoding the second polypeptide comprises, consists of or consists essentially of a mutant ldc2. An example of an ldc2 mutant encodes Ldc2 and may be a codon-optimized ldc2 (e.g., ldc2-co1 (SEQ ID NO: 17)).

[0031] The mutants of Ldc2 are the same as described above. For example, without limitation, the mutants of Ldc2 comprise, consist of, or consist essentially of Ldc2 having a mutation at one or more amino acid positions selected from 111, 262, and 265 (as described above, e.g., SEQ ID NO: 6 (Ldc2S111C), SEQ ID NO: 11 (Ldc2N262T), SEQ ID NO: 12 (Ldc2K265N), SEQ ID NO: 13 (Ldc2S111C / N262T), SEQ ID NO: 14 (Ldc2S111C / K265N), SEQ ID NO: 15 (Ldc2N262T / K265N), SEQ ID NO: 16 (Ldc2S111C / N262T), SEQ ID NO: 17 (Ldc2S111C / K265N), SEQ ID NO: 18 (Ldc2S111C / N262T), SEQ ID NO: 19 (Ldc2S111C / K265N), SEQ ID NO: 20 (Ldc2S111C / K265N), SEQ ID NO: 21 (Ldc2S111C / K265N), SEQ ID NO: 22 (Ldc2S111C / N262T), SEQ ID NO: 23 (Ldc2S111C / K265N), SEQ ID NO: 24 (Ldc2S111C / K265N), SEQ ID NO: 25 (Ldc2S111C / N262T), SEQ ID NO: 26 NO:16(Ldc2S111C / N262T / K265N)、Ldc2S111X、Ldc2N262X'、Ldc2K265X”、Ldc2S111 X / N262X', Ldc2S111X / K265X”, Ldc2N262X’ / K265X” and Ldc2S111X / N262X’ / K265X”).

[0032] In one embodiment, the second polypeptide is a mutant of Ldc2, and the corresponding second polynucleotide encoding the second polypeptide is a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3)), a codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)) comprising one or more suitable nucleotide mutations selected from the following: a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 784, a mutation at nucleotide position 785, a mutation at nucleotide position 786, a mutation at nucleotide position 793, a mutation at nucleotide position 794, and a mutation at nucleotide position 795.

[0033] In another embodiment, the second polypeptide is a mutant of Ldc2, and the corresponding second polynucleotide encoding the second polypeptide is a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), a codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17) comprising one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 332, a mutation at nucleotide position 785, and a mutation at nucleotide position 795. In certain examples, without limitation, the nucleotide at position 332 may be mutated to G, the nucleotide at position 785 may be mutated to C, and the nucleotide at position 795 may be mutated to T or C.

[0034] In another embodiment, the second polypeptide is Ldc2, and the corresponding second polynucleotide is a polynucleotide that is not ldc2 (SEQ ID NO: 3) and encodes Ldc2. Examples of such second polynucleotides include, but are not limited to, codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17).

[0035] In certain embodiments, examples of the second polynucleotide include, but are not limited to, SEQ ID NO: 17 (ldc2-co1), SEQ ID NO: 18 (ldc2-co1C332G), SEQ ID NO: 19 (ldc2-co1A785C), SEQ ID NO: 20 (ldc2-co1A795C), SEQ ID NO: 21 (ldc2-co1C332G / A785C), SEQ ID NO: 22 (ldc2-co1C332G / A795C), SEQ ID NO: 23 (ldc2-co1A785C / A795C), and SEQ ID NO: 24 (ldc2-co1C332G / A785C / A795C).

[0036] In certain embodiments, the first and second polynucleotides may be recombinant or non-naturally occurring polynucleotides. In certain embodiments, the first and second polynucleotides may be cDNAs. In certain embodiments, the first and second polynucleotides are obtained by codon optimization for optimal polypeptide expression in a specific microorganism (e.g., Escherichia coli, Hafnia alvei or Pseudomonas aeruginosa).

[0037] As used herein, nucleotide sequences, polynucleotides and DNA molecules are not limited to functional regions, and may include at least one of expression inhibition regions, coding regions, leader sequences, exons, introns and expression cassettes (see, e.g., Papadakis et al., "Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy," Current Gene Therapy (2004), 4, 89-113). In addition, the nucleotide sequence or polynucleotide may include double-stranded DNA or single-stranded DNA (i.e., the sense strand and antisense strand constituting the double-stranded DNA), or RNA. A polynucleotide comprising a specific polynucleotide sequence may include a fragment and / or mutant of the specific polynucleotide sequence. A fragment of a polynucleotide represents a portion of a polynucleotide encoding a polypeptide that provides substantially the same function as the polypeptide encoded by the complete polynucleotide. Examples of mutants of a specific polynucleotide sequence include naturally occurring allele mutants; artificial mutants; and polynucleotide sequences obtained by deletion, substitution, addition and / or insertion of one or more nucleotides of the specific polynucleotide sequence. It should be understood that such fragments and / or mutants of a specific polynucleotide sequence encode polypeptides having substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence. For example, fragments and / or mutants of ldc2 encode polypeptides having substantially the same function of Ldc2 (e.g., lysine decarboxylase activity).

[0038] Another aspect of the present invention relates to a third polynucleotide encoding one or more third polypeptides, the third polypeptide comprising, consisting of or consisting essentially of one or more fourth polypeptides, the fourth polypeptides being selected from Ldc2, fragments of Ldc2 and mutants of Ldc2; wherein the third polynucleotide comprises one or more fourth polynucleotides encoding one or more fourth polypeptides, respectively; when there are multiple third polypeptides, each third polypeptide may be the same or different; when there are multiple fourth polypeptides, each fourth polypeptide may be the same or different; when there are multiple fourth polynucleotides, each fourth polynucleotide may be the same or different; and the one or more third polypeptides may be expressed alone or as a fusion protein. In certain embodiments, the third and fourth polynucleotides are recombinant or non-naturally occurring polynucleotides. In certain embodiments, the third and fourth polynucleotides are cDNAs. In certain embodiments, the third and fourth polynucleotides may be obtained by codon optimization for optimal polypeptide expression in a specific microorganism (e.g., Escherichia coli, Hafnia alvei or Pseudomonas aeruginosa). An example of a codon-optimized ldc2 encoding Ldc2 is SEQ ID NO: 17 (ldc2-co1). In certain embodiments, the third polynucleotide comprises, consists of, or is substantially composed of Pseudomonas aeruginosa ldc2 (SEQ ID NO: 3), fragments thereof and / or mutants thereof. When the fourth polypeptide is a mutant of Ldc2, they are the same as described above, and the corresponding fourth polynucleotides are the same as described above. Examples of the fourth polynucleotide further include, but are not limited to, polynucleotides encoding Ldc2 (e.g., ldc2 and mutants thereof, such as SEQ ID NO: 17 (ldc2-co1)).

[0039] Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Due to mutation bias and natural selection, different organisms often show a special preference for one of several codons encoding the same amino acid. For example, in fast-growing microorganisms such as Escherichia coli, the best codons reflect the composition of their respective genomic tRNA pools. Therefore, the same amino acid can be used but with a high-frequency codon in a fast-growing microorganism to replace the low-frequency codon of an amino acid. Therefore, the expression of the optimized DNA sequence is improved in a fast-growing microorganism. See, for example, a review of codon optimization techniques http: / / www.guptalab.org / shubhg / pdf / shubhra_codon.pdf, which is incorporated herein by reference in its entirety. As provided herein, a polynucleotide sequence can be codon-optimized for optimal polypeptide expression in a specific microorganism (including but not limited to Escherichia coli, Hafnia alvei and Pseudomonas aeruginosa).

[0040] In certain embodiments, the mutant of polynucleotide can be obtained from the codon optimization of the polynucleotide to reduce its guanine (G) and cytosine (C) polynucleotide content, for improving protein expression. If 50% or more of its bases are G or C, the genome is considered to be GC-rich. The high GC content in the polynucleotide sequence of interest can lead to the formation of secondary structures in mRNA, which can lead to interrupted translation and lower levels of expression. Therefore, changing the G and C residues in the coding sequence to A and T residues without changing the amino acids can provide higher expression levels.

[0041] Pseudomonas aeruginosa ldc2 is considered to be GC-rich because more than 66% of its bases are G or C. In some embodiments, the polynucleotide can be optimized to contain such guanine and cytosine polynucleotide content, which contains less than about 60%, less than about 55%, less than about 50%, less than about 45%, or less than about 40% of the total polynucleotide content of the polynucleotide. In some embodiments, the polynucleotide optimized to contain a specific guanine and cytosine polynucleotide content can be Pseudomonas aeruginosa ldc2.

[0042] As further shown herein, heterologous expression of the Ldc2 mutant Ldc2S111C (SEQ ID NO: 6) has resulted in unexpectedly high cadaverine production, with E. coli expressing Ldc2S111C exhibiting higher cadaverine production than E. coli expressing the E. coli lysine decarboxylase CadA or wild-type Ldc2 (see, e.g., Table 3). Although the serine at amino acid position 111 in Ldc2 is conserved in lysine decarboxylases from different species (see, e.g., Table 4), the serine at position 111 in Ldc2 is conserved in lysine decarboxylases from different species (see, e.g., Table 5). Figure 5), but Ldc2S111C comprises a mutation to cysteine ​​at amino acid position 111. In certain embodiments, the serine at amino acid position 111 in Ldc2 can be mutated to any other amino acid (i.e., Ldc2S111X). For example, without limitation, X can be alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine; preferably, X is alanine, glycine, isoleucine, leucine, methionine, threonine, tyrosine, or valine; more preferably, X is methionine, threonine, or tyrosine.

[0043] In certain embodiments, the polynucleotide encoding Ldc2S111C comprises, consists of, or consists essentially of a polynucleotide encoding Ldc2 having a mutation at nucleotide position 332 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 332 may be mutated to G (e.g., ldc2-co1C332G (SEQ ID NO: 18)).

[0044] In certain embodiments, the polynucleotide encoding Ldc2S111C comprises, consists of, or consists essentially of a polynucleotide encoding Ldc2 having mutations at nucleotide positions 332 and 333 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 332 may be mutated to G and the nucleotide at position 333 may be mutated to T or C.

[0045] In certain embodiments, the polynucleotide encoding Ldc2S111X comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 has one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 331, a mutation at nucleotide position 332, and a mutation at nucleotide position 333.

[0046] In certain embodiments, the polynucleotide encoding Ldc2S111C or Ldc2S111X may be further codon optimized for optimal polypeptide expression in a particular microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0047] As further shown herein, heterologous expression of another Ldc2 mutant, Ldc2N262T (SEQ ID NO: 11), also resulted in unexpectedly high cadaverine production, with E. coli expressing the codon Ldc2N262T exhibiting higher cadaverine production than E. coli expressing the E. coli lysine decarboxylase wild-type Ldc2 (see, e.g., Table 4). Although the asparagine at amino acid position 262 in Ldc2 is conserved in lysine decarboxylases from different species (see, e.g., Table 4), the Figure 6 ), but Ldc2N262T comprises a mutation at amino acid position 262. In certain embodiments, the asparagine at amino acid position 262 in Ldc2 can be mutated to any other amino acid (i.e., Ldc2N262X'). For example, without limitation, X' can be alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In certain embodiments, X' is preferably an amino acid with a polar uncharged side chain, such as, but not limited to, serine, threonine, or glutamine.

[0048] In certain embodiments, the polynucleotide encoding Ldc2N262T comprises, consists of, or consists essentially of a polynucleotide encoding Ldc2 having a mutation at nucleotide position 785 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 785 can be mutated to C (e.g., ldc2-co1A785C (SEQ ID NO: 19)).

[0049] In certain embodiments, the polynucleotide encoding Ldc2N262T comprises, consists of, or consists essentially of a polynucleotide encoding Ldc2 having mutations at nucleotide positions 785 and 786 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 785 can be mutated to C and the nucleotide at position 786 can be mutated to T, C, A, or G.

[0050] In certain embodiments, the polynucleotide encoding Ldc2N262X' comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 has one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 784, a mutation at nucleotide position 785, and a mutation at nucleotide position 786.

[0051] In certain embodiments, the polynucleotide encoding Ldc2N262T or Ldc2N262X' can be further codon-optimized for optimal polypeptide expression in a particular microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0052] As further shown herein, heterologous expression of the Ldc2 mutant Ldc2K265N (SEQ ID NO: 12) also resulted in unexpectedly high cadaverine production, with E. coli expressing Ldc2K265N exhibiting higher cadaverine production than E. coli expressing wild-type Ldc2 (see, e.g., Table 5). Although the lysine at amino acid position 265 in Ldc2 is conserved in lysine decarboxylases from different species (see, e.g., Figure 6 ), but Ldc2K265N comprises a mutation at position 262. In certain embodiments, the lysine at position 265 of Ldc2 can be mutated to any other amino acid (i.e., Ldc2K265X"). For example, without limitation, X' can be alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine; preferably an amino acid with a polar uncharged side chain such as serine, threonine, asparagine, or glutamine.

[0053] In certain embodiments, a polynucleotide encoding Ldc2K265N comprises, consists of, or consists essentially of a polynucleotide encoding Ldc2 having a mutation at nucleotide position 795 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 795 can be mutated to T or C (e.g., ldc2-co1A795C (SEQ ID NO: 20)).

[0054] In certain embodiments, the polynucleotide encoding Ldc2K265X" comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 has one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 793, a mutation at nucleotide position 794, and a mutation at nucleotide position 795.

[0055] In certain embodiments, the polynucleotide encoding Ldc2K265N or Ldc2K265X" can be further codon optimized for optimal polypeptide expression in a specific microorganism (e.g., Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0056] In certain embodiments, a mutant of Ldc2 (also referred to as "Ldc2 mutant") may comprise a mutation at one or more amino acids in the sequence of SEQ ID NO: 4. In certain embodiments, a mutant of Ldc2 is a double mutant comprising SEQ ID NO: 13 (Ldc2S111C / N262T), which has mutations at amino acid positions 111 and 262. In certain embodiments, the serine at amino acid position 111 and the asparagine at amino acid position 262 in Ldc2 may be mutated to any other amino acid (Ldc2S111X / N262X'). X and X' may be the same or different and are the same as described above.

[0057] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 having mutations at nucleotide positions 332 and 785 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 332 can be mutated to a G and the nucleotide at position 785 can be mutated to a C (eg, ldc2-co1 C332G / A785C (SEQ ID NO: 21)).

[0058] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 comprises one or more mutations as described above for the polynucleotide encoding Ldc2S111C and one or more mutations as described above for the polynucleotide encoding Ldc2N262T.

[0059] In certain embodiments, the polynucleotide encoding Ldc2S111X / N262X' comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 comprises one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 784, a mutation at nucleotide position 785, and a mutation at nucleotide position 786.

[0060] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T or Ldc2S111X / N262X′ may be further codon-optimized for optimal polypeptide expression in a particular microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0061] In certain embodiments, the mutant of Ldc2 is a double mutant comprising SEQ ID NO: 14 (Ldc2S111C / K265N), which has mutations at amino acid positions 111 and 265. In certain embodiments, the serine at amino acid position 111 and the lysine at amino acid position 265 can be mutated to any other amino acid (Ldc2S111X / N265X"). X and X" can be the same or different, and are the same as described above.

[0062] In certain embodiments, the polynucleotide encoding Ldc2S111C / K265N comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 having mutations at nucleotide positions 332 and 795 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)).

[0063] In certain embodiments, the polynucleotide encoding Ldc2S111C / K265N comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 comprises one or more mutations as described above for the polynucleotide encoding Ldc2S111C and one or more mutations as described above for the polynucleotide encoding Ldc2K265N. In certain embodiments, the nucleotide at position 332 can be mutated to a G and the nucleotide at position 795 can be mutated to a T or a C (eg, ldc2-co1 C332G / A795C (SEQ ID NO: 22)).

[0064] In certain embodiments, the polynucleotide encoding Ldc2S111X / K265X" comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 has one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 793, a mutation at nucleotide position 794, and a mutation at nucleotide position 795.

[0065] In certain embodiments, the polynucleotide encoding Ldc2S111C / K265N or Ldc2S111X / K265X" can be further codon optimized for optimal polypeptide expression in a specific microorganism (e.g., Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0066] In certain embodiments, the mutant of Ldc2 is a double mutant comprising SEQ ID NO: 15 (Ldc2N262T / K265N), which has mutations at amino acid positions 262 and 265. In certain embodiments, the asparagine at amino acid position 262 and the lysine at amino acid position 265 can be mutated to any other amino acid (Ldc2N262X' / K265X"). X' and X" can be the same or different, and are the same as described above.

[0067] In certain embodiments, the polynucleotide encoding Ldc2N262T / K265N comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 having a mutation at nucleotide positions 785 and 795 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 785 can be mutated to C and the nucleotide at position 795 can be mutated to T or C (eg, ldc2-co1A785C / A795C (SEQ ID NO: 23)).

[0068] In certain embodiments, the polynucleotide encoding Ldc2N262T / K265N comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 comprises one or more mutations as described above for the polynucleotide encoding Ldc2N262T and one or more mutations as described above for the polynucleotide encoding Ldc2K265N.

[0069] In certain embodiments, the polynucleotide encoding Ldc2N262X' / K265X" comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), wherein the polynucleotide encoding Ldc2 comprises one or more suitable nucleotide mutations selected from the group consisting of a mutation at nucleotide position 784, a mutation at nucleotide position 785, a mutation at nucleotide position 786, a mutation at nucleotide position 793, a mutation at nucleotide position 794, and a mutation at nucleotide position 795.

[0070] In certain embodiments, the polynucleotide encoding Ldc2N262T / K265N or Ldc2N262X' / K265X" can be further codon-optimized for optimal polypeptide expression in a specific microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0071] In certain embodiments, the mutant of Ldc2 is a triple mutant comprising SEQ ID NO: 16 (Ldc2S111C / N262T / K265N), which has mutations at amino acid positions 111, 262, and 265. In certain embodiments, the serine at amino acid position 111, the asparagine at amino acid position 262, and the lysine at amino acid position 265 can be mutated to any other amino acid (S111X / N262X' / K265X"). X, X', and X" can be the same or different, and are the same as described above.

[0072] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T / K265N comprises, consists of, or essentially consists of a polynucleotide encoding Ldc2 having mutations at nucleotide positions 332, 785, and 795 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). In certain embodiments, the nucleotide at position 332 can be mutated to a G, the nucleotide at position 785 can be mutated to a C and the nucleotide at position 795 can be mutated to a T or C.

[0073] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T / K265N comprises, consists of, or is essentially composed of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). NO:17)), wherein the polynucleotide encoding Ldc2 comprises one or more mutations as described above for the polynucleotide encoding Ldc2S111C, one or more mutations as described above for the polynucleotide encoding Ldc2N262T, and one or more mutations as described above for the polynucleotide encoding Ldc2K265N (e.g., ldc2-co1C332G / A785C / A795C (SEQ ID NO:24)).

[0074] In certain embodiments, the polynucleotide encoding Ldc2S111X / N262X' / K265X" comprises a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), consists of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)), or consists essentially of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17). NO:17)), the polynucleotide encoding Ldc2 comprises one or more suitable nucleotide mutations selected from the following: a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 784, a mutation at nucleotide position 785, a mutation at nucleotide position 786, a mutation at nucleotide position 793, a mutation at nucleotide position 794 and a mutation at nucleotide position 795.

[0075] In certain embodiments, the polynucleotide encoding Ldc2S111C / N262T / K265N or Ldc2S111X / N262X' / K265X" can be further codon-optimized for optimal polypeptide expression in a specific microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa).

[0076] In certain embodiments, a mutant of Ldc2 (SEQ ID NO: 4) comprises one or more mutations selected from the group consisting of a mutation to X at amino acid position 111, a mutation to X' at amino acid position 262, and a mutation to X" at amino acid position 265 (i.e., "Ldc2111 / 262 / 265"). In certain embodiments, X, X', and X" are independently selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; and with the proviso that X is not serine, X' is not asparagine, and X" is not lysine.

[0077] Thus, the polynucleotide encoding Ldc2 111 / 262 / 265 comprises, consists of, or is substantially composed of a polynucleotide encoding Ldc2 (e.g., ldc2 (SEQ ID NO: 3), codon-optimized ldc2 (e.g., ldc2-co1, SEQ ID NO: 17)). NO:17)), the polynucleotide encoding Ldc2 comprises one or more suitable nucleotide mutations selected from the following: a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 784, a mutation at nucleotide position 785, a mutation at nucleotide position 786, a mutation at nucleotide position 793, a mutation at nucleotide position 794 and a mutation at nucleotide position 795.

[0078] In certain embodiments, the polynucleotides described herein may be recombinant or non-naturally occurring polynucleotide sequences. In certain embodiments, the polynucleotide sequence may be cDNA.

[0079] Another aspect of the present invention relates to a first expression plasmid vector, comprising a fifth polynucleotide encoding one or more fifth polypeptides, consisting of a fifth polynucleotide encoding one or more fifth polypeptides, or consisting essentially of a fifth polynucleotide encoding one or more fifth polypeptides, the fifth polypeptide comprising one or more sixth polypeptides, consisting of one or more sixth polypeptides, or consisting essentially of one or more sixth polypeptides, the sixth polypeptide being selected from Ldc2, a fragment of Ldc2, or a mutant of Ldc2; and a backbone plasmid capable of autonomous replication in a host cell. The sixth polynucleotide encodes the sixth polypeptide. In certain embodiments, the fifth polynucleotide is identical to the first polynucleotide above, the fifth polypeptide is identical to the first polypeptide above, the sixth polynucleotide is identical to the second polypeptide above, and the sixth polypeptide is identical to the second polynucleotide above. In certain embodiments, the host cell is a Pseudomonas aeruginosa cell. In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell.

[0080] In certain embodiments, the first expression plasmid vector further comprises an antitoxin polynucleotide that neutralizes a toxin polypeptide that is harmful to a host cell, and an optional toxin polynucleotide encoding the toxin polypeptide. The toxin and antitoxin polynucleotides and toxin polypeptide are the same as described further below.

[0081] Another aspect of the present invention relates to a second expression plasmid vector, which comprises a seventh polynucleotide encoding one or more seventh polypeptides, consists of a seventh polynucleotide encoding one or more seventh polypeptides, or consists essentially of a seventh polynucleotide encoding one or more seventh polypeptides, the seventh polypeptide comprises one or more eighth polypeptides, consists of one or more eighth polypeptides, or consists essentially of one or more eighth polypeptides, the eighth polypeptide is selected from Ldc2, a fragment of Ldc2, and a mutant of Ldc2; and a backbone plasmid capable of autonomous replication in a host cell. The eighth polynucleotide encodes the eighth polypeptide. In certain embodiments, the seventh polynucleotide is the same as the third polynucleotide described above, the seventh polypeptide is the same as the third polypeptide described above, the eighth polynucleotide is the same as the fourth polypeptide described above, and the eighth polypeptide is the same as the fourth polynucleotide described above. In certain embodiments, the host cell is a Pseudomonas aeruginosa cell. In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell.

[0082] In certain embodiments, heterologous expression of Ldc2 using a second expression plasmid vector comprising a mutant ldc2 (e.g., ldc2-co1 (SEQ ID NO: 17)) results in the same or greater cadaverine production as heterologous expression of Ldc2 using a second expression plasmid vector comprising ldc2. In certain embodiments, fragments or mutants of Ldc2 exhibit lysine decarboxylase activity and / or increase cadaverine production when expressed in a host cell. In certain embodiments, heterologous expression of fragments or mutants of Ldc2 in a host cell results in the same or greater cadaverine production as heterologous expression of Ldc2.

[0083] In certain embodiments, the second expression plasmid vector further comprises an antitoxin polynucleotide that neutralizes a toxin polypeptide that is harmful to the host cell, and an optional toxin polynucleotide encoding the toxin polypeptide. The toxin and antitoxin polynucleotides and toxin polypeptides are the same as described further below.

[0084] As used herein, the term "host cell" refers to a microbial cell, which may be any cell that can be transformed with an expression plasmid vector (e.g., Pseudomonas (e.g., Pseudomonas aeruginosa), Escherichia (e.g., Escherichia coli), Corynebacterium (e.g., Corynebacterium glutamicum), Bacillus, Hafnia (e.g., Hafnia alvei), Brevibacterium, Lactobacillus (e.g., Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus saerimneri), Lactococcus (e.g., Lactococcus lactis, Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. lactis) and Streptococcus (e.g., Streptococcus thermophilus)). In certain embodiments, the host cell may be any cell that is not a Pseudomonas aeruginosa cell.

[0085] The E. coli cell may be any E. coli strain derived from E. coli K12 (eg, MG1655, W3110, DH10b, DH1, BW2952, and strains derived therefrom) or E. coli B, or a strain derived therefrom.

[0086] In certain embodiments, the host cell may comprise one or more endogenous plasmids. In certain embodiments, the host cell does not contain endogenous plasmids. As used herein, the term "cure" means to remove one or more endogenous plasmids from the host cell. In certain embodiments, all endogenous plasmids of the host cell can be "cured" by removing all endogenous plasmids from the host cell. In certain embodiments, one or more endogenous plasmids of the host cell can be "cured" by only removing one or more endogenous plasmids that are targeted for removal from the cell.

[0087] In certain embodiments, the host cell can be a prokaryotic cell (e.g., Hafnia alvei) comprising an endogenous plasmid encoding a specific toxin / antitoxin gene pair. Such toxin / antitoxin gene pairs play a role in maintaining genetic information and stress response. (See Wertz et al. "Chimeric nature of two plasmids of Hafnia alveiencoding the bacteriocins alveicins A and B." Journal of Bacteriology, (2004) 186: 1598-1605). As long as the cell has one or more plasmids comprising antitoxin genes, the antitoxins continuously expressed by the one or more plasmids neutralize the toxins to keep the cells alive. In some prokaryotes, antitoxin proteins degrade faster than toxin proteins. If the plasmid comprising the antitoxin gene is lost from the cell, the toxin protein will exist longer in the cell than the antitoxin protein, and kill or inhibit the growth of the cell. Therefore, it is preferred to maintain a plasmid comprising an antitoxin or toxin / antitoxin gene to keep the host cell alive.

[0088] As used herein, a toxin / antitoxin gene pair has two genes, one is a toxin gene that expresses a polypeptide that is toxic to the host cell, and the other is an antitoxin gene that neutralizes the toxic polypeptide in the host cell. Examples of toxin / antitoxin gene pairs include, but are not limited to, abt / abi gene pairs and aat / aai gene pairs, fragments thereof, and mutants thereof. In some embodiments, the toxin polynucleotide sequence comprises a nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9, a fragment thereof, or a mutant thereof, is composed of a nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9, a fragment thereof, or a mutant thereof, or is substantially composed of a nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9, a fragment thereof, or a mutant thereof. In some embodiments, the antitoxin polynucleotide sequence comprises a nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:10, a fragment thereof, or a mutant thereof, is composed of a nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:10, a fragment thereof, or a mutant thereof, or is substantially composed of a nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:10, a fragment thereof, or a mutant thereof.

[0089] In certain embodiments, the host cell can be any strain of Hafnia alvei, such as a strain of Hafnia alvei that does not contain an endogenous plasmid or a strain of Hafnia alvei that contains an endogenous plasmid. For example, the host cell can be a strain of Hafnia alvei that contains one or more pAlvA plasmids or a cured strain thereof (pAlvA- strain), or a strain of Hafnia alvei that contains one or more pAlvB plasmids and a cured strain thereof (pAlvB- strain).

[0090] In certain embodiments, the expression plasmid vector disclosed herein (e.g., the first or second expression plasmid vector) may further comprise one or more antitoxin genes selected from abi genes, aai genes, mutations and fragments thereof, and / or one or more toxin / antitoxin gene pairs selected from abt / abi gene pairs and aat / aai gene pairs and mutations and fragments thereof. For example, in certain embodiments, the expression plasmid vector (e.g., the first and second expression plasmid vectors) may further comprise an antitoxin polynucleotide neutralizing a toxin polypeptide harmful to a host cell, and a toxin polynucleotide sequence encoding the toxin polypeptide.

[0091] In certain embodiments, the host cell is an industrial strain suitable for industrial scale or large-scale production. For example, industrial strains can be cultured in fermenters. The culture scale can be from hundreds of liters to millions of liters. On the other hand, laboratory strains are usually cultured in a few liters or less. In certain embodiments, industrial strains can be grown in simpler or more economical culture media than laboratory strains.

[0092] The backbone plasmid capable of autonomous replication in a host cell may be any plasmid that can replicate in a host cell. In one embodiment, the expression plasmid vector comprises a backbone plasmid that can replicate in Escherichia coli. In another embodiment, the expression plasmid vector comprises a backbone plasmid that can replicate in Hafnia alvei. Examples of backbone plasmids include, but are not limited to, backbone plasmids that can replicate in Escherichia coli strains, such as pUC (e.g., pUC18 and pUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET, and pSC101 plasmids, and plasmids derived therefrom.

[0093] In certain embodiments, mutants of a polynucleotide can be derived from codon optimization of a polynucleotide for a specific microorganism (eg, Escherichia coli, Hafnia alvei, or Pseudomonas aeruginosa) to enhance polypeptide expression.

[0094] A promoter is a region of DNA that initiates transcription of a specific gene. In certain embodiments, an expression plasmid vector may include one or more promoter polynucleotide sequences. For example, a promoter polynucleotide sequence comprising, consisting of, or consisting essentially of a polynucleotide sequence of SEQ ID NO: 5 may be located upstream of the polynucleotide. In certain embodiments, primers psyn-1 and psyn-2 (see Figure 3 ) synthetic promoter sequence and further inserted into pUC18. In some embodiments, the expression plasmid vector may comprise one or more lac promoter polynucleotide sequences and one or more synthetic promoter polynucleotide sequences. In some embodiments, the expression plasmid vector may comprise only one lac promoter polynucleotide sequence or one synthetic promoter sequence.

[0095] Another aspect of the present invention relates to a first transformant comprising the first expression plasmid vector disclosed herein in a host cell, wherein the first expression plasmid vector comprises, consists of, or consists essentially of:

[0096] a fifth polynucleotide encoding one or more fifth polypeptides comprising, consisting of, or consisting essentially of one or more sixth polypeptides selected from the group consisting of Ldc2, a fragment of Ldc2, and a mutant of Ldc2; and

[0097] A backbone plasmid capable of autonomous replication in host cells.

[0098] The sixth polynucleotide encodes the sixth polypeptide. The host cell, the fifth polynucleotide, the fifth polypeptide, the sixth polynucleotide and the sixth polypeptide are the same as described above.

[0099] As used herein, a transformant is a host cell that has been altered by introducing one or more expression plasmid vectors into the host cell. In certain embodiments, a transformant is obtained by introducing an expression plasmid vector into a host cell that exhibits competence for the plasmid vector.

[0100] In certain embodiments, the host cell is a Pseudomonas aeruginosa cell. In one example, the sixth polypeptide is Ldc2, and the sixth polynucleotide encodes the sixth polypeptide and is a mutant ldc2, such as a codon-optimized ldc2 (e.g., ldc2-co1) for optimal polypeptide expression in a specific microorganism (e.g., Escherichia coli, Hafnia alvei).

[0101] In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell.

[0102] Another aspect of the present invention relates to a second transformant comprising a second expression plasmid vector in a host cell, wherein the expression plasmid vector comprises, consists of, or consists essentially of:

[0103] A seventh polynucleotide encoding one or more seventh polypeptides, wherein the seventh polypeptide comprises, consists of or essentially consists of one or more eighth polypeptides, and the eighth polypeptide is selected from Ldc2, a fragment of Ldc2 and a mutant of Ldc2.

[0104] The eighth polynucleotide encodes the eighth polypeptide. The host cell, the seventh polynucleotide, the seventh polypeptide, the seventh polynucleotide and the eighth polypeptide are the same as described above.

[0105] In certain embodiments, the host cell is a Pseudomonas aeruginosa cell.

[0106] In certain embodiments, the host cell is not a Pseudomonas aeruginosa cell, the eighth polypeptide is Ldc2, and the eighth polynucleotide encodes the eighth polypeptide and is not ldc2, such as a codon-optimized ldc2 (e.g., ldc2-co1) for optimal polypeptide expression in the host cell (e.g., Escherichia coli, Hafnia alvei).

[0107] In certain embodiments, the expression plasmid vector (e.g., the first or second expression plasmid vector) transformed into the transformant disclosed herein (e.g., the first or second transformant) further comprises an antitoxin polynucleotide that neutralizes a toxin polypeptide that is harmful to the host cell, and optionally a toxin polynucleotide encoding a toxin polypeptide, and the transformant can be further altered by introducing an expression plasmid vector (comprising a toxin polynucleotide sequence encoding a toxin polypeptide, and optionally an antitoxin polynucleotide that neutralizes the toxin polypeptide) into a host cell.

[0108] Another aspect of the present invention relates to a first mutant host cell comprising a first or third polynucleotide as disclosed herein, wherein the first or third polynucleotide has been integrated into the chromosome of the host cell. The first or third polynucleotide is the same as described above.

[0109] In certain embodiments, the first or third polynucleotide has been codon-optimized as described above. In certain embodiments, the first or third polynucleotide encodes one or more polypeptides comprising, consisting of, or consisting essentially of Ldc2 and / or one or more Ldc2 mutants, wherein the one or more Ldc2 mutants are selected from SEQ ID NO:6 (Ldc2S111C), SEQ ID NO:11 (Ldc2N262T), SEQ ID NO:12 (Ldc2K265N), SEQ ID NO:13 (Ldc2S111C / N262T), SEQ ID NO:14 (Ldc2S111C / K265N), SEQ ID NO:15 (Ldc2N262T / K265N), SEQ ID NO:16 (Ldc2S111C / N262T / K265N) and Ldc2 111 / 262 / 265.

[0110] In certain embodiments, the first or third polynucleotide can be integrated into the host cell chromosome according to the PCR-mediated gene replacement method (see, for example, Datsenko, 2000, a review of PCR-mediated gene replacement methods, which is incorporated herein by reference in its entirety). The integrated chromosome can also be generated by other suitable methods.

[0111] Another aspect of the present invention relates to a method for preparing Pseudomonas aeruginosa Ldc2, a fragment of Ldc2 or a mutant of Ldc2, the method comprising:

[0112] Obtaining a first transformant, a second transformant and / or a first mutant host cell as disclosed herein (eg, a first and / or a second transformant);

[0113] culturing the first transformant, the second transformant, and / or the first mutant host cell under conditions effective to express the polypeptide; and

[0114] Harvest the peptides.

[0115] The first transformant, the second transformant and / or the first mutant host cell can be cultivated using a culture medium comprising a carbon source and a non-carbon nutrient source. The example of a carbon source includes, but is not limited to, sugar (for example, carbohydrates such as glucose and fructose), oil and / or fat, fatty acid, and / or its derivatives. Oil and fat can include saturated and / or unsaturated fatty acids with 10 or more carbon atoms, such as coconut oil, palm oil, palm kernel oil, etc. Fatty acid can be saturated and / or unsaturated fatty acids, such as caproic acid, sad, capric acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, myristic acid, etc. The example of fatty acid derivatives includes, but is not limited to, esters and salts thereof. The example of a non-carbon source includes, but is not limited to, nitrogen sources, inorganic salts and other organic nutrient sources.

[0116] For example, culture medium can comprise the carbon source that transformant and / or mutant host cell can absorb, optionally have one or more other sources selected from nitrogen source, inorganic salt and another organic nutrient source.In certain embodiments, the weight percentage of nitrogen source is about 0.01%-about 0.1% of culture medium.The example of nitrogen source can comprise ammonia, ammonium salt (such as ammonium chloride, ammonium sulfate and ammonium phosphate), peptone, meat extract, yeast extract etc.The example of inorganic salt includes but is not limited to potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride etc.The example of other organic nutrient sources includes but is not limited to amino acid (such as glycine, alanine, serine, threonine and proline), vitamin (such as vitamin B1, vitamin B12 and vitamin C) etc.

[0117] The culture may be performed at any temperature as long as the cells can grow, and is preferably at about 20° C. to about 40° C. or about 35° C. The culture time may be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0118] In one embodiment, the first transformant, the second transformant and / or the first mutant host cell are cultured in a culture medium comprising peptides, peptones, vitamins (e.g., B vitamins), trace elements (e.g., nitrogen, sulfur, magnesium) and minerals. Examples of such culture media include, but are not limited to, the commonly known Lysogeny broth (LB) culture medium comprising tryptone, yeast extract and NaCl suspended in water (e.g., distilled or deionized).

[0119] Another aspect of the present invention relates to a method for preparing cadaverine (1,5-pentanediamine), the method comprising:

[0120] 1a) culturing a first transformant, a second transformant and / or a first mutant host cell as disclosed herein;

[0121] 1b) producing cadaverine by decarboxylating lysine using the culture obtained in step 1a; and

[0122] 1c) extracting and purifying cadaverine using the culture obtained from step 1b.

[0123] Cultivating the first transformant, the second transformant and / or the first mutant host cell may include the steps of culturing the transformants or mutant host cells as described above.

[0124] As used herein, "using the culture obtained from step 1a" may include further processing of the culture obtained from step 1a. For example, diluting the culture with a buffer solution; centrifuging the culture to collect cells; resuspending the cells in a buffer solution; or lysing the cells into a cell lysate; or / and purifying lysine decarboxylase from the cell lysate.

[0125] In another embodiment, step 1c of the method further comprises the following steps:

[0126] 1c1) separating the solid and liquid components obtained from the reaction of step 1b;

[0127] 1c2) adjusting the pH of the liquid component obtained from step 1c1 to about 14 or higher;

[0128] 1c3) removing water from the liquid component obtained in step 1c2; and

[0129] 1c4) Recovering cadaverine.

[0130] In step 1c1, separation of the solid and liquid components of the reaction of step 1b may be accomplished by conventional centrifugation and / or filtration.

[0131] In step 1c2, the pH of the liquid components of step 1c1 may be adjusted by adding a base such as NaOH. The NaOH may be added as a solid and / or a solution (eg, an aqueous solution).

[0132] In step 1c3, water may be removed by distillation at ambient pressure or under vacuum.

[0133] In step 1c4, cadaverine can be recovered by distillation at ambient pressure or under vacuum.

[0134] Another aspect of the present invention relates to bio-based cadaverine prepared according to the methods disclosed herein.

[0135] As used herein, a "biobased" compound means that the compound is considered biobased under standard ASTM D6866.

[0136] Another aspect of the invention relates to a polyamide having the structure of Structure 1:

[0137]

[0138] Including stereoisomers thereof, wherein:

[0139] m=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22;

[0140] n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22;

[0141] j = about 100 to about 1,000,000; and

[0142] The polyamide is prepared from one or more diamines having a carbon number m and one or more dicarboxylic acids having a carbon number n, at least one of the diamines and dicarboxylic acids contains bio-based carbon under standard ASTM D6866, and m or n for each diamine or dicarboxylic acid may be the same or different.

[0143] In one embodiment, the diamine is bio-based cadaverine, more preferably bio-based cadaverine prepared according to the method disclosed herein. Examples of dicarboxylic acids include, but are not limited to, C 10 Dicarboxylic acid, C 11 Dicarboxylic acid, C 12 Dicarboxylic acid, C 13 Dicarboxylic acid, C 14 Dicarboxylic acid, C 16 Dicarboxylic acid, C 18 Dicarboxylic acids and any combination thereof. In certain embodiments, all or part of C n The dicarboxylic acids are bio-based.

[0144] In another embodiment, the polyamide has the above structure, wherein:

[0145] The polyamide is formed by reacting bio-based cadaverine with one or more dicarboxylic acids, more preferably the bio-based cadaverine is prepared according to the method disclosed herein;

[0146] n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22;

[0147] j = about 100 to about 1,000,000, about 1,000 to about 100,000, or about 1,000 to about 10,000; and

[0148] The dicarboxylic acid contains bio-based carbon under standard ASTM D6866.

[0149] Another aspect of the present invention relates to a method for preparing the polyamide disclosed herein, the method comprising preparing bio-based cadaverine into Cm Diamine.

[0150] In one embodiment, the method further comprises preparing one or more bio-based C n Dicarboxylic acid.

[0151] In another embodiment, the method further comprises reacting the bio-based cadaverine with one or more bio-based C n Polyamides are prepared by reacting dicarboxylic acids.

[0152] Another aspect of the present invention relates to a composition comprising one or more polyamides disclosed herein.

[0153] In one embodiment, the diamine is bio-based cadaverine, more preferably bio-based cadaverine prepared according to the method disclosed herein. Examples of dicarboxylic acids include, but are not limited to, C 10 Dicarboxylic acid, C 11 Dicarboxylic acid, C 12 Dicarboxylic acid, C 13 Dicarboxylic acid, C 14 Dicarboxylic acid, C 16 Dicarboxylic acid, C 18 Dicarboxylic acids and any combination thereof. In certain embodiments, all or part of C n The dicarboxylic acids are bio-based.

[0154] In another embodiment, the polyamide has the above structure, wherein:

[0155] The polyamide is formed by reacting bio-based cadaverine with one or more dicarboxylic acids, more preferably the bio-based cadaverine is prepared according to the method disclosed herein;

[0156] n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22;

[0157] j = about 100 to about 1,000,000, about 1,000 to about 100,000, or about 1,000 to about 10,000; and

[0158] The dicarboxylic acid contains bio-based carbon under standard ASTM D6866.

[0159] Another aspect of the present invention relates to a method for preparing 1,5-pentane diisocyanate, the method comprising:

[0160] 2a) preparing bio-based cadaverine as disclosed herein; and

[0161] 2b) converting the bio-based cadaverine obtained in step 2a into 1,5-pentyl diisocyanate.

[0162] Step 2b may include converting the diamine to an isocyanate / ester using any known method. Examples of such methods are conventional phosgene methods, including a one-step high temperature phosgene method (i.e., mixing phosgene with a diamine at high temperature to obtain an isocyanate / ester), an improved two-step phosgene method, and a triphosgene method, in which triphosgene is used instead of phosgene. There are also other methods that do not use phosgene as a raw material. Examples of such methods are the carbonylation of hexamethylenediamine using CO2 instead of phosgene: CO2 is added to a solution of a primary amine and an organic base, and then an appropriate amount of a phosphorus electrophile is added to the reaction solution to initiate an exothermic dehydration reaction to obtain an isocyanate / ester. Another example is a carbamate thermal decomposition method, in which a primary amine is converted to a carbamate, and then the carbamate is heated to decompose and produce an isocyanate / ester.

[0163] The abbreviations used for amino acids, polypeptides, base sequences and nucleic acids are based on the abbreviations specified in IUPAC-IUB Communication on Biochemical Nomenclature, Eur. J. Biochem., 138:9 (1984), "Guideline for Preparing Specifications Including Base Sequences and Amino Acid Sequences" (United States Patent and Trademark Office), as well as those commonly used in this technical field.

[0164] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise", "include", etc. are to be interpreted in an inclusive sense (i.e., in the sense of "including but not limited to"), as opposed to an exclusive or exhaustive sense. When used in this application, the words "herein", "above", "below", "supra" and words of similar input refer to this application as a whole and not to any particular part of this application. Where the context permits, words used in the singular or plural in the detailed description above may also include the plural or singular, respectively. The words "or" and "and / or" with respect to a list of two or more items encompass all of the following interpretations of the words: any item in the list, all items in the list, and any combination of items in the list.

[0165] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific embodiments discussed should not be construed as limiting the scope of the present invention. It will be clear to those skilled in the art that various equivalences, changes and modifications can be made without departing from the scope of the present invention, and it should be understood that such equivalent embodiments are included herein. In addition, all references cited in this disclosure are incorporated herein by reference in their entirety, as if fully shown herein. Example

[0166] Example 1. The Pseudomonas aeruginosa polypeptide sequence is not similar to the E. coli CadA or LdcC lysine decarboxylase sequence.

[0167] To identify new lysine decarboxylases, the E. coli lysine decarboxylase polypeptides CadA and LdcC were used as BLAST queries to identify similar protein sequences from organisms other than E. coli. A non-redundant (nr) polypeptide sequence database was used, and E. coli (taxid:562) was excluded from the search. BLAST results showing polypeptides with similar sequences are in Figure 1 The protein tree for CadA is shown in Figure 2 The protein tree for LdcC is shown in FIG. Figure 1 and 2 As described in , lysine decarboxylase from Pseudomonas (eg, Pseudomonas aeruginosa) did not appear in the BLAST search results.

[0168] Example 2. Construction of Pseudomonas aeruginosa ldc1 and ldc2 expression plasmid vectors and Escherichia coli strains.

[0169] The genomic DNA of Pseudomonas aeruginosa PAO1 (accession number: NC_002516) was purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) (DSM No. 22644) and used as the template DNA for PCR. PCR primers ldc1-1 and ldc1-2 ( Figure 3 ). The nucleotide sequence of ldc1 is provided in SEQ ID NO: 1 and the Ldc1 protein sequence is provided in SEQ ID NO: 2. The Ldc1-1 and ldc1-2 primers were used to amplify ldc1 (SEQ ID NO: 1).

[0170] PCR primers ldc2-1 and ldc2-2 were designed based on the gene (named ldc2) from Pseudomonas aeruginosa RP73 (accession number: WP_014603046.1). Figure 3 ). The nucleotide sequence of ldc2 is provided in SEQ ID NO:3 and the Ldc2 protein sequence is provided in SEQ ID NO:4. Primers ldc2-1 and ldc2-2 were used to amplify ldc2 (SEQ ID NO:3).

[0171] Construct a recombinant expression plasmid vector containing the ldc1 or ldc2 gene. Insert the respective gene into a pUC18 plasmid vector or a pUC18 plasmid vector containing a synthetic promoter (pCIB10) ( Figure 4 A). PCR primers psyn-1 and psyn-2 ( Figure 3 ) to synthesize a synthetic promoter sequence (SEQ ID NO: 5). Primer psyn-1 contains a promoter sequence and a sequence homologous to pUC18, and primer psyn-2 contains a sequence homologous to pUC18. These two PCR primers are used to amplify a portion of pUC18 that includes a multiple cloning site from a plasmid inserted downstream of the synthetic promoter sequence. Restriction enzymes EcoRI and ScaI are used to digest the amplified DNA containing the synthetic promoter, which is further ligated into pUC18 to construct pCIB10 ( Figure 4 A, synthetic promoter is shown as "Pcp25"). The amplified DNA fragments of ldc1 and ldc2 were digested with restriction enzymes SacI and XbaI and ligated into pUC18 to construct pCIB45 ( Figure 4 B) and pCIB48( Figure 4 E), or ligated into pCIB10 to construct pCIB46 ( Figure 4 C) and pCIB47( Figure 4 D).

[0172] The bacterial strain was constructed by transforming pCIB45-48 into E. coli MG1655K12. pUC18 was transformed into E. coli MG1655K12 as a negative control.

[0173] Example 3. Production of cadaverine by heterologous expression of Pseudomonas aeruginosa Ldc1 and Ldc2 in E. coli.

[0174] A single colony of each E. coli strain containing an empty vector or a Pseudomonas aeruginosa ldc1 or ldc2 recombinant expression plasmid vector (strains CIB45-46 and CIB 47-48, respectively) was grown overnight in 4 mL of culture in Lysogeny broth (LB) medium with ampicillin (100 μg / mL) at 37° C. The next day, each culture was inoculated into 4 mL of fresh LB medium with ampicillin (100 μg / mL) to a final optical density (OD) of 0.05, measured at absorbance 600 nm (OD 600 Each culture was grown for 4 hours at 30°C, and then lysine-HCl and pyridoxal phosphate (PLP) were added to a final concentration of 20 g / L and 0.1 mM, respectively. Each culture was grown for an additional 24 hours, and then the final OD was measured. 600 Nuclear magnetic resonance (NMR) was used to identify and quantify cadaverine (Table 1).

[0175] Table 1. Production of cadaverine by heterologous expression of P. aeruginosa Ldc1 and Ldc2 in E. coli.

[0176]

[0177] As provided in Table 1, the E. coli strains expressing Pseudomonas aeruginosa Ldc2 (CIB47 and 48) showed higher yields of cadaverine compared to the E. coli strains expressing Pseudomonas aeruginosa Ldc1 (CIB45 and CIB46). The strains expressing Ldc1 all showed less cadaverine production than the negative control (NC). Therefore, these results confirm that heterologous expression of Pseudomonas aeruginosa Ldc2 in E. coli affects cadaverine production.

[0178] Example 4. Construction of codon-optimized Pseudomonas aeruginosa ldc2 recombinant expression plasmid vector in Escherichia coli and Hafnia alvei strains.

[0179] The nucleotide sequence of the ldc2 gene was codon optimized for expression in E. coli (ldc2-co1, SEQ ID NO: 17). The codon optimized sequence was cloned into pUC18 and pCIB10 using restriction enzymes SacI and XbaI to produce plasmids pCIB65 and pCIB66, respectively. A positive control was constructed by cloning wild-type E. coli cadA into pUC18 and pCIB10 to produce pCIB60 and pCIB61, respectively. Plasmids pCIB60 and pCIB65 were transformed into E. coli MG1655K12 to produce strains CIB60 and CIB65. Plasmids pCIB61 and pCIB66 were transformed into Hafnia alvei to produce strains CIB61 and CIB66.

[0180] Example 5. Production of cadaverine by P. aeruginosa Ldc2 and E. coli CadA expressed in E. coli and H. alvei.

[0181] Single colonies of each E. coli and H. alvei strain (strains CIB48, CIB60, CIB61, CIB65 and CIB66) were grown overnight in 2.5 mL cultures at 29°C in LB medium with ampicillin (100 μg / mL). E. coli and H. alvei transformed with the empty vector pUC18 were used as negative controls. The next day, minimal medium with ampicillin (100 μg / mL) and lysine-HCl and PLP to final concentrations of 20 g / L and 0.1 mM, respectively. Each culture was incubated at 37°C for 5 hours. A 1 mL sample was collected from each culture to quantify cadaverine production using NMR.

[0182] Table 2. Cadaverine production by P. aeruginosa Ldc2 and E. coli CadA expressed in E. coli and H. alvei.

[0183]

[0184] As provided in Table 2, E. coli cells expressing Ldc2 protein produced from ldc2 or ldc2-co1 DNA showed higher cadaverine production yields (6.02 g / kg and 6.54 g / kg) compared to E. coli cells expressing CadA protein (5.81 g / kg). It is noteworthy that E. coli cells expressing Ldc2 protein produced from ldc2-co1 DNA showed higher cadaverine production yields (6.54 g / kg) compared to E. coli cells expressing Ldc2 protein produced from ldc2 DNA (6.02 g / kg). In addition, Hafnia alvei cells expressing Ldc2 protein (produced from ldc2-co1 DNA) showed higher cadaverine production yields (9.60 g / kg) compared to Hafnia alvei cells expressing CadA protein (6.62 g / kg).

[0185] Example 6. Construction and expression of Pseudomonas aeruginosa Ldc2S111C mutant.

[0186] like Figure 5As shown in the sequence alignment provided in , the serine at amino acid position 111 in Pseudomonas aeruginosa Ldc2 is conserved in lysine decarboxylases from different species including Escherichia coli, Shigella sonnei and Salmonella enterica. The codon-optimized Pseudomonas aeruginosa ldc2 gene (SEQ ID NO: 17) was mutated by generating a point mutation at nucleotide 332 (ldc2-co1C332G (SEQ ID NO: 18)), which resulted in the replacement of the serine at amino acid 111 with cysteine ​​(Ldc2S111C) (SEQ ID NO: 6). ldc2-co1 C332G (SEQ ID NO: 18) was cloned into pUC18 and pCIB10 using restriction enzymes SacI and XbaI to generate plasmids pCIB67 and pCIB68, respectively. Plasmid pCIB67 was transformed into E. coli MG1655K12 to produce strain CIB67, and pCIB68 was transformed into Hafnia alvei to produce strain CIB68. Experiments to determine production were performed as described in Example 5. Briefly, a single colony of each strain was grown overnight in 2.5 mL culture at 29° C. in LB medium with ampicillin (100 μg / mL). The next day, 2.5 mL of minimal medium was supplemented with ampicillin (100 μg / mL) and lysine-HCl and PLP to a final concentration of 20 g / L and 0.1 mM, respectively. Each culture was incubated at 37° C. for 5 hours. A 1 ml sample was collected from each culture to quantify cadaverine production using NMR.

[0187] Table 3. Cadaverine production by P. aeruginosa Ldc2S111C and E. coli CadA expressed in E. coli and H. alvei.

[0188]

[0189] As shown in Table 3, the E. coli cells expressing the Ldc2S111C mutant protein exhibited a much higher cadaverine production yield (9.88 g / kg) compared to the E. coli cells expressing the wild-type Ldc2 protein (6.54 g / kg).

[0190] Example 7. Construction and expression of Pseudomonas aeruginosa Ldc2N262T mutant.

[0191] like Figure 6As shown in the sequence alignment provided in , the asparagine at amino acid position 262 in Pseudomonas aeruginosa Ldc2 is conserved in lysine decarboxylases from different species including Escherichia coli, Shigella sonnei, and Salmonella enterica. Idc2-co1 (SEQ ID NO: 17) was mutated by generating a point mutation at nucleotide 785 (ldc2-co1A785C (SEQ ID NO: 19)), which resulted in substitution of asparagine at amino acid 262 with threonine (Ldc2N262T) (SEQ ID NO: 11). Idc2-co1A785C (SEQ ID NO: 19) was cloned into pUC18 and pCIB10 using restriction enzymes SacI and XbaI to produce plasmid pCIB75. Plasmid pCIB75 was transformed into Escherichia coli MG1655K12 to produce strain CIB75.

[0192] Experiments to determine production were performed as described in Example 5. Briefly, a single colony of each strain was grown overnight in 2.5 mL culture at 29°C in LB medium with ampicillin (100 μg / mL). The next day, 2.5 mL of minimal medium was supplemented with 100 μg / mL ampicillin and lysine-HCl and PLP to final concentrations of 20 g / L and 0.1 mM, respectively. Each culture was incubated at 37°C for 5 hours. A 1 mL sample was collected from each culture to quantify cadaverine production using NMR. Table 4. Cadaverine produced by Pseudomonas aeruginosa Ldc2N262T expressed in Escherichia coli.

[0193]

[0194] As shown in Table 4, the E. coli strain expressing the Ldc2N262T mutant protein exhibited a much higher cadaverine production yield (14.5 g / kg) compared to the E. coli strain expressing the wild-type Ldc2 protein (6.25 g / kg).

[0195] Example 8. Construction and expression of Pseudomonas aeruginosa Ldc2K265N mutant.

[0196] like Figure 6As shown in the sequence alignment provided in , the lysine at amino acid position 265 in Pseudomonas aeruginosa Ldc2 is conserved in lysine decarboxylases from different species including Escherichia coli, Shigella sonnei, and Salmonella enterica. Idc2-co1 (SEQ ID NO: 17) was mutated by generating a point mutation at nucleotide 795 (ldc2-co1A795C (SEQ ID NO: 20)), which resulted in substitution of the lysine at amino acid 265 with asparagine (Ldc2K265N) (SEQ ID NO: 12). Idc2-co1A795C (SEQ ID NO: 20) was cloned into pUC18 and pCIB10 using restriction enzymes SacI and XbaI to produce plasmid pCIB76. Plasmid pCIB76 was transformed into Escherichia coli MG1655K12 to produce strain CIB76.

[0197] Experiments to determine production were performed as described in Example 5. Briefly, a single colony of each strain was grown overnight in 2.5 mL cultures in LB medium with ampicillin (100 μg / mL) at 29°C. The next day, 2.5 mL of minimal medium was supplemented with 100 μg / mL ampicillin and lysine-HCl and PLP to final concentrations of 20 g / L and 0.1 mM, respectively. Each culture was incubated at 37°C for 5 hours. A 1 mL sample was collected from each culture to quantify cadaverine production using NMR.

[0198] Table 5. Cadaverine production by P. aeruginosa Ldc2K265N expressed in E. coli.

[0199]

[0200] As shown in Table 5, the E. coli cells expressing the Ldc2K265N mutant protein exhibited a much higher cadaverine production yield (10.1 g / kg) compared to the cells expressing the wild-type Ldc2 protein (4.73 g / kg).

[0201] Example 9. Integration of ldc2 into the E. coli chromosome and expression of Ldc2.

[0202] According to the PCR-mediated gene replacement method described in Datsenko et al, 2000, ldc2 was integrated into the Escherichia coli chromosome. The ldc2-co1 gene (SEQ ID NO:17) of Pseudomonas aeruginosa was integrated into the Escherichia coli MG1655 chromosome at the locus of the recA gene. This knockout box was constructed by merging 4 fragments together using sewing PCR: 1) 400bp district upstream of recA, 2) ldc2 gene, 3) cat gene that enables the production of chloramphenicol resistance, and 4) 400bp district downstream of recA. The knockout box was transformed into MG1655 comprising the pKD46Red recombinase expression plasmid. Transformant was grown on chloramphenicol plates to identify successful integration, and colony PCR and sequencing were used to verify gene interruption. A colony with the desired integration separated was labeled as CIB96.

[0203] Experiments for expressing Ldc2 from chromosomes to verify integration and confirm cadaverine production were performed as described in Example 5. Briefly, single colonies were grown overnight in 2.5 mL cultures at 29° C. in LB medium with chloramphenicol (25 μg / mL). The next day, 2.5 mL of minimal medium was supplemented with 25 μg / mL chloramphenicol and lysine-HCl and PLP to a final concentration of 20 g / L and 0.1 mM, respectively. Each culture was incubated overnight at 37° C. A sample of 1 ml was collected from each culture to quantify cadaverine production using NMR.

[0204] Table 6. Cadaverine production by P. aeruginosa Ldc2 expressed in E. coli with ldc2 integrated into the chromosome.

[0205]

[0206] As shown in Table 6, E. coli cells containing ldc2-co1 integrated into the chromosome and expressing Ldc2 protein showed much higher cadaverine production yield (5.98 g / kg) compared to the negative control (E. coli cells without chromosome integration) (0.10 g / kg).

[0207] Sequence Listing

[0208] SEQ ID:NO 1 (ldc1 DNA sequence)

[0209]

[0210] SEQ ID: NO 2 (Ldc1 protein sequence)

[0211]

[0212] SEQ ID:NO 3 (ldc2 DNA sequence)

[0213]

[0214] SEQ ID: NO 4 (Ldc2 protein sequence)

[0215] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0216] SEQ ID: NO 5 (Synthetic promoter)

[0217] agtttattcttgacatgtagtgagggggctggtataat

[0218] SEQ ID: NO 6 (Ldc2 S111C protein sequence)

[0219] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAECMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0220] SEQ ID NO:7 (aat DNA sequence)

[0221] >gb|AY271828.1|:385 - 1717 Hafnia alvei plasmid pAlvA, complete sequence

[0222]

[0223] SEQ ID NO:8 (aai DNA sequence)

[0224] >gb|AY271828.1|:1734-2069 Hafnia alvei plasmid pAlvA, complete sequence

[0225]

[0226]

[0227] SEQ ID NO:9 (abt DNA sequence)

[0228] >gb|AY271829.1|:384-1566 Hafnia alvei plasmid pAlvB, complete sequence

[0229]

[0230] SEQ ID NO:10 (abi DNA sequence)

[0231] >gb|AY271829.1|:1583-1918 Hafnia alvei plasmid pAlvB, complete sequence

[0232]

[0233] SEQ ID: NO 11 (Ldc2 N262T protein sequence)

[0234] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRTCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0235] SEQ ID:NO 12 (Ldc2 K265N protein sequence)

[0236] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCH N SILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0237] SEQ ID:NO 13 (Ldc2 S111C / N262T protein sequence)

[0238] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAE CMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDR T CHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0239] SEQ ID:NO 14 (Ldc2 S111C / K265N protein sequence)

[0240] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAE CMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCH N SILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0241] SEQ ID:NO 15 (Ldc2 N262T / K265N protein sequence)

[0242] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRT CH N SILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0243] SEQ ID:NO 16 (Ldc2 S111C / N262T / K265N protein sequence)

[0244] MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAE C MADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVD RT CH NSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPERFERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE

[0245] SEQ ID:NO 17(ldc2-co1 DNA sequence)

[0246]

[0247] SEQ ID: NO 18 (ldc2 - co1 C332G DNA sequence)

[0248] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAAT G

[0249] SEQ ID: NO 19 (ldc2 - co1 A785C DNA sequence)

[0250] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTA C

[0251] SEQ ID: NO 20 (ldc2 - co1 A795C DNA sequence)

[0252] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAA C

[0253] SEQ ID: NO 21 (ldc2 - co1 C332G / A785C DNA sequence)

[0254] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAAT G TATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTA C

[0255] SEQ ID: NO 22 (ldc2 - co1 C332G / A795C DNA sequence)

[0256] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAAT G TATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAA C

[0257] SEQ ID: NO 23 (ldc2 - co1 A785C / A795C DNA sequence)

[0258] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTA C CTGTCACAA C

[0259] SEQ ID: NO 24 (ldc2 - co1 C332G / A785C / A795C DNA sequence)

[0260] ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAAT G TATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTA C CTGTCACAA C

[0261] References

[0262] The references, patents and published patent applications listed below, as well as all references cited in the above specification, are incorporated herein by reference in their entirety, as if fully set forth herein.

[0263] 1. West SE and Iglewski BH (1988). Codon usage in Pseudomonasaeruginosa. Nucleic Acids Res 16:9323-9335.

[0264] 2.Wertz et al.Chimeric nature of two plasmids of H.alvei encoding thebacteriocins alveicins A and B.Journal of Bacteriology,(2004)186:1598-1605.3.Datsenko KA&Wanner BL(2000).One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.PNAS:6640-6645.

[0265] 4.Papadakis et al., Promoters and Control Elements: DesigningExpression Cassettes for Gene Therapy. Current Gene Therapy (2004), 4, 89-113. Sequence Listing <110> Cathay Bio-Industry Co., Ltd. <120> Expression of polypeptides involved in lysine decarboxylation and methods and applications thereof <130> 77705-8005 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 1071 <212> DNA <213> Pseudomonas aeruginosa <400> 1 atgccctacg aagccgatga ctatctttcc cggcacttcc agaccagcgg caccgacctg 60 gcgcggaagg tcgacgaact ggcggccctt gcggctcccg gcgacagccc caatctcgcg 120 ctctaccgcg agatgctctt caccgtgacg cgcatggccc aggccgaccg caaccgctgg 180 gacgccaaga tcatgctgca gaccctgcgc gagatggagc atgccttcag cgtcctcgag 240 cagttcaagc ggcgacgcaa ggtcaccgtg ttcggctcgg cgcgcacgcc ggtcgaacat 300 ccggtctatg ccctggcgcg caagctgggc gaggaactgg cccgctacga cctgatggtg 360 atcaccggcg ccggcggcgg catcatggcc gccgcccacg aaggcgccgg gctggagaac 420 agcctgggct tcaacatcac cctgcccttc gagcagcacg ccaaccatac ggtggacggc 480 agcggcaacc tgctgtcgtt ccactttttc ttcctgcgca agctgttctt cgtcaaggaa 540 gccgacgccc tggtgctctg ccccggcggc ttcggcaccc tcgacgaggc actggaagtg 600 ctgaccctgg tacagaccgg caagagtccg ctggtgccga tcgtgctgct cgaccagccg 660 ggcggccgct actgggaaca cgccctggag ttcatgcagg aacagttgct ggagaatcac 720 tatatcctgc cggccgacat gcgcctgatg cgcctggtgc attcggccga agacgcggtg 780 aaggaaatcg cccagttcta ccgcaacttc cactccagcc gctggctgaa aggcactttc 840 gtgattcgcc tgaaccacgc cctgaacgaa gccgcgctgg cgcacctgca cgaacacttc 900 gccagcctct gcctgagcgg cggcttccag cagcaggcct acagcgagca ggaacaggac 960 gaaccggagt tccgcaacct gacccgcctc gccttcgtgt tcaacgggcg cgaccagggg 1020 cggctgcggg aattgctgga ctacatcaac ctgccggaaa actgggactg a 1071 <210> 2 <211> 438 <212> PRT <213> Pseudomonas aeruginosa <400> 2 Put Ala Ala Leu Asp Glu Leu Arg Gln Val Ala Pro Ser Ile Pro Leu 1 5 10 15 Phe Leu Leu Phe Arg Gln Leu Arg Ile Glu Gln Leu Ser Ser Gln Leu 20 25 30 Leu Asp Glu Val Gln Gly Cys Phe Asn Leu Ala Ala Gly Pro Ala Arg 35 40 45 Phe Ile Ala Glu Arg Ile Asp Ser Asp Leu Arg Glu Trp Arg Ala Pro 50 55 60 Ala Gly Pro Arg Arg Leu Arg Asp Tyr Ala Pro Pro Val Pro Arg Thr 65 70 75 80 Pro Val Ser Ala Arg Tyr Asn Gly Arg Ala Arg Leu Asp Leu Ala Pro 85 90 95 Ala Lys Gln Trp Arg Ile Gly Ser Gly Ser Thr Ala Glu Arg Leu Ala 100 105 110 Thr Pro Leu Asn Asp Leu Ser Thr Ala Tyr Arg Lys Thr Ser Ala Gly 115 120 125 Ala Pro Ala Ala His Ala Gly Asp Ile Ala Glu Ala Phe Arg Arg Ala 130 135 140 Leu Trp Glu Ala Ala Ala Arg Leu Ala Arg Glu Asp Gly Asp Thr Trp 145 150 155 160 Phe Phe Glu Ile Leu Arg Gly Asn Pro Gly Pro Gly Ile Glu Ala Gly 165 170 175 Arg Glu Thr Pro Ala Lys Arg Trp His Gly Leu Ala Glu Thr Leu Asp 180 185 190 Ser Ser Pro Arg Leu Asp Pro Leu Arg Val Ala Leu Ser Ala Pro Gly 195 200 205 Leu Asp Ser Arg Gly Arg Pro Ala Ser Phe Gly Val Pro Ala Ala Val 210 215 220 Val Cys Arg Tyr Leu Arg Arg His Gly Ile Ala Pro Leu Arg Thr Gly 225 230 235 240 Asp Tyr Arg Phe Leu Leu Leu Phe Pro Gln Gly Ala Arg Ala Glu His 245 250 255 Ala Gln Pro Leu Val Asp Arg Leu Cys Glu Phe Lys Arg Arg His Asp 260 265 270 Asp Asp Ala Pro Leu Lys Gln Val Leu Pro Glu Leu Leu Asp Ser Ser 275 280 285 Pro Leu Tyr Arg Tyr Ile Gly Leu Arg Glu Leu Cys Ala Met Ile His 290 295 300 Glu Ala Ser Leu Arg Leu His Leu Thr Ala Leu Ala Asp Ala Ala Ala 305 310 315 320 Arg Thr Ala Gly His Ala Ala Leu Ala Pro Ala Thr Val Tyr Gly His 325 330 335 Leu Val Arg Asp Glu Thr Glu Ala Val Ala Ile Asp Arg Leu Gly Gly 340 345 350 Arg Val Val Ala Ser Leu Val Gly Val His Pro Ala Ala Thr Pro Leu 355 360 365 Leu Leu Pro Gly Glu Arg Val Ala Glu Glu Ser Pro Ala Leu Ile Asp 370 375 380 Tyr Leu Leu Ala Leu Gln Ala Phe Gly Glu His Phe Pro Gly Phe Ala 385,390,395,400 Pro Glu Leu Gln Gly Ile Glu Ile Asp Glu Arg Gly Arg Tyr Arg Val 405 410 415 Arg Cys Val Arg Pro Wing Wing Leu Wing Arg Gly Ser Val Leu Arg Leu 420 425 430 Ala Thr Arg Arg Pro Asp 435 <210> 3 <211> 2256 <212> DNA <213> Pseudomnas aeruginosa <400> 3 atgtataaag acctcaaatt tcccgtcctc atcgtccatc gcgacatcaa ggccgacacc 60 gttgccggcg aacgcgtgcg gggcatcgcc cacgaactgg agcaggacgg cttcagcatt 120 ctctccaccg ccagctccgc cgaggggcgc atcgcgctt ccacccacca cggcctggcc 180 tgcattctgg tcgccgccga aggtgccggg gaaaaccagc gcctgctgca ggatgtggtc 240 gaactgatcc gcgtggcccg cgtgcgggcg ccgcaattgc cgatcttcgc cctcggcgag 300 caggtgacca tcgagaacgc gccggccgag tccatggccg acctgcacca gttgcgcggc 360 atcctctacc tgttcgaaga caccgtgccg ttcctcgccc gccaggtcgc ccgggcggcg 420 cgcaactacc tggccgggct gctgccgcca ttcttccgtg cgctggtcga gcacaccgcg 480 cagtccaact attcctggca tacgccgggc cacggcggcg gtgtcgccta tcgcaagagt 540 ccggtgggac aggcgttcca ccagttcttc ggggagaaca cgctgcgttc cgacctgtcg 600 gtctcggtcc ccgagctggg atcgctgctc gaccataccg gccccctggc cgaggccgag 660 gaccgtgccg cgcgcaattt cggcgccgac cataccttct tcgtgatcaa tggcacttcc 720 accgcgaaca agatcgtctg gcactccatg gtcggtcgcg aagacctggt gctggtggac 780 cgcaactgcc acaagtcgat cctccactcg atcatcatga ccggggcgat accgctctac 840 ctgactccgg aacgcaacga actggggatc atcgggccga tcccgctgag cgaattcagc 900 aagcagtcga tcgccgcgaa gatcgccgcc agcccgctgg cgcgcggccg cgagccgaag 960 gtgaagctgg cggtggtgac taactccacc tacgacggcc tgtgctacaa cgccgagctg 1020 atcaagcaga ccctcggcga cagcgtcgag gtgttgcact tcgacgaggc ttggtacgcc 1080 tatgccgcgt tccacgagtt ctacgacgga cgctatggca tgggcacctc gcgcagcgag 1140 gagggacccc tggtgttcgc cacccactcc acgcacaaga tgctcgccgc cttcagccag 1200 gcctcgatga tccacgtgca ggatggcggg acccggaagc tggacgtggc gcgcttcaac 1260 gaagccttca tgatgcacat ctcgacctcg ccgcagtacg gcatcatcgc ttcgctggac 1320 gtggcttcgg cgatgatgga agggcccgcc gggcgttcgc tgatccagga gaccttcgac 1380 gaggccctca gcttccgccg ggccctggcc aacgtacggc agaacctgga ccggaacgac 1440 tggtggttcg gcgtctggca gccggagcag gtggagggca ccgaccaggt cggcacccat 1500 gactgggtgc tggagccgag cgccgactgg cacggcttcg gcgatatcgc cgaagactac 1560 gtgctgctcg acccgatcaa ggtcaccctg accaccccgg gcctgagcgc tggcggcaag 1620 ctcagcgagc aggggattcc ggccgccatc gtcagccgct tcctctggga gcgcgggctg 1680 gtggtggaga aaaccggtct ctactccttc ctggtgctgt tctcgatggg catcaccaag 1740 ggcaagtgga gcaccctggt caccgaactg ctcgaattca agcgctgtta cgacgccaac 1800 ctgccgctgc ttgacgtctt gccctccgtg gcccaggccg gcggcaagcg ctacaacgga 1860 gtgggcctgc gcgacctcag cgacgccatg cacgccagct accgcgacaa cgccacggcg 1920 aaggccatga agcgcatgta cacggtgctg ccggaggtcg cgatgcggcc gtccgaggcc 1980 tacgacaagc tggtgcgcgg cgaggtcgag gcggtaccga tcgctcggtt ggaagggcgc 2040 atcgcggccg tcatgctggt accctatccg ccgggtatcc cgctgatcat gccgggtgag 2100 cgcttcaccg aggcgacccg ctcgatcctc gactatctcg agttcgcgcg gaccttcgag 2160 cgcgccttcc ctggttcga ctccgatgtg catggcctgc agcatcagga cggaccgtcc 2220 gggcgctgct ataccgttga atgcataaag gaatga 2256 <210> 4 <211> 751 <212> PRT <213> Pseudomonas aeruginosa <400> 4 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ser Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 5 <211> 38 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 5 agtttattct tgacatgtag tgagggggct ggtataat 38 <210> 6 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 6 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Cys Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645,650,655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690,695,700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705,710,715,720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys and Lys Glu 740,745,750 <210> 7 <211> 1333 <212> DNA <213> Hafnia alvei <400> 7 ttgactttgt taaaagtcag gcataagatc aaaatactgt atatataaca atgtatttat 60 atacagtatt ttactttt tatctaacgt cagagagggc aatattatga gtggtggaga 120 tggcaagggt cacaatagtg gagcacatga ttccggtggc agcattaatg gaacttctgg 180 gaaaggtggg ccatcaagcg gaggagcatc agatattct gggtggagtt cggaaaataa 240 cccgtggggc ggtggtact cgggaatgat tggtggcagt caggaggta acggagcta 300 tcatggtggc gaaatacat cttctaacta tgggaagat gtatcacgcc aaatcggtga 360 tgcgatagcc agaaggaag gcatcaatcc gaaaatattc actgggtact ttatccgttc 420 agatggatat ttgatcggaa taacgccact tgtcagtggt gatgctttg gcgttaatct 480 tggcctgttc aaacaatc aaatagtag tagtgaaat aagggatgga atggaagga 540 tggagatggc attaaaaata gtagccaagg tggatggag attaaaacta atgaacttac 600 ttcaaccaa gtagctgctg ctaaatccgt tccagaacct aaaatagta atattataa 660 gtccatgaga gaagctagcg atgaggttat taattctaat ttaaaccaag ggcatggagt 720 tggtgaggca gctagagctg aagagatta cagagaaaa gtaagaacg caatcaatga 780 taatagtccc aatgtgctac aggatgctat taatttaca gcagattttt attaggaagt 840 ttttaacgct tacggagaaa aagccgaaaa actagccaag ttattagctg atcaagctaa 900 aggtaaaag atccgcaatg tagagatgc attgaaatct tatgaaaac aaggctaa 960 cattaacaa aaaatcaatg cgaagatcg cgaagctatc gccaaggctt tggaagctat 1020 ggatgtagaa aaagccgca aaatatatc caagttcagc aaggactag gttgggttgg 1080 cccagctatc gattaactg attggtttac agaattatac aaagcagtga aactgataa 1140 ttggagatct ctttatgtta aaactgaaac tattgcagta gggctagctg caacccatgt 1200 caccgcctta gcattcagtg ctgtcttggg tggcctata gttattttag gttatggttt 1260 gattatggct ggggttgggg cgttagttaa cgagacaata gttgacgagg caataaggt 1320 Cattgggatt at 1333 <210> 8 <211> 336 <212> DNA <213> Hafnia Alvey <400> 8 ctatatttta gcggtcacat ttttattc aaaaaaaaca gaagaacac catay 60 tgatgtcata aaaaaaaa taaaatacaa agtcattaaa tatgttttg gcacaccatc 120 cttaaaaaa cctgttttcc aaaattcttt tttcgtatat ctaagcgctg cttctctat 180 tagaaaccga gagaaaggaa atagaatagc gctagccaaa ccaaagattc tgagcgcaat 240 tattttaggt tcgtcatcac cataactggc gtaaagaata caagcagcca taaagtatcc 300 ccaaaacata ttatgtatgt aatatttcct tgtcat 336 <210> 9 <211> 1077 <212> DNA <213> Hafnia alvei <400> 9 atgagtggtg gagacggtaa aggtcacaat agtggagcac atgattccgg tggcagcatt 60 aatggaactt cggggaaagg tggacctgat tctggtggcg gatattggga caaccatcca 120 catattacaa tcaccggtgg acgggaagta ggtcaagggg gagctggtat caactggggt 180 ggtggttctg gtcatggtaa cggcgggggc tcagttgcca tccaagaata taacacgagt 240 aaatatccta acacgggagg atttcctcct cttggagacg ctagctggct gttaaatcct 300 ccaaaatggt cggttattga agtaaaatca gaaaactcag catggcgctc ttatattact 360 catgttcaag gtcatgttta caaattgact tttgatggta cgggtaagct cattgatacc 420 gcgtatgtta attatgaacc cagtgatgat actcgttgga gcccgcttaa aagttttaaa 480 tataataaag gaaccgctga aaaacaggtt agggatgcca ttaacaatga aaaagaagca 540 gttaaggacg ctgttaaatt tactgcagac ttctataaag aggtttttaa ggtttacgga 600 gaaaaagccg agaagctcgc taagttatta gcagatcaag ctaaaggcaa aaaggttcgc 660 aacgtagaag atgccttgaa atcttatgaa aatataaga ctaacattaa caaaaaaatc 720 aatgcgaaag atcgcgaagc tattgctaaa gccttggagt ctatggatgt aggaaaagcc 780 gcaaaaata tagccaagtt cagtaaagga ctaggttggg ttggccctgc tatcgatata 840 actgattggt ttacagaatt atacaaggca gtggaaactg ataattggag atctttttat 900 gttaaaactg aaactattgc agtagggcta gctgcaaccc atgttgccgc cttggcattc 960 agcgctgtct tgggtgggcc tgtaggtatt ttgggttatg gtttgattat ggctggggtt 1020 ggggcgttag ttaatgagac aatagttgac gaggcaaata aggttattgg gctttaa 1077 <210> 10 <211> 336 <212> DNA <213> Hafnia alvei <400> 10 ctataattta gcggtcacat ttttattc aaaaaaaaca gaataacac ctataggaat 60 tgatgtcata aaaataaaa ttaaatacaa agtcattaaa tatgttttg gcacgccatc 120 cttaaaaaaa ccagttccc aaaatttcttt tttcgtatat ctaagcgcgg ttttctctat 180 taaaaaccga gagaaaggga ataggatagc actagccaaa ccaagattc tgagcgcaat 240 tattttaggt tcgttatccc cataactggc gtaagaata caacagcca taaagtaccc 300 ccaaaacata ttgtatat atattttcct tgtcat 336 <210> 11 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 11 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ser Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Thr Cys His Lys Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 12 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 12 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ser Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Asn Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 13 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 13 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Cys Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Thr Cys His Lys Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 14 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 14 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Cys Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Asn Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 15 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 15 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ser Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Thr Cys His Asn Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 16 <211> 751 <212> PRT <213> artificial <220> <223> synthetic, synthesized <400> 16 Met Tyr Lys Asp Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Gly Ile Ala His Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ser Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Cys Met 100 105 110 Ala Asp Leu His Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Ala Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Asp Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Glu Asp Leu 245 250 255 Val Leu Val Asp Arg Thr Cys His Asn Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Gln Ser Ile 290 295 300 Ala Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Thr Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ser Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Met Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Thr Arg Lys Leu Asp Val 405 410 415 Ala Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Arg Gln Asn Leu Asp Arg Asn Asp 465 470 475 480 Trp Trp Phe Gly Val Trp Gln Pro Glu Gln Val Glu Gly Thr Asp Gln 485 490 495 Val Gly Thr His Asp Trp Val Leu Glu Pro Ser Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Ile Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Thr Thr Pro Gly Leu Ser Ala Gly Gly Lys Leu Ser Glu Gln 530 535 540 Gly Ile Pro Ala Ala Ile Val Ser Arg Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Val Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Cys Tyr Asp Ala Asn Leu Pro Leu Leu Asp Val Leu Pro 595 600 605 Ser Val Ala Gln Ala Gly Gly Lys Arg Tyr Asn Gly Val Gly Leu Arg 610 615 620 Asp Leu Ser Asp Ala Met His Ala Ser Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Lys Arg Met Tyr Thr Val Leu Pro Glu Val Ala Met Arg 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Ala Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Glu Phe Ala Arg Thr Phe Glu 705 710 715 720 Arg Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Gln 725 730 735 Asp Gly Pro Ser Gly Arg Cys Tyr Thr Val Glu Cys Ile Lys Glu 740 745 750 <210> 17 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 17 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tctatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtaactgtc acaaatctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 18 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 18 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tgtatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtaactgtc acaaatctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 19 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 19 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tctatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtacctgtc acaaatctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 20 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 20 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tctatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtaactgtc acaactctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 21 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 21 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tgtatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtacctgtc acaaatctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 22 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 22 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tgtatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtaactgtc acaactctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 23 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 23 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tctatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtacctgtc acaactctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256 <210> 24 <211> 2256 <212> DNA <213> artificial <220> <223> synthetic, synthesized <400> 24 atgtacaaag atctgaaatt ccctgttctg attgtacacc gcgatatcaa ggcggacacg 60 gtagccggcg agcgtgttcg cggtattgcc cacgaactcg aacaagacgg ctttagcatt 120 ctctctacgg cgtcttctgc ggaaggccgc attgtggcta gcacgcacca cggtctcgcc 180 tgcatcctcg tggcagctga gggtgcgggt gagaatcagc gtctgctcca agacgtggtt 240 gagctgatcc gtgtagctcg cgtccgtgcg ccacagctcc cgatcttcgc gctgggcgaa 300 caggtgacta ttgaaaacgc gcctgccgaa tgtatggccg acctgcacca gctccgcggc 360 attctgtatc tcttcgagga tactgtcccg ttcctggcac gtcaggttgc acgcgcagcg 420 cgtaactacc tcgctggcct cctcccgcca ttcttccgtg cactcgtgga gcacacggcc 480 caaagcaatt actcttggca caccccgggt cacggtggtg gtgtcgctta ccgtaaatct 540 ccggtaggtc aagctttcca ccagttcttt ggcgagaata ccctccgctc tgacctgtct 600 gttagcgttc cagagctggg cagcctgctg gatcacactg gccctctcgc ggaagcagag 660 gatcgtgccg ctcgcaattt cggtgcggac cacaccttct ttgtcatcaa tggtacctct 720 actgcgaaca aaatcgtttg gcactctatg gttggtcgcg aggacctggt gctggtcgat 780 cgtacctgtc acaactctat tctgcactcc attatcatga cgggtgctat cccactgtac 840 ctgactccgg aacgcaacga actgggtatt atcggcccta ttccactctc cgagttttct 900 aaacaatcta tcgcagcaaa aattgccgcc tccccactcg cgcgtggtcg tgaaccgaaa 960 gttaaactgg ctgtcgttac caactctacc tatgacggtc tgtgttacaa cgcggaactg 1020 atcaaacaaa ccctcggcga ctctgtcgag gtactgcatt tcgacgaggc ttggtatgct 1080 tatgcggcgt ttcacgagtt ctacgacggc cgctacggta tgggcacttc tcgttccgaa 1140 gagggtccgc tggtctttgc tacccattct acccacaaga tgctcgcggc tttttcccaa 1200 gctagcatga ttcacgttca ggatggtggt acgcgcaagc tggacgtcgc ccgctttaac 1260 gaagccttta tgatgcacat cagcacctct ccacagtacg gcatcattgc gtctctcgat 1320 gtcgcaagcg ctatgatgga aggtcctgcc ggtcgtagcc tgatccaaga gacgttcgat 1380 gaggcgctgt ccttccgtcg tgctctggcg aatgtccgtc agaacctgga ccgtaatgat 1440 tggtggttcg gtgtctggca accggagcag gttgagggca ccgaccaggt aggtactcac 1500 gactgggttc tcgagcctag cgcggactgg catggttttg gtgacattgc ggaggattac 1560 gttctcctcg atcctatcaa agttaccctg accaccccag gtctgagcgc tggcggtaaa 1620 ctctctgaac aaggcatccc ggcagctatc gttagccgtt tcctgtggga acgtggtctg 1680 gtggtcgaga aaacgggtct gtactctttc ctggttctgt tctccatggg tatcacgaaa 1740 ggcaaatggt ctactctggt taccgagctg ctcgaattca aacgctgtta cgacgcgaat 1800 ctgccactcc tggatgtgct gccttctgta gcgcaggcgg gtggtaaacg ctataacggt 1860 gtaggtctgc gtgatctgtc cgatgccatg cacgcttctt atcgtgacaa tgccacggcg 1920 aaggccatga agcgtatgta tacggtgctc ccggaagtag ccatgcgccc gtccgaagct 1980 tatgataagc tcgtacgcgg tgaagtcgaa gctgttccta ttgcacgtct cgagggtcgt 2040 attgcggcgg ttatgctggt tccgtacccg ccaggtatcc cgctcattat gccgggtgaa 2100 cgttttactg aagctacccg ctccattctg gactatctgg agtttgcccg taccttcgag 2160 cgcgcgttcc cgggctttga ctctgatgtt cacggcctcc aacatcaaga tggcccgtct 2220 ggccgttgtt ataccgttga atgcatcaag gaataa 2256

Claims

1. A transformant comprising an expression plasmid vector in a host cell that is not a Pseudomonas aeruginosa cell, the expression plasmid vector comprising: one or more polynucleotides encoding one or more polypeptides selected from mutants of the amino acid sequence of SEQ ID NO: 4 (Ldc2); and A backbone plasmid capable of autonomous replication in the host cell; The mutant of the amino acid sequence of SEQ ID NO:4 (Ldc2) is selected from the amino acid sequences of SEQ ID NO:6 (Ldc2 S111C), SEQ ID NO:11 (Ldc2 N262T) and SEQ ID NO:12 (Ldc2 K265N).

2. The transformant of claim 1, wherein the polynucleotide sequence comprises a mutant of the polynucleotide sequence of SEQ ID NO:3 (ldc2), wherein the mutant comprises a mutation selected from the following: the mutation at nucleotide position 332 is mutated to G, the mutation at nucleotide position 785 is mutated to C, and the mutation at nucleotide position 795 is mutated to T or C.

3. The transformant according to claim 1 or 2, wherein the host cell is an Escherichia coli (E. coli) cell.

4. The transformant of claim 3, wherein the sequence of the polynucleotide is codon-optimized for optimal polypeptide expression in E. coli cells. The transformant of claim 3 , wherein the backbone plasmid is an E. coli expression plasmid vector.

6. The transformant of claim 5, wherein the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101, and any derivative plasmids thereof.

7. The transformant of claim 5, wherein the expression plasmid vector further comprises a promoter polynucleotide sequence, wherein the promoter polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 5, which is located upstream of the polynucleotide sequence.

8. The transformant according to claim 1 or 2, wherein the host cell is a H. alvei cell.

9. An expression plasmid vector comprising: A polynucleotide encoding one or more polypeptides selected from mutants of the amino acid sequence of SEQ ID NO: 4 (Ldc2); and A backbone plasmid capable of autonomous replication in host cells; wherein the host cell is not a Pseudomonas aeruginosa cell; The mutant of the amino acid sequence of SEQ ID NO:4 (Ldc2) is selected from the amino acid sequences of SEQ ID NO:6 (Ldc2S111C), SEQ ID NO:11 (Ldc2 N262T) and SEQ ID NO:12 (Ldc2 K265N).

10. The expression plasmid vector of claim 9, wherein the host cell is an Escherichia coli cell.

11. The expression plasmid vector of claim 10, wherein the sequence of the polynucleotide is a codon-optimized polynucleotide for optimal polypeptide expression in E. coli cells.

12. The expression plasmid vector of claim 11, wherein the backbone plasmid is an Escherichia coli expression plasmid vector.

13. The expression plasmid vector of claim 12, wherein the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101, and any derivative plasmids thereof.

14. The expression plasmid vector of claim 12, wherein the expression plasmid vector further comprises a promoter polynucleotide sequence, the promoter polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 5, which is located upstream of the polynucleotide sequence.

15. The expression plasmid vector of claim 9, wherein the host cell is a Hafnia alvei cell.

16. A mutant host cell comprising a polynucleotide integrated into a host cell chromosome, wherein the polynucleotide encodes a polypeptide comprising one or more polypeptides selected from a mutant of the amino acid sequence of SEQ ID NO: 4 (Ldc2); The mutant of the amino acid sequence of SEQ ID NO:4 (Ldc2) is selected from the amino acid sequences of SEQ ID NO:6 (Ldc2 S111C), SEQ ID NO:11 (Ldc2 N262T) and SEQ ID NO:12 (Ldc2 K265N).

17. The mutant host cell of claim 16, wherein the host cell is an Escherichia coli cell.

18. A method for preparing a polypeptide comprising a mutant of the amino acid sequence of SEQ ID NO: 4 (Ldc2), the method comprising: Obtaining the mutant host cell of claim 16 or 17 and / or the transformant of any one of claims 1 to 8; culturing the mutant host cell under conditions effective to express the polypeptide; and The polypeptide is harvested.

19. A method for preparing cadaverine (1,5-pentanediamine), the method comprising: 1a) culturing the mutant host cell of claim 16 or 17 and / or the transformant of any one of claims 1 to 8; 1b) producing cadaverine by decarboxylating lysine using the culture obtained in step 1a; and 1c) extracting and purifying cadaverine using the culture obtained from step 1b.

20. A polypeptide comprising a mutant of the amino acid sequence of SEQ ID NO:4 (Ldc2), wherein the mutant of the amino acid sequence of SEQ ID NO:4 (Ldc2) is selected from the amino acid sequences of SEQ ID NO:6 (Ldc2 S111C), SEQ ID NO:11 (Ldc2N262T) and SEQ ID NO:12 (Ldc2 K265N).

21. A non-naturally occurring polynucleotide encoding the polypeptide of claim 20.

22. The non-naturally occurring polynucleotide of claim 21, wherein the mutation at nucleotide position 332 is a mutation to a G, the mutation at nucleotide position 785 is a mutation to a C, or the mutation at nucleotide position 795 is a mutation to a T or a C.

23. The non-naturally occurring polynucleotide of claim 21, wherein the polynucleotide sequence encoding Ldc2 is the polynucleotide sequence of SEQ ID NO: 3 or a codon-optimized polynucleotide sequence thereof, wherein the codon-optimized polynucleotide sequence is the polynucleotide sequence of SEQ ID NO:

17.

24. The non-naturally occurring polynucleotide of claim 21, selected from the group consisting of SEQ ID NO: 18 (ldc2-col C332G), SEQ ID NO: 19 (ldc2-col A785C), and SEQ ID NO: 20 (ldc2-col A795C).

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