RU2810730C2 - Use of glycosidases in oligosacharide production - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: invention relates to the production of oligosaccharides. The proposed method of producing 2'-fucosyllactose using a genetically engineered Escherichia coli cell involves providing a genetically engineered Escherichia coli cell that produces 2'-fucosyllactose. Moreover, the Escherichia coli cell is genetically engineered to express at least one heterologous α-1,3-fucosidase which carries out the intracellular degradation of 3-fucosyllactose and 2'3-difucosyllactose formed during the intracellular biosynthesis of 2'-fucosyllactose. Escherichia coli cell reuses such degradation products as lactose and fucose obtained as a result of the enzymatic activity of the specified α-1,3-fucosidase to produce 2'-fucosyllactose, wherein the genetically engineered Escherichia coli cell contains a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase to conserve fucose to produce 2'-fucosyllactose. Genetically engineered Escherichia coli cell is cultivated under conditions and in a medium that are permissive with respect to the production of 2'-fucosyllactose, thereby obtaining 2'-fucosyllactose and possibly isolating 2'-fucosyllactose. Also the following is proposed: a genetically engineered Escherichia coli cell for the production of 2'-fucosyllactose and its use for the production of 2'-fucosyllactose.

EFFECT: invention is aimed at producing 2'-fucosyllactose with fewer non-target by-products.

8 cl, 1 dwg, 2 tbl, 4 ex

Description

The present invention relates to the production of oligosaccharides by microbial fermentation. More specifically, the present invention relates to the use of glycosidases to improve the production of target oligosaccharides by microbial fermentation.

Prior Art

Human breast milk contains a unique mixture of different oligosaccharides called Human Milk Oligosaccharides (HMOs). To date, more than 150 structurally different oligosaccharides have been identified in human breast milk. With very few exceptions, OGMs are characterized by a lactose moiety at their reducing end, and many OGMs contain a fucose moiety and/or an N-acetylneuraminic acid moiety at their non-reducing end. Typically, the monosaccharide residues of OGM are derived from D-glucose, D-galactose, N-acetylglucosamine, L-fucose and N-acetylneuraminic acid. The importance of HMOs in infant nutrition is directly related to their unique biological activities, including protecting the newborn from pathogens and supporting the development of the infant's immune system and cognitive abilities. Thus, there is great interest in producing OGM on a commercial scale.

In addition to the chemical synthesis of individual HMOs, significant progress has been made in developing the production of HMOs through microbial fermentation using genetically modified microorganisms that overexpress a heterologous glycosyltransferase. When such microorganisms are cultivated in a medium and under conditions that are permissive for the microorganisms to express said heterologous glycosyltransferase, OGM can be produced by said microorganism and isolated from the culture medium or cell lysate.

However, glycosyltransferases often have enzymatic side activities such that their overexpression to produce the desired oligosaccharide usually results in by-products that are undesirable. Typically, these by-products are also oligosaccharides, but must be removed from the target oligosaccharide preparation for commercialization of the product. However, removing such by-products from the target oligosaccharide is difficult and time-consuming. One approach to removing such by-products involves the use of glycosidases, which are either exogenously added to the reaction mixture/cell medium containing the desired and undesired oligosaccharides, or produced by a genetically engineered microorganism when induced at a specific point in time at the end of the fermentation process to produce the target oligosaccharide.

International publication WO 2015/032412 A1 relates to the use of fucose and discloses a method in which a genetically modified cell expressing a heterologous fucosyltransferase is cultured in the presence of lactose to produce and secrete a mixture of 2'-fucosyllactose (2'-FL- from English 2 '-fucosyllactose) and difucosyllactose (DFL - from the English difucosyllactose) into the extracellular space of the culture medium with high yield. The saccharides are separated and hydrolyzed by acid or fucosidase to produce fucose in high yield.

International Publication No. WO 2104/090261 A1 discloses a method for forming a mixture containing at least one of 2'-FL and 3-fucosyllactose β-FL), wherein DFL undergoes partial hydrolysis, for example enzymatic hydrolysis or acid hydrolysis. In enzymatic hydrolysis, DFL is exposed to fucosidase, which can release one of the fucose residues from DFL. DFL (10 mM) was incubated with 1,2-α-1_-fucosidase from Xanthomonas manihotis at 37°C in incubation buffer, and DFL hydrolysis was monitored by HPLC (high performance liquid chromatography). After 18 hours, DFL was partially hydrolyzed to 3-FL and fucose. Lactose was not detected.

European Patent Application No. EP 2845905 A1 relates to the preparation of oligosaccharides and discloses the use of one or more glycosidases in a process for the preparation and/or purification of an oligosaccharide. The method includes a) cultivating a host microorganism suitable for the production of the target oligosaccharide, under conditions and in an environment that are permissive for the production of the specified target oligosaccharide, resulting in the production of this oligosaccharide and, where applicable, biosynthetic intermediates - saccharides and/or by-products ; b) the use of glycosidase in the environment in which the host microorganism is cultivated to degrade biosynthetic intermediates - saccharides and/or by-products - saccharides and/or unused saccharide substrates; and c) isolating the target oligosaccharide. In one embodiment, said glycosidase is endogenously produced in a host microorganism, wherein the glycosidase is a glycosidase that does not naturally occur in the host microorganism, and wherein expression of said glycosidase in said host microorganism is inducible such that the expression can be initiated after a sufficient and/or substantially maximum amount of the target oligosaccharide has been produced during cultivation of the host microorganism.

In summary, the prior art discloses the use of glycosidases to remove non-target oligosaccharides from a mixture of target and non-target oligosaccharides by hydrolyzing the non-target oligosaccharides in the reaction mixture/cellular environment. However, these approaches involve the biosynthesis of non-target oligosaccharides by a microorganism, including the use of substrates and energy, and these approaches require the removal of degradation products of non-target oligosaccharides from the target oligosaccharides.

Thus, the object of the present invention was to provide a method for producing a target oligosaccharide by microbial fermentation without the co-production/accumulation of non-target saccharide by-products, namely non-target oligosaccharides, in a cellular environment containing the microorganism to be fermented.

The objective is achieved by providing a genetically engineered microbial host cell capable of producing a target oligosaccharide, wherein said microbial host cell expresses a heterologous glycosidase that is capable of intracellularly degrading metabolic by-products that are formed during intracellular biosynthesis of the target oligosaccharide, thereby preventing the formation of a mixture of target oligosaccharides. and non-target saccharides in the culture medium. These degradation products can then be used in the metabolism of the microbial host cell, for example, for the biosynthesis of the target oligosaccharide.

Table 1 provides a detailed overview of the target oligosaccharides and possible precursors that are added and/or non-target saccharide byproducts that are formed during the production of the target oligosaccharide.

Summary of the invention

In the first aspect, a method for producing a target oligosaccharide is disclosed using a genetically engineered microbial host cell that is capable of producing the target oligosaccharide, wherein said microbial host cell expresses a heterologous glycosidase capable of intracellular degradation of metabolic by-products - saccharides that are formed during the intracellular biosynthesis of the target oligosaccharide.

A second aspect discloses a genetically engineered microbial host cell for producing a target oligosaccharide, wherein said microbial host cell is capable of producing the target oligosaccharide, and wherein said microbial host cell has been genetically engineered to express a heterologous glycosidase that can perform intracellular degradation of metabolic byproducts - saccharides that are formed during the intracellular biosynthesis of the target oligosaccharide.

The third aspect discloses the use of a genetically engineered microbial host cell according to the second aspect to produce a target oligosaccharide.

In a fourth aspect, oligosaccharides are disclosed, namely, desired oligosaccharides that are produced by the method of the first aspect and/or by using a genetically engineered microbial host cell according to the second aspect.

The fifth aspect discloses the use of the target oligosaccharides according to the fourth aspect to produce a nutritional composition.

The sixth aspect discloses nutritional compositions containing the target oligosaccharide according to the fourth aspect.

Brief description of graphic materials

In FIG. 1 shows a schematic representation of an embodiment of a microbial host cell expressing a heterologous glycosidase (e.g., alpha-1,3-fucosidase) that can degrade the metabolic by-products saccharides (e.g., 3-fucosyllactose and 2'3-difucosyllactose) that are produced during intracellular biosynthesis of the target oligosaccharide (2'-fucosyllactose), and where the microbial host cell can reuse degradation products (eg, fucose and lactose) resulting from the enzymatic activity of said glycosidase to produce the target oligosaccharide.

Detailed description

According to the first aspect, a method is provided for producing a target oligosaccharide using a genetically engineered microbial host cell, which includes the following steps:

(i) providing a genetically engineered microbial host cell that is capable of producing the target oligosaccharide, where the microbial host cell is genetically engineered to express a heterologous glycosidase that can perform intracellular degradation of metabolic by-products - saccharides that are formed during the intracellular biosynthesis of the target oligosaccharide, and where the microbial host cell is capable of recycling degradation products resulting from the enzymatic activity of said glycosidase;

(ii) culturing the genetically engineered microbial host cell under conditions and environments that are permissive for the production of the target oligosaccharide, thereby obtaining the target oligosaccharide; And

(iii) the target oligosaccharide can be isolated.

The term “target”, as used herein in relation to oligosaccharides, refers to an oligosaccharide that is intended to be produced by a microbial host cell. The term "target" is used to distinguish between the oligosaccharide being targeted for production and other oligosaccharides that the microbial host cell may produce. These other oligosaccharides are considered "undesirable" regardless of whether or not these other oligosaccharides have a biological function, whether or not they participate in the biosynthesis of other cellular compounds such as glycolipids, glycoproteins or polysaccharides, or whether or not they are metabolic products of saccharides. which are formed during the intracellular biosynthesis of the target oligosaccharide, or due to the reserve (non-target) enzymatic activities of one or more enzymes involved in the biosynthesis of the target oligosaccharide, or due to the enzymatic activity of one or more enzymes that are not directly involved in the biosynthesis of the target oligosaccharide, but use an oligosaccharide as a substrate that is formed as an intermediate in a metabolic pathway, resulting in the target oligosaccharide.

The term "oligosaccharide", as used herein, refers to a saccharide molecule consisting of three to twenty monosaccharide units, wherein each of said monosaccharide units is linked to at least one other of said monosaccharide units by a glycosidic linkage. An oligosaccharide may be a linear chain of monosaccharide residues or a branched chain of monosaccharide residues.

In a further and/or alternative embodiment, the target oligosaccharide is human milk oligosaccharide (HMO).

In an additional and/or alternative embodiment, the target oligosaccharide is an OGM selected from the group consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2'3-difucosyllactose (DFL), lacto- N-triose II, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-I), lacto-N -fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I, lacto-N-difucosylhexaoses II, para-Lacto-N-fucosylhexaoses, fucosyl-lacto-N-sialylpentaoses b, fucosyl-lacto-N-sialylpentaoses c, fucosyl-lacto-N-sialylpentaoses c, disialyl-lacto-N- fucopentaose, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyllactose, lacto-N-neodifucohexaose I, 3'-sialyllactose β-SL), 6'-sialyllactose (6-SL), sialyllacto-N- tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c) and disialyl lacto-N-tetraose.

The method includes providing a genetically engineered microbial host cell that is capable of producing the target oligosaccharide.

The term “genetically engineered,” as used herein, refers to the modification of the genetic makeup of a cell using molecular biology techniques. Modification of the genetic composition of a cell may include gene transfer within and/or across species relationships, insertion, deletion, substitution and/or modification of nucleotides, triplets, genes, open reading frames, promoters, enhancers, terminators and other nucleotide sequences, mediating and/or controlling gene expression. Modification of a cell's genetic composition aims to create a genetically modified organism that has specific, desirable properties. A genetically engineered microbial host cell may contain one or more genes that are not present in the native (non-genetically engineered) form of the cell. Methods for introducing exogenous nucleic acid molecules and/or inserting exogenous nucleic acid molecules (recombinant, heterologous) into the heritable information of a cell to insert, delete or change the nucleotide sequence of the genetic information of a cell are known to a qualified specialist. Genetically engineered cells may contain one or more genes that are found in the native form of the cell, where said genes are modified and reinserted into the cell by artificial means. The term "genetically engineered" also includes cells that contain a nucleic acid molecule that is endogenous to the cell and that is modified without removing the nucleic acid molecule from the cell. Such modifications include modifications resulting from gene replacement, site-specific mutations, and related techniques, including techniques commonly referred to as "genome editing".

The genetically engineered microbial host cell may be a prokaryotic cell or a eukaryotic cell. Suitable microbial host cells include yeast cells, bacterial cells, archaebacterial cells and fungal cells.

In a further and/or alternative embodiment, the prokaryotic cell is a bacterial cell, preferably a bacterial cell selected from a genus of bacteria selected from the group consisting of Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Micromonospora, Pseudomonas, Rhodococcus and Sporolactobacillus. Suitable bacterial species include Bacillus subtilis, B. licheniformis, B. coagulans, B. thermophiius, B. laterosporus, B. megaterium, B. mycoides, B. pumilus, B. lentus, B. cereus, B. circulans, Bifidobacterium longum , B. infantis, B, bifidum, Citrobacter freundii, Clostridium cellulolyticum, C. Ijungdahiii, C, autoethanogenum, C. acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, E. thermophiles, Escherichia coli, Erwinia herbicoia (Pantoea agglomerans), Lactobacillus acidophilus, L. salivarius, L, plantarum, L, helveticus, L. deibrueckii, L, rhamnosus, L. buigaricus, L. crispatus, L. gasseri, L, casei, L. reuteri, L. jensenii, L. lactis, Pantoea citrea , Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, P. aeruginosa, Streptococcus thermophiles and Xanthomonas campestris.

In a further and/or alternative embodiment, the eukaryotic cell is a yeast cell, preferably a yeast cell selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp., in particular Pichia pastoris, Hansenula sp. ., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp, and Schizosaccharomyces sp.

A genetically engineered microbial host cell can produce the target oligosaccharide. The term "can produce" as used herein refers to the ability of a genetically engineered microbial host cell to produce a target oligosaccharide, provided that the microbial host cell is cultured under conditions and media that are permissive for production of the target oligosaccharide by the microbial host cell. Thus, the medium must have a pH value in a certain range, a composition of ions and nutrients, and also contain compounds required to maintain the viability and metabolic activity of the microbial host cell. If necessary to produce the target oligosaccharide, the medium must also contain sufficient amounts of any precursor required for the biosynthesis of the target oligosaccharide by the microbial host cell. Likewise, the conditions (e.g., temperature, pH, oxygen supply, agitation, nutrient supply, etc.) for culturing a microbial host cell to produce the target oligosaccharide must be maintained such that the microbial host cell can be or remain metabolically active to produce the target oligosaccharide.

In a further and/or alternative embodiment, the genetically engineered microbial host cell capable of producing the target oligosaccharide is a microbial host cell that is genetically engineered to have the ability to produce the target oligosaccharide. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express a heterologous glycosyltransferase. The heterologous glycosidase is expressed in the genetically engineered microbial host cell during fermentation, namely during the production or biosynthesis of the target oligosaccharide. In a further and/or alternative embodiment, expression of the heterologous glycosidase is constitutive in the genetically engineered microbial host.

The term "heterologous", as used herein, refers to a nucleotide sequence, nucleic acid molecule or polypeptide that is foreign to a cell or organism, namely, a nucleotide sequence, nucleic acid molecule or polypeptide that do not naturally occur in the specified cell or organism. The term "heterologous sequence" or "heterologous nucleic acid" or "heterologous polypeptide", as used herein, is a sequence or nucleic acid or peptide that is derived from a source that is foreign to a particular host cell (e.g. from a different species), or, if from the same source, modified from its original form. Thus, the heterologous nucleic acid operably linked to a promoter comes from a source different from that of the promoter or, if from the same source, is modified from its original form. The heterologous sequence can be stably introduced, for example, through transfection, transformation, conjugation or transduction, into the genome of a microbial host cell, thereby representing a genetically modified host cell. Techniques can be used that will depend on the host cell, the sequence to be administered. Various techniques are known to one skilled in the art and, for example, are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Accordingly, a “heterologous polypeptide” is a polypeptide that does not naturally occur in the wild-type cell from which the genetically engineered cell is derived, and a “heterologous glycosyltransferase” is a glycosyltransferase that does not naturally occur in the wild-type cell from which the genetically engineered cell is derived. engineered cell.

In a further and/or alternative embodiment, the heterologous glycosyltransferase is selected from the group consisting of fucosyltransferases, preferably α-1,2-fucosyltransferases and α-1,3-fucosyltransferases, glycosyltransferases, galactosyltransferases, preferably β-1,3-galactosyltransferases and β-1 ,4-galactosyltransferases, sialyltransferases, preferably α-2,3-sialyltransferases and α-2,6-sialyltransferases, and N-acetyl-gluchosaminyltransferases.

Fucosyltransferases catalyze the transfer of fucose residues from the donor guanosine diphosphate-activated L-fucose (GDP-fucose) to several acceptor molecules. Fucosyltransferases are expressed in animals, plants, fungi and bacteria, and they are categorized according to their association with fucose on the acceptor substrate. Thus, α-1,2-, α-1,3/4- and α-1,6-fucosyltransferases are different from each other. Suitable fucosyltransferases for heterologous expression in a genetically engineered microbial host cell are disclosed, for example, in European Patent Application No. 17180176.

Sialyltransferases catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) residues from the donor CMP-Neu5Ac to acceptor molecules. Sialyltransferases have been found to be expressed in animals, plants, fungi and bacteria. Sialyltransferases are classified into categories according to the bond that is formed between NeuSAc and the acceptor molecule. Therefore, α-2,3-, α-2,6- and α-2,8-sialyltransferases are different from each other. Suitable sialyltransferases for heterologous expression in a genetically engineered microbial host cell are disclosed, for example, in European Patent Application No. 17183391.

Galactosyltransferases catalyze the transfer of a galactose residue from a donor, UDP (uridine diphosphate)-galactose, to acceptor substrates. Galactosyltransferases are differentiated based on the bond between galactose and the acceptor molecule that is formed. Therefore, β-1,3- and β-1,4-galactosyltransferases are different from each other. A suitable β-1,3-galactosyltransferase for heterologous expression in a genetically engineered microbial host cell is encoded by the Salmonella enterica wbdO gene. A suitable β-1,4-galactosyltransferase for heterologous expression in a genetically engineered microbial host cell is encoded by the lex1 gene of Aggregatibacter aphrophilus.

The genetically engineered microbial host cell is genetically engineered to express a heterologous glycosidase that can perform the intracellular degradation of metabolic byproducts - saccharides that are formed during the intracellular biosynthesis of the target oligosaccharide. Suitable glycosidases are glycosidases that are specific for a glycosidic bond that is hydrolyzed by enzymatic activity and/or for a substrate that is hydrolyzed by the glycosidase. Due to this specificity, the glycosidase hydrolyzes unwanted by-products rather than the target oligosaccharide to be produced. In a further and/or alternative embodiment, the glycosidase does not hydrolyze one or more precursors that are taken up or synthesized by the microbial host cell to produce the target oligosaccharide. Preferably, the glycosidase is an exoglycosidase.

Exoglycosidases are glycoside hydrolase enzymes that break down glycosidic bonds at the terminal residue of the oligosaccharide structure.

In an additional and/or alternative embodiment, the heterologous glycosidase is selected from the group consisting of fucosidases, including α-1,2-fucosidases and α-1,3-fucosidases, sialidases, such as α-2,3-sialidases, α-2, 6-sialidases, α-2,8-sialidases, galactosidases such as β-1,3-galactosidases, β-1,4-galactosidases and β-1,6-galactosidases, β-N-acetylhexosaminidases and glucosidases such as β-1,3-glucosidases.

A suitable fucosidase is α-1,2-fucosidase. α-1,2-fucosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of linear alpha-1,2-linked L-fucopyranosyl residues from oligosaccharides. A preferred α-1,2-fucosidase is Bifidobacterium bifidum AfcA (SEQ ID NO: 2).

In an additional and/or alternative embodiment, a genetically engineered microbial host cell is provided that can produce 3-FL, wherein said genetically engineered microbial host cell expresses α-1,2-fucosidase. In order to be able to produce 3-FL, the genetically engineered microbial host cell expresses alpha-1,3-fucosyltransferase. This alpha-1,3-fucosyltransferase can transfer a fucose residue from GDP-fucose to the glucose moiety of lactose as an acceptor substrate, thereby synthesizing 3-FL as the target oligosaccharide. 2'-FL and 2'3-DFL are undesirable saccharide by-products in the preparation of 3-FL.

By expressing a heterologous α-1,2-fucosidase in a genetically engineered microbial host cell that can produce 3-FL, the production of 2'-FL and 2'3-DFL by-products can be stopped or at least reduced, since these by-products the products are hydrolyzed within the genetically engineered microbial host cell under the action of heterologous α-1,2-fucosidase. The resulting degradation products are fucose and lactose. Both fucose and lactose can be used by the genetically engineered microbial host cell to produce the target 3-FL.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express α-1,2-fucosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding an α-1,2-fucosidase for its expression. Preferably, the nucleotide sequence encoding α-1,2-fucosidase is a nucleotide sequence selected from the group consisting of:

- nucleotide sequence as represented by SEQ ID NO: 1;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 1, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 1 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 2; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 2, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 2.

The term “hybridizes” or “hybridization” as used herein means hybridization under conventional conditions as described in Sambrook et al. (1989) "Molecular Cloning, A Laboratory Manual" (Cold Spring Harbor Laboratory Press, New York), preferably under harsh conditions. Stringent hybridization conditions, for example, are as follows: hybridization in 4× SSC at 65°C followed by multiple washes in 0.1× SSC at 65°C for a total of approximately 1 hour. Less stringent hybridization conditions are, for example, as follows: hybridization in 4 × SSC at 37°C and subsequent multiple washes in 1 × SSC at room temperature (approximately 21°C). “Stringent hybridization conditions” may also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7% SDS (sodium dodecyl sulphate), 1 mM EDTA and 1% BSA (Bovine Serum Albumin) for 16 hours followed by two washes with 2 x SSC and 0.1% SDS at 68°C.

For the expression of a nucleotide sequence encoding α-1,2-fucosidase or a functional variant thereof, the nucleotide sequence is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding α-1,2-fucosidase or a functional variant thereof in the genetically engineered microbial host cell.

“Expression control sequences” are regulatory nucleotide sequences that are not part of the protein-coding nucleotide sequence, but mediate the expression of the protein-coding nucleotide sequence. Nucleotide sequences of regulatory elements include promoters, cis-regulatory elements, enhancers, introns and terminators. Depending on the type of regulatory element, it is located on the nucleic acid molecule before the nucleotide sequence encoding the protein (namely 3'), or behind the nucleotide sequence encoding the protein (namely 5'). Regulatory elements are functional in the microbial host cell.

The term "operably linked" means that the regulatory element is connected in such a way to the nucleotide sequence encoding the protein, namely, is located in such a way relative to the nucleotide sequence encoding the protein, for example, a nucleic acid molecule, that the expression of the nucleotide sequence encoding the protein is under the control of this regulatory element. element can occur in a living cell.

For purposes of the present invention, a “promoter” is a nucleotide sequence that regulates gene expression, which is typically located at the 5' end of a gene and, through interaction with specific DNA-binding proteins, mediates the initiation of transcription by RNA polymerase.

In addition, suitable promoters include synthetic promoters. There are promoters that have been created through molecular biology techniques that are not found in nature in a given configuration. A synthetic promoter is a minimal promoter containing only one or more selected specific cis elements in addition to the minimal promoter. These cis-elements represent binding sites for DNA-binding proteins such as transcription factors, and are isolated from natural promoters derived from previously isolated cis-elements, or technically obtained through random recombination techniques and selected by appropriate methods; Compared to a natural promoter, due to its less complex design, the synthetic promoter is activated by only a few exogenous and endogenous factors and is thus more specifically regulated.

A "minimal promoter" or "core promoter" is a nucleotide sequence that contains binding sites for a complex of basal transcription factors and allows precise initiation of transcription by RNA polymerase II. The characteristic sequence motifs of the minimal promoter are the TATA box, the initiator element (Inr), the “TFBII recognition element” (BRE) and the “downstream core promoter element” (OPE). In a minimal promoter, these elements may occur alone or in combination. The minimal promoter or its sequence motifs are available, for example, from a gene of a bacterium, fungus or virus.

"Cis elements" are nucleotide sequences that are located on the same nucleic acid molecule as the nucleotide sequence encoding the protein to be expressed. Cis elements do not have to encode RNA or protein and in the direction of transcription may be located before or after the nucleotide sequence encoding the protein to be expressed. Upstream cis elements upstream of the nucleotide sequence encoding the protein to be expressed often provide the necessary binding motifs, in particular for transcription factors that participate as trans-acting elements at the molecular level, on the other hand aspects in the regulation of transcription of this gene. If, in addition, cis elements lead to transcriptional inhibition, they are called silencers. Cis elements that lead to increased transcription are called enhancers. The combination of cis/trans activities in the promoter determines the intensity with which RNA polymerase performs transcription.

In addition, the promoter may be a chimeric promoter and/or a promoter that has been modified with cis elements. Modification of a promoter may also mean the additional inclusion of a cis element in a promoter that, for example, already naturally has a cis element. In addition, the modification also includes multimerization of a cis element, in particular multimerization of a naturally occurring cis element. Compared to the native version, such a modified promoter may, for example, have altered properties in terms of specificity, expression level or background activity.

Terminators are nucleotide sequences on DNA that typically mark the end of a gene and cause transcription to terminate.

Another suitable fucosidase is α-1,3-fucosidase. α-1,3-fucosidase is a highly specific glycosidase that catalyzes the hydrolysis of α-1,3-linked L-fucopyranosyl residues from oligosaccharides. A preferred α-1,3-fucosidase is AfcB from Bifidobacterium bifidum (SEQ ID NO: 4).

In an additional and/or alternative embodiment, a genetically engineered microbial host cell is provided that is capable of producing 2'-FL, wherein said genetically engineered host microorganism expresses α-1,3-fucosidase. In order to be able to produce 2'-FL, the genetically engineered microbial host cell expresses α-1,2-fucosyltransferase. This alpha-1,2-fucosyltransferase can transfer a fucose residue from GDP-fucose to the galactose moiety of lactose as an acceptor substrate, thereby synthesizing 2'-FL as the target oligosaccharide. 3-FL and 2'3-DFL are undesirable saccharide by-products in 2-FL products.

By expressing a heterologous α-1,3-fucosidase in a genetically engineered microbial host cell that can produce 2'-FL, the production of 3-FL and 2'3-DFL by-products can be stopped or at least reduced because that these by-products are hydrolyzed in the genetically engineered microbial host cell via heterologous α-1,3-fucosidase. The resulting degradation products are fucose and lactose. Both fucose and lactose can be used by the genetically engineered host microorganism to produce the target 2'-FL.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express α-1,3-fucosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding α-1,3-fucosidase for its expression. Preferably, the nucleotide sequence encoding α-1,3-fucosidase is a nucleotide sequence selected from the group consisting of:

- nucleotide sequence as represented by SEQ ID NO: 3;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 3, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 3 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 4; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 4, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 4.

For the expression of a nucleotide sequence encoding α-1,3-fucosidase or a functional variant thereof, said nucleotide sequence is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding α-1,3-fucosidase or a functional variant thereof in a genetically engineered microbial host cell.

In an additional and/or alternative embodiment, a genetically engineered microbial host cell is provided that can produce LNFP-I, wherein said genetically engineered microbial host cell expresses α-1,3-fucosidase. In order to be able to produce LNFP-I, the genetically engineered microbial cell expresses β-1,3-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase. This β-1,3-N-acetylglucosaminyltransferase can transfer the GlcNAc residue from UDP-GlcNAc to the galactose moiety of lactose, thereby synthesizing lacto-N-triose-11 (LNT-II). This β-1,3-galactosyltransferase can transfer a galactose residue from UDP-galactose to the GlcNAc moiety of LNT-II, thereby synthesizing lacto-N-tetraose (LNT). This α-1,2-fucosyltransferase can transfer a fucose residue from GDP-fucose to the terminal galactose moiety of LNT, thereby synthesizing LNFP-I. 3-FL and 2′3-DFL would be undesirable by-products in the production of LNFP-I. By expressing α-1,3-fucosidase in a genetically engineered microbial host cell capable of producing LNFP-I, the production of 3-FL and 2'3-DFL by-products can be stopped or at least reduced, for the reason that data the by-products are hydrolyzed by heterologous α-1,3-fucosidase within the genetically engineered microbial host cell. The resulting degradation products are fucose, lactose and 2'-FL. Fucose and lactose can be used by the genetically engineered host microorganism to produce the target LNFP-I.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express α-1,3-fucosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding α-1,3-fucosidase for its expression. Preferably, the nucleotide sequence encoding α-1,3-fucosidase is a nucleotide sequence selected from the group consisting of:

- nucleotide sequence as represented by SEQ ID NO: 3;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 3, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 3 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 4; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 4, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 4.

For the expression of a nucleotide sequence encoding α-1,3-fucosidase or a functional variant thereof, the nucleotide sequence is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding α-1,3-fucosidase or a functional variant thereof in a genetically engineered microbial cell -the owner.

A suitable sialidase is α-2,3-sialidase. α-2,3-Sialidase is a highly specific exoglycosidase that catalyzes the hydrolysis of linear α-2,3-linked L-sialyl residues from oligosaccharides. A preferred α-2,3-sialidase is Streptococcus pneumoniae NanB (SEQ ID NO: 6).

In an additional and/or alternative embodiment, a genetically engineered microbial host cell is provided that is capable of producing 6'-SL, wherein said genetically engineered microbial host cell expresses α-2,3 sialidase. In order to be able to produce 6'-SL, the genetically engineered microbial host cell expresses α-2,6-sialyltransferase. This 2,6-sialyltransferase can transfer the NeuSAc residue from CMP-Neu5Ac to the galactose moiety of lactose as a substrate, thereby synthesizing 6'-SL. 3'-SL is an off-target by-product in the production of 6'-SL.

By expressing α-2,3-sialidase in a genetically engineered microbial host cell that can produce 6'SL, production of the 3'-SL byproduct can be stopped or at least reduced because the byproduct is hydrolyzed by α-2,3-sialidases in a genetically modified microbial host cell. The resulting degradation products are N-acetylneuraminic acid and lactose. Both N-acetylneuraminic acid and lactose can be used by the genetically engineered host microorganism to produce the target 6'-SL.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express α-2,3-sialidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding an α-2,3-sialidase for its expression. Preferably, the nucleotide sequence encoding the α-2,3-sialidase is a nucleotide sequence selected from the group consisting of:

- the nucleotide sequence as represented by SEQ ID NO: 5;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 5, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 5 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 6; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 6, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 6.

For expression of a nucleotide sequence encoding α-2,3-sialidase or a functional variant thereof, said nucleotide sequence is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding α-2,3-sialidase or a functional variant thereof in a genetically engineered microbial host cell.

A suitable galactosidase is β-1,3-galactosidase. β-1,3-Galactosidase is an enzyme that catalyzes the hydrolysis of the β-1,3-linked galactose residue from oligosaccharides. A preferred β-1,3-galactosidase is Bifidobacterium longum Bga42A (SEQ ID NO: 8).

In an additional and/or alternative embodiment, a genetically engineered microbial host cell is provided that can produce LNnT, wherein the genetically engineered microbial host cell expresses β-1,3-galactosidase. In order to have the ability to produce LNnT, the genetically engineered microbial host cell expresses β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase. This β-1,3-N-acetylglucosaminyltransferase can transfer the GlcNAc residue from UDP-GlcNAc to the galactose moiety of lactose, thereby synthesizing LNT-II. This β-1,4-galactosyltransferase can transfer a galactose residue from UDP-galactose to the GlcNAc moiety of LNT-II, thereby synthesizing LNnT as the target oligosaccharide.

LNT is an unintended by-product in LNnT products. By expressing β-1,3-galactosidase in a genetically engineered microbial host cell capable of producing LNnT, production of the LNT byproduct can be stopped or at least reduced because the byproduct is hydrolyzed in the genetically engineered microbial host cell under action of heterologous β-1,3-galactosidase. The resulting degradation products are galactose and LNT-II. Galactose as well as LNT-II can be used by the genetically engineered host microorganism to produce the target LNnT.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express β-1,3-galactosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding a β-1,3-galactosidase for its expression.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express β-1,3-galactosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding a β-1,3-galactosidase for its expression. Preferably, the nucleotide sequence encoding β-1,3-galactosidase is a nucleotide sequence selected from the group consisting of:

- the nucleotide sequence as represented by SEQ ID NO: 7;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 7, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 7 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 8; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 8, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 8.

For the expression of a nucleotide sequence encoding a β-1,3-galactosidase or a functional variant thereof, the nucleotide sequence is operably linked to expression control sequences that mediate the expression of a nucleotide sequence encoding a β-1,3-glucosidase or a functional variant thereof in a genetically engineered microbial host cell.

Another suitable galactosidase is galactan β-1,3-galactosidase. Galactan β-1,3-galactosidase is an enzyme that catalyzes the hydrolysis of the β-1,3-linked galactose residue from oligosaccharide chains bearing galactose. A preferred galactan β-1,3-galactosidase is Clostridium thermocellum Ct1,3Gal43A (SEQ ID NO: 10).

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express galactan β-1,3-galactosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding galactan β-1,3-galactosidase for its expression. Preferably, the nucleotide sequence encoding galactan β-1,3-galactosidase is a nucleotide sequence selected from the group consisting of:

- the nucleotide sequence as represented by SEQ ID NO: 9;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 9, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 9 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 10; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 10, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 10.

For the expression of a nucleotide sequence encoding galactan β-1,3-galactosidase or a functional variant thereof, the nucleotide sequence is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding galactan β-1,3-glucosidase or a functional variant thereof, in genetically engineered microbial host cell.

A suitable glucosidase is β-1,3-glucosidase. β-1,3-Glucosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of the β-1,3-linked glucose moiety from oligosaccharides. A preferred β-1,3-glucosidase is PgIA Paenibacillus sp. (SEQ ID NO: 12).

In an additional and/or alternative embodiment, there is provided a genetically engineered microbial host that can produce LNT or LNnT, wherein the genetically engineered microbial host cell expresses β1,3-glucosidase and/or β-1,3-galactosidase. In order to be able to produce LNT, the genetically engineered microbial host cell expresses β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase. This β-1,3-N-acetylglucosaminyltransferase can transfer the GlcNAc residue from UDP-GlcNAc to the galactose moiety of lactose, thereby synthesizing lacto-N-triose-II (LNT-II). This β-1,3-galactosyltransferase can transfer a galactose residue from UDP-galactose to the GlcNAc moiety of LNT-II, thereby synthesizing lacto-N-tetraose (LNT). In order to be able to produce LNnT, the genetically engineered microbial host cell expresses β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase. This β-1,3-N-acetylglucosaminyltransferase can synthesize LNT-II. This β-1,4-galactosyltransferase can transfer a galactose residue from UDP-galactose to the GlcNAc moiety of LNT-II, thereby synthesizing LNnT as the target oligosaccharide.

One skilled in the art will recognize that β-1,3-N-acetylglucosaminyltransferases, like Neisseria meningitides LgtA, accept a wide range of donor substrates. Typically, when transferring GlcNAc from UDP-GlcNAc to the corresponding acceptor saccharide, LgtA can also use UDP-galactose or UDP-glucose as donor substrates. Using a genetically engineered host microorganism capable of producing LNT or LNnT as described, said β-1,3-N-acetylglucosaminyltransferase can also transfer a galactose moiety from UDP-galactose, as well as a glucose moiety from UDP-glucose to the galactose moiety of lactose, synthesizing , thus the unwanted by-products Gal(β1,3)Gal(β1,4)Glc and Glc(β1,3)Gal(β1,4)Glc, respectively.

By expressing galactan β-1,3-galactosidase and/or β-1,3-glucosidase in a genetically engineered microbial host cell capable of producing LNT or LNnT, the production of Gal(β1,3)Gal(β1,4)Glc by-products and Glc(β1,3)Gal(β1,4)Glc may be discontinued or at least reduced due to the fact that these by-products are hydrolyzed in the genetically engineered microbial host cell by the action of galactan β-1,3-galactosidase and/ or β1,3-glucosidases. The resulting degradation products are galactose and/or glucose and lactose. Both monosaccharides, as well as lactose, can be used by a genetically engineered microbial host cell to produce the target LNT or LNnT.

In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to express β-1,3-glucosidase. In a further and/or alternative embodiment, the genetically engineered microbial host cell is genetically engineered to contain a nucleic acid molecule comprising a nucleotide sequence encoding a β-1,3-glucosidase for its expression. Preferably, the nucleotide sequence encoding the β-1,3-glucosidase is a nucleotide sequence selected from the group consisting of:

- the nucleotide sequence as represented by SEQ ID NO: 11;

- nucleotide sequences that are complementary to a nucleotide sequence that hybridizes to the nucleotide sequence as represented by SEQ ID NO: 11, under stringent conditions;

- nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence as represented by SEQ ID NO: 11 ;

- nucleotide sequences encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 12; And

- nucleotide sequences encoding a functional variant of polypeptide sequences as represented by SEQ ID NO: 12, where the amino acid sequence of the functional variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% sequence identity, 97%, 98% or 99% with the amino acid sequence as represented by SEQ ID NO: 10.

For the expression of a nucleotide sequence encoding a β-1,3-glucosidase or a functional variant thereof, the nucleotide sequence is operably linked to expression control sequences that mediate the expression of a nucleotide sequence encoding a β-1,3-glucosidase or a functional variant thereof in a genetically engineered microbial host cell.

The genetically engineered microbial host cell may reuse at least one of the degradation products resulting from the enzymatic activity of the heterologous glycosidase in the genetically engineered microbial host cell. Thus, the genetically engineered microbial host cell can utilize at least one of the degradation products produced by the enzymatic activity of the heterologous glycosidase to produce the target oligosaccharide. For example, a monosaccharide residue released from a non-target saccharide by-product by a heterologous glycosidase can be reactivated, namely bound to a nucleotide, transferred from the resulting nucleotide-activated monosaccharide to an acceptor substrate via an appropriate glycosyltransferase to produce the target oligosaccharide or a precursor of the target oligosaccharide .

The method includes the step of cultivating a genetically engineered microbial host cell in an environment that is permissive for the production of a target oligosaccharide by the specified genetically engineered host microorganism, and under conditions that are permissive for the production of the target oligosaccharide by the specified genetically engineered host microorganism.

The medium, which is permissive to production of the target oligosaccharide by the genetically engineered microbial host cell, contains nutrients, at least one energy source, essential metals and minerals, and a buffering agent. The medium optionally contains a precursor of the target oligosaccharide, which precursor can be taken up by the genetically engineered microbial host cell and used to produce the target oligosaccharide, provided that the genetically engineered microbial host cell cannot synthesize the precursor on its own. Next, the genetically engineered microbial host cell ingests the precursor and subjects the precursor to biosynthesis of the target oligosaccharide. For example, lactose can be considered a precursor to 2'-fucosyllactose.

Permissive conditions are maintained during the cultivation of genetically engineered microbial host cells to produce the target oligosaccharide. Conditions are “permissive” if the genetically engineered microbial host cells that are cultured under the conditions remain alive and produce the target oligosaccharide. Preferably, permissive culture conditions allow the proliferation of genetically engineered microbial host cells. Conditions that must be maintained at a certain value or within a certain range include pH, temperature, oxygen, and concentrations of nutrients, energy sources, and essential metals and minerals.

In an additional and/or alternative embodiment, the method includes the step of isolating the target oligosaccharide. The target oligosaccharide can be isolated from the fermentation broth and/or from a genetically engineered microorganism α host.

The method as previously described herein is advantageous in that fewer or no off-target byproducts are produced during production of the target oligosaccharide. Thus, it is less laborious and costly to isolate and purify the target oligosaccharide from the fermentation broth or cell lysate.

In addition, much more substrate is specifically used to produce the target oligosaccharide, rather than becoming unavailable for production of the target oligosaccharide because it is incorporated into unwanted byproducts that cannot be metabolized by the microbial host cell.

According to a second aspect, there are provided genetically engineered microbial host cells to produce a target oligosaccharide, wherein the microbial host cell can produce the target oligosaccharide, and where the microbial host cell is genetically engineered to express a heterologous glycosidase that can perform intracellular degradation of metabolic byproducts that are produced during intracellular biosynthesis of the target oligosaccharide.

In a third aspect, genetically engineered microbial host cells, as previously described herein, are used to produce the target oligosaccharide. The use of genetically engineered microbial host cells to produce a target oligosaccharide through fermentation is advantageous because the production of non-target saccharide by-products is prevented or even eliminated. Thus, it saves resources and is less labor intensive to isolate the target oligosaccharide from the fermentation broth, since separation of the target oligosaccharide from non-target oligosaccharide by-products can be avoided. In addition, much more of the recovery product and energy sources provided to the genetically engineered microbial host cells of the present invention are converted to the target product compared to a native microbial host cell that has not been genetically engineered to express a heterologous glycosidase.

According to a fourth aspect, the target oligosaccharides that are obtained by the method and/or use of genetically engineered microbial host cells described hereinbefore are preferably selected from the group of OGMs.

Target oligosaccharides that are obtained by the method and/or use of genetically engineered microbial host cells described herein can be used to prepare a nutritional composition.

The nutritional composition is a medicinal composition, a dietary composition, an infant formula, or the like.

The present invention will be described with respect to specific embodiments and with reference to drawings, but the invention is not limited thereto, but only by the claims. In addition, the terms first, second, and the like in the specification and claims are used to distinguish between like elements and not necessarily to describe sequence, in time, space, rank, or any other manner. It should be understood that the terms used in this manner are interchangeable in appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

It should be understood that the term “comprising” as used in the claims should not be considered limited to the means listed hereinafter; it does not exclude other elements or stages. Thus, it should be considered to determine the presence of the claimed features, integers, steps or components referred to, but it does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that for the purposes of the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to “one embodiment” or “embodiment” means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in different places throughout this specification do not necessarily all refer to the same embodiment, but may. Moreover, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Likewise, it should be understood that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in one single embodiment, figure, or description thereof for the purpose of simplifying the disclosure and aiding in the understanding of one or more different aspects of the invention. This manner of disclosure, however, should not be taken to imply that the claimed invention requires more features than are expressly listed in each claim. Rather, as reflected in the following claims, aspects of the invention are comprised of less than all of the features of one of the above disclosed embodiments. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing alone as a separate embodiment of the invention.

In addition, while some embodiments described herein include some, but not all, of the features included in other embodiments, it is understood that combinations of features of different embodiments are within the scope of the invention and form different embodiments, as will be appreciated by those skilled in the art. areas. For example, in the following claims, any of the claimed embodiments may be used in any combination.

In addition, some of the embodiments are described herein as a method or combination of method elements that may be implemented by a computer system processor or other means of performing a function. Thus, a processor with the necessary instructions for implementing such method or method element constitutes means for implementing the method or method element. In addition, the apparatus embodiment described herein is an example of a means of implementing the function performed by the apparatus for the purpose of carrying out the invention.

Numerous specific details are set forth in the descriptions and graphics provided herein. However, it will be understood that embodiments of the invention may be practiced without these specific details. In other examples, well-known methods, structures and techniques have not been shown in detail so as not to obscure the understanding of this description.

The invention will now be described by way of detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be put together in accordance with the knowledge of those skilled in the art without deviating from the true spirit or technical idea of the invention, the invention being limited only by the terms of the appended claims.

Example 1: Metabolic engineering of E. coli strain BL21(DE3) for production of 2'-fucosyllactose

E. coli BL21(DE3) (Novagen) was used as the starting strain to construct the α host strain for 2′-FL production. Genetic engineering of the parent strain involved gene disruption and deletion events and integration of heterologous genes.

Since 2'-fucosyllactose is synthesized from lactose, it is used for bacterial culture, and from GDP-L-fucose, which is produced from living cells, first, the wild-type copy of the lacZ gene encoding endogenous β-galactosidase was inactivated by mismatch mutagenesis oligonucleotides (Ellis et al., “High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides,” Proc. Natl. Acad. Sci. USA 98: 6742-6746 (2001)). Using the same method, the arabinose isomerase gene araA was disrupted.

The lacZΩ gene fragment was introduced under the control of the temperature-sensitive transcriptional repressor cl857. The lacZα gene fragment is expressed under the control of the PgbA BL21 (DE3) E coli promoter in this strain, revealing a LacZ ⁺ strain.

Genomic deletions were carried out by Red-dependent recombination of phage λ according to the method of Datsenko and Warner (“One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, Proc. Natl. Acad. Sci. USA 97:6640- 6645 (2000)). The fuel and fucK genes, encoding L-fucose isomerase and L-fuculose kinase, respectively, were deleted to prevent L-fucose degradation. The wzxC-wcaJ genes were also deleted. WcaJ possibly encodes UDP-glucose: undecaprenylphosphate glucose-1-phosphate transferase, which catalyzes the first step in the synthesis of colanic acid (Stevenson et al., “Organization of the Escherichia coli K-12 gene cluster responsible for the production of the extracellular polysaccharide colonic acid,” J. Bacterid 178:4885-4893 (1996)); colanic acid production will compete for GDP-fucose with the fucosyltransferase reaction.

Integration of heterologous genes into the genome was carried out through transposition. Large clusters of genes have been integrated into the genome through the mediation of a hyperactive C9 mutant of the mariner Himarl transposase (Lampe et al., “Hyperactive transposase mutants of the Himarl mariner transposon,” Proc. Natl. Acad. Sci. USA 96:11428-11433 (1999)) , which was inserted into the pEcomar plasmid under the transcriptional control of the P _ara promoter. To enhance de novo synthesis of GDP-fucose, genes encoding phosphomannomutase (manB), mannose-1-phosphate guanosyltransferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG), from DH5α K12 E, coli was overexpressed in strain BL21(DE3) E, coli; the manCB operon was placed under the control of the constitutive promoter P _tet , the gmd, wcaG operon is transcribed from the constitutive promoter P _T5 - Transposon cassette <P _tet -manCB-P _T5 -gmd, wcaG-FRT-dhfr-FRT> (SEQ ID NO: 13), including the dihydrofolate reductase gene for resistance to trimethoprim, flanked by inverted terminal repeats specifically recognized by the transposase of mariner-like elements Himar1, was inserted into the genome of E, coli from pEcomar CQ-manCB-gmd, wcaG-dhfr.

For chromosomal integration of single genes, transposase EZ-Tn5™ (Epicentre, USA) was used. To obtain EZ-Tn5 transposomes, the gene under study, together with the FRT-site-flanked antibiotic resistance cassette, was amplified using primers that carried 19-bp recognition sites in both regions. Mosaic End (5'-CTGTCTCTTATACACATCT, SEQ ID NO: 21) for EZ-Tn5 transposase. Using the transposase EZ-Tn5TM, the lactose importer gene LacY from K12 TG1 E, coli (account no. ABN72583), the 2-fucosyltransferase gene wbgL from E. coli: O126 (account no. ADN43847), and the gene yberc0001_9420, encoding the sugar efflux transporter superfamily membrane transporters from Yersinia bercovieri ATCC 43970 (account no. EEQ08298), were integrated using the appropriate integration cassettes: <P _tet -lacY-FRT-aadA-FRT> (SEQ ID NO: 14), <P _tet -wbgL.co-FRT -neo-FRT> (SEQ ID NO: 15) and <P _tet -yberc0001_9420co-FRT-cat-FRT> (SEQ ID NO: 16). The wbgL and yberc0001_9420 genes were synthetically synthesized and codon optimized by (co) GenScript Cooperation (USA). After successful integration of the lacY gene, the resistance gene was eliminated from streptomycin-resistant clones using FLP recombinase encoded on plasmid pCP20 (Datsenko and Warner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products,” Proc. Natl Acad Sci USA 97:6640-6645 (2000).

Since BL21(DE3) E, coli does not have a functional gal operon, a natively regulated copy of the galETKM operon from E, coli K was integrated into strain B via EZ transposition using the integration cassette <P _gal -galE-galT-galK-galM> ( SEQ ID NO: 17). Integrants were selected from MacConkey agar containing 1% galactose as red colonies. The resulting strain can metabolize monosaccharides - glucose and galactose, resulting from the hydrolysis of lactose.

An additional improvement regarding the synthesis of 2'-fucosyllactose by the E coli strain was achieved by deleting the pfkA gene encoding phosphofructokinase A. When E coli is cultured on a gluconeogenic substrate like glycerol, phosphorylation of fructose-6-phosphate by PfkA is a treadmill reaction with high ATP consumption, and, in addition, competes with ManA for substrate. The pfkA gene was deleted by homologous recombination according to Datsenko and Wanner (2000) using a gentamicin resistance cassette (aacC1) that is flanked by lox71/66 sites (Lambert, JM et al. (2007) Cre-lox-based system for multiple gene deletions and selectable - marker removal in Lactobacillus plantarum. Appl. Environ. Microbiol 73: 1126-1135). After successful deletion of the pfkA gene, the antibiotic resistance gene was removed from the E, colic genome using Cre recombinase (Abremski, K et al. (1983) Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell 32: 1301-1311), which was cloned under the control of the P _ara promoter into the pKD46 framework (Datsenko and Wanner, 2000).

In the case of various fucosyltransferases, in addition to transferase activity, GDP-L-fucose hydrolase activity has been demonstrated. In addition, in the case of wbgL, the α-1,2-fucosyltransferase used herein for the synthesis of 2'-fucosyllactose, this hydrolytic activity was shown (see EP 3 050 973 A1). To conserve free L-fucose for the production of 2'-fucosyllactose and to eliminate contaminating L-fucose from the culture broth, the fkp gene encoding the bifunctional L-fucokinase/L-fucose-1-phosphate guanylyltransferase of Bacteroides fragilis, under the transcriptional control of the P _tet promoter, is combined the lox71/66-flanked aacC1 gene was chromosomally integrated via transposition using the EZ-Tn5™ transposase, <P _tet -fkp-lox-aacC1-lox> (SEQ ID NO: 18). After successful integration, the gentamicin resistance gene was removed from the genome as described above.

To enhance the flow of the metabolized carbon source glycerol through the gluconeogenic pathway from triose phosphates to fructose-6-phosphate to fuel the biosynthesis of GDP-L-fucose, genes encoding fructose-1,6-bisphosphate aldolase (fbaB) and heterologous fructose-1,6- bisphosphate phosphatase (fbpase), from Pisum sativum. The fbaB gene from E. coli BL21 (DE3) was fused to the P _tet promoter. P. sativum chloroplast FBPase activity was allosterically regulated through disulfide-dithiol exchange via reduction with thioredoxins. Replacement of cysteine residue 153 with a serine results in a constitutively active enzyme. The gene encoding chloroplast FBPase from P. sativum (Accession No. AAD10213) was acquired, codon optimized for expression in E. coli, tagged with an N-terminal hexahistidine tag, and modified to encode the C153S variant of the enzyme from Genescript. The fbpase gene is transcribed from promoter 17. The <P _tet -fbaB-P ₁₇ -His ₆ -fbpase-lox-aacC1-lox> cassette (SEQ ID NO: 19) was used for EZ-Tn5™ transposase-mediated integration in the host strain. After removal of the gentamicin resistance gene from the E. coli genome, the strain was used to produce 2'-fucosyllactose. Subsequently, this strain is called “strain A”.

Example 2: Construction of E. coli strain BL21(DE3) to produce 2'-fucosyllactose with high purity

Fed-batch cultures using strain A to produce 2'-fucosyllactose revealed the presence of by-products β-fucosyllactose and 2'3-difucosyllactose) in the culture broth. To minimize the production of these by-products, as well as to improve carbon yield, α-1,3-fucosidase was subcloned behind the constitutive promoter and integrated into the genome of strain A. Thus, the afcB gene of Bifidobacterium bifidum (Accession No. AB474964) was fused to the constitutive promoter P _and and the gentamicin resistance genome. The resulting transposon cassette <P _and -afcS-lox-aacC7-lox> (SEQ ID NO: 20), flanked by inverted terminal repeats specifically recognized by the transposase of mariner-like elements Himar1, was inserted into the E, coli genome from pEcomar afcB-aacC1, forming "strain B".

Example 3: HPLC Analysis to Detect 2'-Fucosyllactose in Culture Supernatant

HPLC analyzes were performed using a refractometric detector (RID-10A) (Shimadzu, Germany) and a Waters XBridge Amide 3.5 μm (250 × 4.6 mm) column (Eschborn, Germany) coupled to an HPLC system (Shimadzu, Germany) . Elution was carried out isocratically using 30% A: 50% (v/v) ACN (from English acetonitrile - acetonitrile) in ddH ₂ O, 0.1% (v/v) NH ₄ OH and 70% B : 80% (v/v) ACN in ddH ₂ O, 0.1% (v/v) NH ₄ OH (v/v) as eluent at 35°C and flow rate 1, 4 ml min ^-1 . HPLC samples were sterile filtered (0.22 μm pore size) and purified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). 10 μL of samples were applied to the column, and the concentration of 2′-fucosyllactose was calculated according to the standard curve. Other sugars such as L-fucose and/or other monosaccharides, lactose and/or other disaccharides, 3-fucosyllactose and/or other trisaccharides, 2'3-difucosyllactose and/or other tetrasaccharides, and glycerol may also be detected with using these analysis conditions. The relative amounts of identified Sugars can be determined by comparing the AUC (from the English area under the curve - area under the curve) of all peaks in the chromatogram. Peaks also present in the water control are excluded from this calculation.

Example 4: Production of 2'-fucosyllactose by fermentation

Fermentations were carried out in 3L fermenters at 33°C (New Brunswick, Edison, USA), starting with 1000 ml of medium with mineral salts containing 3 g/l KH ₂ PO ₄ , 12 g/l K ₂ HPO ₄ , 5 g/l l (NH ₄ ) ₂ SO ₄ , 0.3 g/l citric acid, 2 g/l MgSO ₄ × 7⋅H ₂ O, 0.1 g/l NaCl and 0.015 g/l CaCl ₂ × 6⋅H ₂ O with the addition of 1 g/l solution of trace elements (54.4 g ^⋅ l ^-1 iron (III)-ammonium citrate, 9.8 g/l MnCl ₂ × 4⋅H ₂ O, 1.6 g/l CoCl ₂ × 6⋅H ₂ O, 1 g/l CuCl ₂ × 2⋅H ₂ O, 1.9 g/l H ₃ VO ₃ , 9 g/l ZnSO ₄ × 7⋅H ₂ O, 1.1 g/l Na ₂ MoO ₄ × 2⋅H ₂ O, 1.5 g/l Na ₂ SeO ₃ , 1.5 g/l NiSO ₄ × 6⋅H ₂ O) and containing 2% (v/v) glycerol in as a carbon source, 60 mM lactose and the antibiotic kanamycin (25 μg/ml). Aeration was maintained at 3 L/min. Dissolved oxygen was maintained at a saturation level of 20-30% by controlling the stirring speed. The pH was maintained at 7.0 by adding 25% ammonium solution. Cultivation was started with a 2.5% (v/v) inoculum from a preculture growing in the same medium containing glycerol but not lactose. After passing the batch phase, as indicated by an increase in the level of dissolved oxygen, glycerol was supplied (60% (v/v) with the addition of 2 g/l MgSO ₄ × 7⋅H ₂ O, 0.015 g/l CaCl ₂ × 6⋅ H ₂ O and 1 ml/l of microelements solution) at flow rates of 7.0-8.0 ml/h, relative to the original volume. The supply of lactose (0.66 M) was carried out throughout the cultivation and was adjusted intuitively to realize a constant supply of lactose in the culture broth. The supply of lactose was stopped towards the end of fermentation and cultivation continued until the lactose was completely converted to 2′-fucosyllactose. Approximately 94 hours after seeding the fermenter, a titer of 2'-fucosyllactose in the cell medium of approximately 150 g/l was achieved using the strain described in example 1 (strain A). A 2'-fucosyllactose-producing strain genetically modified as described in Example 2 (strain B) was cultured similarly and also resulted in a 2'-fucosyllactose titer of approximately 150 g/L. However, the amount of by-products was significantly lower than after cultivation of strain A (Table 2). While the saccharide content in the culture supernatant of strain A was only 94.22% 2'-fucosyllactose, it increased by 5.50% in the culture supernatant of strain B, showing a purity of 99.72%.

--->

LIST OF SEQUENCES

<110> Jennewein Biotechnologie GmbH

<120> APPLICATION OF GLYCOSIDASES IN OLIGOSACHARIDE PRODUCTION

<130>P 1804 WO

<160> 20

<170> Patent In version 3.5

<210> 1

<211> 5880

<212> DNA

<213> Bifidobacterium bifidum

<400> 1

atgaaacata gagcgatgtc atcgcgtctg atgccactgg tggcgtcctg cgcgacggtc 60

ggcatgctgc tggccggact acctgtgtcg gccgtcgcgg tcggcacgac gagagcggca 120

gcgtccgacg cctcgtcctc caccacagca accatcaccc cctccgccga taccacgttg 180

cagacatgga cgagcgagaa gaattcctca atggcgtcca agccgtacat cggcacactg 240

caagggccct cgcaaggcgt gttcggcgag aagttcgagt ccacggatgc cgcggacacc 300

accgatctga agaccggcct gctgacgttc gacctgagcg cctacgacca tgcccccgat 360

tccgcaacgt tcgagatgac gtacctcggc taccgcggca acccgacggc caccgacacc 420

gacaccatca aggtgacccc cgtcgacacc accgtgtgca ccaataacgc cacagactgc 480

ggcgcgaatg tcgcgaccgg cgcgaccaag ccgaagttca gcatcaacga ctcctcattc 540

gtcgccgagt ccaagccgtt cgagtacggt acgacggttt acacgggcga cgccatcacc 600

gtggttcccg ccaataccaa gaaggtcacc gtagatgtga ccgaaatcgt gcgccagcag 660

ttcgccgaag gcaagaaggt catcaccctg gccgtgggcg agaccaagaa gaccgaggtt 720

cgtttcgcca gttccgaagg cacgacgtcc ctgaacggcg cgaccgcaga catggctccg 780

aagctgaccg tttccgtgtc caccaaggac gatctcaagc cctccgccga caccacgttg 840

caggcatggg ccagcgagaa gaacgagaag aagaacactg cggcctatgt cggcgcgctg 900

cagccggaag gcgattacgg cgacttcggt gagaagttca agtccaccga cgtccacgat 960

gtcacagacg ccaagatggg tctgatgacg ttcgacctgt ccgattacac cgcggcgccc 1020

gagcactcca tcctcacctt gacgtatctg ggctacgccg gtgcagacaa gaccgccacg 1080

gccaccgata aggtcaaggt ggtcgctgtt gacacgtcgc ggtgcaccgg caccgctccc 1140

tgcgacacca acaatgccac gtgggcgaac cgcccggact tcgaggtgac cgataccacg 1200

aagaccgcga cgtcccatgc gttcgcttat ggatctaaga agtattccga tggcatgacc 1260

gtcgaatcgg gcaacgccaa gaaggtcctg ctcgacgtgt ccgatgtcat caaggcagag 1320

ttcgccaagt tcagcgccgg cgccaccgag aagaagatca cgctggccct gggcgagctc 1380

aacaagtccg acatgcgttt cggcagcaag gaagtcacct cgctgaccgg cgccaccgaa 1440

gccatgcagc cgaccttgtc cgtcaccaag aagccgaagg catacacgct gagcatcgaa 1500

ggcccgacca aggtcaagta ccagaagggc gaggcgttcg acaaggccgg actcgtggtc 1560

aaggccacca gcacggctga cggcacggtc aagacgctga ccgaaggcaa cggtgaggat 1620

aactacacca tcgacaccag cgctttcgat agtgccagca tcggcgtata ccctgttacc 1680

gtgaagtaca acaaggaccc cgaaatcgcc gcttcgttca acgcctatgt catcgccagt 1740

gtcgaggacg gcggagacgg cgacaccagc aaagacgact ggctgtggta caagcagccc 1800

gcgtcgcaga ccgacgccac cgccaccgcc ggcggcaatt acggcaaccc cgacaacaac 1860

cgttggcagc agaccacctt gccgttcggc aacggcaaga tcggcggcac cgtctggggc 1920

gaggtcagcc gtgaacgcgt caccttcaac gaggacgc tgtggaccgg cggccccgga 1980

tcctcgacca gctacaacgg cggcaacaac gagaccaagg gtcagaacgg cgccacgctg 2040

cgcgcgctca acaagcagct cgcgaacggc gccgagacgg tcaatcccgg caacctgacc 2100

ggcggcgaga acgcggccga gcagggcaac tacctgaact ggggcgacat ctacctcgac 2160

tacgggttca acgatacgac cgtcaccgaa taccgccgcg acctgaacct gagcaagggc 2220

aaggccgacg tcacgttcaa gcatgacggc gtcacctaca cgcgcgaata cttcgcgtcg 2280

aaccccgaca atgtcatggt cgcccgcctc acggccagca aagccggcaa gctgaacttc 2340

aacgtcagca tgccgaccaa cacgaactac tccaagaccg gcgaaaccac gacggtcaag 2400

ggtgacacgc tcaccgtcaa gggcgctctc ggcaacaacg gcctgctgta caactcgcag 2460

atcaaggtcg tcctcgacaa cggtgagggc acgctctccg aaggctccga cggcgcttcg 2520

ctgaaggtct ccgacgcgaa ggcggtcacg ctgtacatcg ccgccgcgac ggactacaag 2580

cagaagtatc cgtcctaccg caccggcgaa accgccgccg aggtgaacac ccgcgtcgcc 2640

aaggtcgtgc aggacgccgc caacaagggc tacaccgccg tcaagaaagc gcacatcgac 2700

gatcattccg ccatctacga ccgcgtgaag atcgatttgg gccagtccgg ccacagctcc 2760

gacggcgccg tcgccaccga cgcgctgctc aaggcgtacc agagaggctc cgcaaccacc 2820

gcgcagaagc gcgagctgga gacgctggtg tacaagtacg gccgctactt gaccatcggc 2880

tcctcccgtg agaacagcca gctgcccagc aacctgcagg gcatctggtc ggtcaccgcg 2940

ggcgacaacg cccacggcaa cacgccttgg ggctccgact tccacatgaa cgtgaacctc 3000

cagatgaact actggccgac ctattcggcc aacatgggag agctcgccga gccgctcatc 3060

gagtatgtgg agggtctggt caagcccggc cgtgtgaccg ccaaggtcta cgcgggcgcg 3120

gagacgacga accccgagac cacgccgatc ggcgagggcg agggctacat ggcccacacc 3180

gagaacaccg cctacggctg gaccgcaccc ggtcaatcgt tctcgtgggg ttggagcccg 3240

gccgccgtgc cgtggatcct gcagaacgtg tacgaggcgt acgagtactc cggcgaccct 3300

gccctgcttg atcgcgtgta cgcgctgctc aagggaggaat cgcacttcta cgtcaactac 3360

atgctgcaca aggccggctc cagctccggt gaccgcctga ctaccggcgt cgcgtactcg 3420

cccgaacagg gcccgctggg caccgacggc aacacgtacg agagctcgct cgtgtggcag 3480

atgctcaacg acgccatcga ggcggccaag gccaagggag atccggacgg tctggtcggc 3540

aataccaccg actgctcggc cgacaactgg gccaagaatg acagcggcaa cttcaccgat 3600

gcgaacgcca accgttcctg gagctgcgcc aagagcctgc tcaagccgat cgaggtcggc 3660

gactccggcc agatcaagga atggtacttc gaaggtgcgc tcggcaagaa gaaggatgga 3720

tccaccatca gcggctacca ggcggacaac cagcaccgtc acatgtccca cctgctcgga 3780

ctgttccccg gtgatttgat caccatcgac aactccgagt acatggatgc ggccaagacc 3840

tcgctgaggt accgctgctt caagggcaac gtgctgcagt ccaacaccgg ctgggccatt 3900

ggccagcgca tcaattcgtg ggctcgcacc ggcgacggca acaccacgta ccagctggtc 3960

gagctgcagc tcaagaacgc gatgtatgca aacctgttcg attaccatgc gccgttccag 4020

atcgacggca acttcggcaa cacctccggt gtcgacgaaa tgctgctgca gtccaactcc 4080

accttcaccg acaccgccgg caagaagtac gtgaactaca cgaacatcct gcccgccctg 4140

cccgatgcct gggcgggcgg ctcggtgagc ggcctcgtgg cccgcggcaa cttcaccgtc 4200

ggcacgacat ggaagaacgg caaggccacc gaagtcaggc tgacctccaa caagggcaag 4260

caggcggccg tcaagatcac cgccggcggc gcccagaact acgaggtcaa gaacggtgac 4320

accgccgtga acgccaaggt cgtgaccaac gcggacggcg cctcgctgct cgtgttcgat 4380

accaccgcag gcaccacgta cacgatcacg aagaaggcga gcgccaacgt gcccgtcacc 4440

ggcgtgaccg tgaccggcgc caacaccgcc accgcaggcg acaccgtcac tcttacggct 4500

accgtcgccc cggccaatgc gaccgacaag tccgtcacct ggtcgacctc cgacgccgcc 4560

gtagctacgg tcaacgccaa cggcgtggtg accacgaaga aggccggcaa ggtgaccatc 4620

accgccacgt cgaacggcga caagacgaag ttcggttcca tcgagatcac cgtctccgcc 4680

gcgaccgtgc ccgtcaccag cgtcaccgtt gccggcgacg ccgcgatgac cgtcgatgga 4740

gagcagaccc tgacggcgac cgtcgccccg gccactgcga ccgacaagac ggtcacgtgg 4800

aagtcctccg acgccactgt ggcgacggtt gacgccaacg gcaaggtcgt cgcgaagaag 4860

gccggcgaag tgacgatcac cgccacggcc ggtggcgtgt ccggcacgct gaagatcacg 4920

gtgagcgaca aggccccgac cgtcatcccg gtccagtccg tgaccgtgac aggcaagcag 4980

gagctcgtcg aaggcgcctc cacgaccctg acggcgaccg tcgccccggc tgacgcgacc 5040

gacaagacgg ttacgtggaa gtcgagcgac gagtccgtcg ccacggtcga caaggacggc 5100

gtcgtgaccg ccaagaaggc cggcacggtg accatcaccg ccacggccgg tggcgtgtcc 5160

ggcacgctcc acatcaccgt gacggccaag cccgtcgaga ccgtccccgt caccagcgtg 5220

gaggtcaccg tcgaggccgg caccaccgtc tccgtcggca agacactcca ggccaccgcg 5280

accgtcaagc ccggcaacgc caccaacaag aaggtgacgt ggaagtcgag cgacgaatcc 5340

atcgcgacgg tcgacgccaa cggcgtcatc accgcgaaga aggccggcaa ggtcgtcatc 5400

acggccacct cgaccgacgg cacggacaag tccggcagcg tcgagatcac cgtcgtggat 5460

gagaccaagc cgacgcccga ccacaagtcc gtcaaggccg ataccggcga cgtgaccgcc 5520

ggcaagaccg gtacggtcac cgagccgaag gacgtggcgg gctggaagag ccgctccatc 5580

atcaagcaag gcaagctcgg caaggccgaa atcgccgacg gcacgctcgt gtatgcggcc 5640

ggcgacaaga ccggtgacga cagcttcgtc gtgcagtaca cgatggccga cggcacggtc 5700

atcgacgtga cctacagcgt cacggtcaag gccgccgaaa ccggcaagaa cgacggcgac 5760

ggcaagggcg acggtgtcgc gaagaccggc gccgccgtcg gcgcgctcgc cggcctcggc 5820

ttgatgctgc tcgccgtcgg agtgagcgtg gtgatgattc gccgcaagca ctccgcctga 5880

<210> 2

<211> 1959

<212> PRT

<213> Bifidobacterium bifidum

<400> 2

Met Lys His Arg Ala Met Ser Ser Arg Leu Met Pro Leu Val Ala Ser

1 5 10 15

Cys Ala Thr Val Gly Met Leu Leu Ala Gly Leu Pro Val Ser Ala Val

20 25 30

Ala Val Gly Thr Thr Arg Ala Ala Ala Ser Asp Ala Ser Ser Ser Thr

35 40 45

Thr Ala Thr Ile Thr Pro Ser Ala Asp Thr Thr Leu Gln Thr Trp Thr

50 55 60

Ser Glu Lys Asn Ser Ser Met Ala Ser Lys Pro Tyr Ile Gly Thr Leu

65 70 75 80

Gln Gly Pro Ser Gln Gly Val Phe Gly Glu Lys Phe Glu Ser Thr Asp

85 90 95

Ala Ala Asp Thr Thr Asp Leu Lys Thr Gly Leu Leu Thr Phe Asp Leu

100 105 110

Ser Ala Tyr Asp His Ala Pro Asp Ser Ala Thr Phe Glu Met Thr Tyr

115 120 125

Leu Gly Tyr Arg Gly Asn Pro Thr Ala Thr Asp Thr Asp Thr Ile Lys

130 135 140

Val Thr Pro Val Asp Thr Thr Val Cys Thr Asn Asn Ala Thr Asp Cys

145 150 155 160

Gly Ala Asn Val Ala Thr Gly Ala Thr Lys Pro Lys Phe Ser Ile Asn

165 170 175

Asp Ser Ser Phe Val Ala Glu Ser Lys Pro Phe Glu Tyr Gly Thr Thr

180 185 190

Val Tyr Thr Gly Asp Ala Ile Thr Val Val Pro Ala Asn Thr Lys Lys

195 200 205

Val Thr Val Asp Val Thr Glu Ile Val Arg Gln Gln Phe Ala Glu Gly

210 215 220

Lys Lys Val Ile Thr Leu Ala Val Gly Glu Thr Lys Lys Thr Glu Val

225 230 235 240

Arg Phe Ala Ser Ser Glu Gly Thr Thr Ser Leu Asn Gly Ala Thr Ala

245 250 255

Asp Met Ala Pro Lys Leu Thr Val Ser Val Ser Thr Lys Asp Asp Leu

260 265 270

Lys Pro Ser Ala Asp Thr Thr Leu Gln Ala Trp Ala Ser Glu Lys Asn

275 280 285

Glu Lys Lys Asn Thr Ala Ala Tyr Val Gly Ala Leu Gln Pro Glu Gly

290 295 300

Asp Tyr Gly Asp Phe Gly Glu Lys Phe Lys Ser Thr Asp Val His Asp

305 310 315 320

Val Thr Asp Ala Lys Met Gly Leu Met Thr Phe Asp Leu Ser Asp Tyr

325 330 335

Thr Ala Ala Pro Glu His Ser Ile Leu Thr Leu Thr Tyr Leu Gly Tyr

340 345 350

Ala Gly Ala Asp Lys Thr Ala Thr Ala Thr Asp Lys Val Lys Val Val

355 360 365

Ala Val Asp Thr Ser Arg Cys Thr Gly Thr Ala Pro Cys Asp Thr Asn

370 375 380

Asn Ala Thr Trp Ala Asn Arg Pro Asp Phe Glu Val Thr Asp Thr Thr

385 390 395 400

Lys Thr Ala Thr Ser His Ala Phe Ala Tyr Gly Ser Lys Lys Tyr Ser

405 410 415

Asp Gly Met Thr Val Glu Ser Gly Asn Ala Lys Lys Val Leu Leu Asp

420 425 430

Val Ser Asp Val Ile Lys Ala Glu Phe Ala Lys Phe Ser Ala Gly Ala

435 440 445

Thr Glu Lys Lys Ile Thr Leu Ala Leu Gly Glu Leu Asn Lys Ser Asp

450 455 460

Met Arg Phe Gly Ser Lys Glu Val Thr Ser Leu Thr Gly Ala Thr Glu

465 470 475 480

Ala Met Gln Pro Thr Leu Ser Val Thr Lys Lys Pro Lys Ala Tyr Thr

485 490 495

Leu Ser Ile Glu Gly Pro Thr Lys Val Lys Tyr Gln Lys Gly Glu Ala

500 505 510

Phe Asp Lys Ala Gly Leu Val Val Lys Ala Thr Ser Thr Ala Asp Gly

515 520 525

Thr Val Lys Thr Leu Thr Glu Gly Asn Gly Glu Asp Asn Tyr Thr Ile

530 535 540

Asp Thr Ser Ala Phe Asp Ser Ala Ser Ile Gly Val Tyr Pro Val Thr

545 550 555 560

Val Lys Tyr Asn Lys Asp Pro Glu Ile Ala Ala Ser Phe Asn Ala Tyr

565 570 575

Val Ile Ala Ser Val Glu Asp Gly Gly Asp Gly Asp Thr Ser Lys Asp

580 585 590

Asp Trp Leu Trp Tyr Lys Gln Pro Ala Ser Gln Thr Asp Ala Thr Ala

595 600 605

Thr Ala Gly Gly Asn Tyr Gly Asn Pro Asp Asn Asn Arg Trp Gln Gln

610 615 620

Thr Thr Leu Pro Phe Gly Asn Gly Lys Ile Gly Gly Thr Val Trp Gly

625 630 635 640

Glu Val Ser Arg Glu Arg Val Thr Phe Asn Glu Glu Thr Leu Trp Thr

645 650 655

Gly Gly Pro Gly Ser Ser Thr Ser Tyr Asn Gly Gly Asn Asn Glu Thr

660 665 670

Lys Gly Gln Asn Gly Ala Thr Leu Arg Ala Leu Asn Lys Gln Leu Ala

675 680 685

Asn Gly Ala Glu Thr Val Asn Pro Gly Asn Leu Thr Gly Gly Glu Asn

690 695 700

Ala Ala Glu Gln Gly Asn Tyr Leu Asn Trp Gly Asp Ile Tyr Leu Asp

705 710 715 720

Tyr Gly Phe Asn Asp Thr Thr Val Thr Glu Tyr Arg Arg Asp Leu Asn

725 730 735

Leu Ser Lys Gly Lys Ala Asp Val Thr Phe Lys His Asp Gly Val Thr

740 745 750

Tyr Thr Arg Glu Tyr Phe Ala Ser Asn Pro Asp Asn Val Met Val Ala

755 760 765

Arg Leu Thr Ala Ser Lys Ala Gly Lys Leu Asn Phe Asn Val Ser Met

770 775 780

Pro Thr Asn Thr Asn Tyr Ser Lys Thr Gly Glu Thr Thr Thr Val Lys

785 790 795 800

Gly Asp Thr Leu Thr Val Lys Gly Ala Leu Gly Asn Asn Gly Leu Leu

805 810 815

Tyr Asn Ser Gln Ile Lys Val Val Leu Asp Asn Gly Glu Gly Thr Leu

820 825 830

Ser Glu Gly Ser Asp Gly Ala Ser Leu Lys Val Ser Asp Ala Lys Ala

835 840 845

Val Thr Leu Tyr Ile Ala Ala Ala Thr Asp Tyr Lys Gln Lys Tyr Pro

850 855 860

Ser Tyr Arg Thr Gly Glu Thr Ala Ala Glu Val Asn Thr Arg Val Ala

865 870 875 880

Lys Val Val Gln Asp Ala Ala Asn Lys Gly Tyr Thr Ala Val Lys Lys

885 890 895

Ala His Ile Asp Asp His Ser Ala Ile Tyr Asp Arg Val Lys Ile Asp

900 905 910

Leu Gly Gln Ser Gly His Ser Ser Asp Gly Ala Val Ala Thr Asp Ala

915 920 925

Leu Leu Lys Ala Tyr Gln Arg Gly Ser Ala Thr Thr Ala Gln Lys Arg

930 935 940

Glu Leu Glu Thr Leu Val Tyr Lys Tyr Gly Arg Tyr Leu Thr Ile Gly

945 950 955 960

Ser Ser Arg Glu Asn Ser Gln Leu Pro Ser Asn Leu Gln Gly Ile Trp

965 970 975

Ser Val Thr Ala Gly Asp Asn Ala His Gly Asn Thr Pro Trp Gly Ser

980 985 990

Asp Phe His Met Asn Val Asn Leu Gln Met Asn Tyr Trp Pro Thr Tyr

995 1000 1005

Ser Ala Asn Met Gly Glu Leu Ala Glu Pro Leu Ile Glu Tyr Val

1010 1015 1020

Glu Gly Leu Val Lys Pro Gly Arg Val Thr Ala Lys Val Tyr Ala

1025 1030 1035

Gly Ala Glu Thr Thr Asn Pro Glu Thr Thr Pro Ile Gly Glu Gly

1040 1045 1050

Glu Gly Tyr Met Ala His Thr Glu Asn Thr Ala Tyr Gly Trp Thr

1055 1060 1065

Ala Pro Gly Gln Ser Phe Ser Trp Gly Trp Ser Pro Ala Ala Val

1070 1075 1080

Pro Trp Ile Leu Gln Asn Val Tyr Glu Ala Tyr Glu Tyr Ser Gly

1085 1090 1095

Asp Pro Ala Leu Leu Asp Arg Val Tyr Ala Leu Leu Lys Glu Glu

1100 1105 1110

Ser His Phe Tyr Val Asn Tyr Met Leu His Lys Ala Gly Ser Ser

1115 1120 1125

Ser Gly Asp Arg Leu Thr Thr Gly Val Ala Tyr Ser Pro Glu Gln

1130 1135 1140

Gly Pro Leu Gly Thr Asp Gly Asn Thr Tyr Glu Ser Ser Leu Val

1145 1150 1155

Trp Gln Met Leu Asn Asp Ala Ile Glu Ala Ala Lys Ala Lys Gly

1160 1165 1170

Asp Pro Asp Gly Leu Val Gly Asn Thr Thr Asp Cys Ser Ala Asp

1175 1180 1185

Asn Trp Ala Lys Asn Asp Ser Gly Asn Phe Thr Asp Ala Asn Ala

1190 1195 1200

Asn Arg Ser Trp Ser Cys Ala Lys Ser Leu Leu Lys Pro Ile Glu

1205 1210 1215

Val Gly Asp Ser Gly Gln Ile Lys Glu Trp Tyr Phe Glu Gly Ala

1220 1225 1230

Leu Gly Lys Lys Lys Asp Gly Ser Thr Ile Ser Gly Tyr Gln Ala

1235 1240 1245

Asp Asn Gln His Arg His Met Ser His Leu Leu Gly Leu Phe Pro

1250 1255 1260

Gly Asp Leu Ile Thr Ile Asp Asn Ser Glu Tyr Met Asp Ala Ala

1265 1270 1275

Lys Thr Ser Leu Arg Tyr Arg Cys Phe Lys Gly Asn Val Leu Gln

1280 1285 1290

Ser Asn Thr Gly Trp Ala Ile Gly Gln Arg Ile Asn Ser Trp Ala

1295 1300 1305

Arg Thr Gly Asp Gly Asn Thr Thr Tyr Gln Leu Val Glu Leu Gln

1310 1315 1320

Leu Lys Asn Ala Met Tyr Ala Asn Leu Phe Asp Tyr His Ala Pro

1325 1330 1335

Phe Gln Ile Asp Gly Asn Phe Gly Asn Thr Ser Gly Val Asp Glu

1340 1345 1350

Met Leu Leu Gln Ser Asn Ser Thr Phe Thr Asp Thr Ala Gly Lys

1355 1360 1365

Lys Tyr Val Asn Tyr Thr Asn Ile Leu Pro Ala Leu Pro Asp Ala

1370 1375 1380

Trp Ala Gly Gly Ser Val Ser Gly Leu Val Ala Arg Gly Asn Phe

1385 1390 1395

Thr Val Gly Thr Thr Trp Lys Asn Gly Lys Ala Thr Glu Val Arg

1400 1405 1410

Leu Thr Ser Asn Lys Gly Lys Gln Ala Ala Val Lys Ile Thr Ala

1415 1420 1425

Gly Gly Ala Gln Asn Tyr Glu Val Lys Asn Gly Asp Thr Ala Val

1430 1435 1440

Asn Ala Lys Val Val Thr Asn Ala Asp Gly Ala Ser Leu Leu Val

1445 1450 1455

Phe Asp Thr Thr Ala Gly Thr Thr Tyr Thr Ile Thr Lys Lys Ala

1460 1465 1470

Ser Ala Asn Val Pro Val Thr Gly Val Thr Val Thr Gly Ala Asn

1475 1480 1485

Thr Ala Thr Ala Gly Asp Thr Val Thr Leu Thr Ala Thr Val Ala

1490 1495 1500

Pro Ala Asn Ala Thr Asp Lys Ser Val Thr Trp Ser Thr Ser Asp

1505 1510 1515

Ala Ala Val Ala Thr Val Asn Ala Asn Gly Val Val Thr Thr Lys

1520 1525 1530

Lys Ala Gly Lys Val Thr Ile Thr Ala Thr Ser Asn Gly Asp Lys

1535 1540 1545

Thr Lys Phe Gly Ser Ile Glu Ile Thr Val Ser Ala Ala Thr Val

1550 1555 1560

Pro Val Thr Ser Val Thr Val Ala Gly Asp Ala Ala Met Thr Val

1565 1570 1575

Asp Gly Glu Gln Thr Leu Thr Ala Thr Val Ala Pro Ala Thr Ala

1580 1585 1590

Thr Asp Lys Thr Val Thr Trp Lys Ser Ser Asp Ala Thr Val Ala

1595 1600 1605

Thr Val Asp Ala Asn Gly Lys Val Val Ala Lys Lys Ala Gly Glu

1610 1615 1620

Val Thr Ile Thr Ala Thr Ala Gly Gly Val Ser Gly Thr Leu Lys

1625 1630 1635

Ile Thr Val Ser Asp Lys Ala Pro Thr Val Ile Pro Val Gln Ser

1640 1645 1650

Val Thr Val Thr Gly Lys Gln Glu Leu Val Glu Gly Ala Ser Thr

1655 1660 1665

Thr Leu Thr Ala Thr Val Ala Pro Ala Asp Ala Thr Asp Lys Thr

1670 1675 1680

Val Thr Trp Lys Ser Ser Asp Glu Ser Val Ala Thr Val Asp Lys

1685 1690 1695

Asp Gly Val Val Thr Ala Lys Lys Ala Gly Thr Val Thr Ile Thr

1700 1705 1710

Ala Thr Ala Gly Gly Val Ser Gly Thr Leu His Ile Thr Val Thr

1715 1720 1725

Ala Lys Pro Val Glu Thr Val Pro Val Thr Ser Val Glu Val Thr

1730 1735 1740

Val Glu Ala Gly Thr Thr Val Ser Val Gly Lys Thr Leu Gln Ala

1745 1750 1755

Thr Ala Thr Val Lys Pro Gly Asn Ala Thr Asn Lys Lys Val Thr

1760 1765 1770

Trp Lys Ser Ser Asp Glu Ser Ile Ala Thr Val Asp Ala Asn Gly

1775 1780 1785

Val Ile Thr Ala Lys Lys Ala Gly Lys Val Val Ile Thr Ala Thr

1790 1795 1800

Ser Thr Asp Gly Thr Asp Lys Ser Gly Ser Val Glu Ile Thr Val

1805 1810 1815

Val Asp Glu Thr Lys Pro Thr Pro Asp His Lys Ser Val Lys Ala

1820 1825 1830

Asp Thr Gly Asp Val Thr Ala Gly Lys Thr Gly Thr Val Thr Glu

1835 1840 1845

Pro Lys Asp Val Ala Gly Trp Lys Ser Arg Ser Ile Ile Lys Gln

1850 1855 1860

Gly Lys Leu Gly Lys Ala Glu Ile Ala Asp Gly Thr Leu Val Tyr

1865 1870 1875

Ala Ala Gly Asp Lys Thr Gly Asp Asp Ser Phe Val Val Gln Tyr

1880 1885 1890

Thr Met Ala Asp Gly Thr Val Ile Asp Val Thr Tyr Ser Val Thr

1895 1900 1905

Val Lys Ala Ala Glu Thr Gly Lys Asn Asp Gly Asp Gly Lys Gly

1910 1915 1920

Asp Gly Val Ala Lys Thr Gly Ala Ala Val Gly Ala Leu Ala Gly

1925 1930 1935

Leu Gly Leu Met Leu Leu Ala Val Gly Val Ser Val Val Met Ile

1940 1945 1950

Arg Arg Lys His Ser Ala

1955

<210> 3

<211> 4482

<212> DNA

<213> Bifidobacterium bifidum

<400> 3

atgctacaca cagcatcaag aggatgctcg cgttcgtggc tgcgcagact caccgcattg 60

atagcggtct cggcgctcgc gttcgtggca ttgccgaacg tcgcggtggc ggcggatccg 120

atggaatacc tcgatgtgtc gttcggcggc acgttcgctg cagacaccta caccacaggt 180

ggcgacgagg tggcgaaggg ccccgtgacc aagcacggca gcataccgac caagcttgac 240

ggcggcggca tcaccctcgc tggcggcacc aacggcgtga cattcacctc gaccgcgagc 300

ttcagcgaga gtgggaaggt gaacaaggga ttccgcgccg aaatggagta ccgtacgacg 360

cagacgccca gcaacctcgc cacattgttc tccgccatgg gcaacatctt cgtgcgggcg 420

aacggcagca acctcgaata cggcttctcc acgaaccctt ccggcagtac atggaacgac 480

tacacaaagt ccgtgacgct gccttccaac aatgtgaagc acatcatcca gctgacatat 540

ctgccgggag ccgacggcgc tgcctcgacg ttgcagttgt cggtggatgg cgtggccggc 600

gagaccgcca cctccgcggc cggcgagctc gcggccgtca gcgattccgt cgggaacaag 660

ttcgggatcg gctacgaggt gaaccccgct tccggcgcgg cgagccgcgg tcttgccggt 720

gacgtgttcc gcgcgcgtgt cgccgattcg gacgccccgt gggagattct tgacgcatcc 780

cagctgctgc atgtcaattt caacggcacg ttcagcggca cctcatatac cgcggcgagc 840

ggcgagcaga tgctgggctc gctggtgtcg cgctcggcca atccgtccat ctcgaactcc 900

gccgtcacgc tgggcggcgg cacggccgga ttcgatttca cgcccacgga cttcaccctc 960

ggtgacaacg aggccatcac ccgcccgctg gtcgcggagc tgcgcttcac cccgacgcag 1020

accggcgaca accagaccct gttcggcgcg ggcggcaacc tgttcctgcg ctacgagtcg 1080

aacaagctcg tgttcggcgc ctccaccaag tccggcgata attggaccga ccacaagatc 1140

gagtccgcgg ccgccacggg tgcggagcac gtcgtgtcgg tggcgtacgt gcccaataag 1200

gccggcaccg gcgcgaagct tgtcatgcgc gtggatggcg gcgacgccca gaccaaggac 1260

atcactggtc tggcttacct gaattcgagc atcaagggca aggtcggctt cggcaacgac 1320

gtgcataccg acgcgctcag ccgcggcttc gtcggctcgc tgagcgagat ccgcctggcc 1380

gaaacctccg cgaacttcac caccaacgaa ttcaagctgg tctactctca ggtcagctgc 1440

gacacgtcgg gcatcaagga ggcgaatacc ttcgacgtgg agcccgccga gtgcgaggcc 1500

gcgcttaaga ccaagctgtc caagctgcgt ccgaccgaag ggcaggccga ctacatcgac 1560

tggggtcaga tcggattcct ccattacggc atcaacacgt actacaacca ggagtggggt 1620

cacggtaacg aggatccctc ccgcatcaac ccgaccggcc tcgacaccga ccagtgggcg 1680

aagtccttcg ccgacggtgg cttcaagatg atcatggtga cggtcaagca ccatgacggt 1740

ttcgagctgt acgactcgcg gtacaacacc gagcacgact gggcaaacac cgccgtcgcc 1800

aagcgcacgg gggagaagga cctgttccgc aagattgtcg cctcggcgaa gaaatacggc 1860

ctgaaggtcg gcatctacta ttcgccggcc gattcctaca tggagaggaa gggcgtctgg 1920

ggcaacaact ccgcacgcgt cgagcgcacg atccccacgc tggtggagaa cgacgaccgc 1980

gccggcaagg tggcttccgg caaactgccc acgttcaagt acaaggccac ggattacggc 2040

gcctacatgc tcaaccagct ctatgagctg ctgactgagt acggcgacat ctccgaggtc 2100

tggttcgacg gtgcccaagg caacaccgca ggcactgagc attacgacta tggcgtgttc 2160

tacgagatga tccgccggct tcagccccag gcaattcagg ccaacgccgc atacgatgcc 2220

cgatgggtgg gcaacgagga cggctgggcc cgtcagaccg agtggagccc gcaggcggca 2280

tacaacgacg gcgtggacaa ggtgtcgctc aagcctggcc agatggcccc cgacggtaag 2340

cttggcagca tgtcgagcgt gctgtccgag atccgcagcg gcgccgccaa ccagctgcac 2400

tggtatccgg ccgaagtcga cgccaagaac cggcccggat ggttctaccg tgccagccaa 2460

tcgccggcgt ccgtagccga agtcgtgaag tactacgagc agtccacggg acgcaactcg 2520

cagtatctgc tgaacgtccc accgtccgat accggcaagc tcgccgatgc ggatgccgcg 2580

ggacttaagg ggctgggcga ggagctcgcc cgacgctacg gcaccgatct tgccctgggc 2640

aagagcgcga ccgtcgccgc gtccgcgaac gacactgcgg tagcggcccc gaagctgacc 2700

gacggttcga agctctcctc cgacaaggcc gtgggcaata cgccgacgta caccatcgat 2760

ctgggcagca ctgtcgccgt ggatgcagtg aagatctccg aggacgtgcg caatgccggc 2820

cagcagatcg aaagcgccac tctgcaggga cgagtcaatg gaacatggac gaatctggcg 2880

actatgacga cggtcgggca gcagcgcgac cttcgcttca cgtcccagaa catcgatgcc 2940

atccgtctgg tggtcaactc ctcccgcggt ccggtgcgtc tgagccgtct tgaggtgttc 3000

cacaccgaat ccgagattca gaccggcgcc cgcgcctact acatcgatcc gacggcgcag 3060

accgcgggag atggattcac gaaggacaag cccatgacgt cgatcgagca gctgcacgat 3120

gtgaccgtcg cgccaggctc cgtgatcttc gtcaaggcgg gcaccgagct gaccggggac 3180

ttcgccgtct tcggctacgg caccaaggac gagcccatca ccgtgacgac atacggcgaa 3240

agcgacaaag ccaccaccgc gagcttcgac ggcatgaccg ccgggctgac gctgaagcag 3300

gcgctgaagg cgctcggcaa ggacgacgcc ggctgggtcg tggccgattc cgccactgca 3360

ccggcctccc gcgtgtatgt cccgcaggat gagatcagcg tgcacgccca gtcgtcgcag 3420

aactccggcg cagaggcggc gagggcgctc gacggcgact cgtcgacgag ctggcactcc 3480

cagtacagcc cgaccaccgc gtctgctccg cattgggtga ctctcgatct cggcaaatcg 3540

cgtgagaacg tcgcctactt cgactacctc gcccgtatcg acggcaacaa taacggtgcc 3600

gccaaggatt acgaggtgta tgtctccgac gatcccaacg attttggagc ccctgtggcc 3660

tcgggcacgt tgaagaacgt cgcctacacg cagcgcatca agctgacccc caagaacgga 3720

cggtacgtca agttcgtcat caagaccgat tattccggat cgaacttcgg ctccgcggcg 3780

gaaatgaatg tcgagttgct gcccacggcc gtagaggagg acaaggtcgc caccccgcag 3840

aagccgacag tggacgatga tgccgataca tacaccatcc ccgacatcga gggagtcgtg 3900

tacaaggtcg acggcaaggt gttggccgct ggttccgtag tgaacgtggg cgatgaggac 3960

gtgaccgtca cggtcaccgc cgagcccgcc gacggatacc gcttcccgga tggtgtgacg 4020

tccccagtca cgtatgagct gacgttcacc aagaagggtg gcgagaagcc tccgaccgaa 4080

gtcaacaagg acaagctgca cgccacgatc accaaggctc aggcgatcga ccgttccgcc 4140

tatacggacg agtcgctcaa ggtgcttgat gacaagctcg ccgcagcgct caaggtctat 4200

gacgatgaca aggtgagcca ggatgatgtc gatgccgccg aggcggctct gtctgcggcg 4260

atcgacgcgc tgaagaccaa gccgacgacc cccggcggtg aaggtgagaa gcctggtgaa 4320

ggtgaaaagc ccggtgacgg caacaagccc ggtgacggca agaagcccgg cgacgtgatc 4380

gcaaagaccg gcgcctccac aatgggcgtt gtcttcgctg cactcgcgat ggtagcgggt 4440

gcggtcgtga cgcttgaagc caagcgtaag tccaaccggt aa 4482

<210> 4

<211> 1493

<212>PRT

<213> Bifidobacterium bifidum

<400> 4

Met Leu His Thr Ala Ser Arg Gly Cys Ser Arg Ser Trp Leu Arg Arg

1 5 10 15

Leu Thr Ala Leu Ile Ala Val Ser Ala Leu Ala Phe Val Ala Leu Pro

20 25 30

Asn Val Ala Val Ala Ala Asp Pro Met Glu Tyr Leu Asp Val Ser Phe

35 40 45

Gly Gly Thr Phe Ala Ala Asp Thr Tyr Thr Thr Gly Gly Asp Glu Val

50 55 60

Ala Lys Gly Pro Val Thr Lys His Gly Ser Ile Pro Thr Lys Leu Asp

65 70 75 80

Gly Gly Gly Ile Thr Leu Ala Gly Gly Thr Asn Gly Val Thr Phe Thr

85 90 95

Ser Thr Ala Ser Phe Ser Glu Ser Gly Lys Val Asn Lys Gly Phe Arg

100 105 110

Ala Glu Met Glu Tyr Arg Thr Thr Gln Thr Pro Ser Asn Leu Ala Thr

115 120 125

Leu Phe Ser Ala Met Gly Asn Ile Phe Val Arg Ala Asn Gly Ser Asn

130 135 140

Leu Glu Tyr Gly Phe Ser Thr Asn Pro Ser Gly Ser Thr Trp Asn Asp

145 150 155 160

Tyr Thr Lys Ser Val Thr Leu Pro Ser Asn Asn Val Lys His Ile Ile

165 170 175

Gln Leu Thr Tyr Leu Pro Gly Ala Asp Gly Ala Ala Ser Thr Leu Gln

180 185 190

Leu Ser Val Asp Gly Val Ala Gly Glu Thr Ala Thr Ser Ala Ala Gly

195 200 205

Glu Leu Ala Ala Val Ser Asp Ser Val Gly Asn Lys Phe Gly Ile Gly

210 215 220

Tyr Glu Val Asn Pro Ala Ser Gly Ala Ala Ser Arg Gly Leu Ala Gly

225 230 235 240

Asp Val Phe Arg Ala Arg Val Ala Asp Ser Asp Ala Pro Trp Glu Ile

245 250 255

Leu Asp Ala Ser Gln Leu Leu His Val Asn Phe Asn Gly Thr Phe Ser

260 265 270

Gly Thr Ser Tyr Thr Ala Ala Ser Gly Glu Gln Met Leu Gly Ser Leu

275 280 285

Val Ser Arg Ser Ala Asn Pro Ser Ile Ser Asn Ser Ala Val Thr Leu

290 295 300

Gly Gly Gly Thr Ala Gly Phe Asp Phe Thr Pro Thr Asp Phe Thr Leu

305 310 315 320

Gly Asp Asn Glu Ala Ile Thr Arg Pro Leu Val Ala Glu Leu Arg Phe

325 330 335

Thr Pro Thr Gln Thr Gly Asp Asn Gln Thr Leu Phe Gly Ala Gly Gly

340 345 350

Asn Leu Phe Leu Arg Tyr Glu Ser Asn Lys Leu Val Phe Gly Ala Ser

355 360 365

Thr Lys Ser Gly Asp Asn Trp Thr Asp His Lys Ile Glu Ser Ala Ala

370 375 380

Ala Thr Gly Ala Glu His Val Val Ser Val Ala Tyr Val Pro Asn Lys

385 390 395 400

Ala Gly Thr Gly Ala Lys Leu Val Met Arg Val Asp Gly Gly Asp Ala

405 410 415

Gln Thr Lys Asp Ile Thr Gly Leu Ala Tyr Leu Asn Ser Ser Ile Lys

420 425 430

Gly Lys Val Gly Phe Gly Asn Asp Val His Thr Asp Ala Leu Ser Arg

435 440 445

Gly Phe Val Gly Ser Leu Ser Glu Ile Arg Leu Ala Glu Thr Ser Ala

450 455 460

Asn Phe Thr Thr Asn Glu Phe Lys Leu Val Tyr Ser Gln Val Ser Cys

465 470 475 480

Asp Thr Ser Gly Ile Lys Glu Ala Asn Thr Phe Asp Val Glu Pro Ala

485 490 495

Glu Cys Glu Ala Ala Leu Lys Thr Lys Leu Ser Lys Leu Arg Pro Thr

500 505 510

Glu Gly Gln Ala Asp Tyr Ile Asp Trp Gly Gln Ile Gly Phe Leu His

515 520 525

Tyr Gly Ile Asn Thr Tyr Tyr Asn Gln Glu Trp Gly His Gly Asn Glu

530 535 540

Asp Pro Ser Arg Ile Asn Pro Thr Gly Leu Asp Thr Asp Gln Trp Ala

545 550 555 560

Lys Ser Phe Ala Asp Gly Gly Phe Lys Met Ile Met Val Thr Val Lys

565 570 575

His His Asp Gly Phe Glu Leu Tyr Asp Ser Arg Tyr Asn Thr Glu His

580 585 590

Asp Trp Ala Asn Thr Ala Val Ala Lys Arg Thr Gly Glu Lys Asp Leu

595 600 605

Phe Arg Lys Ile Val Ala Ser Ala Lys Lys Tyr Gly Leu Lys Val Gly

610 615 620

Ile Tyr Tyr Ser Pro Ala Asp Ser Tyr Met Glu Arg Lys Gly Val Trp

625 630 635 640

Gly Asn Asn Ser Ala Arg Val Glu Arg Thr Ile Pro Thr Leu Val Glu

645 650 655

Asn Asp Asp Arg Ala Gly Lys Val Ala Ser Gly Lys Leu Pro Thr Phe

660 665 670

Lys Tyr Lys Ala Thr Asp Tyr Gly Ala Tyr Met Leu Asn Gln Leu Tyr

675 680 685

Glu Leu Leu Thr Glu Tyr Gly Asp Ile Ser Glu Val Trp Phe Asp Gly

690 695 700

Ala Gln Gly Asn Thr Ala Gly Thr Glu His Tyr Asp Tyr Gly Val Phe

705 710 715 720

Tyr Glu Met Ile Arg Arg Leu Gln Pro Gln Ala Ile Gln Ala Asn Ala

725 730 735

Ala Tyr Asp Ala Arg Trp Val Gly Asn Glu Asp Gly Trp Ala Arg Gln

740 745 750

Thr Glu Trp Ser Pro Gln Ala Ala Tyr Asn Asp Gly Val Asp Lys Val

755 760 765

Ser Leu Lys Pro Gly Gln Met Ala Pro Asp Gly Lys Leu Gly Ser Met

770 775 780

Ser Ser Val Leu Ser Glu Ile Arg Ser Gly Ala Ala Asn Gln Leu His

785 790 795 800

Trp Tyr Pro Ala Glu Val Asp Ala Lys Asn Arg Pro Gly Trp Phe Tyr

805 810 815

Arg Ala Ser Gln Ser Pro Ala Ser Val Ala Glu Val Val Lys Tyr Tyr

820 825 830

Glu Gln Ser Thr Gly Arg Asn Ser Gln Tyr Leu Leu Asn Val Pro Pro

835 840 845

Ser Asp Thr Gly Lys Leu Ala Asp Ala Asp Ala Ala Gly Leu Lys Gly

850 855 860

Leu Gly Glu Glu Leu Ala Arg Arg Tyr Gly Thr Asp Leu Ala Leu Gly

865 870 875 880

Lys Ser Ala Thr Val Ala Ala Ser Ala Asn Asp Thr Ala Val Ala Ala

885 890 895

Pro Lys Leu Thr Asp Gly Ser Lys Leu Ser Ser Asp Lys Ala Val Gly

900 905 910

Asn Thr Pro Thr Tyr Thr Ile Asp Leu Gly Ser Thr Val Ala Val Asp

915 920 925

Ala Val Lys Ile Ser Glu Asp Val Arg Asn Ala Gly Gln Gln Ile Glu

930 935 940

Ser Ala Thr Leu Gln Gly Arg Val Asn Gly Thr Trp Thr Asn Leu Ala

945 950 955 960

Thr Met Thr Thr Val Gly Gln Gln Arg Asp Leu Arg Phe Thr Ser Gln

965 970 975

Asn Ile Asp Ala Ile Arg Leu Val Val Asn Ser Ser Arg Gly Pro Val

980 985 990

Arg Leu Ser Arg Leu Glu Val Phe His Thr Glu Ser Glu Ile Gln Thr

995 1000 1005

Gly Ala Arg Ala Tyr Tyr Ile Asp Pro Thr Ala Gln Thr Ala Gly

1010 1015 1020

Asp Gly Phe Thr Lys Asp Lys Pro Met Thr Ser Ile Glu Gln Leu

1025 1030 1035

His Asp Val Thr Val Ala Pro Gly Ser Val Ile Phe Val Lys Ala

1040 1045 1050

Gly Thr Glu Leu Thr Gly Asp Phe Ala Val Phe Gly Tyr Gly Thr

1055 1060 1065

Lys Asp Glu Pro Ile Thr Val Thr Thr Tyr Gly Glu Ser Asp Lys

1070 1075 1080

Ala Thr Thr Ala Ser Phe Asp Gly Met Thr Ala Gly Leu Thr Leu

1085 1090 1095

Lys Gln Ala Leu Lys Ala Leu Gly Lys Asp Asp Ala Gly Trp Val

1100 1105 1110

Val Ala Asp Ser Ala Thr Ala Pro Ala Ser Arg Val Tyr Val Pro

1115 1120 1125

Gln Asp Glu Ile Ser Val His Ala Gln Ser Ser Gln Asn Ser Gly

1130 1135 1140

Ala Glu Ala Ala Arg Ala Leu Asp Gly Asp Ser Ser Thr Ser Trp

1145 1150 1155

His Ser Gln Tyr Ser Pro Thr Thr Ala Ser Ala Pro His Trp Val

1160 1165 1170

Thr Leu Asp Leu Gly Lys Ser Arg Glu Asn Val Ala Tyr Phe Asp

1175 1180 1185

Tyr Leu Ala Arg Ile Asp Gly Asn Asn Asn Gly Ala Ala Lys Asp

1190 1195 1200

Tyr Glu Val Tyr Val Ser Asp Asp Pro Asn Asp Phe Gly Ala Pro

1205 1210 1215

Val Ala Ser Gly Thr Leu Lys Asn Val Ala Tyr Thr Gln Arg Ile

1220 1225 1230

Lys Leu Thr Pro Lys Asn Gly Arg Tyr Val Lys Phe Val Ile Lys

1235 1240 1245

Thr Asp Tyr Ser Gly Ser Asn Phe Gly Ser Ala Ala Glu Met Asn

1250 1255 1260

Val Glu Leu Leu Pro Thr Ala Val Glu Glu Asp Lys Val Ala Thr

1265 1270 1275

Pro Gln Lys Pro Thr Val Asp Asp Asp Ala Asp Thr Tyr Thr Ile

1280 1285 1290

Pro Asp Ile Glu Gly Val Val Tyr Lys Val Asp Gly Lys Val Leu

1295 1300 1305

Ala Ala Gly Ser Val Val Asn Val Gly Asp Glu Asp Val Thr Val

1310 1315 1320

Thr Val Thr Ala Glu Pro Ala Asp Gly Tyr Arg Phe Pro Asp Gly

1325 1330 1335

Val Thr Ser Pro Val Thr Tyr Glu Leu Thr Phe Thr Lys Lys Gly

1340 1345 1350

Gly Glu Lys Pro Pro Thr Glu Val Asn Lys Asp Lys Leu His Ala

1355 1360 1365

Thr Ile Thr Lys Ala Gln Ala Ile Asp Arg Ser Ala Tyr Thr Asp

1370 1375 1380

Glu Ser Leu Lys Val Leu Asp Asp Lys Leu Ala Ala Ala Leu Lys

1385 1390 1395

Val Tyr Asp Asp Asp Lys Val Ser Gln Asp Asp Val Asp Ala Ala

1400 1405 1410

Glu Ala Ala Leu Ser Ala Ala Ile Asp Ala Leu Lys Thr Lys Pro

1415 1420 1425

Thr Thr Pro Gly Gly Glu Gly Glu Lys Pro Gly Glu Gly Glu Lys

1430 1435 1440

Pro Gly Asp Gly Asn Lys Pro Gly Asp Gly Lys Lys Pro Gly Asp

1445 1450 1455

Val Ile Ala Lys Thr Gly Ala Ser Thr Met Gly Val Val Phe Ala

1460 1465 1470

Ala Leu Ala Met Val Ala Gly Ala Val Val Thr Leu Glu Ala Lys

1475 1480 1485

Arg Lys Ser Asn Arg

1490

<210> 5

<211> 2094

<212> DNA

<213> Streptococcus pneumoniae

<400> 5

atgaataaaa gaggtcttta ttcaaaacta ggaatttctg ttgtaggcat tagtctttta 60

atgggagtcc ccactttgat tcatgcgaat gaattaaact atggtcaact gtccatatct 120

cctatttttc aaggaggttc atatcaactg aacaataaga gtatagatat cagctctttg 180

ttattagata aattgtctgg agagagtcag acagtagtaa tgaaatttaa agcagataaa 240

ccaaactctc ttcaagcttt gtttggccta tctaatagta aagcaggctt taaaaataat 300

tacttttcaa ttttcatgag agattctggt gagataggtg tagaaataag agacgcccaa 360

gagggaataa attatttatt ttctagacca gcttcattat ggggaaagca taaaggacag 420

gcagttgaaa atacactagt atttgtatct gattctaaag ataaaacata cacaatgtat 480

gttaatggaa tagaagtgtt ctctgaaaca gttgatacat ttttgccaat ttcaaatata 540

aatggtatag ataaggcaac actaggagct gttaatcgtg aaggtaagga acattacctc 600

gcaaaaggaa gtattggtga aatcagtcta tttaacaaag caattagtga tcaggaagtt 660

tcaaatattc ccttgtcaaa tccatttcag ttaattttcc aatcaggaga ttctactcaa 720

gctaactatt ttagaatacc gacactatat acattaagta gtggaagagt tctatcaagt 780

attgatgcac gttatggtgg gactcatgat tctaaaagta agattaatat tgccacttct 840

tatagtgatg ataatgggaa aacgtggagt gagccaattt ttgctatgaa gtttaatgac 900

tatgaggagc agttagttta ctggccacga gataataaat taaagaatag tcaaattagt 960

ggaagtgctt cattcataga ttcatccatt gttgaagata aaaaatctgg gaaaacgata 1020

ttactagctg atgttatgcc tgcgggtatt ggaaataata atgcaaataa agccgactca 1080

ggttttaaag aaataaatgg tcattattat ttaaaactaa agaagaatgg agataacgat 1140

ttccgttata cagttagaga aaatggtgtc gtttatgatg aaacaactaa taaacctaca 1200

aattatacta taaatgataa gtatgaagtt ttggagggag gaaagtcttt aacagtcgaa 1260

caatattcgg ttgattttga tagtggctct ttaagagaaa ggcataatgg aaacaggtt 1320

cctatgaatg ttttctacaa agattcgtta tttaaagtga ctcctactaa ttatatagca 1380

atgacaacta gtcagaatag aggagagagt tgggaacaat ttaagttgtt gcctccgttc 1440

ttaggagaaa aacataatgg aacttacttg tgtcctggac aaggtttagc attaaaatca 1500

agtaacagat tgatttttgc aacatatact agtggagaac taacctatct catttcggat 1560

gatagtggtc aaacatggaa gaaatcctca gcttcaattc cgtttaaaaa tgcaacagca 1620

gaagcacaaa tggttgaact gagagatggt gtgattagaa cattctttag aaccactaca 1680

ggtaagatag cttatatgac tagtagagat tctggagaaa catggtcgaa agtttcgtat 1740

attgatggaa ttcaacaaac ttcatatggc acacaagtat ctgcaattaa atactctcaa 1800

ttaattgatg gaaaagaagc agtcattttg agtacaccaa attctagaag tggccgtaag 1860

ggaggccaat tagttgtcgg tttggtcaat aaagaagatg atagtattga ttggagatac 1920

cactatgata ttgatttgcc ttcgtatggt tatgcctatt ctgcgattac agaattgcca 1980

aatcatcaca taggtgtact gtttgaaaaa tatgattcgt ggtcgagaaa tgaattgcat 2040

ttaagcaatg tagttcagta tatagatttg gaaattaatg atttaacaaa ataa 2094

<210> 6

<211> 697

<212> PRT

<213> Streptococcus pneumoniae

<400> 6

Met Asn Lys Arg Gly Leu Tyr Ser Lys Leu Gly Ile Ser Val Val Gly

1 5 10 15

Ile Ser Leu Leu Met Gly Val Pro Thr Leu Ile His Ala Asn Glu Leu

20 25 30

Asn Tyr Gly Gln Leu Ser Ile Ser Pro Ile Phe Gln Gly Gly Ser Tyr

35 40 45

Gln Leu Asn Asn Lys Ser Ile Asp Ile Ser Ser Leu Leu Leu Asp Lys

50 55 60

Leu Ser Gly Glu Ser Gln Thr Val Val Met Lys Phe Lys Ala Asp Lys

65 70 75 80

Pro Asn Ser Leu Gln Ala Leu Phe Gly Leu Ser Asn Ser Lys Ala Gly

85 90 95

Phe Lys Asn Asn Tyr Phe Ser Ile Phe Met Arg Asp Ser Gly Glu Ile

100 105 110

Gly Val Glu Ile Arg Asp Ala Gln Lys Gly Ile Asn Tyr Leu Phe Ser

115 120 125

Arg Pro Ala Ser Leu Trp Gly Lys His Lys Gly Gln Ala Val Glu Asn

130 135 140

Thr Leu Val Phe Val Ser Asp Ser Lys Asp Lys Thr Tyr Thr Met Tyr

145 150 155 160

Val Asn Gly Ile Glu Val Phe Ser Glu Thr Val Asp Thr Phe Leu Pro

165 170 175

Ile Ser Asn Ile Asn Gly Ile Asp Lys Ala Thr Leu Gly Ala Val Asn

180 185 190

Arg Glu Gly Lys Glu His Tyr Leu Ala Lys Gly Ser Ile Asp Glu Ile

195 200 205

Ser Leu Phe Asn Lys Ala Ile Ser Asp Gln Glu Val Ser Thr Ile Pro

210 215 220

Leu Ser Asn Pro Phe Gln Leu Ile Phe Gln Ser Gly Asp Ser Thr Gln

225 230 235 240

Ala Asn Tyr Phe Arg Ile Pro Thr Leu Tyr Thr Leu Ser Ser Gly Arg

245 250 255

Val Leu Ser Ser Ile Asp Ala Arg Tyr Gly Gly Thr His Asp Ser Lys

260 265 270

Ser Lys Ile Asn Ile Ala Thr Ser Tyr Ser Asp Asp Asn Gly Lys Thr

275 280 285

Trp Ser Glu Pro Ile Phe Ala Met Lys Phe Asn Asp Tyr Glu Glu Gln

290 295 300

Leu Val Tyr Trp Pro Arg Asp Asn Lys Leu Lys Asn Ser Gln Ile Ser

305 310 315 320

Gly Ser Ala Ser Phe Ile Asp Ser Ser Ile Val Glu Asp Lys Lys Ser

325 330 335

Gly Lys Thr Ile Leu Leu Ala Asp Val Met Pro Ala Gly Ile Gly Asn

340 345 350

Asn Asn Ala Asn Lys Ala Asp Ser Gly Phe Lys Glu Ile Asn Gly His

355 360 365

Tyr Tyr Leu Lys Leu Lys Lys Asn Gly Asp Asn Asp Phe Arg Tyr Thr

370 375 380

Val Arg Glu Asn Gly Val Val Tyr Asn Glu Thr Thr Asn Lys Pro Thr

385 390 395 400

Asn Tyr Thr Ile Asn Asp Lys Tyr Glu Val Leu Glu Gly Gly Lys Ser

405 410 415

Leu Thr Val Glu Gln Tyr Ser Val Asp Phe Asp Ser Gly Ser Leu Arg

420 425 430

Glu Arg His Asn Gly Lys Gln Val Pro Met Asn Val Phe Tyr Lys Asp

435 440 445

Ser Leu Phe Lys Val Thr Pro Thr Asn Tyr Ile Ala Met Thr Thr Ser

450 455 460

Gln Asn Arg Gly Glu Ser Trp Glu Gln Phe Lys Leu Leu Pro Pro Phe

465 470 475 480

Leu Gly Glu Lys His Asn Gly Thr Tyr Leu Cys Pro Gly Gln Gly Leu

485 490 495

Ala Leu Lys Ser Ser Asn Arg Leu Ile Phe Ala Thr Tyr Thr Ser Gly

500 505 510

Glu Leu Thr Tyr Leu Ile Ser Asp Asp Ser Gly Gln Thr Trp Lys Lys

515 520 525

Ser Ser Ala Ser Ile Pro Phe Lys Asn Ala Thr Ala Glu Ala Gln Met

530 535 540

Val Glu Leu Arg Asp Gly Val Ile Arg Thr Phe Phe Arg Thr Thr Thr

545 550 555 560

Gly Lys Ile Ala Tyr Met Thr Ser Arg Asp Ser Gly Glu Thr Trp Ser

565 570 575

Lys Val Ser Tyr Ile Asp Gly Ile Gln Gln Thr Ser Tyr Gly Thr Gln

580 585 590

Val Ser Ala Ile Lys Tyr Ser Gln Leu Ile Asp Gly Lys Glu Ala Val

595 600 605

Ile Leu Ser Thr Pro Asn Ser Arg Ser Gly Arg Lys Gly Gly Gln Leu

610 615 620

Val Val Gly Leu Val Asn Lys Glu Asp Asp Ser Ile Asp Trp Lys Tyr

625 630 635 640

His Tyr Asp Ile Asp Leu Pro Ser Tyr Gly Tyr Ala Tyr Ser Ala Ile

645 650 655

Thr Glu Leu Pro Asn His His Ile Gly Val Leu Phe Glu Lys Tyr Asp

660 665 670

Ser Trp Ser Arg Asn Glu Leu His Leu Ser Asn Val Val Gln Tyr Ile

675 680 685

Asp Leu Glu Ile Asn Asp Leu Thr Lys

690 695

<210> 7

<211> 2076

<212> DNA

<213> Bifidobacterium longum

<400> 7

atggaacata gagcgttcaa gtggccgcag ccacttgcgg gcaacaagcc ccgcatctgg 60

tacggcggcg attacaaccc cgaccaatgg cctgaggaag tgtgggacga agatgtagcc 120

ctcatgcagc aggccggcgt caacctcgtc tccgtagcca tcttctcctg ggccaagctt 180

gagcccgaag aaggcgtgta cgacttcgat tggctcgacc gcgtcatcga caagctcggc 240

aaggccggca tcgccgtcga tctcgcctcc ggcaccgcat ccccgccgat gtggatgacc 300

caggcccacc cggagatcct ctgggtcgac taccgcggcg acgtctgcca gcccggtgcc 360

cgccagcact ggcgcgccac cagcccggtc ttccttgact acgcgctcaa cctgtgccgc 420

aagatggccg agcactacaa ggacaacccc tatgtggtct cttggcatgt gagcaacgag 480

tacggctgcc acaaccgctt cgactattcc gaagacgccg agcgcgcctt ccagaagtgg 540

tgcgagaaga agtacggcac catcgacgct gtcaacgacg cctggggcac cgccttctgg 600

gcgcagcgca tgaacaattt ctccgagatc atcccgccgc gattcatcgg cgacggcaac 660

ttcatgaacc cgggcaagct gcttgattgg aagcgtttca gctccgacgc gctgctggac 720

ttctacaagg ccgagcgcga cgccctgctc gagatcgccc ccaagccgca gaccaccaac 780

ttcatggtct ccgcgggctg caccgtcctc gactacgaca agtggggtca tgacgtggac 840

ttcgtgtcca acgaccatta cttctcgccc ggcgaggccc acttcgacga gatggcctac 900

gcggcctgcc tcaccgacgg catcgcccgc aagaacccgt ggttcctcat ggaacattcc 960

acgtccgccg tcaactggcg cccgaccaac taccggctcg agcccggcga gctggtgcgc 1020

gactccctgg cccatctggc catgggcgcc gacgccatct gctacttcca gtggcgtcag 1080

tccaaggccg gcgccgagaa gtggcattcc gccatggtgc cccacgcagg ccccgactcc 1140

cagatcttcc gcgatgtgtg cgagctgggt gccgacctca acaagcttgc tgacgagggc 1200

ctgctgagca ccaagctggt caagtccaag gtcgccatcg tcttcgacta cgagtcccag 1260

tgggccaccg agcacaccgc cacccccacg caggaggtgc gccactggac cgagccgctg 1320

gactggttcc gcgcgctggc ggacaatggc ctgaccgccg acgtggtgcc ggtccgcggt 1380

ccttgggatg agtacgaggc cgtcgtgttg ccgagcctgg ccatcctgtc cgagcagacc 1440

acgcgccgcg tgcgcgagta tgtggcgaac ggcggcaagc tgttcgtgac ctactacacc 1500

ggtctggtgg acgacaggga tcacgtctgg ctgggcggct accccggctc cattcgcgac 1560

gtggtgggcg tgcgcgtcga ggaattcgcc ccgatgggca ccgacgcccc cggcaccatg 1620

gaccaccttg acttggacaa cggaaccgtg gcgcacgatt tcgccgacgt gatcacctcc 1680

gtggccgata ccgctcacgt ggtcgcctcc ttcaaggcag ataagtggac cggtttcgac 1740

ggcgctcccg ccatcaccgt caacgacttc ggcgacggca aggccgcata cgtcggtgcc 1800

cgtctcgggc gtgagggctt ggccaagagc ctgcccgcgc tgctggagga actcggcatc 1860

gagacttcgg ctgaggacga tcgtggtgaa gtgctgcgcg tcgagcgtgc ggacgaaact 1920

ggcgagaacc acttcgtgtt cctgttcaac cgcacccacg atgttgcggt cgtggacgtg 1980

gaaggcgaac cgctggtcgc ctcgctggcc caggtcaacg agtccgagca cacggccgcc 2040

atccagccca acggcgtact cgtcgtcaag ctgtaa 2076

<210> 8

<211> 691

<212> PRT

<213> Bifidobacterium longum

<400> 8

Met Glu His Arg Ala Phe Lys Trp Pro Gln Pro Leu Ala Gly Asn Lys

1 5 10 15

Pro Arg Ile Trp Tyr Gly Gly Asp Tyr Asn Pro Asp Gln Trp Pro Glu

20 25 30

Glu Val Trp Asp Glu Asp Val Ala Leu Met Gln Gln Ala Gly Val Asn

35 40 45

Leu Val Ser Val Ala Ile Phe Ser Trp Ala Lys Leu Glu Pro Glu Glu

50 55 60

Gly Val Tyr Asp Phe Asp Trp Leu Asp Arg Val Ile Asp Lys Leu Gly

65 70 75 80

Lys Ala Gly Ile Ala Val Asp Leu Ala Ser Gly Thr Ala Ser Pro Pro

85 90 95

Met Trp Met Thr Gln Ala His Pro Glu Ile Leu Trp Val Asp Tyr Arg

100 105 110

Gly Asp Val Cys Gln Pro Gly Ala Arg Gln His Trp Arg Ala Thr Ser

115 120 125

Pro Val Phe Leu Asp Tyr Ala Leu Asn Leu Cys Arg Lys Met Ala Glu

130 135 140

His Tyr Lys Asp Asn Pro Tyr Val Val Ser Trp His Val Ser Asn Glu

145 150 155 160

Tyr Gly Cys His Asn Arg Phe Asp Tyr Ser Glu Asp Ala Glu Arg Ala

165 170 175

Phe Gln Lys Trp Cys Glu Lys Lys Tyr Gly Thr Ile Asp Ala Val Asn

180 185 190

Asp Ala Trp Gly Thr Ala Phe Trp Ala Gln Arg Met Asn Asn Phe Ser

195 200 205

Glu Ile Ile Pro Pro Arg Phe Ile Gly Asp Gly Asn Phe Met Asn Pro

210 215 220

Gly Lys Leu Leu Asp Trp Lys Arg Phe Ser Ser Asp Ala Leu Leu Asp

225 230 235 240

Phe Tyr Lys Ala Glu Arg Asp Ala Leu Leu Glu Ile Ala Pro Lys Pro

245 250 255

Gln Thr Thr Asn Phe Met Val Ser Ala Gly Cys Thr Val Leu Asp Tyr

260 265 270

Asp Lys Trp Gly His Asp Val Asp Phe Val Ser Asn Asp His Tyr Phe

275 280 285

Ser Pro Gly Glu Ala His Phe Asp Glu Met Ala Tyr Ala Ala Cys Leu

290 295 300

Thr Asp Gly Ile Ala Arg Lys Asn Pro Trp Phe Leu Met Glu His Ser

305 310 315 320

Thr Ser Ala Val Asn Trp Arg Pro Thr Asn Tyr Arg Leu Glu Pro Gly

325 330 335

Glu Leu Val Arg Asp Ser Leu Ala His Leu Ala Met Gly Ala Asp Ala

340 345 350

Ile Cys Tyr Phe Gln Trp Arg Gln Ser Lys Ala Gly Ala Glu Lys Trp

355 360 365

His Ser Ala Met Val Pro His Ala Gly Pro Asp Ser Gln Ile Phe Arg

370 375 380

Asp Val Cys Glu Leu Gly Ala Asp Leu Asn Lys Leu Ala Asp Glu Gly

385 390 395 400

Leu Leu Ser Thr Lys Leu Val Lys Ser Lys Val Ala Ile Val Phe Asp

405 410 415

Tyr Glu Ser Gln Trp Ala Thr Glu His Thr Ala Thr Pro Thr Gln Glu

420 425 430

Val Arg His Trp Thr Glu Pro Leu Asp Trp Phe Arg Ala Leu Ala Asp

435 440 445

Asn Gly Leu Thr Ala Asp Val Val Pro Val Arg Gly Pro Trp Asp Glu

450 455 460

Tyr Glu Ala Val Val Leu Pro Ser Leu Ala Ile Leu Ser Glu Gln Thr

465 470 475 480

Thr Arg Arg Val Arg Glu Tyr Val Ala Asn Gly Gly Lys Leu Phe Val

485 490 495

Thr Tyr Tyr Thr Gly Leu Val Asp Asp Arg Asp His Val Trp Leu Gly

500 505 510

Gly Tyr Pro Gly Ser Ile Arg Asp Val Val Gly Val Arg Val Glu Glu

515 520 525

Phe Ala Pro Met Gly Thr Asp Ala Pro Gly Thr Met Asp His Leu Asp

530 535 540

Leu Asp Asn Gly Thr Val Ala His Asp Phe Ala Asp Val Ile Thr Ser

545 550 555 560

Val Ala Asp Thr Ala His Val Val Ala Ser Phe Lys Ala Asp Lys Trp

565 570 575

Thr Gly Phe Asp Gly Ala Pro Ala Ile Thr Val Asn Asp Phe Gly Asp

580 585 590

Gly Lys Ala Ala Tyr Val Gly Ala Arg Leu Gly Arg Glu Gly Leu Ala

595 600 605

Lys Ser Leu Pro Ala Leu Leu Glu Glu Leu Gly Ile Glu Thr Ser Ala

610 615 620

Glu Asp Asp Arg Gly Glu Val Leu Arg Val Glu Arg Ala Asp Glu Thr

625 630 635 640

Gly Glu Asn His Phe Val Phe Leu Phe Asn Arg Thr His Asp Val Ala

645 650 655

Val Val Asp Val Glu Gly Glu Pro Leu Val Ala Ser Leu Ala Gln Val

660 665 670

Asn Glu Ser Glu His Thr Ala Ala Ile Gln Pro Asn Gly Val Leu Val

675 680 685

Val Lys Leu

690

<210> 9

<211> 1626

<212> DNA

<213> Clostridium thermocellum

<400> 9

atggcagaag gggttatagt caacggaact cagtttaaag acacatcggg aaatgtgata 60

catgcccatg ggggaggcat gttaaagcat ggtgactatt attactggta cggtgaatac 120

cgggacgact ccaacttgtt tttgggtgta agttgctaca ggtcaaaaga tcttgtaaac 180

tgggaataca gaggagaagt gctgagccga aattccgctc ctgaactgaa tcactgcaat 240

attgaaagac cgaaagtcat gtacaacgca tcaaccggtg aatttgtcat gtggatgcac 300

tggggagaacg gcataaacta cggtcaggca agagcagctg ttgcgtattc caaaacgccc 360

gacggcaaat tcacatacat tcgaagcttt cgtcccatgc aggataccgg cgttatggat 420

catggccttc cgggatatat gtcaagggac tgcaatgtat ttgtggacac tgacggcaag 480

ggatatttta tatccgcagc caatgagaac atggacctgc acctttatga gctgacacct 540

gactataaaa atattgcatc ccttaaggca aagctgtttg tcggacagca gagggaagca 600

ccatgcctta taaagagaaa cggctactat taccttatta cttccggttg tacaggttgg 660

aacccgaatc aggctaaata cgcatattcc aaagatttgg ccagtggctg gtcccagctt 720

tacaatcttg gtaattcaac cacctacagg tcacagccga cttttatcat tcccgttcag 780

ggaagctcgg gaaccagtta tctttatatg ggtgaccgtt gggccggtgc ctggggagga 840

aaggttaatg actcccaata tgtatggctt cccttaaact tcatatccga tacaacactt 900

gaactgccct attatgactc tgtaaagatt gatgcttctt caggaataat ttccgagtac 960

ataccggaca ctacacgcta caagctggta aacaaaaaca gcggaaaagt cctggatgtt 1020

cttgacggtt ctgtcgataa tgcagcccag atagtccaat ggaccgataa cgggtctttg 1080

agtcaacagt ggtaccttgt ggacgtgggc ggtggttata aaaagattgt aaatgtaaag 1140

agcggaagag ccttggatgt aaaagacgaa tccaaggaag acggtggagt attaatacaa 1200

tataccagca acggcggata taatcagcac tggaaattca cagacatagg tgacgggtat 1260

tacaagattt ccagccgcca ctgcggaaaa cttatagatg tgcgaaaatg gtcaacggaa 1320

gacggcggaa taattcagca gtggtccgat gccggaggaa caaatcagca ttggaagctg 1380

gtgcttgtat caagtcccga gccttcacca tcaccttctc cccaagtggt taaaggagat 1440

gtaaacggcg acttgaaagt aaattcaacg gatttttcca tgttaagaag atatttactt 1500

aaaaccattg acaattttcc gacagaaaac ggaaaacagg ctgccgattt gaacggagac 1560

ggcagaataa actcttcgga tcttacaatg ctgaaaagat acttgcttat ggaagtggat 1620

ttgtaa 1626

<210> 10

<211> 541

<212> PRT

<213> Clostridium thermocellum

<400> 10

Met Ala Glu Gly Val Ile Val Asn Gly Thr Gln Phe Lys Asp Thr Ser

1 5 10 15

Gly Asn Val Ile His Ala His Gly Gly Gly Met Leu Lys His Gly Asp

20 25 30

Tyr Tyr Tyr Trp Tyr Gly Glu Tyr Arg Asp Asp Ser Asn Leu Phe Leu

35 40 45

Gly Val Ser Cys Tyr Arg Ser Lys Asp Leu Val Asn Trp Glu Tyr Arg

50 55 60

Gly Glu Val Leu Ser Arg Asn Ser Ala Pro Glu Leu Asn His Cys Asn

65 70 75 80

Ile Glu Arg Pro Lys Val Met Tyr Asn Ala Ser Thr Gly Glu Phe Val

85 90 95

Met Trp Met His Trp Glu Asn Gly Ile Asn Tyr Gly Gln Ala Arg Ala

100 105 110

Ala Val Ala Tyr Ser Lys Thr Pro Asp Gly Lys Phe Thr Tyr Ile Arg

115 120 125

Ser Phe Arg Pro Met Gln Asp Thr Gly Val Met Asp His Gly Leu Pro

130 135 140

Gly Tyr Met Ser Arg Asp Cys Asn Val Phe Val Asp Thr Asp Gly Lys

145 150 155 160

Gly Tyr Phe Ile Ser Ala Ala Asn Glu Asn Met Asp Leu His Leu Tyr

165 170 175

Glu Leu Thr Pro Asp Tyr Lys Asn Ile Ala Ser Leu Lys Ala Lys Leu

180 185 190

Phe Val Gly Gln Gln Arg Glu Ala Pro Cys Leu Ile Lys Arg Asn Gly

195 200 205

Tyr Tyr Tyr Leu Ile Thr Ser Gly Cys Thr Gly Trp Asn Pro Asn Gln

210 215 220

Ala Lys Tyr Ala Tyr Ser Lys Asp Leu Ala Ser Gly Trp Ser Gln Leu

225 230 235 240

Tyr Asn Leu Gly Asn Ser Thr Thr Tyr Arg Ser Gln Pro Thr Phe Ile

245 250 255

Ile Pro Val Gln Gly Ser Ser Gly Thr Ser Tyr Leu Tyr Met Gly Asp

260 265 270

Arg Trp Ala Gly Ala Trp Gly Gly Lys Val Asn Asp Ser Gln Tyr Val

275 280 285

Trp Leu Pro Leu Asn Phe Ile Ser Asp Thr Thr Leu Glu Leu Pro Tyr

290 295 300

Tyr Asp Ser Val Lys Ile Asp Ala Ser Ser Gly Ile Ile Ser Glu Tyr

305 310 315 320

Ile Pro Asp Thr Thr Arg Tyr Lys Leu Val Asn Lys Asn Ser Gly Lys

325 330 335

Val Leu Asp Val Leu Asp Gly Ser Val Asp Asn Ala Ala Gln Ile Val

340 345 350

Gln Trp Thr Asp Asn Gly Ser Leu Ser Gln Gln Trp Tyr Leu Val Asp

355 360 365

Val Gly Gly Gly Tyr Lys Lys Ile Val Asn Val Lys Ser Gly Arg Ala

370 375 380

Leu Asp Val Lys Asp Glu Ser Lys Glu Asp Gly Gly Val Leu Ile Gln

385 390 395 400

Tyr Thr Ser Asn Gly Gly Tyr Asn Gln His Trp Lys Phe Thr Asp Ile

405 410 415

Gly Asp Gly Tyr Tyr Lys Ile Ser Ser Arg His Cys Gly Lys Leu Ile

420 425 430

Asp Val Arg Lys Trp Ser Thr Glu Asp Gly Gly Ile Ile Gln Gln Trp

435 440 445

Ser Asp Ala Gly Gly Thr Asn Gln His Trp Lys Leu Val Leu Val Ser

450 455 460

Ser Pro Glu Pro Ser Pro Ser Pro Ser Pro Gln Val Val Lys Gly Asp

465 470 475 480

Val Asn Gly Asp Leu Lys Val Asn Ser Thr Asp Phe Ser Met Leu Arg

485 490 495

Arg Tyr Leu Leu Lys Thr Ile Asp Asn Phe Pro Thr Glu Asn Gly Lys

500 505 510

Gln Ala Ala Asp Leu Asn Gly Asp Gly Arg Ile Asn Ser Ser Asp Leu

515 520 525

Thr Met Leu Lys Arg Tyr Leu Leu Met Glu Val Asp Leu

530 535 540

<210> 11

<211> 2631

<212> DNA

<213> Paenibacillus sp.

<400> 11

atgaatcgac acgtcctgct tcatccgtat ctccaccgga aggcgttgcc tctgctcctg 60

gccttgacgc tgctgacggg catcgccctg ttcccggcct ccaccgcgca ggcggcgacg 120

accgtgacgt cgatgacgta cttctctgcc aatgacggtc ccgtcatctc caaatccggc 180

gtcgggcaag ccagctacgg tttcgtcatg ccgatcttca acggaggcgc tgcgacctgg 240

aacgatgtcg ccgatgacgt cggcgttcgc gtcaaggtcg gcggcagctg ggtcgacatt 300

gacagcgttg gcggctatgt gtacaaccag aactggggcc attggaacga cagcggcacc 360

tatggctact ggttcaccct ctccgccacg accgagctgc agctctactc caaggcgaac 420

agcagcgtca cactcaacta cacgctcgtc ttccagaatg tcaatgaaac gaccattacc 480

tcgatgacac cgacccaggg cccgcaattg accgcagggt ataccggcgg cgcaggcttc 540

acctatccgg tcttcaacaa cgatccctcc atcccgtatg cagccgtagc cggcgatctg 600

aaggtgtacg tcaagccagt cgccagcagt acctggatcg atatcgacaa caacgcggcg 660

agcggctgga tctacgacag caacttcggc cagttcaccg aaggcggcgg cggctactgg 720

ttcaccgtca ccgagtcgat caacgtcaag ctcgagtcca ggacgtcctc ggccaacgtc 780

gtctatacga tcaacttccc gcagccgacg cgcagcagct acacactctc cgcctatgac 840

ggcacgacct acagcgccga tgcgagcggc gcgatcggta tcccgctgcc gcggatcgac 900

ggcaccccgg cgatcggcag cgagctcggc aacttcgtct accagatcta ccggaacggc 960

cagtgggtcg agatgagcaa ctcggcgcag agcagcttcg tctactcggc caatggctac 1020

aacaacatgt ccgacgccaa tcaatggggc tactgggccg actacatcta cggcctctgg 1080

ttccggccga tccaggagga tatgcagatc cgcatcggct atccgctgaa tggccagtcc 1140

ggcggcagcg tcggcagcaa cttcgtcacc tatacgctga tcggcaaccc gaacgcgccg 1200

cgacccgatg tgagcgacca gggcgacgtc gagatcggca cgcccaccga tccggccatc 1260

gcaggatgga atctgtattg gcaggatgaa ttcgccggca gcgcgctcga tctgaacaag 1320

tggaactacg agaccggcta ctacatcggc aacgacccca atctgtgggg ctggggcaac 1380

gccgagatgc agcactatac gacgagcacg caaaatgtct tcgtcgctga cggcaaactc 1440

aacatccgag cgctccacga ttaccaatcg ttcccgcagg acccgaaccg ctacgcgacc 1500

tactcctccg gcaagatcaa caccaaggac aacatgtcgc tgcagtacgg ccgcgtcgat 1560

atccgcgcca agctgccgac tggcgatggc gtctggccgg cactgtggat gctgccggag 1620

gactccgtct acggcgcatg ggcggcatca ggagagatcg acatcatgga ggcgaagggc 1680

cgtctgcccg gcacgacgag cggcgcgatc cactacggcg gccaatggcc ggtcaaccgc 1740

tacctcgccg gagaatgcta cctcccgcaa ggtacgacat tcgccgacga ctttaatgtg 1800

tacacgatga tctgggaaga ggacaacatg aagtggtacg ttaacggtga gtttttcttc 1860

aaggtgacgc gcgagcagtg gtactccgtc gccgccccca acaatccgga cgcgccgttc 1920

gaccagccgt tctatctgat catgaacctg gcggtcggcg gccacttcga cggcgggcgt 1980

acgcccgacc cgtccgacat cccggcgacg atgcagatcg actacgtgcg ggtgtacaaa 2040

gagggcgcgg gcggcggtcc gggcaacccg ggcggcaacg tcgcggtgac cggcgttagc 2100

gtgaccccgg caacggcgca ggtgcaggtc ggtcagaccg tctcgctgag cgccaacgtc 2160

gcgccagcca atgcaacgaa caagcaagtg acctggtcag tcgccaatgg cagcatcgcc 2220

tcggtgagcg ccagcggcgt cgtcagtgga ctcgctgctg gcacgacgac cgtaaccgcc 2280

acgaccgcag acggcaaccg caccgcctcg gcgacgatca ccgtcgtgcc gccaccgacg 2340

acgaccgtca tcatcggcga tagcgtgcgc ggcatccgaa agaccggcga caacctgctc 2400

ttctacgtca acggcgcaac ctacgccgac ctgcactaca aggtgaacgg cggcggtcag 2460

cctaatgtcg cgatgacgca cacaggaggc ggcaactaca cctacccggt gcatggcctc 2520

caacaaggcg ataccgtcga atacttcttc acctacaacc ccggcaacgg cgcgctagac 2580

acgccttggc agacttatgt gcatggggta acacaaggtg ttgttgagta a 2631

<210> 12

<211> 876

<212> PRT

<213> Paenibacillus sp.

<400> 12

Met Asn Arg His Val Leu Leu His Pro Tyr Leu His Arg Lys Ala Leu

1 5 10 15

Pro Leu Leu Leu Ala Leu Thr Leu Leu Thr Gly Ile Ala Leu Phe Pro

20 25 30

Ala Ser Thr Ala Gln Ala Ala Thr Thr Val Thr Ser Met Thr Tyr Phe

35 40 45

Ser Ala Asn Asp Gly Pro Val Ile Ser Lys Ser Gly Val Gly Gln Ala

50 55 60

Ser Tyr Gly Phe Val Met Pro Ile Phe Asn Gly Gly Ala Ala Thr Trp

65 70 75 80

Asn Asp Val Ala Asp Asp Val Gly Val Arg Val Lys Val Gly Gly Ser

85 90 95

Trp Val Asp Ile Asp Ser Val Gly Gly Tyr Val Tyr Asn Gln Asn Trp

100 105 110

Gly His Trp Asn Asp Ser Gly Thr Tyr Gly Tyr Trp Phe Thr Leu Ser

115 120 125

Ala Thr Thr Glu Leu Gln Leu Tyr Ser Lys Ala Asn Ser Ser Val Thr

130 135 140

Leu Asn Tyr Thr Leu Val Phe Gln Asn Val Asn Glu Thr Thr Ile Thr

145 150 155 160

Ser Met Thr Pro Thr Gln Gly Pro Gln Leu Thr Ala Gly Tyr Thr Gly

165 170 175

Gly Ala Gly Phe Thr Tyr Pro Val Phe Asn Asn Asp Pro Ser Ile Pro

180 185 190

Tyr Ala Ala Val Ala Gly Asp Leu Lys Val Tyr Val Lys Pro Val Ala

195 200 205

Ser Ser Thr Trp Ile Asp Ile Asp Asn Asn Ala Ala Ser Gly Trp Ile

210 215 220

Tyr Asp Ser Asn Phe Gly Gln Phe Thr Glu Gly Gly Gly Gly Tyr Trp

225 230 235 240

Phe Thr Val Thr Glu Ser Ile Asn Val Lys Leu Glu Ser Arg Thr Ser

245 250 255

Ser Ala Asn Val Val Tyr Thr Ile Asn Phe Pro Gln Pro Thr Arg Ser

260 265 270

Ser Tyr Thr Leu Ser Ala Tyr Asp Gly Thr Thr Tyr Ser Ala Asp Ala

275 280 285

Ser Gly Ala Ile Gly Ile Pro Leu Pro Arg Ile Asp Gly Thr Pro Ala

290 295 300

Ile Gly Ser Glu Leu Gly Asn Phe Val Tyr Gln Ile Tyr Arg Asn Gly

305 310 315 320

Gln Trp Val Glu Met Ser Asn Ser Ala Gln Ser Ser Phe Val Tyr Ser

325 330 335

Ala Asn Gly Tyr Asn Asn Met Ser Asp Ala Asn Gln Trp Gly Tyr Trp

340 345 350

Ala Asp Tyr Ile Tyr Gly Leu Trp Phe Arg Pro Ile Gln Glu Asp Met

355 360 365

Gln Ile Arg Ile Gly Tyr Pro Leu Asn Gly Gln Ser Gly Gly Ser Val

370 375 380

Gly Ser Asn Phe Val Thr Tyr Thr Leu Ile Gly Asn Pro Asn Ala Pro

385 390 395 400

Arg Pro Asp Val Ser Asp Gln Gly Asp Val Glu Ile Gly Thr Pro Thr

405 410 415

Asp Pro Ala Ile Ala Gly Trp Asn Leu Tyr Trp Gln Asp Glu Phe Ala

420 425 430

Gly Ser Ala Leu Asp Leu Asn Lys Trp Asn Tyr Glu Thr Gly Tyr Tyr

435 440 445

Ile Gly Asn Asp Pro Asn Leu Trp Gly Trp Gly Asn Ala Glu Met Gln

450 455 460

His Tyr Thr Thr Ser Thr Gln Asn Val Phe Val Ala Asp Gly Lys Leu

465 470 475 480

Asn Ile Arg Ala Leu His Asp Tyr Gln Ser Phe Pro Gln Asp Pro Asn

485 490 495

Arg Tyr Ala Thr Tyr Ser Ser Gly Lys Ile Asn Thr Lys Asp Asn Met

500 505 510

Ser Leu Gln Tyr Gly Arg Val Asp Ile Arg Ala Lys Leu Pro Thr Gly

515 520 525

Asp Gly Val Trp Pro Ala Leu Trp Met Leu Pro Glu Asp Ser Val Tyr

530 535 540

Gly Ala Trp Ala Ala Ser Gly Glu Ile Asp Ile Met Glu Ala Lys Gly

545 550 555 560

Arg Leu Pro Gly Thr Thr Ser Gly Ala Ile His Tyr Gly Gly Gln Trp

565 570 575

Pro Val Asn Arg Tyr Leu Ala Gly Glu Cys Tyr Leu Pro Gln Gly Thr

580 585 590

Thr Phe Ala Asp Asp Phe Asn Val Tyr Thr Met Ile Trp Glu Glu Asp

595 600 605

Asn Met Lys Trp Tyr Val Asn Gly Glu Phe Phe Phe Lys Val Thr Arg

610 615 620

Glu Gln Trp Tyr Ser Val Ala Ala Pro Asn Asn Pro Asp Ala Pro Phe

625 630 635 640

Asp Gln Pro Phe Tyr Leu Ile Met Asn Leu Ala Val Gly Gly His Phe

645 650 655

Asp Gly Gly Arg Thr Pro Asp Pro Ser Asp Ile Pro Ala Thr Met Gln

660 665 670

Ile Asp Tyr Val Arg Val Tyr Lys Glu Gly Ala Gly Gly Gly Pro Gly

675 680 685

Asn Pro Gly Gly Asn Val Ala Val Thr Gly Val Ser Val Thr Pro Ala

690 695 700

Thr Ala Gln Val Gln Val Gly Gln Thr Val Ser Leu Ser Ala Asn Val

705 710 715 720

Ala Pro Ala Asn Ala Thr Asn Lys Gln Val Thr Trp Ser Val Ala Asn

725 730 735

Gly Ser Ile Ala Ser Val Ser Ala Ser Gly Val Val Ser Gly Leu Ala

740 745 750

Ala Gly Thr Thr Val Thr Ala Thr Thr Ala Asp Gly Asn Arg Thr

755 760 765

Ala Ser Ala Thr Ile Thr Val Val Pro Pro Pro Thr Thr Thr Val Ile

770 775 780

Ile Gly Asp Ser Val Arg Gly Ile Arg Lys Thr Gly Asp Asn Leu Leu

785 790 795 800

Phe Tyr Val Asn Gly Ala Thr Tyr Ala Asp Leu His Tyr Lys Val Asn

805 810 815

Gly Gly Gly Gln Pro Asn Val Ala Met Thr His Thr Gly Gly Gly Asn

820 825 830

Tyr Thr Tyr Pro Val His Gly Leu Gln Gln Gly Asp Thr Val Glu Tyr

835 840 845

Phe Phe Thr Tyr Asn Pro Gly Asn Gly Ala Leu Asp Thr Pro Trp Gln

850 855 860

Thr Tyr Val His Gly Val Thr Gln Gly Val Val Glu

865 870 875

<210> 13

<211> 6783

<212> DNA

<213> Artificial sequence

<220>

<223> Transposon cassette

<400> 13

gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt tattttacca 60

ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct agaaataatt 120

ttgtttaact ttaagaagga gatatacaat ttcgtcgaca cacaggaaac atattaaaaa 180

ttaaaacctg caggagtttg aaggagatag aaccatggcg cagtcgaaac tctatccagt 240

tgtgatggca ggtggctccg gtagccgctt atggccgctt tcccgcgtac tttatcccaa 300

gcagttttta tgcctgaaag gcgatctcac catgctgcaa accaccatct gccgcctgaa 360

cggcgtggag tgcgaaagcc cggtggtgat ttgcaatgag cagcaccgct ttattgtcgc 420

ggaacagctg cgtcaactga acaaacttac cgagaacatt attctcgaac cggcagggcg 480

aaacacggca cctgccattg cgctggcggc gctggcggca aaacgtcata gcccggagag 540

cgacccgtta atgctggtat tggcggcgga tcatgtgatt gccgatgaag acgcgttccg 600

tgccgccgtg cgtaatgcca tgccatatgc cgaagcgggc aagctggtga ccttcggcat 660

tgtgccggat ctaccagaaa ccggttatgg ctatattcgt cgcggtgaag tgtctgcggg 720

tgagcaggat atggtggcct ttgaagtggc gcagtttgtc gaaaaaccga atctggaaac 780

cgctcaggcc tatgtggcaa gcggcgaata ttactggaac agcggtatgt tcctgttccg 840

cgccggacgc tatctcgaag aactgaaaaa atatcgcccg gatatcctcg atgcctgtga 900

aaaagcgatg agcgccgtcg atccggatct caattttatt cgcgtggatg aagaagcgtt 960

tctcgcctgc ccggaagagt cggtggatta cgcggtcatg gaacgtacgg cagatgctgt 1020

tgtggtgccg atggatgcgg gctggagcga tgttggctcc tggtcttcat tatggggagat 1080

cagcgcccac accgccgagg gcaacgtttg ccacggcgat gtgattaatc acaaaactga 1140

aaacagctat gtgtatgctg aatctggcct ggtcaccacc gtcggggtga aagatctggt 1200

agtggtgcag accaaagatg cggtgctgat tgccgaccgt aacgcggtac aggatgtgaa 1260

aaaagtggtc gagcagatca aagccgatgg tcgccatgag catcgggtgc atcgcgaagt 1320

gtatcgtccg tggggcaaat atgactctat cgacgcgggc gaccgctacc aggtgaaacg 1380

catcaccgtg aaaccgggcg agggcttgtc ggtacagatg caccatcacc gcgcggaaca 1440

ctgggtggtt gtcgcgggaa cggcaaaagt caccattgat ggtgatatca aactgcttgg 1500

tgaaaacgag tccatttata ttccgctggg ggcgacgcat tgcctggaaa acccggggaa 1560

aattccgctc gatttaattg aagtgcgctc cggctcttat ctcgaagagg atgatgtggt 1620

gcgtttcgcg gatcgctacg gacgggtgta aacgtcgcat caggcaatga atgcgaaacc 1680

gcggtgtaaa taacgacaaa aataaaattg gccgcttcgg tcagggccaa ctattgcctg 1740

aaaaagggta acgatatgaa aaaattaacc tgctttaaag cctatgatat tcgcgggaaa 1800

ttaggcgaag aactgaatga agatatcgcc tggcgcattg gtcgcgccta tggcgaattt 1860

ctcaaaccga aaaccattgt gttaggcggt gatgtccgcc tcaccagcga aaccttaaaa 1920

ctggcgctgg cgaaaggttt acaggatgcg ggcgttgacg tgctggatat tggtatgtcc 1980

ggcaccgaag agatctattt cgccacgttc catctcggcg tggatggcgg cattgaagtt 2040

accgccagcc ataatccgat ggattataac ggcatgaagc tggttcgcga gggggctcgc 2100

ccgatcagcg gagataccgg actgcgcgac gtccagcgtc tggctgaagc caacgacttt 2160

cctcccgtcg atgaaaccaa acgcggtcgc tatcagcaaa tcaacctgcg tgacgcttac 2220

gttgatcacc tgttcggtta tatcaatgtc aaaaacctca cgccgctcaa gctggtgatc 2280

aactccggga acggcgcagc gggtccggtg gtggacgcca ttgaagcccg ctttaaagcc 2340

ctcggcgcgc ccgtggaatt aatcaaagtg cacaacacgc cggacggcaa tttccccaac 2400

ggtattccta acccactact gccggaatgc cgcgacgaca cccgcaatgc ggtcatcaaa 2460

cacggcgcgg atatgggcat tgcttttgat ggcgattttg accgctgttt cctgtttgac 2520

gaaaaagggc agtttattga gggctactac attgtcggcc tgttggcaga agcattcctc 2580

gaaaaaaatc ccggcgcgaa gatcatccac gatccacgtc tctcctggaa caccgttgat 2640

gtggtgactg ccgcaggtgg cacgccggta atgtcgaaaa ccggacacgc ctttattaaa 2700

gaacgtatgc gcaaggaaga cgccatctat ggtggcgaaa tgagcgccca ccattacttc 2760

cgtgatttcg cttactgcga cagcggcatg atcccgtggc tgctggtcgc cgaactggtg 2820

tgcctgaaag ataaaacgct gggcgaactg gtacgcgacc ggatggcggc gtttccggca 2880

agcggtgaga tcaacagcaa actggcgcaa cccgttgagg cgattaaccg cgtggaacag 2940

cattttagcc gtgaggcgct ggcggtggat cgcaccgatg gcatcagcat gacctttgcc 3000

gactggcgct ttaacctgcg cacctccaat accgaaccgg tggtgcgcct gaatgtggaa 3060

tcgcgcggtg atgtgccgct gatggaagcg cgaacgcgaa ctctgctgac gttgctgaac 3120

gagtaaaaac gcggccgcga tatcgttgta aaacgacggc cagtgcaaga atcataaaaa 3180

atttatttgc tttcaggaaa atttttctgt ataatagatt cataaatttg agagaggagt 3240

ttttgtgagc ggataacaat tccccatctt agtatattag ttaagtataa atacaccgcg 3300

gaggacgaag gagatagaac catgtcaaaa gtcgctctca tcaccggtgt aaccggacaa 3360

gacggttctt acctggcaga gtttctgctg gaaaaaggtt acgaggtgca tggtattaag 3420

cgtcgcgcat cgtcattcaa caccgagcgc gtggatcaca tttatcagga tccgcacacc 3480

tgcaacccga aattccatct gcattatggc gacctgagtg atacctctaa cctgacgcgc 3540

attttgcgtg aagtacagcc ggatgaagtg tacaacctgg gcgcaatgag ccacgttgcg 3600

gtctcttttg agtcaccaga atataccgct gacgtcgacg cgatgggtac gctgcgcctg 3660

ctggaggcga tccgcttcct cggtctggaa aagaaaactc gtttctatca ggcttccacc 3720

tctgaactgt atggtctggt gcaggaaatt ccgcagaaag agaccacgcc gttctacccg 3780

cgatctccgt atgcggtcgc caaactgtac gcctactgga tcaccgttaa ctaccgtgaa 3840

tcctacggca tgtacgcctg taacggaatt ctcttcaacc atgaatcccc gcgccgcggc 3900

gaaaccttcg ttacccgcaa aatcacccgc gcaatcgcca acatcgccca ggggctggag 3960

tcgtgcctgt acctcggcaa tatggattcc ctgcgtgact ggggccacgc caaagactac 4020

gtaaaaatgc agtggatgat gctgcagcag gaacagccgg aagatttcgt tatcgcgacc 4080

ggcgttcagt actccgtgcg tcagttcgtg gaaatggcgg cagcacagct gggcatcaaa 4140

ctgcgctttg aaggcacggg cgttgaagag aagggcattg tggtttccgt caccgggcat 4200

gacgcgccgg gcgttaaacc gggtgatgtg attatcgctg ttgacccgcg ttacttccgt 4260

ccggctgaag ttgaaacgct gctcggcgac ccgaccaaag cgcacgaaaa actgggctgg 4320

aaaccggaaa tcaccctcag agagatggtg tctgaaatgg tggctaatga cctcgaagcg 4380

gcgaaaaaac actctctgct gaaatctcac ggctacgacg tggcgatcgc gctggagtca 4440

taagcatgag taaacaacga gtttttattg ctggtcatcg cgggatggtc ggttccgcca 4500

tcaggcggca gctcgaacag cgcggtgatg tggaactggt attacgcacc cgcgacgagc 4560

tgaacctgct ggacagccgc gccgtgcatg atttctttgc cagcgaacgt attgaccagg 4620

tctatctggc ggcggcgaaa gtgggcggca ttgttgccaa caacacctat ccggcggatt 4680

tcatctacca gaacatgatg attgagagca acatcattca cgccgcgcat cagaacgacg 4740

tgaacaaact gctgtttctc ggatcgtcct gcatctaccc gaaactggca aaacagccga 4800

tggcagaaag cgagttgttg cagggcacgc tggagccgac taacgagcct tatgctattg 4860

ccaaaatcgc cgggatcaaa ctgtgcgaat catacaaccg ccagtacgga cgcgattacc 4920

gctcagtcat gccgaccaac ctgtacgggc cacacgacaa cttccacccg agtaattcgc 4980

atgtgatccc agcattgctg cgtcgcttcc acgaggcgac ggcacagaat gcgccggacg 5040

tggtggtatg gggcagcggt acaccgatgc gcgaatttct gcacgtcgat gatatggcgg 5100

cggcgagcat tcatgtcatg gagctggcgc atgaagtctg gctggagaac acccagccga 5160

tgttgtcgca cattaacgtc ggcacggggcg ttgactgcac tatccgcgag ctggcgcaaa 5220

ccatcgccaa agtggtgggt tacaaaggcc gggtggtttt tgatgccagc aaaccggatg 5280

gcacgccgcg caaactgctg gatgtgacgc gcctgcatca gcttggctgg tatcacgaaa 5340

tctcactgga agcggggctt gccagcactt accagtggtt ccttgagaat caagaccgct 5400

ttcgggggggg gagctaacgc gccatttaaa tcaacctcag cggtcatagc tgtttcctgt 5460

gactgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 5520

gctgaaacca atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 5580

tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 5640

aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgggatcc 5700

aggccggcct gttaacgaat taatcttccg cggcggtatc gataagcttg atatcgaatt 5760

ccgaagttcc tattctctag aaagtatagg aacttcaggt ctgaagagga gtttacgtcc 5820

agccaagcta gcttggctgc aggtcgtcga aattctaccg ggtaggggag gcgcttttcc 5880

caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg 5940

gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg 6000

gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc 6060

agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga 6120

tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc 6180

tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca 6240

ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca 6300

cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga 6360

cctgcagcct gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 6420

ggtgaggaac taaaccatgg gtcaaagtag cgatgaagcc aacgctcccg ttgcagggca 6480

gtttgcgctt cccctgagtg ccacctttgg cttaggggat cgcgtacgca agaaatctgg 6540

tgccgcttgg cagggtcaag tcgtcggttg gtattgcaca aaactcactc ctgaaggcta 6600

tgcggtcgag tccgaatccc acccaggctc agtgcaaatt tatcctgtgg ctgcacttga 6660

acgtgtggcc taatgagggg atcaattctc tagagctcgc tgatcagaag ttcctattct 6720

ctagaaagta taggaacttc gatggcgcct catccctgaa gccaataggg ataacagggt 6780

aat 6783

<210> 14

<211> 2851

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 14

tggccagatg attaattcct aatttttgtt gacactctat cattgataga gttattttac 60

cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat ctagaaataa 120

ttttgtttaa ctttaagaag gagatataca aatgtactat ttaaaaaaca caaacttttg 180

gatgttcggt ttattctttt tcttttactt ttttatcatg ggagcctact tcccgttttt 240

cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc 300

cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact 360

cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt ttgcgccgtt 420

ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat cgattgttgg 480

tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg catttattga 540

gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg 600

ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc agtttgtttt 660

ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg ccaaaacgga 720

tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg catttagcct 780

taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac tgtatgttat 840

tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct ttacttcgtt 900

ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa tgggcgaatt 960

acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa 1020

cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat cgttcgccac 1080

ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac cgttcctgct 1140

ggtgggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag cgacgattta 1200

tctggtctgt ttctgcttct ttaagcaact ggcgatgatt tttatgtctg tactggcggg 1260

caatatgtat gaaagcatcg gtttccaggg cgcttatctg gtgctgggtc tggtggcgct 1320

gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg 1380

tcgtcaggtg aatgaagtcg ctgggagcta agcggccgcg tcgacacgca aaaaggccat 1440

ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc 1500

gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac 1560

tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag 1620

cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggaga cccccacacta 1680

ccatcatgta tgaatatcct ccttagttcc tattccgaag ttcctattct ctagaaagta 1740

taggaacttc ggcgcgtcct acctgtgaca cgcgtgccgc agtctcacgc ccggagcgta 1800

gcgaccgagt gagctagcta tttgtttatt tttctaaata cattcaaata tgtatccgct 1860

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgaggga 1920

agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca 1980

tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa 2040

gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg 2100

gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct 2160

ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc 2220

taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga 2280

gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt 2340

tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt 2400

tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga 2460

gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc 2520

gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt 2580

catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc 2640

agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa 2700

ataatgtcta acaattcgtt caagccgagg ggccgcaaga tccggccacg atgacccggt 2760

cgtcgggtac cggcagggcg gggcgtaagg cgcgccattt aaatgaagtt cctattccga 2820

agttcctatt ctctagaaag tataggaact t 2851

<210> 15

<211> 2858

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 15

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 60

actccctatc agtgatagag aaaagtgaaa tgaatagttc gacaaaaatc tagaaataat 120

tttgtttaac tttaagaagg agatatacaa atgggcagca ttattcgtct gcagggtggt 180

ctgggtaatc agctgtttca gtttagcttt ggttatgccc tgagcaaaat taatggtaca 240

ccgctgtatt tcgacattag ccattatgcc gaaaacgatg atcatggtgg ttatcgtctg 300

aataatctgc agattccgga agaatatctg cagtattata ccccgaaaat taataatatt 360

tataaactgc tggtgcgtgg cagccgtctg tatccggata tttttctgtt tctgggcttt 420

tgcaacgaat ttcatgccta tggctacgat tttgaatata ttgcccagaa atggaaaagc 480

aaaaaataca ttggctactg gcagagcgaa cacttttttc ataaacatat tctggacctg 540

aaagaatttt ttattccgaa aaatgtgagc gaacaggcaa atctgctggc agcaaaaatt 600

ctggaaagcc agagcagcct gagcattcat attcgtcgtg gcgattatat taaaaacaaa 660

accgcaaccc tgacacatgg tgtttgtagc ctggaatatt ataaaaaagc cctgaacaaa 720

atccgcgatc tggcaatgat tcgtgatgtg tttatcttta gcgacgatat cttctggtgc 780

aaagaaaata ttgaaaccct gctgagcaaa aaatataata tttattatag cgaagatctg 840

agccaagaag aggatctgtg gctgatgagc ctggcaaatc atcatattat tgccaatagc 900

agctttagtt ggtggggtgc atatctgggt agcagcgcaa gccagattgt tatttatccg 960

accccgtggt atgatattac cccgaaaaac acctatatcc cgattgtgaa ccattggatc 1020

aacgttgata aacatagcag ctgctaagcg gccgcgtcga cacgcaaaaa ggccatccgt 1080

caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca 1140

ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg tcctactcag 1200

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 1260

tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc acactaccat 1320

catgtatgaa tatcctcctt agttcctatt ccgaagttcc tattctctag aaagtatagg 1380

aacttcggcg cgtcctacct gtgacacgcg tcaagatccc ctcacgctgc cgcaagcact 1440

cagggcgcaa gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt 1500

gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa 1560

agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat 1620

ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct 1680

gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat 1740

ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 1800

gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 1860

gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 1920

agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 1980

tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 2040

actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 2100

ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 2160

cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 2220

ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 2280

tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 2340

atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 2400

gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 2460

aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 2520

attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 2580

gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 2640

cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 2700

ccagcgcggg gatctcatgc tggagttctt cgcccacccc agcttcaaaa gcgctctcgg 2760

taccggcagg gcggggcgta aggcgcgcca tttaaatgaa gttcctattc cgaagttcct 2820

attctctaga aagtatagga acttcgaagc agctccag 2858

<210> 16

<211> 2631

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 16

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 60

actccctatc agtgatagag aaaagtgaaa tgaatagttc gacaaaaatc tagaaataat 120

tttgtttaac tttaagaagg agatatacaa atgaagtcgg cactgacctt ttcccgtcgc 180

atcaatccgg tgtttctggc gttctttgtc gttgcttttc tgagcggtat cgcaggcgca 240

ctgcaggctc cgaccctgag tctgtttctg tccacggaag tgaaagttcg tccgctgtgg 300

gttggtctgt tctataccgt caacgcaatc gctggcatta cggttagctt tatcctggcg 360

aaacgttcag attcgcgcgg tgaccgtcgc aagctgatta tggtgtgcta tctgatggcg 420

gttggcaact gtctgctgtt tgccttcaat cgtgattacc tgaccctgat cacggcaggt 480

gtgctgctgg cgagcgttgc caacaccgca atgccgcaga ttttcgcgct ggcccgtgaa 540

tatgccgaca gctctgcacg cgaagtggtt atgtttagtt ccatcatgcg cgctcaactg 600

agtctggcat gggtgattgg tccgccgctg tcctttatgc tggcgctgaa ttacggtttt 660

accctgatgt tctcaatcgc ggccggcatt ttcgttctgt cggccctggt cgtgtggttt 720

atcctgccga gtgtcccgcg tgcagaaccg gttgtcgatg caccggtggt tgtccaggt 780

tcactgttcg cagacaaaaa cgttctgctg ctgtttatcg cgtcgatgct gatgtggacc 840

tgcaatacga tgtatattat cgatatgccg ctgtacatta ccgcaagcct gggtctgccg 900

gaacgtctgg ctggtctgct gatgggtacc gcagctggcc tggaaattcc gatcatgctg 960

ctggcgggtt attctgtgcg ttactttggc aaacgcaaga ttatgctgtt cgctgttctg 1020

gcgggtgtcc tgttttatac cggcctggtt ctgtttaaat tcaagacggc cctgatgctg 1080

ctgcagatct ttaacgcaat tttcatcggt attgtggctg gcattggtat gctgtacttc 1140

caagatctga tgccgggtcg tgcaggtgca gcaaccacgc tgtttaccaa tagcatctct 1200

acgggtgtca ttctggcagg cgtgctgcaa ggcggtctga ccgaaacgtg gggccatgac 1260

agcgtctatg tgatggcgat ggtcctgtct attctggccc tgattatctg tgcacgtgtg 1320

cgcgaagctt aaatcgatac tagcataacc ccttggggcc tctaaacgcg tcgacacgca 1380

aaaaggccat ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg 1440

cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga 1500

tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct 1560

ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggaga 1620

ccccacacta ccatcatgta tgaatatcct ccttagttcc tattccgaag ttcctattct 1680

ctagaaagta taggaacttc ggcgcgtcct acctgtgacg gaagatcact tcgcagaata 1740

aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt ggcgaaaatg 1800

agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc 1860

gggcgtattt tttgagttgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa 1920

aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc 1980

atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt 2040

tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc 2100

ccgcctgatg aatgctcatc cggaattacg tatggcaatg aaagacggtg agctggtgat 2160

atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc 2220

gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt 2280

ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt 2340

cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga 2400

caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct 2460

gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagatg 2520

cttaatgaat acaacagtac tgcgatgagt ggcagggcgg ggcgtaaggc gcgccattta 2580

aatgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt c 2631

<210> 17

<211> 4259

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 17

ttactcagca ataaactgat attccgtcag gctggaatac tcttcgccag gacgcaggaa 60

gcagtccggt tgcggccatt cagggtggtt cgggctgtcc ggtagaaact cgctttccag 120

agccagccct tgccagtcgg cgtaaggttc ggttccccgc gacggtgtgc cgccgaggaa 180

gttgccggag tagaattgca gagccggagc ggtggtgtag accttcagct gcaatttttc 240

atctgctgac cagacatgcg ccgccacttt cttgccatcg cctttggcct gtaacaagaa 300

tgcgtgatcg taacctttca ctttgcgctg atcgtcgtcg gcaagaaact cactggcgat 360

gattttggcg ctgcggaaat caaaagacgt tccggcgaca gatttcaggc cgtcgtgcgg 420

aatgccgcct tcatcaaccg gcagatattc gtccgccaga atctgcaact tgtgattgcg 480

cacgtcagac tgctcgccgt caagattgaa atagacgtga ttagtcatat tcaccgggca 540

aggtttatca actgtggcgc gataagtaat ggagatacgg ttatcgtcgg tcagacgata 600

ttgcaccgtc gcgccgagat tacccgggaa gccctgatca ccatcatctg aactcagggc 660

aaacagcacc tgacgatcgt tctggttcac aatctgccag cgacgtttgt cgaacccttc 720

cggcccgccg tgcagctggt taacgccctg acttggcgaa agcgtcacgg tttcaccgtc 780

aaaggtataa cggctattgg cgatacggtt ggcataacga ccaatagagg cccccagaaa 840

cgcggcctga tcctgatagc attccgggct ggcacagccg agcagcgcct cgcggacgct 900

gccatcggaa agcggaatac gggcggaaag taaagtcgca ccccagtcca tcagcgtgac 960

taccatccct gcgttgttac gcaaagttaa cagtcggtac ggctgaccat cgggtgccag 1020

tgcgggagtt tcgttcagca ctgtcctgct ccttgtgatg gtttacaaac gtaaaaagtc 1080

tctttaatac ctgtttttgc ttcatattgt tcagcgacag cttgctgtac ggcaggcacc 1140

agctcttccg ggatcagcgc gacgatacag ccgccaaatc cgccgccggt catgcgtacg 1200

ccacctttgt cgccaatcac agctttgacg atttctacca gagtgtcaat ttgcggcacg 1260

gtgatttcga aatcatcgcg catagaggca tgagactccg ccatcaactc gcccatacgt 1320

ttcaggtcgc cttgctccag cgcgctggca gcttcaacgg tgcgggcgtt ttcagtcagt 1380

atatgacgca cgcgttttgc cacgatcggg tccagttcat gcgcaacagc gttgaactct 1440

tcaatggtga catcacgcag ggctggctgc tggaagaaac gcgcaccggt ttcgcactgt 1500

tcacgacggg tgttgtattc gctgccaacc agggtacgtt tgaagttact gttgatgatg 1560

acgacagcca cacctttggg catggaaact gctttggtcc ccagtgagcg gcaatcgatc 1620

agcaaggcat gatctttctt gccgagcgcg gaaattagct gatccatgat cccgcagtta 1680

cagcctacaa actggttttc tgcttcctga ccgttaagcg cgatttgtgc gccgtccagc 1740

ggcagatgat aaagctgctg caatacggtt ccgaccgcga cttccagtga agcggaagaa 1800

cttaacccgg caccctgcgg cacattgccg ctgatcacca tgtccacgcc gccgaagctg 1860

ttgttacgca gttgcagatg tttcaccacg ccacgaacgt agttagccca ttgatagttt 1920

tcatgtgcga caatgggcgc atcgagggaa aactcgtcga gctgattttc ataatcggct 1980

gccatcacgc gaactttacg gtcatcgcgt ggtgcacaac tgatcacggt ttgataatca 2040

atcgcgcagg gcagaacgaa accgtcgttg tagtcggtgt gttcaccaat caaattcacg 2100

cggccaggcg cctgaatggt gtgagtggca gggtagccaa atgcgttggc aaacagagat 2160

tgtgtttttt ctttcagact catttcttac actccggatt cgcgaaaatg gatatcgctg 2220

actgcgcgca aacgctctgc tgcctgttct gcggtcaggt ctcgctgggt ctctgccagc 2280

atttcataac caaccataaa tttacgtacg gtggcggagc gcagcagagg cggataaaag 2340

tgcgcgtgca gctgccagtg ttgattctct tcgccattaa atggcgcgcc gtgccagccc 2400

atagagtagg ggaaggagca ctggaagagg ttgtcataac gactggtcag ctttttcaac 2460

gccagcgcca gatcgctgcg ctgggcgtcg gtcaaatcgg tgatccgtaa aacgtgggct 2520

ttgggcagca gtagcgtttc gaacggccag gcagcccagt aaggcacgac ggctaaccag 2580

tgttcggttt cgacaacggt acggctaccg tctgccagct cgcgctgaac ataatccacc 2640

agcattggtg atttctgttc ggcaaaatat tctttttgca ggcggtcttc gcgctcagct 2700

tcgttaggca ggaagctatt tgcccaaatc tgaccgtgcg gatgcgggtt agagcagccc 2760

atcgccgcgc ctttgttttc aaaaacctgc acccatgggt acgttttccc cagttctgcg 2820

gtttgctcct gccaggtttt gacgatttcc gtcaatgctg caacgctgag ctctggcagc 2880

gttttactgt gatccggtga aaagcagatc acccggctgg tgccgcgcgc gctctggcaa 2940

cgcatcagcg gatcgtgact ttctggcgca tctggcgtgt cagacatcaa agccgcaaag 3000

tcattagtga aaacgtaagt cccggtgtaa tcggggtttt tatcgcctgt cacccgcaca 3060

ttacctgcgc agaggaagca atctggatcg tgcgcaggta acacctgttt ggctggcgtt 3120

tcctgcgccc cctgccaggg gcgcttagcg cggtgcggtg aaaccagaat ccattgcccg 3180

gtgagcgggt tgtagcggcg atgtggatga tcaacgggat taaattgcgt catggtcgtt 3240

ccttaatcgg gatatccctg tggatggcgt gactgccagt gccaggtgtc ctgcgccatt 3300

tcatcgagtg tgcgcgttac gcgccagttc agttcacggt cggctttgct ggcgtccgcc 3360

cagtaggccg gaaggtcgcc ctcgcgacgc ggtgcaaaat gataattaac cggtttgccg 3420

caggctttgc tgaaggcatt aaccacgtcc agcacgctgt tgcctacgcc agcgccgagg 3480

ttgtagatgt gtacgcctgg cttgttcgcc agtttttcca tcgccacgac gtgaccgtcc 3540

gccagatcca ttacgtggat gtaatcgcgt acgccagtac catcttcggt cggataatcg 3600

ttaccaaaaa tcgccagcga gtcgcgacgg cctacagcaa cctgggcgat gtatggcatc 3660

aggttattcg gaatgccttg cggatcttcg cccatatcgc ccgacggatg cgcgccaacc 3720

gggttgaagt agcgcagcag ggcaatgctc cagtccggct gggctttttg cagatcggtg 3780

aggatctgtt caccatcag cttgcttttg ccgtaagggc tttgcggtgt gccggtcggg 3840

aagctttcaa cgtatggaat tttgggctga tcgccataaa cggtggcgga ggagctaaaa 3900

ataaagtttt tgacgttagc ggcgcgcatg gcgctaatca ggcgcagagt gccgttgaca 3960

ttgttgtcgt aatattccag cggtttttgt accgattcgc ccacggcttt cagcccggcg 4020

aagtggatca cggtgtcgat agcgtgatcg tgcaggatct cggtcatcaa cgcttcgtta 4080

cgaatatcgc cttcaacaaa cgttggatgt ttgccgccta aacgctcgat aacaggcagt 4140

acgctgcgct tactgttaca gaggttatca agaatgatga catcatgacc gttttgcagt 4200

aattgcacac aggtatgact tccaatgtaa ccgctaccac cggtaaccag aactctcat 4259

<210> 18

<211> 4223

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 18

tggccagatg attaattcct aatttttgtt gacactctat cattgataga gttattttac 60

cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat ctagaaataa 120

ttttgtttaa ctttaagaag gagatataca aatgcaaaaa ctactatctt taccgtccaa 180

tctggttcag tcttttcatg aactggagag ggtgaatcgt accgattggt tttgtacttc 240

cgacccggta ggtaagaaac ttggttccgg tggtggaaca tcctggctgc ttgaagaatg 300

ttataatgaa tattcagatg gtgctacttt tggagagtgg cttgaaaaag aaaaaagaat 360

tcttcttcat gcgggtgggc aaagccgtcg tttacccggc tatgcacctt ctggaaagat 420

tctcactccg gttcctgtgt tccggtggga gagagggcaa catctgggac aaaatctgct 480

ttctctgcaa cttcccctat atgaaaaaat catgtctttg gctccggata aactccatac 540

actgattgcg agtggtgatg tctatattcg ttcggagaaa cctttgcaga gtattcccga 600

agcggatgtg gtttgttatg gactgtgggt agatccgtct ctggctaccc atcatggcgt 660

gtttgcttcc gatcgcaaac atcccgaaca actcgacttt atgcttcaga agccttcgtt 720

ggcagaattg gaatctttat cgaagaccca tttgttcctg atggacatcg gtatatggct 780

tttgagtgac cgtgccgtag aaatcttgat gaaacgttct cataaagaaa gctctgaaga 840

actaaagtat tatgatcttt attccgattt tggattagct ttgggaactc atccccgtat 900

tgaagacgaa gaggtcaata cgctatccgt tgctattctg cctttgccgg gaggagtt 960

ctatcattac gggaccagta aagaactgat ttcttcaact ctttccgtac agaataaggt 1020

ttacgatcag cgtcgtatca tgcaccgtaa agtaaagccc aatccggcta tgtttgtcca 1080

aaatgctgtc gtgcggatac ctctttgtgc cgagaatgct gatttatgga tcgagaacag 1140

tcatatcgga ccaaagtgga agattgcttc acgacatatt attaccgggg ttccggaaaa 1200

tgactggtca ttggctgtgc ctgccggagt gtgtgtagat gtggttccga tgggtgataa 1260

gggctttgtt gcccgtccat acggtctgga cgatgttttc aaaggagatt tgagagattc 1320

caaaacaacc ctgacgggta ttccttttgg tgaatggatg tccaaacgcg gtttgtcata 1380

tacagatttg aaaggacgta cggacgattt acaggcagtt tccgtattcc ctatggttaa 1440

ttctgtagaa gagttgggat tggtgttgag gtggatgttg tccgaacccg aactggagga 1500

aggaaagaat atctggttac gttccgaaca tttttctgcg gacgaaattt cggcaggtgc 1560

caatctgaag cgtttgtatg cacaacgtga agagttcaga aaaggaaact ggaaagcatt 1620

ggccgttaat catgaaaaaa gtgtttttta tcaacttgat ttggccgatg cagctgaaga 1680

ttttgtacgt cttggtttgg atatgcctga attattgcct gaggatgctc tgcagatgtc 1740

acgcatccat aaccggatgt tgcgtgcgcg tattttgaaa ttagacggga aagattatcg 1800

tccggaagaa caggctgctt ttgatttgct tcgtgacggc ttgctggacg ggatcagtaa 1860

tcgtaagagt accccaaaat tggatgtata ttccgatcag attgtttggg gacgtagccc 1920

cgtgcgcatc gatatggcag gtggatggac cgatactcct ccttattcac tttattcggg 1980

aggaaatgtg gtgaatctag ccattgagtt gaacggacaa cctcccttac aggtctatgt 2040

gaagccgtgt aaagacttcc atatcgtcct gcgttctatc gatatgggtg ctatggaaat 2100

agtatctacg tttgatgaat tgcaagatta taagaagatc ggttcacctt tctctattcc 2160

gaaagccgct ctgtcattgg caggctttgc acctgcgttt tctgctgtat cttatgcttc 2220

attagaggaa cagcttaaag atttcggtgc aggtattgaa gtgactttat tggctgctat 2280

tcctgccggt tccggtttgg gcaccagttc cattctggct tctaccgtac ttggtgccat 2340

taacgatttc tgtggtttag cctgggataa aaatgagatt tgtcaacgta ctcttgttct 2400

tgaacaattg ctgactaccg gaggtggatg gcaggatcag tatggaggtg tgttgcaggg 2460

tgtgaagctt cttcagaccg aggccggctt tgctcaaagt ccattggtgc gttggctacc 2520

cgatcattta tttacgcatc ctgaatacaa agactgtcac ttgctttatt ataccggtat 2580

aactcgtacg gcaaaaggga tcttggcaga aatagtcagt tccatgttcc tcaattcatc 2640

gttgcatctc aatttacttt cggaaatgaa ggcgcatgca ttggatatga atgaagctat 2700

acagcgtgga agttttgttg agtttggccg tttggtagga aaaacctggg aacaaaacaa 2760

agcattggat agcggaacaa atcctccggc tgtggaggca attatcgatc tgataaaaga 2820

ttataccttg ggatataaat tgccgggagc cggtggtggc gggtacttat atatggtagc 2880

gaaagatccg caagctgctg ttcgtattcg taagatactg acagaaaacg ctccgaatcc 2940

gcgggcacgt tttgtcgaaa tgacgttatc tgataaggga ttccaagtat cacgatcata 3000

actgaaacca atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 3060

tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 3120

aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgggatcc 3180

aggccggcct gttaagacgg ccagtgaatt cgagctcggt acctaccgtt cgtataatgt 3240

atgctatacg aagttatcga gctctagaga atgatcccct cattaggcca cacgttcaag 3300

tgcagcgcac accgtggaaa cggatgaagg cacgaaccca gttgacataa gcctgttcgg 3360

ttcgtaaact gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg 3420

aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg 3480

tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga 3540

tgttatggag cagcaacgat gttacgcagc agcaacgatg ttacgcagca gggcagtcgc 3600

cctaaaacaa agttaggtgg ctcaagtatg ggcatcattc gcacatgtag gctcggccct 3660

gaccaagtca aatccatgcg ggctgctctt gatcttttcg gtcgtgagtt cggagacgta 3720

gccacctact cccaacatca gccggactcc gattacctcg ggaacttgct ccgtagtaag 3780

acattcatcg cgcttgctgc cttcgaccaa gaagcggttg ttggcgctct cgcggcttac 3840

gttctgccca ggtttgagca gccgcgtagt gagatctata tctatgatct cgcagtctcc 3900

ggcgagcacc ggaggcaggg cattgccacc gcgctcatca atctcctcaa gcatgaggcc 3960

aacgcgcttg gtgcttatgt gatctacgtg caagcagatt acggtgacga tcccgcagtg 4020

gctctctata caaagttggg catacgggaa gaagtgatgc actttgatat cgacccaagt 4080

accgccacct aacaattcgt tcaagccgag atcgtagaat ttcgacgacc tgcagccaag 4140

cataacttcg tataatgtat gctatacgaa cggtaggatc ctctagagtc gacctgcagg 4200

catgagatgt gtataagaga cag 4223

<210> 19

<211> 3792

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 19

gggaattgat tctggtacca aatgagtcga ccggccagat gattaattcc taatttttgt 60

tgacactcta tcattgatag agttatttta ccactcccta tcagtgatag agaaaagtga 120

aatgaatagt tcgacaaaaa tctagaaata attttgttta actttaagaa ggagatatac 180

aaatgattac ccgcaaaagg cgggccagga caatccatag ccgatatcca atcggaattt 240

acgggagcat agtaatgaca gatattgcac agttgcttgg caaagacgcc gacaaccttt 300

tacagcaccg ttgtatgact attccttctg accagcttta tctccccgga catgactacg 360

tagaccgcgt gatgattgac aataatcgcc cgccagcggt gttacgtaat atgcagacgt 420

tgtacaacac tgggcgtctg gctggcacag gatatctttc tattctgccg gttgaccagg 480

gcgttgagca ctctgccgga gcttcatttg ctgctaaccc gctctacttt gacccgaaaa 540

acattgttga actggcgatc gaagcgggct gtaactgtgt ggcatcaact tacggcgtgt 600

tggcgtcggt atcgcggcgc tatgcgcatc gcattccat cctcgtcaaa cttaatcaca 660

acgagacgct aagttacccg aacacctacg atcaaacgct gtatgccagc gtggagcagg 720

ccttcaacat gggcgcggtg gcggttggtg cgactatcta ttttggttcg gaagagtcac 780

gtcgccagat tgaagaaatt tctgcggctt ttgaacgtgc gcacgagctg ggcatggtga 840

cagtgctgtg ggcctatttg cgtaactccg cctttaagaa agatggcgtt gattaccatg 900

tttccgccga cctgaccggt caggcaaacc atctggcggc gaccataggt gcagatatcg 960

tcaaacaaaa aatggcggaa aataacggcg gctataaagc aattaattac ggttataccg 1020

acgatcgcgt gtacagcaag ttaaccagcg aaaacccgat tgatctggtg cgttatcagt 1080

tagctaactg ctatatgggc cgggccgggt tgataaactc cggcggtgct gcaggcggtg 1140

aaactgacct cagcgatgca gtgcgtactg cggttatcaa caaacgcgct ggcggaatgg 1200

ggctgattct tggacgtaag gcgttcaaga aatcgatggc tgacggcgtg aaactgatta 1260

acgccgtgca ggatgtttat ctcgatagca aaattactat cgcctaagag gatcgagatc 1320

tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1380

ctctagaaat aattttgttt aactttaaga aggagatata ccatggggcca tcatcatcat 1440

catcatcatc atcatcacag cagcggccat atcgaaggtc gtcatatggc ggtgaaagaa 1500

gcgaccagcg agaccaagaa gcgtagcggt tacgagatca ttaccctgac cagctggctg 1560

ctgcaacaag aacagaaggg tatcattgac gcggaactga ccatcgttct gagcagcatt 1620

agcatggcgt gcaaacagat cgcgagcctg gtgcaacgtg cgaacattag caacctgacc 1680

ggtacccaag gcgcggttaa catccagggt gaagaccaaa agaaactgga tgttattagc 1740

aacgaggtgt tcagcaactg cctgcgtagc agcggtcgta ccggcatcat tgcgagcgag 1800

gaagaggacg tggcggttgc ggtggaagag agctacagcg gtaactatat cgtggttttt 1860

gacccgctgg atggcagcag caacctggat gcggctgtga gcaccggtag catcttcggc 1920

atttacagcc cgaacgacga gagcctgccg gattttggtg acgatagcga cgataacacc 1980

ctgggcaccg aagagcaacg ttgcatcgtt aacgtgtgcc aaccgggtag caacctgctg 2040

gcggcgggct actgcatgta tagcagcagc gttgcgttcg tgctgaccat tggcaagggc 2100

gttttcgtgt ttaccctgga cccgctgtac ggtgaattcg tgctgaccca ggagaacctg 2160

caaatcccga agagcggtga aatttacagc tttaacgagg gcaactataa actgtgggat 2220

gaaaacctga agaaatatat cgacgatctg aaggaaccgg gtccgagcgg taaaccgtac 2280

agcgcgcgtt atatcggtag cctggttggc gacttccacc gtaccctgct gtacggtggc 2340

atttacggtt atccgcgtga taagaaaagc aagaacggca aactgcgtct gctgtatgaa 2400

tgcgcgccga tgagctttat tgttgagcag gcgggtggca aaggtagcga cggccaccag 2460

cgtgtgctgg atatccaacc gaccgaaatt caccagcgtg ttccgctgta cattggtagc 2520

accgaagagg ttgaaaaagt tgaaaagtat ctggcgtaat cgagtctggt aaagaaaccg 2580

ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct 2640

aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 2700

tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg 2760

tagcggcgca ttaagcgcgg cgggtggacg gccagtgaat tcgagctcgg tacctaccgt 2820

tcgtataatg tatgctatac gaagttatcg agctctagag aatgatcccc tcattaggcc 2880

acacgttcaa gtgcagcgca caccgtggaa acggatgaag gcacgaaccc agttgacata 2940

agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca actggtccag 3000

aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga 3060

ctgttttttt gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg 3120

tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagcaacgat gttacgcagc 3180

agggcagtcg ccctaaaaca aagttaggtg gctcaagtat gggcatcatt cgcacatgta 3240

ggctcggccc tgaccaagtc aaatccatgc gggctgctct tgatcttttc ggtcgtgagt 3300

tcggagacgt agccacctac tcccaacatc agccggactc cgattacctc gggaacttgc 3360

tccgtagtaa gacattcatc gcgcttgctg ccttcgacca agaagcggtt gttggcgctc 3420

tcgcggctta cgttctgccc aggtttgagc agccgcgtag tgagatctat atctatgatc 3480

tcgcagtctc cggcgagcac cggaggcagg gcattgccac cgcgctcatc aatctcctca 3540

agcatgaggc caacgcgctt ggtgcttatg tgatctacgt gcaagcagat tacggtgacg 3600

atcccgcagt ggctctctat acaaagttgg gcatacggga agaagtgatg cactttgata 3660

tcgacccaag taccgccacc taacaattcg ttcaagccga gatcgtagaa tttcgacgac 3720

ctgcagccaa gcataacttc gtataatgta tgctatacga acggtaggat cctctagagt 3780

cgacctgcag gc 3792

<210> 20

<211> 5917

<212> DNA

<213> Artificial sequence

<220>

<223> Transposon cassette

<400> 20

acaggttggc tgataagtcc ccggtctagc ttgcatgcag attgcagcat tacacgtctt 60

gatttgacgg ctagctcagt cctaggtaca gtgctagcac tgctttgtgg aaggagatag 120

acttatggcg gatccgatgg aatacctcga tgtgtcgttc ggcggcacgt tcgctgcaga 180

cacctacacc acaggtggcg acgaggtggc gaagggcccc gtgaccaagc acggcagcat 240

accgaccaag cttgacggcg gcggcatcac cctcgctggc ggcaccaacg gcgtgacatt 300

cacctcgacc gcgagcttca gcgagagtgg gaaggtgaac aagggattcc gcgccgaaat 360

ggagtaccgt acgacgcaga cgcccagcaa cctcgccaca ttgttctccg ccatgggcaa 420

catcttcgtg cgggcgaacg gcagcaacct cgaatacggc ttctccacga acccttccgg 480

cagtacatgg aacgactaca caaagtccgt gacgctgcct tccaacaatg tgaagcacat 540

catccagctg acatatctgc cgggagccga cggcgctgcc tcgacgttgc agttgtcggt 600

ggatggcgtg gccggcgaga ccgccacctc cgcggccggc gagctcgcgg ccgtcagcga 660

ttccgtcggg aacaagttcg ggatcggcta cgaggtgaac cccgcttccg gcgcggcgag 720

ccgcggtctt gccggtgacg tgttccgcgc gcgtgtcgcc gattcggacg ccccgtggga 780

gattcttgac gcatcccagc tgctgcatgt caatttcaac ggcacgttca gcggcacctc 840

atataccgcg gcgagcggcg agcagatgct gggctcgctg gtgtcgcgct cggccaatcc 900

gtccatctcg aactccgccg tcacgctggg cggcggcacg gccggattcg atttcacgcc 960

cacggacttc accctcggtg acaacgaggc catcacccgc ccgctggtcg cggagctgcg 1020

cttcaccccg acgcagaccg gcgacaacca gaccctgttc ggcgcgggcg gcaacctgtt 1080

cctgcgctac gagtcgaaca agctcgtgtt cggcgcctcc accaagtccg gcgataattg 1140

gaccgaccac aagatcgagt ccgcggccgc cacgggtgcg gagcacgtcg tgtcggtggc 1200

gtacgtgccc aataaggccg gcaccggcgc gaagcttgtc atgcgcgtgg atggcggcga 1260

cgcccagacc aaggacatca ctggtctggc ttacctgaat tcgagcatca agggcaaggt 1320

cggcttcggc aacgacgtgc ataccgacgc gctcagccgc ggcttcgtcg gctcgctgag 1380

cgagatccgc ctggccgaaa cctccgcgaa cttcaccacc aacgaattca agctggtcta 1440

ctctcaggtc agctgcgaca cgtcgggcat caaggaggcg aataccttcg acgtggagcc 1500

cgccgagtgc gaggccgcgc ttaagaccaa gctgtccaag ctgcgtccga ccgaagggca 1560

ggccgactac atcgactggg gtcagatcgg attcctccat tacggcatca acacgtacta 1620

caaccaggag tggggtcacg gtaacgagga tccctcccgc atcaacccga ccggcctcga 1680

caccgaccag tgggcgaagt ccttcgccga cggtggcttc aagatgatca tggtgacggt 1740

caagcaccat gacggtttcg agctgtacga ctcgcggtac aacaccgagc acgactgggc 1800

aaacaccgcc gtcgccaagc gcacggggga gaaggacctg ttccgcaaga ttgtcgcctc 1860

ggcgaagaaa tacggcctga aggtcggcat ctactattcg ccggccgatt cctacatgga 1920

gaggaagggc gtctggggca acaactccgc acgcgtcgag cgcacgatcc ccacgctggt 1980

ggagaacgac gaccgcgccg gcaaggtggc ttccggcaaa ctgcccacgt tcaagtacaa 2040

ggccacggat tacggcgcct acatgctcaa ccagctctat gagctgctga ctgagtacgg 2100

cgacatctcc gaggtctggt tcgacggtgc ccaaggcaac accgcaggca ctgagcatta 2160

cgactatggc gtgttctacg agatgatccg ccggcttcag ccccaggcaa ttcaggccaa 2220

cgccgcatac gatgcccgat gggtgggcaa cgaggacggc tgggcccgtc agaccgagtg 2280

gagcccgcag gcggcataca acgacggcgt ggacaaggtg tcgctcaagc ctggccagat 2340

ggcccccgac ggtaagcttg gcagcatgtc gagcgtgctg tccgagatcc gcagcggcgc 2400

cgccaaccag ctgcactggt atccggccga agtcgacgcc aagaaccggc ccggatggtt 2460

ctaccgtgcc agccaatcgc cggcgtccgt agccgaagtc gtgaagtact acgagcagtc 2520

cacgggacgc aactcgcagt atctgctgaa cgtcccaccg tccgataccg gcaagctcgc 2580

cgatgcggat gccgcgggac ttaaggggct gggcgaggag ctcgcccgac gctacggcac 2640

cgatcttgcc ctgggcaaga gcgcgaccgt cgccgcgtcc gcgaacgaca ctgcggtagc 2700

ggccccgaag ctgaccgacg gttcgaagct ctcctccgac aaggccgtgg gcaatacgcc 2760

gacgtacacc atcgatctgg gcagcactgt cgccgtggat gcagtgaaga tctccgagga 2820

cgtgcgcaat gccggccagc agatcgaaag cgccactctg cagggacgag tcaatggaac 2880

atggacgaat ctggcgacta tgacgacggt cgggcagcag cgcgaccttc gcttcacgtc 2940

ccagaacatc gatgccatcc gtctggtggt caactcctcc cgcggtccgg tgcgtctgag 3000

ccgtcttgag gtgttccaca ccgaatccga gattcagacc ggcgcccgcg cctactacat 3060

cgatccgacg gcgcagaccg cgggagatgg attcacgaag gacaagccca tgacgtcgat 3120

cgagcagctg cacgatgtga ccgtcgcgcc aggctccgtg atcttcgtca aggcgggcac 3180

cgagctgacc ggggacttcg ccgtcttcgg ctacggcacc aaggacgagc ccatcaccgt 3240

gacgacatac ggcgaaagcg acaaagccac caccgcgagc ttcgacggca tgaccgccgg 3300

gctgacgctg aagcaggcgc tgaaggcgct cggcaaggac gacgccggct gggtcgtggc 3360

cgattccgcc actgcaccgg cctcccgcgt gtatgtcccg caggatgaga tcagcgtgca 3420

cgcccagtcg tcgcagaact ccggcgcaga ggcggcgagg gcgctcgacg gcgactcgtc 3480

gacgagctgg cactcccagt acagcccgac caccgcgtct gctccgcatt gggtgactct 3540

cgatctcggc aaatcgcgtg agaacgtcgc ctacttcgac tacctcgccc gtatcgacgg 3600

caacaataac ggtgccgcca aggattacga ggtgtatgtc tccgacgatc ccaacgattt 3660

tggagcccct gtggcctcgg gcacgttgaa gaacgtcgcc tacacgcagc gcatcaagct 3720

gacccccaag aacggacggt acgtcaagtt cgtcatcaag accgattatt ccggatcgaa 3780

cttcggctcc gcggcggaaa tgaatgtcga gttgctgccc acggccgtag aggaggacaa 3840

ggtcgccacc ccgcagaagc cgacagtgga cgatgatgcc gatacataca ccatccccga 3900

catcgaggga gtcgtgtaca aggtcgacgg caaggtgttg gccgctggtt ccgtagtgaa 3960

cgtgggcgat gaggacgtga ccgtcacggt caccgccgag cccgccgacg gataccgctt 4020

cccggatggt gtgacgtccc cagtcacgta tgagctgacg ttcaccaaga agggtggcga 4080

gaagcctccg accgaagtca acaaggacaa gctgcacgcc acgatcacca aggctcaggc 4140

gatcgaccgt tccgcctata cggacgagtc gctcaaggtg cttgatgaca agctcgccgc 4200

agcgctcaag gtctatgacg atgacaaggt gagccaggat gatgtcgatg ccgccgaggc 4260

ggctctgtct gcggcgatcg acgcgctgaa gaccaagccg acgacccccg gcggtgaagg 4320

tgagaagcct ggtgaaggtg aaaagcccgg tgacggcaac aagcccggtg acggcaagaa 4380

gcccggcgac gtgatcgcaa agaccggcgc ctccacaatg taactagcat aaccccttgg 4440

ggcctctaaa cgggtcttga ggggtttttt gctgaaacca atttgcctgg cggcagtagc 4500

gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 4560

agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 4620

tcagtcgaaa gactgggcct ttcgggatcc aggccggcct gttaacgaat taatcttccg 4680

cggcggtatc gataagcttg atatcgaatt ccgaagttcc tattctctag acgccattca 4740

ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 4800

cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 4860

gacgttgtaa aacgacggcc agtgaattcg agctcggtac ctaccgttcg tataatgtat 4920

gctatacgaa gttatcgagc tctagagaat gatcccctca ttaggccaca cgttcaagtg 4980

cagcgcacac cgtggaaacg gatgaaggca cgaacccagt tgacataagc ctgttcggtt 5040

cgtaaactgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa 5100

cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttgta 5160

cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg 5220

ttatggagca gcaacgatgt tacgcagcag caacgatgtt acgcagcagg gcagtcgccc 5280

taaaacaaag ttaggtggct caagtatggg catcattcgc acatgtaggc tcggccctga 5340

ccaagtcaaa tccatgcggg ctgctcttga tcttttcggt cgtgagttcg gagacgtagc 5400

cacctactcc caacatcagc cggactccga ttacctcggg aacttgctcc gtagtaagac 5460

attcatcgcg cttgctgcct tcgaccaaga agcggttgtt ggcgctctcg cggcttacgt 5520

tctgcccaga tttgagcagc cgcgtagtga gatctatatc tatgatctcg cagtctccgg 5580

cgagcaccgg aggcagggca ttgccaccgc gctcatcaat ctcctcaagc atgaggccaa 5640

cgcgcttggt gcttatgtga tctacgtgca agcagattac ggtgacgatc ccgcagtggc 5700

tctctataca aagttgggca tacgggaaga agtgatgcac tttgatatcg acccaagtac 5760

cgccacctaa caattcgttc aagccgagat cgtagaattt cgacgacctg cagccaagca 5820

taacttcgta taatgtatgc tatacgaacg gtaggatcct ctagagtcga ccaggtggca 5880

cttttcgggc agaccgggga cttatcagcc aacctgt 5917

<---

Claims (15)

1. A method for producing 2'-fucosyllactose using a genetically engineered Escherichia coli cell, which includes the following steps: (i) providing a genetically engineered Escherichia coli cell that produces 2'-fucosyllactose, wherein the Escherichia coli cell is genetically engineered to express at least one heterologous α-1,3-fucosidase that performs intracellular degradation of 3-fucosyllactose and 2'3-difucosyllactose , which are formed during the intracellular biosynthesis of 2'-fucosyllactose, and at the same time the Escherichia coli cell reuses degradation products - lactose and fucose, obtained as a result of the enzymatic activity of the specified α-1,3-fucosidase, to produce 2'-fucosyllactose, and genetically the engineered Escherichia coli cell contains a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase to store fucose to produce 2'-fucosyllactose; (ii) culturing the genetically engineered Escherichia coli cell under conditions and media that are permissive for the production of 2'-fucosyllactose, thereby producing 2'-fucosyllactose; And (iii) isolation of 2'-fucosyllactose is possible. 2. A genetically engineered Escherichia coli cell to produce 2'-fucosyllactose, where the Escherichia coli cell: a) produces 2'-fucosyllactose intracellularly; b) genetically engineered to express at least one heterologous α-1,3-fucosidase, which carries out the intracellular degradation of 3-fucosyllactose and 2'3-difucosyllactose, which are formed during the intracellular biosynthesis of 2'-fucosyllactose; c) genetically engineered to contain bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase to retain free fucose for the production of 2'-fucosyllactose; And d) reuses the degradation products lactose and fucose obtained from the enzymatic activity of said α-1,3-fucosidase to produce 2'-fucosyllactose. 3. The method according to claim 1 or the genetically engineered Escherichia coli cell according to claim 2, where the genetically engineered Escherichia coli cell is genetically engineered to express a heterologous glycosyltransferase, preferably a glycosyltransferase selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, N-acetyl- glucosaminyltransferases and glucosyltransferases. 4. Method according to any one of paragraphs. 1 and 3 or a genetically engineered Escherichia coli cell according to any one of paragraphs. 2, 3, wherein a genetically engineered Escherichia coli cell is genetically engineered to express a heterologous α-1,2-fucosyltransferase and a heterologous α-1,3-fucosidase. 5. Method according to any one of paragraphs. 1 and 3 or a genetically engineered Escherichia coli cell according to any one of paragraphs. 2, 3, wherein a genetically engineered Escherichia coli cell is genetically engineered to express the α-1,2-fucosyltransferase 2-fucosyltransferase gene wbgL from E. coli:O126. 6. Method or genetically engineered Escherichia coli cell according to any one of paragraphs. 4 or 5, wherein the genetically engineered Escherichia coli cell is genetically engineered to express the α-1,3-fucosidase AfcB from Bifidobacterium bifidum. 7. Method according to any one of paragraphs. 1, 3-6 or a genetically engineered Escherichia coli cell according to any one of paragraphs. 2-6, wherein the genetically engineered Escherichia coli cell is genetically engineered to contain an fkp gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyltransferase, preferably encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyltransferase from Bacteroides fragilis. 8. Use of a genetically engineered Escherichia coli cell according to any one of paragraphs. 2-7 to obtain 2'-fucosyllactose.

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