RU2810729C2 - Production of fucosylated oligosaccharides in bacillus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: non-spore-forming cell of Bacillus subtilis has been proposed for the production of 2'-fucosyllactose. Sporulation is impaired in this cell as a result of deletion or functional inactivation of one or more genes encoding Spo0A, sigma E and sigma F. Also, this cell contains lactose permease LacY E. coli andα-1,2-fucosyltransferase WbgL of E. coli. The cell is also genetically engineered to express de novo genes from the GDP-fucose biosynthesis pathway, which are the E. coli manA, E. coli manB, E. coli manC, E. coli gmd and E. coli wcaG genes; and wherein the said Bacillus cell does not possess anyβ-galactosidase activity or has reducedβ-galactosidase activity compared to a wild-type Bacillus progenitor cell of the same species as a result of deletion or functional inactivation of gan A. Also the following is proposed: the use of the said Bacillus cell to produce 2'-fucosyllactose and a method of producing 2'-fucosyllactose using the said Bacillus cell.

EFFECT: invention can be used for efficient production of 2'-fucosyllactose.

4 cl, 1 tbl, 5 ex

Description

The present invention relates to the fields of genetic engineering, in particular to the genetic engineering of Bacillus cells for the production of fucosylated oligosaccharides and to the microbial production of fucosylated oligosaccharides using these genetically engineered Bacillus cells.

Prior Art

Human breast milk has optimal nutritional properties for infants. Sugars, which are present in human breast milk, are the main component of human milk, superior to fats and proteins. In addition to lactose, which serves as a source of energy, human breast milk contains from about 5 to 25 grams per liter of complex sugar molecules, namely oligosaccharides. These oligosaccharides are found in significant concentrations only in human milk, and they are collectively known as human milk oligosaccharides (HMOs).

To date, approximately 200 structurally distinct OGMs have been identified. These HGMs are based on the disaccharide lactose (consisting of a glucose group (Glc) and a galactose group (Gal)) and carry additional monosaccharide residues that are based on N-acetyl-glucosamine (GlcNAc), fucose (Fuc), salicylic acid (NeuNAc) and galactose (Gal). The concentration and composition of HMOs in human breast milk varies among individuals and throughout the lactation period, from up to 20 g/L in colostrum to 5-10 g/L in mature milk.

The milk of women belonging to the so-called “secretor phenotype” contains high levels of α-1,2-fucosylated HGMs. These women express the FUT2 gene, which encodes the so-called “fucosyltransferase 2”. The most common HGMs in their milk are 2'-fucosyllactose (2'-FL - from the English 2'-fucosyllactose; Fuc(α1-2)Gal(β1-4)Glc) and lacto-N-fucopentaose-I (LNPF- I - from English lacto-N-fucopentaose-I; Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc).

Breast milk oligosaccharides are not digested as they pass through the intestines of infants. Due to their stability in the intestines of the infant, they demonstrate beneficial effects. More specifically, HMOs have been shown to be prebiotic because they serve as a carbon source for commensal microorganisms of the genera Bifidobacterium, Bacteroides, and Lactobacillus. Thus, OGM support the proliferation of these microorganisms in the intestines of infants.

Breast milk oligosaccharides also directly reduce pathogen colonization of the infant's intestine by preventing said pathogens from adhering to glycan structures on the surface of the intestinal mucosa. OGMs function as decoys due to their structural similarity to epithelial surface glycans and inhibit pathogen invasion, thereby reducing the risk of infections.

Alpha-1,2-fucosylated HMOs have been shown to protect against infections by Campylobacter jejuni, the causative agent of the most common bacterial diarrhea. α-1,2-Fucosylated HMOs are also associated with protection against heat-stable toxin-induced diarrhea in Escherichia coli. In addition, the risk of infections with caliciviruses that mediate diarrhea is reduced by the high content of α-1,2-fucosylated HMOs in breast milk. OGM, mainly fucosylated OGM lacto-N-fucopentaose V (LNFP-V; Gal(β1-3)GlcNAc (β1-3)Gal(β1-4)[Fucα1-3]Glc), binds to the binding site toxin A from Clostridium difficile with carbohydrate, the most common cause of hospital-acquired diarrhea. Thus, OGMs appear to interfere with the interaction of toxin A from C. difficile with cellular receptors.

In addition, the adherence of Pseudomonas aeruginosa to epithelial cells was significantly inhibited by 2-FL and 3-fucosyllactose (3-FL - from English 3-fucosyllactose; Gal(β1-4)[Fucα1-3]Glc). The binding of noroviruses (Norwalk viruses, NLV - from the English Norwalk-like virus), the main cause of acute gastroenteritis, with histo-blood group antigens is prevented by α-1,2-fucosylated OGM, as well as α-1,3-fucosylated OGM. This indicates the potential of these OGMs to inhibit the binding of the norovirus capsid to host receptor glycans.

Due to the known benefits of HMOs and primarily fucosylated HMOs, an economically feasible method for their synthesis is desirable such that these oligosaccharides, or at least some of these oligosaccharides, become available as an additive to infant formula.

Due to limited supply and difficulties in obtaining pure fractions of individual human milk oligosaccharides, chemical routes have been developed to synthesize some of these complex molecules. However, both chemical synthesis and biocatalytic approaches have proven to be commercially unsustainable. In addition, the chemical synthesis of human milk oligosaccharides in particular involves the use of several harmful chemicals that pose a risk of contamination of the final product.

Due to problems encountered in the chemical synthesis of human milk oligosaccharides, enzymatic approaches to the production of HMOs have been developed. To date, the microbial production of several HGMs, such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose II, 3'- sialyllactose and 6'-sialyllactose, using mainly genetically engineered Escherichia coli strains.

Today, recombinant E. coli cells are used for microbial production of OGM on an industrial scale. However, the genus Escherichia coli contains pathogenic members as well as non-pathogenic members. Although non-pathogenic E. coli strains are used for microbial production of HGM, such non-pathogenic E. coli are not considered safe for the manufacture of products intended for human consumption in a variety of applications. Thus, microbial cells of genera that would be regarded as safe in such areas are required for the manufacture of compounds that are intended for human consumption in order to obtain regulatory approval of said products for their commercialization in said areas.

The problem is solved by using bacterial cells of the genus Bacillus, which are considered safe for human consumption and which are capable of producing fucosylated oligosaccharide.

Bacteria of the genus Bacillus are gram-positive, rod-shaped, endospore-forming microbial cells of either aerobic or facultative anaerobic species. The genus Bacillus belongs to the phylum Firmicutes. The genome of members of the genus Bacillus tends toward A-T base pairs in its codon usage frequency. Bacillus species are almost ubiquitous in nature. For example, they can be found in soil (B. subtilis) and are also found in extreme environmental conditions such as high pH (B. alcalophilus), high temperature (B. thermophilus) or high salt content (B. halodurans).

The genus Bacillus contains 266 named species, which include free-living species as well as parasitic pathogenic species. Two species of Bacillus are considered medically significant: B. anthracis, which causes anthrax, and B. cereus, which causes food poisoning. The third species, B. thuringiensis, is an important insect pathogen that produces a toxin that can kill insects. Thus, it is used as an insecticide to control insect pests.

Due to their GRAS (generally recognized as safe) status, several Bacillus species, such as B. amyloliquefaciens, B. licheniformis and B. subtilis, are used in biotechnological production of various proteins and compounds used in food and pharmaceutical industry.

B. amyloliquefaciens is a source of the BamHI restriction enzyme and also synthesizes a natural protein, the antibiotic barnase. In addition, B. amyloliquefaciens produces plantazolicin, an antibiotic with selective activity against B. anthracis. Alpha-amylase from B. amyloliquefaciens is often used in starch hydrolysis. B. amyloliquefaciens is also a source of subtilisin, which catalyzes the breakdown of proteins.

B. amyloliquefaciens is a root colonizing bacterium that is used to control some plant root pathogens in agriculture, aquaculture and hydroponics because it has effects against bacterial and fungal pathogens and can prevent infection through competitive exclusion or displacement of an unwanted pathogen in competition.

Its high ability to secrete alkaline serine protease has made B. licheniformis one of the most important bacteria in the industrial production of the enzyme. Carlsberg subtilisin, secreted by B. licheniformis, is used as a protease for detergents and is sold under the trade name Alcalase®.

B. subtilis is a catalase-positive bacterium that is found in the soil and gastrointestinal tract of ruminants and humans. B. subtilis and substances derived from this endotoxin-free bacterium have been evaluated by various authorities for their safety and benefit in food applications. In the United States, the carbohydrase and protease enzymes from B. subtilis are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). Bacillus subtilis has also been given "qualified presumption of safety" status by the European Food Safety Authority.

In addition, non-toxigenic and non-pathogenic strains of B. subtilis are commonly used in the food industry. For example, fermented soybeans in the form of natto, which are commonly consumed in Japan, contain up to 10 viable B. subtilis cells per gram. Fermented beans are recognized for their contribution to healthy gut flora and vitamin _K2 absorption. The product of natto and B. subtilis var. natto as its main component are FOSHU (Foods for Specified Health Use), approved by the Ministry of Health, Labor and Welfare of Japan as effective for maintaining health.

B. subtilis is easy to work with, grows quickly, and culture conditions are simple. Recombinant strains of B. subtilis are used in the production of polyhydroxyalkanolates, hyaluronic acid and various enzymes such as amylase and proteases.

Natural isolates of wild-type B. subtilis are difficult to work with compared to laboratory strains that have been subjected to domestication processes based on mutagenesis and selection. These domesticated strains often have improved abilities to develop natural competence (uptake and integration of environmental DNA), growth, and loss of abilities required "in the wild." In B. subtilis, linear DNA, as well as multimeric forms of plasmid DNA, are actively taken up by natural competent cells.

Under certain physiological conditions, a small subpopulation of B. subtilis cells becomes competent. In B. subtilis, natural competence is regulated by a complex regulatory network. Key regulators in this network are, among others, the master competence regulator ComK and the transcriptional master sporulation regulator Spo0A. The efficiency of transformation of B. subtilis cells and possibly the efficiency of DNA integration into their genome can be improved through genetic engineering. This can be achieved by ectopic integration of an expression cassette containing a regulated promoter (eg, the P _mtlA mannitol-inducible promoter) and the cellK and comS genes into the B. subtilis genome. Additionally, this strategy enables transformation of B. subtilis through natural competence using a complex environment (eg, LB).

To produce fucosylated saccharides, B. subtilis can be genetically modified in a variety of ways.

Gene integration and/or (simultaneous) gene inactivation through disruption or deletion can be achieved through homologous recombination. For efficient homologous recombination, at least 400-500 bp. homologous arms are required in B. subtilis.

Another method for targeted genome design is the modern CRISPR-Cas9 system. This fast, marker-free genome editing tool can be used for large-scale genomic deletions, small and large DNA insertions, gene silencing through stop codon insertion, and the introduction of point mutations. No prior genomic modifications are required for flawless genome editing via CRISPR-Cas9.

Random chromosomal gene integration and insertional mutagenesis can be performed using a modified mariner transposon. This system is not associated with hot spots in B. subtilis, while demonstrating high efficiency in random ectopic integration.

Although Bacillus species are used for industrial enzyme production, Bacillus cells are not currently available for industrial production of oligosaccharides, particularly fucosylated oligosaccharides.

Chinese patent application CN 108 410 787 A discloses recombinant Bacillus subtilis cells that synthesize lactyl-N-neotetraose. These recombinant B. subtilis cells have a lactopermease gene, which is integrated into the cell genome. In addition, said Bacillus cell which carries a plasmid containing a beta-1,3-N-glucosamine transferase gene and a beta-1,4-galactosyltransferase gene. B. subtilis cells can be cultured in the presence of exogenous lactose and synthesize lactyl-N-neotetraose at titers as low as about 1 g/L, which is too low for economical industrial production.

Although numerous patent applications mention Bacillus as a genus that is suitable for the production of oligosaccharides such as lacto-N-neotetraose, no commercial application of Bacillus for the production of fucosylated oligosaccharides, in particular human milk fucosylated oligosaccharides, has yet been realized. presumably due to the significant metabolic modification effort required to implement the necessary biosynthetic pathways for OGM production in Bacillus. While the above B. subtilis production of LNnT depends on donor substrates that occur naturally in B. subtilis cells, the production of fucosylated oligosaccharide in Bacillus requires the implementation of a heterologous metabolic pathway in the cell to provide the required donor substrate - HDP (guanosine diphosphate) - fucose.

Chinese patent application CN 109 735 479 A discloses recombinant Bacillus subtilis cells to carry out the synthesis of 2'-fucosyllactose, where the expression level of lactose transporter enzyme is increased, and where the cell expresses fucosokinase, phosphate guanine transferase and fucosyl transferase. The yield of 2'-fucosyllactose in the fermentation medium was reported to range from 0.424 g/L to 1.042 g/L.

However, the object of the present invention was to provide a bacterial cell of the genus Bacillus for the production of fucosylated oligosaccharides.

The goal is achieved by proposing a Bacillus cell that has a lactose permease to import exogenous lactose into the cell, a de novo GDP-fucose biosynthetic pathway to provide GDP-fucose, and a fucosyltransferase to transfer the fucose moiety from GDP-fucose to lactose. Said Bacillus cell can be cultured in the presence of exogenous lactose to produce a fucosylated oligosaccharide.

SUMMARY OF THE INVENTION

According to the first aspect, a non-spore-forming bacterial cell of the genus Bacillus is proposed for producing a fucosylated oligosaccharide, wherein the Bacillus cell has a lactose permease, a GDP-fucose biosynthetic pathway, and a fucosyltransferase.

According to the second aspect, the use of a non-spore-forming bacterial cell of the genus Bacillus according to the first aspect is proposed for the production of a fucosylated oligosaccharide.

According to a third aspect, there is provided a method for producing a fucosylated oligosaccharide, comprising:

- providing a non-spore-forming bacterial cell of the genus Bacillus, wherein said Bacillus cell contains lactose permease, a pathway for the biosynthesis of GDP-fucose and a fucosyltransferase that accepts lactose;

- culturing the Bacillus cell in a culture medium containing lactose and under conditions that are permissive to the Bacillus cell, producing a fucosylated oligosaccharide, and

- possibly extracting the fucosylated oligosaccharide from the culture medium and/or Bacillus cells.

According to a fourth aspect, fucosylated oligosaccharides are provided that have been produced by a Bacillus cell that contains lactose permease, the GDP-fucose biosynthetic pathway, and fucosyltransferase.

According to a fifth aspect, there is proposed the use of a fucosylated oligosaccharide that has been produced by a Bacillus cell in which lactose permease, the GDP-fucose biosynthetic pathway and fucosyltransferase are present, to produce a nutritional composition.

According to a sixth aspect, nutritional compositions are provided containing at least one fucosylated oligosaccharide that is produced by a Bacillus cell that contains lactose permease, the GDP-fucose biosynthetic pathway, and fucosyltransferase.

Detailed description

The present invention relates to Bacillus cells for the production of fucosylated oligosaccharides, their use and methods for producing fucosylated oligosaccharides by culturing said Bacillus cells in a culture medium containing lactose and under conditions that are permissive for the production of said fucosylated oligosaccharide by said Bacillus cell.

In order to have the ability to produce a fucosylated oligosaccharide, the Bacillus cell must provide the fucosyltransferase with a donor substrate containing a fucose moiety and an acceptor substrate, which is a disaccharide or oligosaccharide, such that the fucosyltransferase can transfer the fucose moiety from the donor substrate to said substrate -acceptor, resulting in the formation of a fucosylated oligosaccharide.

It should be understood that the fucosylated oligosaccharide that said Bacillus cell is designed to produce is the desired fucosylated oligosaccharide, while other fucosylated oligosaccharides that may be formed by fucosyltransferase heterogeneity during production of the desired fucosylated oligosaccharide are considered undesired fucosylated oligosaccharides or by-products. A suitable donor substrate is GDP-L-fucose, and a suitable acceptor substrate for the formation of fucosyllactose is the disaccharide lactose. The resulting desired fucosylated oligosaccharide is 2'-fucosyllactose, 3-fucosyllactose or 2',3-difucosyllactose.

Wild-type Bacillus cells neither synthesize lactose intracellularly nor absorb or metabolize exogenous lactose. However, lactose is a substrate acceptor for the moiety of fucose by lactose-accepting fucosyltransferase in the formation of some fucosylated oligosaccharides. Therefore, with the ability to produce a fucosylated oligosaccharide such as 2'-fucosyllactose, 3-fucosyllactose or 2',3-difucosyllactose, the Bacillus cell must have the ability to provide lactose to lactose-accepting fucosyltransferase, either by producing lactose intracellularly and/or by uptake of lactose externally .

In one embodiment, a Bacillus cell for producing fucosylated oligosaccharide can take up exogenous lactose by having lactose permease. The term "exogenous" in relation to lactose, as used herein, refers to lactose that does not originate from the Bacillus cell, namely, being intracellularly synthesized by the Bacillus cell, but that originates externally to the Bacillus cell, and which is added to the culture medium in which the Bacillus cell is grown to produce a fucosylated oligosaccharide.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to take up exogenous lactose, namely to have lactose permease. Thus, the Bacillus cell requires a heterologous lactose permease to produce fucosylated oligosaccharide. A suitable lactose permease is E. coli LacY or a functional variant thereof.

The term “heterologous”, as used herein, in relation to proteins, polypeptides, enzymes and transporters, as well as in relation to nucleic acid molecules and/or nucleotide sequences, refers to a molecule that is not native to the type of cell that contains the specified molecule. The term "non-native" indicates that the molecule is not present in a naturally occurring or wild-type progenitor cell, namely a cell of the same species that is most commonly found in nature, the Bacillus cell.

The term "functional variant", as used herein, in relation to enzymes and/or transport molecules, refers to proteins or polypeptides having the same activity (enzymatic, catalytic or translocation) as the enzyme or transporter , which is referred to, but which has an amino acid sequence different from the amino acid sequence of the enzyme or transporter molecule being referred to. Thus, an exemplary protein/polypeptide variant differs in amino acid sequence from the referenced protein/polypeptide. The variant and referenced protein/polypeptide may differ in amino acid sequence by one or more substitutions, additions and/or deletions in any combination. Therefore, the term “functional variant” includes truncated versions of the referenced protein/polypeptide that have the same activity as the referenced protein/polypeptide. The substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. The protein/polypeptide variant may be a naturally occurring variant, such as an allelic variant, or it may be a variant that is not known to occur in nature. Variants of proteins/polypeptides not found in nature can be created through mutagenesis techniques, through direct synthesis, or through other genetic engineering techniques known to those skilled in the art. Within the scope of the present disclosure, the term “variant” also includes proteins and interspecies homologs that have an amino acid/nucleic acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75 %, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000 or more amino acids, with the reference polypeptide.

The term “same activity”, as used herein, refers to the enzymatic, catalytic or transport activity of a protein/polypeptide only from a quality standpoint. Therefore, a “functional variant” also includes variants that have increased or decreased activity relative to the activity of the referenced protein/polypeptide.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain or express a nucleotide sequence that encodes a lactose permease, preferably a nucleotide sequence that encodes E. coli LacY lactose permease or a functional variant thereof.

E. coli LacY lactose permease is encoded by the protein coding region (namely, open reading frame) of the E. coli lacY gene (Gen Bank accession number: NP_414877.1). Therefore, in one embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli LacY lactose permease or a functional variant thereof.

In one additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous lactose permease is adapted to the codon frequency of Bacillus. The codon usage frequency of B. subtilis, for example, is unusual in that the total GC content is below about 45%, the GC content of the first letter of these codons is above about 51%, the CG content of the second letter of these codons is below about 36.1%, and the CG content of the third letter of data codons below approximately 46%.

To express lactose permease, the Bacillus cell contains a recombinant lactose permease gene, wherein the nucleotide sequence that encodes the lactose permease is operably linked to expression control sequences that mediate expression of the lactose permease open reading frame.

The term "operably linked", as used herein, will mean a functional relationship between a nucleic acid/gene expression control sequence (such as a promoter, a signal sequence, or a series of transcription factor binding sites) and a second nucleic acid sequence ( commonly referred to as a "protein coding region", an "open reading frame" and sometimes even a "genome"), where an expression control sequence influences the transcription and/or translation of a nucleotide sequence corresponding to a second sequence. Accordingly, the term “promoter” refers to the DNA sequences that typically “precede” the open reading frame in the DNA polymer and provide the transcription initiation site in the mRNA. "Regulatory" DNA sequences, also usually located "upstream" (that is, preceding) the open reading frame in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcription initiation. Collectively referred to as a "promoter/regulator" or "control" DNA sequence, the sequence data that precedes a selected open reading frame (or series of open reading frames) in a functional DNA polymer helps determine whether transcription will occur (and ultimately expression) open reading frame. DNA sequences that “follow” a gene in a DNA polymer and provide a signal to terminate transcription in mRNA are called transcription termination sequences.

The recombinant lactose permease gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

Expression of a heterologous lactose permease gene in a Bacillus cell allows the resulting Bacillus cell to take up externally supplied lactose from the culture medium. The absorbed lactose can then serve as an acceptor substrate for fucosyltransferase (see later in this document).

For the Bacillus cell to produce fucosylated oligosaccharide, it must have the ability to provide a donor substrate for the transfer of the fucose moiety to the acceptor substrate. The donor substrate for the fucose moiety is GDP-fucose. Therefore, the Bacillus cell must have the ability to produce GDP-fucose intracellularly. For the intracellular biosynthesis of GDP-fucose, the Bacillus cell has a pathway for the biosynthesis of GDP-fucose. Bacillus is genetically engineered to have a GDP-fucose biosynthetic pathway. The GDP-fucose biosynthetic pathway may be either a de novo GDP-fucose biosynthetic pathway and/or a GDP-fucose recycling pathway.

In one embodiment, the Bacillus cell has a de novo GDP-fucose biosynthetic pathway for intracellular biosynthesis of GDP-fucose. The Bacillus cell is genetically engineered to have a de novo pathway for the biosynthesis of GDP-fucose.

The de novo biosynthesis pathway of GDP-fucose includes the activities of the following enzymes:

1. fructose-6-phosphate isomerase;

2. phosphomannomutase;

3. GDP: mannose-1-phosphate guanylyltransferase;

4. GDP-mannose-4,6-dehydratase; And

5. GDP fucose synthase.

Therefore, the Bacillus cell has fructose-6-phosphate isomerase (commonly also referred to as mannose-6-phosphate isomerase), phosphomannomutase, GDP:mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase and GDP-fucose synthase to produce fucosylated oligosaccharide.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to have fructose-6-phosphate isomerase, phosphomannomutase, GDP:mannose-1-phosphate-guanylyltransferase, GDP-mannose-4,6-dehydratase and GDP-fucose synthase.

The de novo biosynthesis pathway of GDP-fucose begins with the isomerization of fructose 6-phosphate to mannose 6-phosphate, a reaction that is catalyzed by fructose 6-phosphate isomerase. In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes fructose-6-phosphate isomerase. Suitable fructose 6-phosphate is E. coli ManA or a functional variant thereof. A typical nucleotide sequence is the nucleotide sequence that encodes E. coli ManA.

E. coli fructose-6-phosphate isomerase ManA is encoded by the protein-coding region of the E. coli manA gene (GenBank accession number: NP_416130.3). Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli ManA or a functional variant thereof.

The E. coli fructose-6-phosphate isomerase ManA is encoded by the open reading frame of the E. coli manA gene (GenBank accession number: NP_416130.3). Thus, in an additional and/or alternative embodiment, the Bacillus cell contains the E. coli tapA gene or a functional variant thereof.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding fructose-6-phosphate isomerase is adapted to the codon frequency of Bacillus.

Other suitable fructose-6-phosphate isomerases are B. subtilis ManA and the paralog proteins B. subtilis GmuF (YdhS) and B. subtilis Pmi (Yvyl) or functional variants thereof. Exemplary nucleotide sequences are nucleotide sequences that encode B. subtilis ManA, B. subtilis GmuF, or B. subtilis Pmi. The B. subtilis fructose-6-phosphate isomerase ManA is encoded by the open reading frame of the B. subtilis manA gene (Gen Bank accession number: NP_389084.1).

B. subtilis fructose-6-phosphate isomerase ManA is encoded by the protein coding region of the B. subtilis manA gene (GenBank accession number: (NP_389084.1)). Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes B. subtilis ManA or a functional variant thereof. Thus, in a further and/or alternative embodiment, the Bacillus cell contains the B. subtilis tapA gene or a functional variant thereof. In yet another additional and/or alternative embodiment, the Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes the gmuF gene B. subtilis or a functional variant thereof, and/or a nucleotide sequence that encodes the B. subtilis pmi gene or a functional variant thereof.

In a further and/or alternative embodiment, expression of the native tapA gene, native gmuF gene and/or native pmi gene may be increased. The expression of the native tapA gene, the native gmuF gene and/or the native pmi gene may be increased because the endogenous promoter of at least one of the native tapA gene, the native gmuF gene and the native pmi gene is replaced by a stronger promoter, namely a promoter mediating increased expression level compared to the native promoter of the tapA gene, gmuF gene and pmi gene, respectively. In yet another additional or alternative embodiment, expression of the native tapA gene, native gmuF gene, and/or native pmi gene may be enhanced because additional copies of the native tapA gene, native gmuF gene, and/or native pmi gene are propagated in the Bacillus cell.

In yet another additional and/or alternative embodiment, expression of the native tapA gene may be enhanced by deletion or functional inactivation of the tapP gene encoding the mannose transporter, resulting in constitutive expression of the native tapA gene.

In yet another additional or alternative embodiment, expression of the native gmuF gene may be enhanced by deletion or functional inactivation of the gmuR gene encoding the corresponding transcriptional repressor.

For expression of fructose-6-phosphate isomerase, the Bacillus cell contains a recombinant fructose-6-phosphate isomerase gene in which the nucleotide sequence that encodes fructose-6-phosphate isomerase is operably linked to expression control sequences.

The recombinant fructose-6-phosphate isomerase gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In the second step of de novo biosynthesis of GDP-fucose, mannose-6-phosphate is converted to mannose-1-phosphate by the enzymatic activity of phosphomannomutase. In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes a phosphomannomutase.

A suitable phosphomannomutase is E. coii phosphomannomutase ManB. Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli ManB or a functional variant thereof.

E. coli phosphomannomutase ManB is encoded by the nucleotide sequence of the protein-coding region of the E. coli manB gene (Gen Bank accession number: NP_416552.1). Thus, in an additional and/or alternative embodiment, the Bacillus cell contains the E. coli tapB gene or a functional variant thereof.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding phosphomannomutase is adapted to the codon frequency of Bacillus.

To express phosphomannomutase, the Bacillus cell contains a recombinant phosphomannomutase gene in which the nucleotide sequence that encodes the phosphomannomutase is operably linked to expression control sequences.

The recombinant phosphomannomutase gene may be integrated into the Bacillus chromosome or present in the Bacillus cell as an episomal version on a plasmid.

The next step in the cfe novo GDP-fucose biosynthetic pathway is the formation of GDP-mannose through the following reaction:

α-D-mannose-1-phosphate+GTP+H ⁺ =>diphosphate+GDP-α-D-mannose

The conversion of mannose-1-phosphate to GDP-mannose is mediated by GDP:mannose-1-phosphate guanylyltransferase. Thus, in the Bacillus cell, a heterologous GDP:mannose-1-phosphate guanylyltransferase is available for the production of fucosylated oligosaccharide. A suitable GDP:mannose-1-phosphate guanylyl transferase is E. coli ManC or a functional variant thereof.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes a GDP:mannose-1-phosphate guanylyltransferase. An exemplary nucleotide sequence is a nucleotide sequence that encodes E. coli ManC. Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli ManC or a functional variant thereof.

The E. coli GDP:mannose-1-phosphate guanylyltransferase ManC is encoded by the open reading frame of the E. coli tapC gene (Gen Bank accession number: NP_416553.1). Thus, in an additional and/or alternative embodiment, the Bacillus cell contains the E. coli taC gene or a functional variant thereof.

In an additional and/or alternative embodiment, the codon usage frequency of the nucleotide sequence encoding GDP:mannose-1-phosphate guanylyltransferase is adapted to the codon usage frequency of Bacillus.

When GDP:mannose-1-phosphate guanylyltransferase is expressed, the Bacillus cell contains a recombinant GDP:mannose-1-phosphate guanylyltranserase gene in which the nucleotide sequence that encodes the GDP:mannose-1-phosphate guanylyltransferase is operably linked to expression control sequences.

The recombinant GDP:mannose-1-phosphate guanylyltransferase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In a subsequent step in the de novo GDP-fucose pathway, catalyzed by GDP-mannose-4,6-dehydratase, GDP-D-mannose is converted to GDP-4-dehydro-6-deoxy-D-mannose.

Thus, in the Bacillus cell, GDP-mannose-4,6-dehydratase is available to produce fucosylated oligosaccharide. A suitable GDP-mannose-4,6-dehydratase is Gmd E. coll.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes GDP-mannose-4,6-dehydratase. An exemplary nucleotide sequence is a nucleotide sequence that encodes E. coll Gmd. Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli Gmd or a functional variant thereof.

The E. coli GDP-mannose 4,6-dehydratase Gmd is encoded by the open reading frame of the E. coli gmd gene (Gen Bank accession number: NP_416557.1). Thus, in an additional and/or alternative embodiment, the Bacillus cell contains the E. coli gmd gene or a functional variant thereof.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding GDP-mannose-4,6-dehydratase is adapted to the codon frequency of Bacillus.

In the case of expression of GDP-mannose-4,6-dehydratase, the Bacillus cell contains a recombinant GDP-mannose-4,6-dehydratase gene in which the nucleotide sequence that encodes GDP-mannose-4,6-dehydratase is operably linked to the sequences expression control.

The recombinant GDP-mannose-4,6-dehydratase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In the final reaction of the de novo GDP-fucose biosynthesis pathway, GDP-4-dehydro-6-deoxy-D-mannose is converted to GDP-fucose as a result of a two-step NADPH (reduced nicotinamide adenine dinucleotide phosphate)-dependent reaction. This conversion is mediated by GDP fucose synthase and includes an epimerase reaction and a reductase reaction. This epimerase reaction converts GDP-4-keto-6-deoxymannose to GDP-4-keto-6-deoxygalactose, which is then reduced to GDP-fucose.

Thus, the Bacillus cell has GDP-fucose synthase for the production of fucosylated oligosaccharide. A suitable GDP fucose synthase is E. coli WcaG or a functional variant thereof.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes GDP fucose synthase. An exemplary nucleotide sequence is a nucleotide sequence that encodes E. coli WcaG. Thus, in a further embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli WcaG or a functional variant thereof.

The E. coli GDP-fucose synthase WcaG is encoded by the open reading frame of the E. coli wcaG gene (Gen Bank accession number: NP_416556.1). Thus, in an additional and/or alternative embodiment, the Bacillus cell contains the E. coli wcaG gene or a functional variant thereof.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding GDP fucose synthase is adapted to the codon frequency of Bacillus.

In the case of expression of GDP fucose synthase, the Bacillus cell contains a recombinant GDP fucose synthase gene in which the nucleotide sequence that encodes GDP fucose synthase is operably linked to expression control sequences.

The recombinant GDP fucose synthase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In an additional and/or alternative embodiment, the Bacillus cell has a GDP-fucose recycling pathway for intracellular biosynthesis of GDP-fucose. In the GDP-L-fucose recycling pathway, free cytoplasmic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose.

The recycling pathway for the biosynthesis of GDP-fucose includes the following enzymes:

I. fucosokinase; And

II. L-fucose-1-phosphate guanylyltransferase.

Therefore, in the Bacillus cell, fucosokinase and L-fucose-1-phosphate guanylyltransferase are available for the production of fucosylated oligosaccharide, which has a GDP-fucose recycling pathway.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to have fucosokinase and L-fucose-1-phosphate guanylyl transferase.

Fucosokinase, also called fucokinase, ATP:6-deoxy-L-galactose-1-phosphotransferase, ATP:β-L-fucose-1-phosphotransferase or L-fucokinase activity, L-fucose kinase activity catalyzes the reaction L-fucose+ATP → β -L-fucose-1-phosphate + ADP + 2 H ⁺ .

L-fucose-1-phosphate guanylyltransferase or GDP-L-fucose pyrophosphorylase then converts said β-L-fucose-1-phosphate into GDP-L-fucose.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes fucosokinase or a functional variant thereof, and L-fucose-1-phosphate guanylyltransferase or a functional variant thereof.

In an additional and/or alternative embodiment, fucosokinase and L-fucose-1-phosphate guanylyl transferase are combined in one single polypeptide. Suitable transformation genes encoding fucosokinase, fucose-1-phosphate guanylyltransferase and/or bifunctional L-fucose-1-phosphate guanylyltransferase can be obtained from the genera Bacteroides, Lentisphaera, Ruminococcus, Solibacter, Arabidopsis, Oryza, Physcomitrella, Vitis, Danio, Bos, Equus, Macaca, Pan, Homo, Rattus, Mus and Xenopus. An example of a bifunctional fucose kinase/L-fucose-1-phosphate guanylyltransferase is found in Bactericides fragilis.

In B. fragilis, the bifunctional fucose kinase/L-fucose-1-phosphate guanylyltransferase Fkp is encoded by the B. fragilis fkp gene (GenBank accession no. AY849806).

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes B. fragilis Fkp or a functional variant thereof.

In an additional and/or alternative embodiment, the codon usage frequency of nucleotide sequences encoding fucosokinase, fucose-1-phosphate guanylyltransferase and/or bifunctional fucosokinase/L-fucose-1-phosphate guanylyltransferase is adjusted to the codon usage frequency of Bacillus.

In the case of expression of fucosokinase and fucose-1-phosphate guanylyltransferase, the Bacillus cell contains at least one recombinant gene in which the protein coding region encoding fucosokinase and L-fucose-1-phosphate guanylyltransferase and/or bifunctional fucosokinase/L-fucose-1 -phosphate guanylyl transferase fucosokinase, functionally linked to expression control sequences.

The recombinant fucosokinase gene, the fucose-1-phosphate guanylyltransferase gene and/or the bifunctional fucosokinase/L-fucose-1-phosphate guanylyltransferase gene, can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In the case of intracellular formation of fucosyllactose, the Bacillus cell contains fucosyltransferase. The enzymatic activity of fucosyltransferase transfers the fucose moiety from the donor substrate to the acceptor substrate. For the biosynthesis of fucosyllactose, said acceptor substrate is lactose. Therefore, fucosyltransferase is a lactose-accepting fucosyltransferase.

The fucosyltransferase is selected from the group consisting of α-1,2-fucosyltransferases for the biosynthesis of 2'-fucosyllactose and α-1,3-fucosyltransferases for the biosynthesis of 3-fucosyllactose.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express at least one nucleotide sequence that encodes a fucosyltransferase.

For the production of 2'-fucosyllactose (2'-FL) α-1,2-fucosyltransferase WbgL from E. coli O126 and FucT2 from Helicobacter pylori (EP 2 479 263 B1), α-1,2-fucosyltransferase WblA from Vibrio cholera O22 , FutD from H. bilis ATCC 437879, FutE from H cinaede CCUG 18818, FutN from Bacteroides vulgatus ATCC 8482, FutO from Bacteroides ovatus ATCC 8483, WbgN from E. coli O55:H7, Bft1 and Bft3 from Bacteroides fragilis NCTC 934 3 (WO 2014 /018596 A2), and α-1,2-fucosyltransferases FucT2 from H. pylori for the synthesis of Lewis Y and Lewis B saccharides (US 6670160 B2) have been described and are suitable α-1,2-fucosyltransferases for the biosynthesis of 2'-FL in Bacillus cells.

To obtain 3-fucosyllactose, α-1,3-fucosyltransferase Amuc from Akkermansia muciniphila and FucT6 and FucT7 from Bacteroides fragilis (EP 2 439 264 A1), α-1,3-fucosyltransferase FutA from H. pylori (US 2014/0120611 A1) are described ), and they are suitable α-1,3-fucosyltransferases for 3-FL biosynthesis in Bacillus cells. In addition, WO 2016/040531 A1 discloses α-1,3-fucosyltransferase CafC from B. nordii CL02T12C05 for the synthesis of 3-fucosyllactose and lactodifucotetraose, and CafD from H. hepaticus ATCC51449 for the production of LNnFP-III.

Additional fucosyltransferases that can be expressed in Bacillus cells to produce fucosylated saccharides are disclosed in WO 2019/0088133 A1, which is incorporated herein by reference.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the fucosyltransferase is adapted to the codon frequency of Bacillus.

In the case of fucosyltransferase expression, the Bacillus cell contains at least one recombinant gene in which the protein-coding region encoding the fucosyltransferase is operably linked to expression control sequences.

The recombinant fucosyltransferase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In a further and/or alternative embodiment, the Bacillus cell may ingest L-fucose to produce fucosylated oligosaccharide. The ability to absorb L-fucose is an advantage of Bacillus cells, which have a GDP-fucose recycling pathway.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to enable or improve the uptake of L-fucose. Thus, the Bacillus cell contains heterologous L-fucose permease. A suitable L-fucose permease is E. coli FucP or a functional variant thereof.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes L-fucose permease, preferably a nucleotide sequence that encodes E. coli FucP or a functional variant thereof.

E. coli L-fucose permease is encoded by the protein coding region of the E. coli fucP gene (Gen Bank accession number: NP_417281.1). Therefore, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes E. coli FucP or a functional variant thereof.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding L-fucose permease is adapted to the codon frequency of Bacillus.

To express L-fucose permease, the Bacillus cell contains a recombinant L-fucose permease gene in which the nucleotide sequence that encodes L-fucose permease is operably linked to expression control sequences.

The recombinant L-fucose permease gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid.

In a further and/or alternative embodiment, a fucosylated oligosaccharide-producing Bacillus cell has no or reduced β-galactosidase activity compared to a wild-type cell of the same species.

Intracellular biosynthesis of fucosylated oligosaccharides requires the import of lactose as an acceptor substrate for lactose-accepting fucosyltransferase. Any intracellular enzymatic activity that hydrolyzes ingested lactose will affect the efficiency of fucosyllactose formation because the pool of intracellular lactose will be reduced. Thus, it will be advantageous if the Bacillus cell to produce fucosylated oligosaccharide has no or at least reduced beta-galactosidase activity compared to a wild-type Bacillus cell.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to eliminate or at least reduce the cell's β-galactosidase activity.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered through deletion or functional inactivation of the dapA gene. In yet another embodiment, a Bacillus cell is genetically engineered to reduce the expression level of the dapA gene compared to a wild-type Bacillus cell.

The Bacillus ganA gene is also called yvfH or 1acA. It is a GanR regulon gene that contains genes encoding enzymes involved in galactan utilization. The ganA gene encodes beta-galactosidase, which is involved in the utilization of Bacillus galactan.

Deletion or functional inactivation of the ganA gene eliminates GanA-mediated β-galactosidase activity in the Bacillus cell, while reducing the level of ganA expression reduces the amount of GanA in the Bacillus cell and therefore β-galactosidase activity, which would interfere with fucosylated oligosaccharide biosynthesis.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered through deletion or functional inactivation of the yesZ gene. The Bacillus yesZ gene encodes YesZ beta-galactosidase, which plays a role in the degradation of rhamnogalacturonan derived from plant cell walls. Expression of the Bacillus yesZ gene is induced by rhamnogalacturonan I. In yet another embodiment, a Bacillus cell is genetically engineered to reduce the level of expression of the yesZ gene relative to a wild-type Bacillus cell.

Deletion or functional inactivation of the yesZ gene eliminates YesZ-mediated β-galactosidase activity in the Bacillus cell, whereas reducing the level of yesZ expression reduces the amount of YesZ in the Bacillus cell and, therefore, β-galactosidase activity, which would interfere with the biosynthesis of fucosylated oligosaccharide.

When B. subtilis enters the post-exponential growth phase, they (begin to) produce large amounts of extracellular proteases. Foreign proteins are often sensitive to proteases. Thus, an exoprotease-free strain is desirable to enhance the stability of heterologous proteins and to allow the accumulation of high levels of foreign proteins. The Bacillus genome encodes at least eight extracellular proteases, namely, nprE, aprE, epr, bpr, mpr, nprB, vpr and wprA. Thus, in a further and/or alternative embodiment, the cell is genetically engineered in the sense that at least one gene encoding an extracellular protease has been deleted or functionally inactivated, preferably at least one of the genes selected from the group consisting of nprE , aprE, epr, bpr, mpr, nprB, vpr and wprA. Preferably, two, three, four, five, six, seven or eight genes selected from the group consisting of nprE, aprE, epr, bpr, mpr, nprB, vpr and wprA are deleted or functionally inactivated.

B. subtilis synthesizes pulcherrimic acid when growing in media containing carbohydrates such as glucose or lactose. The released pulcherrimic acid forms the red pigment pulcherrimin, a salt of pulcherrimic acid (iron(III) chelate), where the iron is present in the growth medium. The formation of this undesirable by-product during fermentation processes can be prevented/abrogated by deletion or disruption of the yvmC and/or surX genes. The yvmC gene encodes cyclodipeptide synthase, and the surX gene encodes cytochrome P450 cyclo-l-leucyl-l-leucyl dipeptide oxidase.

Thus, in a further and/or alternative embodiment, the Bacillus cell is genetically engineered such that at least one of the yvmC and surX genes is deleted or functionally inactivated.

The rhizobacterium B. subtilis contains genes for the synthesis of more than 20 antibiotics. Among them there are peptide antibiotics, like the lantibiotics of Bacillus subtilis and lantibiotic-like peptides (subtilin, ericin S, mersacidin, sublancin 168, subtilosin A), and non-ribosomally synthesized (peptide) antibiotics (surfactin, iturin, bacillomycin, mycosubtilin, corynebactin/bacillibactin, fengycin, plipastatin, mycobacillin, TL-119, bacilysin, bacilisocin, amicumacin, 3,3'-neotrehalosadiamine, difficidin, rhizocticin).

For the production of fucosylated oligosaccharide, it is preferable to use a Bacillus cell that does not produce the antibiotic. Thus, in an additional and/or alternative embodiment, the Bacillus cell does not synthesize one or more antibiotics selected from the group consisting of lantibiotics and lantibiotic-like peptides, such as subtilin, ericin S, mersacidin, sublancin 168, subtilosin A; non-ribosomally synthesized (peptide) antibiotics such as surfactin, iturin, bacillomycin, mycosubtilin, corynebactin/bacillibactin, fengycin, plipastatin, mycobacillin, TL-119, bacilysin, bacilizocin, amicoumacin, 3,3'-neotrehalosadiamine, difficidin and rhizocticin. A Bacillus cell can be genetically engineered to produce a Bacillus cell that does not synthesize one or more of these antibiotics.

Wild-type Bacillus cells can form spores. Sporulation, namely the process of spore formation, in bacteria is considered as a reaction of a bacterial cell that initiates a developmental program leading to the formation of daughter cells of different morphology and developmental direction. Bacillus sporulation has been studied as a major model of cell differentiation. During sporulation, the rod-shaped Bacillus cell divides asymmetrically, resulting in two genetically identical cells with different morphologies and developmental directions.

However, in industrial production, it is undesirable if the producing bacterial strain forms spores during fermentation. Thus, it is preferable to use Bacillus cells to produce fucosylated oligosaccharides, which are not capable of spore formation. Such Bacillus cells are called "non-spore-forming".

Preferably, the non-sporulating Bacillus cell capable of producing fucosylated oligosaccharide is derived from one of the B. subtilis strains listed in Table 1.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered through deletion or functional inactivation of Spo0A. Suitable functional inactivation of Spo0A involves deletion of the phosphorylation site where the phosphotransferases Spo0F and Spo0B phosphorylate Spo0A.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered by deletion or functional inactivation of genes encoding the sigma factor SigE (sigE) and/or the sigma factor SigF (sigF).

The Bacillus cell of the invention is capable of producing fucosylated oligosaccharide when cultured in the presence of lactose in a medium and under conditions that are permissive for the Bacillus cell to produce fucosylated oligosaccharide.

A second aspect provides the use of a Bacillus cell, as previously described herein, for the production of a fucosylated oligosaccharide, preferably a human milk fucosylated oligosaccharide such as 2'-FL, 3-FL or 2',3-DiFL.

Because Bacillus is generally regarded as safe, the production of fucosylated oligosaccharides for human consumption using a producing organism generally considered safe will also be considered safe, provided that changes in the Bacillus cells to produce the fucosylated oligosaccharide do not affect the safety of the producing strain in regarding human health and the environment. Therefore, it is expected that there will be fewer problems affecting the approval of a fucosylated oligosaccharide for human consumption, in particular, fucosylated human milk oligosaccharides for human consumption, more particularly for an infant formula that consists of or contains a fucosylated oligosaccharide that has been produced by using Bacillus cells as described herein. Thus, the acceptance of microbially produced fucosylated oligosaccharides in infant formula and nutritional compositions by regulatory authorities as well as by consumers should be improved.

According to a third aspect, there is provided a method for producing a fucosylated oligosaccharide, which includes:

- providing a non-sporulating Bacillus cell that is genetically engineered to have a lactose permease, a GDP-fucose biosynthetic pathway and a fucosyltransferase;

- culturing the Bacillus cell in a culture medium containing lactose and under conditions that are permissive for the production of fucosylated oligosaccharide, and

it is possible to extract the fucosylated oligosaccharide from the culture medium and/or from the Bacillus cell.

The non-sporulating Bacillus cell that is provided is a Bacillus cell as described herein.

In an additional and/or alternative embodiment, the fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 2',3-fucosyllactose (DiFL - from English 2', 3-difucosyllactose), lacto-N-fucopentaose I (LNFP I - from the English lacto-N-fucopentaose I), lacto-N-neofucopentaose I (LNnFP I - from the English lacto-N-neofucopentaose I), lacto-N -fucopentaose II (LNFP II - from English lacto-N-fucopentaose II), lacto-N-fucopentaose III (LNFP III - from English lacto-N-fucopentaose III), lacto-N-fucopentaose V (LNFP V - from English lacto-N-fucopentaose V), lacto-N-neofucopentaose V (LNnFP V - from English lacto-N-neofucopentaose V), lacto-N-difucohexaose I (LNDH I - from English lacto-N-difucohexaose I ) and lacto-N-difucohexaose II (LND - from English lacto-N-difucohexaose II).

In a further and/or alternative embodiment, the culture medium contains L-fucose, in particular for culturing a Bacillus cell to produce a fucosylated oligosaccharide that has a GDP-fucose recycling pathway to provide GDP-fucose as a donor substrate for fucosyltransferase.

The invention also relates to fucosylated oligosaccharides that have been produced by a Bacillus cell and/or to a method as described herein for the use of said fucosylated oligosaccharides for the manufacture of a nutritional composition, preferably an infant formula, dietary supplement or health food product. In addition, the present invention also relates to nutritional compositions containing a fucosylated oligosaccharide that has been produced by a Bacillus cell and/or a method as described herein.

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 noted that the term “comprising” as used in the claims should not be considered limited to the means listed below; 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 understanding one or more of the various 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 claims below, aspects of the invention are comprised of less than all of the features of the one disclosed embodiment set forth above. 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 claims below, any of the claimed embodiments can 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.

EXAMPLES

Example 1: Transformation of Bacillus subtilis

Bacillus subtilis can be genetically manipulated through various techniques. For transformation of B, subtilis competent cells were prepared by a modified two-step protocol (Anagnostopoulos, C. and Spizizen, J. (1961) J Bacteriol 81 (5): 741-746). The overnight culture was inoculated in MG1 medium and shaken at 37°C. MG1 medium is Spitzeisen's minimal medium supplemented with 0.5% glucose, 5 mM MgSO ₄ and 0.02% casamino acids (possibly additionally supplemented with biotin and/or L-tryptophan). The next morning, this culture was diluted 1:20 in fresh MG1 medium and incubated at 37°C for approximately 6 hours. 1 ml of culture was diluted in 8 ml of MG2 medium, which differs from MG1 medium in the concentration of casamino acids (0.01% instead of 0.02%). In the shortened protocol, the overnight culture is directly propagated in MG2 medium. After incubation for another 90 min, a 1 ml portion of the culture was mixed with 1-3 μg of multimeric plasmid DNA or linear DNA and incubated at 37°C for 30-60 min with shaking. Multimeric plasmid DNA was prepared either by using E. coli strain NM538 to propagate the plasmid DNA or by linearizing the plasmid by digestion with a single cut restriction enzyme that cleaves within the framework followed by re-ligation with T4 DNA ligase.

The cells were then plated with 2xYT and agar containing the appropriate antibiotic. Antibiotics were added in appropriate concentrations: 5 μg⋅ml ^-1 erythromycin, 5 μg⋅ml ^-1 chloramphenicol, 10 μg⋅ml ^-1 kanamycin, 100 μg⋅ml ^-1 pectinomycin.

Alternatively, for protoplast transformation (Romero, D. et al. (2006) Journal of Microbiological Methods 66:556-559), cells were grown in 20 ml Penassay broth (PAB) at 37°C before stationary growth phase (OD ₆₀₀ (from the English optical density - optical phase) is 1.7-2). Cells were then pelleted by centrifugation and resuspended in 10 ml SMPP medium (0.3% bovine serum albumin, 5% 2 M sucrose, 25% 4×PAB, 50% 2×SMM) consisting of 2×SMM representing consists of 1 M sucrose, 0.04 M disodium maleic acid hydrate and 0.04 M MgCl ₂ (pH 6.5). After adding lysozyme (10 mg ml ^-1 ) and mutanolysin (75 U ml ^-1 ), the mixture was incubated at 37°C with shaking to form protoplasts. The formation of protoplasts was checked using a microscope. The protoplasts were then carefully collected by centrifugation at 5200 × g and 4°C for 5 min, washed twice with ice-cold electrotransformation wash buffer (1 × SMM), and finally suspended in this solution. Plasmid DNA (1-3 μg) was added to 120 μl of protoplast suspension, and the mixture was kept on ice for at least 5 minutes. The transformation mixture was transferred into a 0.2 cm cuvette, and one electroporation pulse was applied at 25 μF, 400 Ω, and 0.7 kV. Immediately after the electroporation discharge, 1 ml of reducing medium (equal volumes of 4×PAB and 2×SMM, freshly prepared before use) was added to the cuvette. The transformation reaction mixture was then transferred to a 2 ml tube and incubated at 37°C with shaking for 12 hours. For regeneration, the cell suspension was distributed onto DM3 and agar plates (Chang, S. and Cohen, S. (1979) MGG 168( 1):111-115) and incubated at 37°C for 48 hours. The regenerating mixture DM3 contained the following sterile solutions per liter: 200 ml of 4% agar, 100 ml of 5% casamino acids, 50 ml of 10% agar of yeast extract, 100 ml of 3.5% K ₂ HPO ₄ and 1.5% KH ₂ PO ₄ , 25 ml of 20% glucose, 20 ml of 1 M MgCl ₂ , 500 ml of 0.5 M sorbitol and 5 ml of filter-sterilized 2% bovine serum albumin (added to the mixture when the temperature is below 55°C), and it was supplemented with the appropriate antibiotic.

Electroporation of B. subtilis was performed according to a modified protocol from Zhang et al. (2011) provided by MoBiTec GmbH (Zhang, G., Bao, P., Zhang. Y., Deng, A. Chen. N. and Wen, T. (2011) Anal. Biochem., 409:130-137) . The overnight 2xYT culture was diluted 100-fold with fresh 2xYT medium and the culture was grown to an OD ₆₀₀ of 0.2 at 37°C on a rotary shaker. The culture was then supplemented with 1% DL-threonine, 2% glycine, 0.1% tryptophan, and 0.03% Tween 80. After culturing for an additional 60 min, the cell suspension was cooled on ice for 20 min. centrifuged at 5000×g for 10 min at 4°C and washed twice with electroporation buffer (0.5 M trehalose, 0.5 M sorbitol, 0.5 M mannitol, 0.5 mM MgCl ₂ , 0.5 mM K ₂ HPO ₄ , 0.5 mM KH ₂ PO ₄ , pH 7.4, filter sterilized and stored frozen). Finally, the cells were resuspended in electroporation buffer at a ratio of 1/100 of the original culture volume, and 100 μl of the cell suspension was mixed with DNA. The transformation mixture was transferred to a 0.1 cm cuvette and electroporation was performed at 1.8 kV with a single pulse delivered by a MicroPulser™ instrument (Bio-Rad). Immediately after pulse transfer, 1 ml of 2×YT broth containing 0.5 M sorbitol and 0.38 M mannitol was added to the cuvette. The transformation suspension was transferred into a 2 ml test tube and incubated at 37°C for 3 hours on a rotary shaker. Cells were spread on a 2×YT agar plate and incubated at 37°C overnight.

Using an alternative electroporation protocol (Xue, G. P., J. S. Johnson, and B. P. Dalrymple: 1999; Journal of Microbiological Methods 34:183-191), 5 ml of LB containing 0.5 M glucite was inoculated with B. subtilis and incubated in overnight at 37°C. Next, the overnight culture was diluted (1:16) with 75 ml of LB containing 0.5 M glucite and incubated until an OD ₆₀₀ of 0.85 - 0.95 was obtained. Cells were then pelleted by centrifugation for 10 min at 4°C at 5,000×g and washed four times with ice-cold electroporation buffer (10% glycerol, 0.5 M glucose, 0.5 M mannitol). Finally, the cells were resuspended in 1–2 ml of electroporation buffer. Electroporation was performed using 60 μl of competent cells with DNA in a chilled electroporation cuvette (distance between electrodes 1 mm). The cell-DNA mixture was exposed to a single electrical pulse at 25 μF, 200 Ω, and 21 kV/cm. Finally, 1 ml of recovery broth (LB containing 0.5 M glucite and 0.38 M mannitol) was added to the electropermeabilized cells, and the bacterial culture was incubated for 3 h at 37°C, followed by plating on LB agar supplemented with antibiotic. .

Two different enriched media were used, namely Luria broth (LB) and 2×YT. Luria broth (LB) medium consisted of 1% tryptone, 0.5% yeast extract, and 0.5% NaCl (pH 7.2).

2×YT medium consisted of 1.6% tryptone, 1% yeast extract, and 0.5% NaCl (pH 7.5).

To obtain plates with enriched agar medium, 15 g l ^-1 agar was added.

For shake flask experiments, Spizizen's minimal medium was used (Spizizen, J. 1958 Proc. Natl. Acad. Sci. U.S.A. 44(10):1072-1078).

Spitzeisen's minimal medium contains the following salts: 2 g/l (NH ₄ ) ₂ SO ₄ , 14 g/l K ₂ HPO ₄ , 6 g/l KH ₂ PO ₄ , 1 g/l citrate Na ₃ × 2⋅H ₂ O and 0.2 g/l MgSO ₄ × 7⋅H ₂ O.

Preculture medium consisting of Spitzen salts supplemented with 2% D-glucose, 0.05% casamino acids and MgSO ₄ to a final concentration of 2 mM (optionally supplemented with biotin and/or L-tryptophan).

The main culture medium consisted of minimal Spitzeisen salts, supplemented with 2% D-glucose, 0.05% casamino acids, MgSO ₄ to a final concentration of 2 mM and 0.5 ml⋅l ^-1 1000× trace element solution (possibly additional supplemented with biotin and/or L-tryptophan).

The solution of trace elements (1000x) consisted of 100.6 g⋅l ^-1 C ₆ H ₉ NO ₆ , 56.4 g⋅l ^-1 iron (III)-ammonium citrate, 9.8 g⋅l ^-1 MnCl ₂ × 4⋅H ₂ O, 1.6 g⋅l ^-1 CoCl ₂ × 6 H ₂ O, 1 g⋅l ^-1 CuCl ₂ × 2⋅H ₂ O, 1.9 g⋅l ^-1 H ₃ VO ₃ , 9 g ⋅l ^-1 ZnSO ₄ × 7⋅H ₂ O, 1.1 g⋅l ^-1 Na ₂ MoO ₄ × 2⋅H ₂ O, 1.5 g⋅l ^-1 Na ₂ SeO ₃ , 1.5 g⋅ l ^-1 NiSO ₄ × 6⋅H ₂ O.

If necessary, appropriate antibiotic(s) were added to the medium in order to make it selective.

B. subtilis strains were initially grown on enriched agar plates to produce single colonies. These dishes were grown for 1 day at 30-37°C. For shake flask experiments, 20 mL of preculture was inoculated with a single colony and grown overnight at 30–37°C on a rotary shaker. The next 20 ml of main cultures were inoculated with this preculture to an initial OD ₆₀₀ of approximately 0.1 and incubated at 30-37°C on a rotary shaker. If induction was required, 40–60 ml of the main culture was divided into 20 ml portions at the induction time point. The volume of the culture did not exceed 20% of the capacity of the shake flask.

Example 2: Construction of a Bacillus subtilis producer strain for 2'-fucosyllactose

Modification of the metabolism of a sporulation-defective Bacillus subtilis strain (Table 1) was achieved by integrating the heterologous E. coli tapC, E. coli tapB, and E. coli tapA genes and simultaneous removal of the endogenous lacA gene through homologous recombination. The dapA gene (yvfN, lacA) of B. subtilis, which is located within the galactose operon, encodes beta-galactosidase.

For the synthesis of GDP-mannose, the open reading frames tapC, tapB and tapA are functionally linked to the constitutive promoter P43 B, subtilis (part of the iGem repository: sequence ID: BBa_K143013) in the form of an operon. The tapC gene (Gen Bank accession number: NP_416553.1) encodes GDP:mannose-1-phosphate guanylyltransferase from E. coli. The tapB gene (Gen Bank accession number: NP_416552.1) encodes E. coli phosphomannomutase and the tapA gene (Gen Bank accession number: NP_389084.1) encodes B. subtilis fructose-6-phosphate isomerase. Each of the genes mentioned above was fused in silico to the RBS sequence of B. subtilis. The <P43-tapCBA> expression cassette described herein was codon optimized for expression in B. subtilis and produced synthetically through GenScript Corp. Next, the full integration cassette was assembled and cloned into pBR322 (New England Biolabs GmbH, Frankfurt, Germany) to create the suicide plasmid <pBR322 flank ganA up-lox71-erm-lox66-P43-manCBA-flank ganA down> (SEQ ID NO: 1 ). Then, B. subtilis was transformed with this plasmid due to its natural competence. The cells were spread on 2x YT and agar plates containing the appropriate antibiotic (5 μg ml ^-1 erythromycin). Integration of the <P43-tapCBA> expression cassette into the ganA locus of the B. subtilis genome, resulting in strain A, was verified by colony PCR (polymerase chain reaction). Gene expression was confirmed by targeted proteomics and/or real-time PCR.

To produce 2'-fucosyllactose from lactose and GDP-mannose in the resulting strain for the integration of <P43-tapCBA>, an expression plasmid (SEQ ID NO: 2) was constructed, which contains all the necessary genes under the control of the inducible promoter P _grac100 . Therefore, the B. subtilis expression vector pHT253 (MoBiTec GmbH, Göttingen, Germany) was used as a scaffold. The open reading frame of the E. coli lacY gene (Gen Bank accession number: NP_414877.1) was amplified by PCR from chromosomal DNA. The open reading frame of the wbgL gene encoding E. coli O126 α-1,2-fucosyltransferases was codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp. In addition, the open reading frames of the E. coli genes gmd (Gen Bank accession number: NP_416557.1; encoding GDP-mannose-4,6-dehydratase) and wcaG (Gen Bank accession number: NP_416556.1; encoding GDP-fucose synthase) were subjected to codon optimization for expression in B. subtilis and produced synthetically by GenScript Corp. Each gene contained in the inducible expression cassette was associated with an RBS sequence B, subtilis. In addition, the appropriate B. subtilis termination sequence from part of the iGem repository (Sequence ID: BBa_B0015) was introduced into the final expression plasmid <pHT253 P _grac100 -wbgL-gmd-wcaG-lacY-terminator> (SEQ ID NO: 2). The integration strain <P43-manCBA> was transformed with this expression plasmid (SEQ ID NO: 2) due to its natural competence. Gene expression was confirmed by targeted proteomics and/or real-time PCR. Transformants were cultured under conditions that are permissive for B. subtilis to produce 2'-fucosyllactose in the presence of exogenous lactose.

Example 3: Construction of a Bacillus subtilis producer strain for 2'-fucosyllactose

Strain A, which is described in example 2, was used as the parent strain. To produce 2'-fucosyllactose from lactose and GDP-mannose, additionally, the E. coli lacY, gmd, wcaG and wbgL genes were integrated into the endogenous atuE locus (atuA) of strain A (encoding alpha-amylase).

The open reading frame of the wbgL gene (encoding α-1,2-fucosyltransferase) was codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp. In addition, the open reading frames of the genes gmd (Gen Bank accession number: NP_416557.1; encoding GDP-mannose-4,6-dehydratase) and wcaG (Gen Bank accession number: NP_416556.1; encoding GDP-fucose synthase) were subjected to codon optimization for expression in B. subtilis (conducted by GenScript Corp), placed under the control of the strong constitutive B. subtilis P _lepA promoter as a two-gene operon and produced synthetically by GenScript Corp. The open reading frame of the E. coli lacY gene (Gen Bank accession number: NP_414877.1), encoding lactose permease, was amplified by PCR from chromosomal DNA.

All necessary genes for the production of 2'-fucosyllactose from lactose and GDP-mannose were assembled into a constitutive expression cassette <P43-wbgL-lacY-P _lepA -gmd-wcaG>, in which each gene was linked to the RBS sequence of B. subtilis. Additionally, a suitable B. subtilis termination sequence from part of the iGem repository (sequence ID: BBa_B0015) was placed downstream of this expression cassette. The construct described above was cloned into pBR322 (New England Biolabs GmbH, Frankfurt, Germany) to produce <pBR322 flank amyE up-lox71-aacd9-lox66-P43-wbgL-lacY-P _lepA -gmd wcaG-ferminator-flank amyE down> (SEQ ID NO: 3).

The resulting amyE integration vector (SEQ ID NO: 3) was used to transform B. subtilis due to its natural competence. Selection of transformants was carried out using spectinomycin (100 μg ml ^-1 ). Integration of the expression cassette <P43-wbgL-lacY-P _lepA -gmd wcaG-terminator> into the atuE locus of the B. subtilis genome was verified by colony PCR. Gene expression was confirmed by targeted proteomics and/or real-time PCR.

Example 4: Construction of a Bacillus subtilis producer strain for 2'-fucosyllactose

To produce 2'-fucosyllactose from GDP-fucose and exogenous lactose, initially, the E. coli lacY gene was integrated into the endogenous atuE (atuA) locus (encoding alpha-amylase). The open reading frame of the E. coli lacY gene (Gen Bank accession number: NP_414877.1), encoding lactose permease, was amplified by PCR from chromosomal DNA. The <flank amyE up-lox71-aad9-lox66-P43-lacY-flank amyE down> integration cassette (SEQ ID NO: 4) was constructed and cloned into pBR322 (New England Biolabs GmbH, Frankfurt, Germany). B. subtilis was transformed with the resulting plasmid due to its natural competence. Integration of the <P43-lacY> expression cassette into the atyE locus of the B. subtilis genome, resulting in strain B, was verified by colony PCR. Gene expression was confirmed by targeted proteomics and/or real-time PCR.

To synthesize GDP-fucose from exogenous L-fucose, a B. subtilis expression plasmid containing all the necessary genes was constructed. Plasmid pDW1 was used as a scaffold. This plasmid was produced synthetically by GenScript Corp and contains the bla and erm genes for selection in E. coli and B. subtilis, respectively. Additionally, it contains the pMB1 replicon for propagation in E. coli and the pUB110 replicon for propagation in B. subtilis.

The B. fragilis fkp gene (GenBank accession number AY849806) encodes a bifunctional fucokinase/L-fucose-1-phosphate guanylyltransferase, the E. coli wbgL gene encodes α-1,2-fucosyltransferases, and the E. coli fucP gene (Gen Bank accession number: NP_417281. 1) encodes L-fucose permease. Each of the open reading frames of these genes was codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp. An expression cassette was constructed that contains the fkp, wbgL and fucP genes under the control of the strong constitutive promoter P436. subtilis.

Each gene contained in the expression cassette was linked to the RBS sequence of B. subtilis. In addition, the appropriate B. subtilis terminator sequence from part of the iGem repository (Sequence ID: BBa_B0015) was introduced into the final expression plasmid <pDW1-P43-fkp-wbgL-fucP-terminator> (SEQ ID NO: 5). Strain B was transformed with this expression plasmid (SEQ ID NO: 5) due to its natural competence. Gene expression was confirmed by targeted proteomics and/or real-time PCR. Transformants were cultured under conditions that are permissive for B. subtilis to produce 2'-fucosyllactose in the presence of exogenous lactose and L-fucose.

Example 5: Production of 2'-fucosyllactose using metabolically modified Bacillus subtilis strains

The preculture was inoculated with a metabolically modified Bacillus subtilis strain suitable for the biosynthesis of 2'-fucosyllactose (as described in Examples 2, 3 and 4, respectively).

The preculture was incubated at 30-37°C overnight and then diluted to an initial OD ₆₀₀ of approximately 0.1 in fresh basal culture medium. When the main culture reached an OD ₆₀₀ of approximately 0.5, 2 mM lactose was added to the growth medium. When the inducible P _grac100 promoter was used for gene expression, induction was carried out by lactose (2 mM) or both lactose (2 mM) and IPTG (isopropylthiogalactoside) (1 mM). For utilization biosynthesis of GDP-fucose, 2 mM L-fucose was additionally added. Culture was stopped after 24 h/48 h and intracellular and extracellular 2'-fucosyllactose was analyzed by thin layer chromatography and/or HPLC and/or mass spectrometry (as described in WO 2017/042382 A or WO 2019/008133 A). The biosynthesis of significant amounts of 2'-fucosyllactose was detected.

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LIST OF SEQUENCES

<110> Jennewein Biotechnologie GmbH

<120> PRODUCTION OF FUCOSYLATED OLIGOSACCHARIDES IN BACILLUS

<130>P 1903 WO

<160> 5

<170> Patent In version 3.5

<210> 1

<211> 11270

<212> DNA

<213> Artificial sequence

<220>

<223> Suicide plasmid

<400> 1

ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa

60

ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg

120

caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt

180

gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata

240

tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg

300

ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac

360

cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac

420

aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca

480

cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg

540

actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct

600

caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat

660

gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt

720

cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct

780

ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct

840

tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa

900

acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt

960

cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc

1020

cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca

1080

tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc

1140

gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat

1200

tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg

1260

ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga

1320

attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac

1380

atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg

1440

tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg

1500

ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc

1560

tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg

1620

taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca

1680

ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga

1740

ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa

1800

cgttccagta acggggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt

1860

ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc

1920

aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt

1980

ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac

2040

gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac

2100

ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc

2160

agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc

2220

cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg

2280

tactgagagt gcaccagcgg gcaaggaaag ccttcaatat gtgcagtggt atgtcaactc

2340

tatgaagatc agcctgttta caatggcagg gtctttgctc tgtgtgacgt ttacggccta

2400

tgcgttttcg cgctttcggt ttaaagggag gaaatacgct ttaacgctct ttttattgct

2460

gcagatgatt cctcagtttt cagctttaat tgccttgttt gtgctggcgc aaatcttggg

2520

aatgatcaat agccactggc tgctaatctt gctttatatc ggcggcctga tcccgatgaa

2580

tacgtatttg atgaaagggt acatggattc cattccgatg gatttagacg aaagcgccaa

2640

gattgacgga gccagcagca ccagaatctt cttccagatc attctgccat tatcaaaacc

2700

gatggcggca gtcgtggcca tgaacggctt taccggtccg ctcggagatt ttgtgctgtc

2760

ctcaaccata ttgagaacgc ctgaatcata tacattgccc gtcggtctat tcaatttagt

2820

gaatgatgtc atggggggcca gctatacgac atttgcggcc ggagccctgc ttatcagcat

2880

accggttgcc gtcatcttta ttatgctgca aaagaatttt gtgtccggat taaccgcagg

2940

cggaacgaag ggctaagaga acaagggagga gaatgtgatg tcaaagcttg aaaaaacgca

3000

cgtaacaaaa gcaaaattta tgctccatgg gggagactac aaccccgatc agtggctgga

3060

tcggcccgat attttagctg acgatatcaa actgatgaag ctttctcata cgaatacgtt

3120

ttctgtcggc accagtgaat tcgagctcgg tacctaccgt tcgtataatg tatgctatac

3180

gaagttatta cgcgttaacc cgggcccgcg gatgcatatg atcagatcct ttaactctgg

3240

caaccctcaa aattgaatga gacatgctac acctccggat aataaatata tataaacgta

3300

tatagatttc ataaagtcta acacactaga cttatttact tcgtaattaa gtcgttaaac

3360

cgtgtgctct acgaccaaaa ctataaaacc tttaagaact ttcttttttt acaagaaaaa

3420

agaaattaga taaatctctc atatctttta ttcaataatc gcatccgatt gcagtataaa

3480

tttaacgatc actcatcatg ttcatattta tcagagctcg tgctataatt atactaattt

3540

tataaggagg aaaaaatatg ggcattttta gtatttttgt aatcagcaca gttcattatc

3600

aaccaaacaa aaaataagtg gttataatga atcgttaata agcaaaattc atataaccaa

3660

attaaagagg gttataatga acgagaaaaa tataaaacac agtcaaaact ttattacttc

3720

aaaacataat atagataaaa taatgacaaa tataagatta aatgaacatg ataatatctt

3780

tgaaatcggc tcaggaaaag gccattttac ccttgaatta gtaaagaggt gtaatttcgt

3840

aactgccatt gaaatagacc ataaattatg caaaactaca gaaaataaac ttgttgatca

3900

cgataatttc caagttttaa acaaggatat attgcagttt aaatttccta aaaaccaatc

3960

ctataaaata tatggtaata taccttataa cataagtacg gatataatac gcaaaattgt

4020

ttttgatagt atagctaatg agatttattt aatcgtggaa tacgggtttg ctaaaagatt

4080

attaaataca aaacgctcat tggcattact tttaatggca gaagttgata tttctatatt

4140

aagtatggtt ccaagagaat attttcatcc taaacctaaa gtgaatagct cacttatcag

4200

attaagtaga aaaaaatcaa gaatatcaca caaagataaa caaaagtata attatttcgt

4260

tatgaaatgg gttaacaaag aatacaagaa aatatttaca aaaaatcaat ttaacaattc

4320

cttaaaacat gcaggaattg acgatttaaa caatattagc tttgaacaat tcttatctct

4380

tttcaatagc tataaattat ttaataagta agttaaggga tgcataaact gcatccctta

4440

acttgttttt cgtgtgccta ttttttgtga atcgattata acttcgtata atgtatgcta

4500

tacgaacggt aggatcctct agagtcgacc tgcaggcatt ttacattttt agaaatgggc

4560

gtgaaaaaaa gcgcgcgatt atgtaaaata taaagattaa ctaataagga ggacaaacat

4620

ggcacaatct aaactgtatc cggttgtgat ggcgggcgga tctggatcaa gactgtggcc

4680

gctttcacgc gttctttacc cgaaacaatt tctgtgtctg aaaggagatt taacaatgct

4740

gcagacaaca atttgcagat taaatggcgt ggaatgtgaa tctccggtcg ttatttgcaa

4800

cgaacagcat agatttatcg tcgctgaaca acttcgccag ctgaacaaac tgacagaaaa

4860

catcatcctg gaaccggccg gcagaaatac agcaccggcg attgctcttg cagcgttagc

4920

tgccaaacgc cattcaccgg aaagcgatcc gcttatgtta gtgctggcag cggatcatgt

4980

cattgcggat gaagatgctt ttagagctgc cgttcgcaac gctatgccgt atgccgaagc

5040

aggcaaactt gtgacatttg gaattgtccc ggatttaccg gaaacaggct atggatatat

5100

cagacgcggc gaagtgtcag cgggagaaca agatatggtt gcctttgaag tggcacagtt

5160

tgtcgaaaaa ccgaatctgg aaacagcgca agcgtatgtt gcttcaggcg aatattattg

5220

gaacagcgga atgtttcttt ttagagccgg ccgctatctg gaagaactta aaaaatacag

5280

accggatatt ttagatgcat gtgaaaaagc gatgtcagct gtcgatccgg atttaaactt

5340

tatcagagtt gatgaagaag cctttctggc atgcccggaa gaaagcgttg attatgcagt

5400

gatggaacgc acagccgatg cagtggtcgt tccgatggat gcgggctggt ctgatgttgg

5460

atcatggtca agcctttggg aaattagcgc gcatacagct gaaggcaatg tttgtcatgg

5520

agatgtgatc aaccataaaa cagaaaacag ctatgtttac gcggaatctg gcctggtgac

5580

aacagtcgga gttaaagatt tagtggtcgt tcagacaaaa gatgcggtgt taattgctga

5640

tagaaacgcc gtgcaagatg ttaagaaagt tgtcgaacag atcaaagcag atggaagaca

5700

tgaacataga gtccatcgcg aagtttatcg cccgtgggga aaatatgata gcattgatgc

5760

gggcgatcgc tatcaagtca aacgcatcac agttaaaccg ggcgaaggac tgtctgttca

5820

gatgcatcat catcgcgcag aacattgggt tgtggtcgcc ggcacagcaa aagtgacaat

5880

tgatggcgat attaaactgc ttggagaaaa cgaatctatt tatatcccgt taggagctac

5940

acattgcctg gaaaacccgg gcaaaattcc gcttgattta atcgaagtca gaagcggatc

6000

ttatcttgaa gaagatgatg ttgtgcgctt tgccgataga tatggccgcg tttaagctct

6060

taaggaggat tttagaatga aaaaacttac atgctttaaa gcgtatgata ttcgcggcaa

6120

acttggagaa gaattaaacg aagatattgc ttggagaatc ggccgcgcct atggagaatt

6180

tctgaaaccg aaaacaatcg tgctgggcgg agatgtcaga cttacatcag aaacacttaa

6240

actggctctg gccaaaggcc tgcaagatgc tggagttgat gtgcttgata ttggcatgag

6300

cggaacagaa gaaatctatt ttgccacatt tcatttagga gtcgatggcg gaatcgaagt

6360

tacagcaagc cataatccga tggattataa cggaatgaaa ctggttagag aaggcgcaag

6420

accgatttct ggcgatacag gattaagaga tgtgcagcgc ctggcagaag cgaacgattt

6480

tccgccggtc gatgaaacaa aaagaggacg ctatcaacag attaatttac gcgatgcgta

6540

tgttgatcat ctttttggct acatcaacgt taaaaacctg acaccgctta aactggtgat

6600

caattctggc aacggagcag cgggaccggt tgtggatgct attgaagcca gatttaaagc

6660

acttggcgca ccggttgaat taatcaaagt gcataataca ccggatggca attttccgaa

6720

cggcattccg aacccgctgc ttccggaatg cagagatgat acacgcaatg cggttattaa

6780

acatggcgct gatatgggaa tcgcctttga tggagattt gatcgctgtt ttctttttga

6840

tgaaaaaggc caatttatcg aaggatacta catcgtgggc ttactggctg aagcatttct

6900

ggagaaaaat ccgggagcga aaattatcca tgatccgaga ctttcttgga atacagtcga

6960

tgtcgttaca gctgccggcg gaacaccggt tatgtcaaaa acaggccatg cttttatcaa

7020

agaaagaatg cgcaaagaag atgcaatcta tggcggagaa atgagcgcgc atcattactt

7080

tagagattt gcttactgcg attctggcat gatcccgtgg cttttagtcg cggaactggt

7140

ttgtcttaaa gataaaacac tgggagaact ggtcagagat cgcatggcag cgtttccggc

7200

ttcaggcgaa attaatagca aactggcaca accggtggaa gcgatcaaca gagtcgaaca

7260

gcatttttca cgcgaagcac tggcggttga tagaacagat ggaattagca tgacatttgc

7320

cgattggaga tttaatcttc gcacatctaa cacagaaccg gtggtcagac ttaacgttga

7380

atcacgcggc gatgtgccgt taatggaagc aagaacacgc acactgctta cattactgaa

7440

tgaataaaaa ggaggaacta ctatgacaac agaaccgctg tttttcaaac cggtctttaa

7500

agaaagaatt tggggcggaa cagctctggc cgattttggc tatacaatcc cgtctcaacg

7560

cacaggagaa tgctgggctt ttgcagcgca tcaaaatggc cagagcgttg tgcagaacgg

7620

catgtacaaa ggatttacac tgtctgaact gtgggaacat catagacatc tttttggcca

7680

attagaagga gatcgctttc cgctgcttac aaaaatttta gatgctgatc aggatctgtc

7740

agttcaggtt catccgaatg atgaatatgc caatatccat gaaaacggcg aacttggaaa

7800

aacagaatgc tggtatatta tcgattgtca gaaagatgct gaaattatct atggccataa

7860

cgccacaaca aaagaagaat taacaacaat gatcgaaaga ggagaatggg atgaattact

7920

gagacgcgtg aaagtcaaac cgggcgattt cttttatgtc ccgtcaggaa cagttcatgc

7980

aattggcaaa ggaatccttg cgttagaaac acaacagaat agcgatacaa catacagact

8040

gtacgattac gatcgcaaag atgcagaagg caaacttaga gaactgcatc ttaaaaaatc

8100

aatcgaagtt atcgaagtgc cgagcattcc ggaacgccat acagttcatc atgaacaaat

8160

cgaagattta ctgacaacaa cactgatcga atgcgcatat tttagcgtgg gcaaatggaa

8220

cctttcagga agcgcgtctt taaaacaaca gaaaccgttt ctgcttattt ctgtgatcga

8280

aggcgaagga cgcatgattt caggcgaata tgtctatccg tttaagaaag gcgatcacat

8340

gttactgccg tatggcctgg gagaatttaa acttgaagga tatgcggaat gtatcgtttc

8400

tcatctttaa gcgaggcagg atcaggacaa tgattatatt tttgtcatga atttcacgga

8460

agaaaaacag ctggtcacgt ttgatcagag tgtgaaggac ataatgacag gagacatatt

8520

gtcaggcgac ctgacgatgg aaaagtatga agtgagaatt gtcgtaaaca cacattaggc

8580

tgatgctccg ctcgatatgg gcggattctt ttttctatag aatgaaaacg cttgctaagt

8640

cttgggggga tgaaatcatg aaaagcaaag tgaaaatgtt ctttgcggct gccatcgtgt

8700

ggagtgcatg tagttcaaca ggatatgccg ctgccattga gaaggagaag cacgtgtcag

8760

agcttcgggc agaggatctt tttgttaaaa aagtagaggg gatgaacaag gattttatca

8820

aaggggcaga tgtatccagc gttattgctt tggaaaacag cggtgtcacc ttttacaata

8880

caaacggaaa acgccaggat atctttacaa ctttaaaaca ggctggggtc aactatgttc

8940

gcgtccgcat ctggaatcac ccgtatgatt caaatggcaa cgggtatggc gggggaaaca

9000

atgatgttca aaaagccatc gaaatcggaa aaagagcgac agcgaacgga atgaaggtgc

9060

tggccgactt tcactactct gatttctggg ccgatccagc gaaacaaaag gtgcccaaag

9120

cctgggcgaa tctcagcttt gaagcaaaaa aagcaaagct ctatgagtat acgaaacaaa

9180

gcctgcaaaa gatgatcaag gaaggcgtgc ggtgtgaaat accgcacaga tgcgtaagga

9240

gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg

9300

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat

9360

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta

9420

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa

9480

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc

9540

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt

9600

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca

9660

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg

9720

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat

9780

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta

9840

cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct

9900

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac

9960

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa

10020

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa

10080

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt

10140

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca

10200

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca

10260

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc

10320

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa

10380

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc

10440

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca

10500

acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat

10560

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag

10620

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac

10680

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt

10740

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt

10800

gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc

10860

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat

10920

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca

10980

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga

11040

cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg

11100

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg

11160

ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga

11220

cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa

11270

<210> 2

<211> 12427

<212> DNA

<213> Artificial sequence

<220>

<223> expression plasmid

<400> 2

ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa

60

tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa

120

gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat

180

aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt

240

tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat

300

ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag

360

gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt

420

aaaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt

480

tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc

540

cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga

600

aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa

660

tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct

720

cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa

780

tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc

840

ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat

900

ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa

960

aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct

1020

tatcttgata ataagggtaa ctattgccga tcgtccattc cgacagcatc gccagtcact

1080

atggcgtgct gctagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg

1140

gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta

1200

agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc

1260

gagctcaggc cttaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga

1320

aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt

1380

attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt

1440

caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg

1500

aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc

1560

gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat

1620

tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt

1680

cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc

1740

tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc

1800

cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag

1860

atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt

1920

ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat

1980

ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag

2040

attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac

2100

gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg

2160

caggggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg

2220

tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt

2280

tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc

2340

ggcatactct gcgacatcgt ataacgttac tggtttcatc aaaatcgtct ccctccgttt

2400

gaatatttga ttgatcgtaa cgatgaag cactctttcc actatcccta cagtgttatg

2460

gcttgaacaa tcacgaaaca ataattggta cgtacgatct ttcagccgac tcaaacatca

2520

aatcttacaa atgtagtctt tgaaagtatt acatatgtaa gatttaaatg caaccgtttt

2580

ttcggaagga aatgatgacc tcgtttccac cggaattagc ttggtaccaa aggaggtaag

2640

gatcactaga aaatttttta aaaaatctct tgacattgga agggagatat gttattataa

2700

gaattgcgga attgtgagcg gataacaatt cccatataga ttaactaata aggaggacaa

2760

acatgggttc tatcatcaga cttcagggtg gtttaggcaa tcaactgttt caatttagct

2820

ttggttatgc tctttcaaaa atcaatggca caccgctgta ctttgatatt tcacattacg

2880

cagaaaacga tgatcatggc ggatatagac tgaataacct tcaaatcccg gaagaatatc

2940

ttcagtacta cacaccgaaa atcaacaaca tctacaaact gcttgtcaga ggcagccgct

3000

tatatccgga tatttttctg tttcttggct tttgcaacga atttcatgcg tatggctatg

3060

attttgaata catcgcgcaa aaatggaaaa gcaaaaaata catcggatac tggcagtctg

3120

aacatttctt tcataaacat attctggatc tgaaagaatt tttcatcccg aaaaacgtta

3180

gcgaacaagc taacttactg gcagcgaaaa tcttagaatc tcagtcaagc ctgtcaattc

3240

atatcagacg cggcgattac atcaaaaaca aaacagccac actgacacat ggagtttgct

3300

ctcttgaata ctacaagaaa gcactgaaca aaatcagaga tttagccatg attcgcgatg

3360

tgtttatctt ttctgatgat attttctggt gtaaagaaaa catcgaaaca cttctgagca

3420

aaaaatacaa catctactac tcagaagatt taagccaaga agaagattta tggctgatgt

3480

ctctggctaa tcatcatatt atcgccaact cttcattttc atggtggggc gcatatcttg

3540

gaagctctgc gagccagatt gttatctatc cgacaccgtg gtatgatatt acaccgaaaa

3600

acacatacat tcctatcgtg aaccattgga tcaacgtgga caaacattcc tcctgctaag

3660

gagaaaagct agcgattaac taataaggag gaactgaaat gtcaaaagtg gcgcttatta

3720

caggcgtcac aggacaagat ggcagctatt tagctgaatt tctgcttgaa aaaggatatg

3780

aagtgcatgg cattaaaaga cgcgcatcat catttaacac agaaagagtc gatcatatct

3840

atcaagatcc gcatacatgc aacccgaaat ttcatctgca ttatggcgat ctgagcgata

3900

catctaacct gacaagaatt ttacgcgaag tccagccgga tgaagtttat aacctgggcg

3960

cgatgtctca tgtcgctgtt tcatttgaaa gcccggaata tacagccgat gtcgatgcaa

4020

tgggaacact tagattactg gaagccattc gctttttagg cctggaaaag aaaacacgct

4080

tttatcaggc atctacatca gaactgtatg gacttgttca agaaatcccg cagaaagaaa

4140

caacaccgtt ttatccgaga agcccgtatg cggtcgctaa actttacgcg tactggatca

4200

cagttaatta tcgcgaatct tatggaatgt atgcttgcaa tggcatctta tttaaccatg

4260

aatcaccgag acgcggagaa acatttgtta caagaaaaat cacacgcgcc attgcaaata

4320

tcgcccaagg actggaaagc tgtctttatt taggcaacat ggattctctt agagattggg

4380

gccatgcaaa agattacgtt aaaatgcaat ggatgatgtt acaacaggaa cagccggaag

4440

attttgtgat cgccacagga gtgcaatatt cagtcagaca gtttgttgaa atggcagcgg

4500

ctcagcttgg cattaaactg cgctttgaag gcacaggagt ggaagaaaaa ggaatcgttg

4560

tgagcgttac aggccatgat gcgccgggag tgaaaccggg cgatgtgatt atcgccgtcg

4620

atccgagata ttttcgcccg gcagaagtcg aaacactttt aggagatccg acaaaagcgc

4680

atgaaaaact gggctggaaa ccggaaatta cactgagaga aatggttagc gaaatggtgg

4740

ctaacgattt agaagccgcg aaaaaacatt ctctgcttaa atcacatggc tatgatgtgg

4800

cgatcgctct ggaatcttaa aaaggaggaa ctactatgag caaacaaaga gtttttatcg

4860

ctggccatcg cggaatggtg ggctctgcca ttagacgcca attagaacag agaggagatg

4920

tggaacttgt cttaagaaca cgcgatgaac tgaatctgct tgattcaaga gctgtccatg

4980

atttctttgc cagcgaacgc atcgatcagg tttatcttgc agcggctaaa gttggcggaa

5040

ttgtggcaaa taacacatat ccggcggatt ttatctacca aaacatgatg atcgaatcta

5100

acattatcca tgccgcacat cagaatgatg ttaacaaact gctgtttctg ggatcaagct

5160

gcatctatcc gaaacttgca aaacaaccga tggcggaatc agaactttta cagggcacac

5220

tggaaccgac aaatgaaccg tatgcaattg cgaaaatcgc gggaatcaaa ctttgtgaat

5280

cttacaacag acaatacggc agagattatc gctcagtgat gccgacaaac ttatatggac

5340

cgcatgataa ttttcatccg tctaactcac atgtcattcc ggcactgctt agacgctttc

5400

atgaagctac agcccagaac gcgccggatg ttgtggtctg gggctcagga acaccgatgc

5460

gcgaatttct gcatgttgat gatatggcgg ctgccagcat ccatgtgatg gaacttgctc

5520

atgaagtctg gttagaaaat acacaaccga tgcttagcca tattaacgtc ggcacaggag

5580

ttgattgcac aatcagagaa ttagctcaga caattgccaa agttgtggga tataaaggcc

5640

gcgtcgtttt tgatgcctct aaaccggatg gcacaccgag aaaattactg gatgtgacac

5700

gcctgcatca acttggatgg tatcatgaaa ttagcttaga agcaggcctg gcgtctacat

5760

accaatggtt tttagaaaac caggatagat ttcgcggcgg atcataaagt gatagcggta

5820

ccattatagg taagagagga atgtacacat gtactattta aaaaacacaa acttttggat

5880

gttcggttta ttctttttct tttacttttt tatcatggga gcctacttcc cgtttttccc

5940

gatttggcta catgacatca accatatcag caaaagtgat acgggtatta tttttgccgc

6000

tatttctctg ttctcgctat tattccaacc gctgtttggt ctgctttctg acaaactcgg

6060

gctgcgcaaa tacctgctgt ggattattac cggcatgtta gtgatgtttg cgccgttctt

6120

tatttttatc ttcggggccac tgttacaata caacatttta gtaggatcga ttgttggtgg

6180

tatttatcta ggcttttgtt ttaacgccgg tgcgccagca gtagaggcat ttattgagaa

6240

agtcagccgt cgcagtaatt tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg

6300

ggcgctgtgt gcctcgattg tcggcatcat gttcaccatc aataatcagt ttgttttctg

6360

gctgggctct ggctgtgcac tcatcctcgc cgttttactc tttttcgcca aaacggatgc

6420

gccctcttct gccacggttg ccaatgcggt aggtgccaac cattcggcat ttagccttaa

6480

gctggcactg gaactgttca gacagccaaa actgtggttt ttgtcactgt atgttattgg

6540

cgtttcctgc acctacgatg tttttgacca acagtttgct aatttcttta cttcgttctt

6600

tgctaccggt gaacagggta cgcgggtatt tggctacgta acgacaatgg gcgaattact

6660

taacgcctcg attatgttct ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc

6720

cctgctgctg gctggcacta ttatgtctgt acgtattatt ggctcatcgt tcgccacctc

6780

agcgctggaa gtggttattc tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt

6840

gggctgcttt aaatatatta ccagccagtt tgaagtgcgt ttttcagcga cgatttatct

6900

ggtctgtttc tgcttcttta agcaactggc gatgattttt atgtctgtac tggcgggcaa

6960

tatgtatgaa agcatcggtt tccagggcgc ttatctggtg ctgggtctgg tggcgctggg

7020

cttcacctta atttccgtgt tcacgcttag cggccccggc ccgctttccc tgctgcgtcg

7080

tcaggtgaat gaagtcgctt aaggatccat gtctagagtc gacgtccccg gggcagcccg

7140

cctaatgagc gggctttttt cacgtcccag gcatcaaata aaacgaaagg ctcagtcgaa

7200

agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac

7260

tggctcacct tcgggtgggc ctttctgcgt ttatacccgg ggcagcccgc ctaatgagcg

7320

ggcttttttc acgtcacgcg tccatggaga tctttgtctg caactgaaaa gtttatacct

7380

tacctggaac aaatggttga aacatacgag gctaatatcg gcttattagg aatagtccct

7440

gtactaataa aatcaggtgg atcagttgat cagtatattt tggacgaagc tcggaaagaa

7500

tttggagatg acttgcttaa ttccacaatt aaattaaggg aaagaataaa gcgatttgat

7560

gttcaaggaa tcacggaaga agatactcat gataaagaag ctctaaaact attcaataac

7620

cttacaatgg aattgatcga aagggtggaa ggttaatggt acgaaaatta ggggatctac

7680

ctagaaagcc acaaggcgat aggtcaagct taaagaaccc ttacatggat cttacagatt

7740

ctgaaagtaa agaaacaaca gaggttaaac aaacagaacc aaaaagaaaa aaagcattgt

7800

tgaaaacaat gaaagttgat gtttcaatcc ataataagat taaatcgctg cacgaaattc

7860

tggcagcatc cgaagggaat tcatattact tagaggatac tattgagaga gctattgata

7920

agatggttga gacattacct gagagccaaa aaacttttta tgaatatgaa ttaaaaaaaa

7980

gaaccaacaa aggctgagac agactccaaa cgagtctgtt tttttaaaaa aaatattagg

8040

agcattgaat atatattaga gaattaagaa agacatggga ataaaaatat tttaaatcca

8100

gtaaaaatat gataagatta tttcagaata tgaagaactc tgtttgtttt tgatgaaaaa

8160

acaaacaaaa aaaatccacc taacggaatc tcaatttaac taacagcggc caaactgaga

8220

agttaaattt gagaagggga aaaggcggat ttatacttgt atttaactat ctccatttta

8280

acattttatt aaaccccata caagtgaaaa tcctctttta cactgttcct ttaggtgatc

8340

gcggagggac attatgagtg aagtaaacct aaaaggaaat acagatgaat tagtgtatta

8400

tcgacagcaa accactggaa ataaaatcgc caggaagaga atcaaaaaag ggaaagaaga

8460

agtttattat gttgctgaaa cggaagagaa gatatggaca gaagagcaaa taaaaaactt

8520

ttctttagac aaatttggta cgcatatacc ttacatagaa ggtcattata caatcttaaa

8580

taattacttc tttgattttt ggggctattt tttaggtgct gaaggaattg cgctctatgc

8640

tcacctaact cgttatgcat acggcagcaa agacttttgc tttcctagtc tacaaacaat

8700

cgctaaaaaa atggacaaga ctcctgttac agttagaggc tacttgaaac tgcttgaaag

8760

gtacggtttt atttggaagg taaacgtccg taataaaacc aaggataaca cagaggaatc

8820

cccgattttt aagattagac gtaaggttcc tttgctttca gaagaacttt taaatggaaa

8880

ccctaatatt gaaattccag atgacgagga agcacatgta aagaaggctt taaaaaagga

8940

aaaagaggt cttccaaagg ttttgaaaaa agagcacgat gaatttgtta aaaaaatgat

9000

ggatgagtca gaaacaatta atattccaga ggccttacaa tatgacacaa tgtatgaaga

9060

tatactcagt aaaggagaaa ttcgaaaaga aatcaaaaaa caaataccta atcctacaac

9120

atcttttgag agtatatcaa tgacaactga agaggaaaaa gtcgacagta ctttaaaaag

9180

cgaaatgcaa aatcgtgtct ctaagccttc ttttgatacc tggtttaaaa acactaagat

9240

caaaattgaa aataaaaatt gtttattact tgtaccgagt gaatttgcat ttgaatggat

9300

taagaaaaga tatttagaaa caattaaaac agtccttgaa gaagctggat atgttttcga

9360

aaaaatcgaa ctaagaaaag tgcaataaac tgctgaagta tttcagcagt ttttttttatt

9420

tagaaatagt gaaaaaaata taatcaggga ggtatcaata tttaatgagt actgatttaa

9480

atttatttag actggaatta ataattaaca cgtagactaa ttaaaattta atgagggata

9540

aagaggatac aaaaatatta atttcaatcc ctattaaatt ttaacaaggg ggggattaaa

9600

atttaattag aggtttatcc acaagaaaag accctaataa aatttttact agggttataa

9660

cactgattaa tttcttaatg ggggagggat taaaatttaa tgacaaagaa aacaatcttt

9720

taagaaaagc ttttaaaaga taataataaa aagagctttg cgattaagca aaactcttta

9780

ctttttcatt gacattatca aattcatcga tttcaaattg ttgttgtatc ataaagttaa

9840

ttctgttttg cacaaccttt tcaggaatat aaaacacatc tgaggcttgt tttataaact

9900

cagggtcgct aaagtcaatg taacgtagca tatgatatgg tatagcttcc acccaagtta

9960

gcctttctgc ttcttctgaa tgtttttcat atacttccat gggtatctct aaatgatttt

10020

cctcatgtag caaggtatga gcaaaaagtt tatggaattg atagttcctc tctttttctt

10080

caactttttt atctaaaaca aacactttaa catctgagtc aatgtaagca taagatgttt

10140

ttccagtcat aatttcaatc ccaaatcttt tagacagaaa ttctggacgt aaatcttttg

10200

gtgaaagaat ttttttatgt agcaatatat ccgatacagc accttctaaa agcgttggtg

10260

aatagggcat tttacctatc tcctctcatt ttgtggaata aaaatagtca tattcgtcca

10320

tctacctatc ctattatcga acagttgaac tttttaatca aggatcagtc ctttttttca

10380

ttattcttaa actgtgctct taactttaac aactcgattt gtttttccag atctcgaggg

10440

taactagcct cgccgatccc gcaagaggcc cggcagtcag gtggcacttt tcggggaaat

10500

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg

10560

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa

10620

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac

10680

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac

10740

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt

10800

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc

10860

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca

10920

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc

10980

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag

11040

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa

11100

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg

11160

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa

11220

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg

11280

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt

11340

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt

11400

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag

11460

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat

11520

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct

11580

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct

11640

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca

11700

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc

11760

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc

11820

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct

11880

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag

11940

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc

12000

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg

12060

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag

12120

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt

12180

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac

12240

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg

12300

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc

12360

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata

12420

cgcatgc

12427

<210> 3

<211> 12330

<212> DNA

<213> Artificial sequence

<220>

<223> integration vector

<400> 3

ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa

60

ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg

120

caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt

180

gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata

240

tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg

300

ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac

360

cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac

420

aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca

480

cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg

540

actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct

600

caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat

660

gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt

720

cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct

780

ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct

840

tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa

900

acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt

960

cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc

1020

cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca

1080

tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc

1140

gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat

1200

tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg

1260

ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga

1320

attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac

1380

atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg

1440

tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg

1500

ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc

1560

tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg

1620

taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca

1680

ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga

1740

ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa

1800

cgttccagta acggggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt

1860

ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc

1920

aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt

1980

ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac

2040

gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac

2100

ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc

2160

agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc

2220

cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg

2280

tactgagagt gcaccatatg acccgacatc cggcgttctc atggcggtgc ttgccgccag

2340

cggtattccg tatgtcaagt ggctgcggtt tatggtgccg cttgctctga tttggttctt

2400

gatcgggctt gtctttatcg tgatcggagt catgatcaat tgggggccgt tttaacgatt

2460

gctgcccgcc ggcttgtacg gcgggctttt gagttattca ttgcagaagc gcaggctgtt

2520

attgtaacat gtaagccata agccattcgt aaaagtgcgg gaggaaggtc atgaataatc

2580

tgcgtaatag actttcaggc gtgaatggga aaaataagag agtaaaagaa aaagaacaaa

2640

aaatctggtc ggagattggg atgatagcgg gagcatttgc gctgcttgat gtgatcatcc

2700

gcggcattat gtttgaattt ccgtttaaag aatgggctgc aagccttgtg tttttgttca

2760

tcattatctt atattactgc atcagggctg cggcatccgg aatgctcatg ccgagaatag

2820

acaccaaaga agaactgcaa aaacgggtga agcagcagcg aatagaatca attgcggtcg

2880

cctttgcggt agtggtgctt acgatgtacg acagggggat tccccataca ttcttcgctt

2940

ggctgaaaat gattcttctt tttatcgtct gcggcggcgt tctgtttctg cttcggtatg

3000

tgattgtgaa gctggcttac agaagagcgg taaaagaaga aataaaaaag aaatcatctt

3060

ttttgtttgg aaagcgaggg aagcgttcac agtttcgggc agcttttttt ataggaacat

3120

tgatttgtat tcactctgcc aagttgtttt gatagagtga ttgtgataat tttaaatgta

3180

agcgttaaca aaattctcca gtcttcacat cggtttgaaa ggaggaagcg gaagaatgaa

3240

gtaagaggga tttttgactc cgaagtaagt cttcaaaaaa tcaaataagg agtgtcaaga

3300

ccagtgaatt cgagctcggt acctaccgtt cgtataatgt atgctatacg aagttatgat

3360

aaaaaattta gaagccaatg aaatctataa ataaactaaa ttaagtttat ttaattaaca

3420

actatggata taaaataggt actaatcaaa atagtgagga ggatatattt gaatacatac

3480

gaacaaatta ataaagtgaa aaaaatactt cggaaacatt taaaaaataa ccttattggt

3540

acttacatgt ttggatcagg agttgagagt ggactaaaac caaatagtga tcttgacttt

3600

ttagtcgtcg tatctgaacc attgacagat caaagtaaag aaatacttat acaaaaaatt

3660

agacctattt caaaaaaaat aggagataaa agcaacttac gatatattga attaacaatt

3720

attattcagc aagaaatggt accgtggaat catcctccca aacaagaatt tatttatgga

3780

gaatggttac aagagcttta tgaacaagga tacattcctc agaaggaatt aaattcagat

3840

ttaaccataa tgctttacca agcaaaacga aaaaataaaa gaatatacgg aaattatgac

3900

ttagaggaat tactacctga tattccatt tctgatgtga gaagagccat tatggattcg

3960

tcagaggaat taatagataa ttatcaggat gatgaaacca actctatatt aactttatgc

4020

cgtatgattt taactatgga cacgggtaaa atcataccaa aagatattgc gggaaatgca

4080

gtggctgaat cttctccatt agaacatagg gagagaattt tgttagcagt tcgtagttat

4140

cttggagaga atattgaatg gactaatgaa aatgtaaatt taactataaa ctatttaaat

4200

aacagattaa aaaaattata aaaaaattga aaaaatggtg gaaacacttt tttcaatttt

4260

tttgttttat tatttaatat ttgggaaata ttcattctaa tataacttcg tataatgtat

4320

gctatacgaa cggtaggatc ctctagagtc gacctgcagg cattttacat ttttagaaat

4380

gggcgtgaaa aaaagcgcgc gattatgtaa aatataaaga ttaactaata aggaggacaa

4440

acatgggttc tatcatcaga cttcagggtg gtttaggcaa tcaactgttt caatttagct

4500

ttggttatgc tctttcaaaa atcaatggca caccgctgta ctttgatatt tcacattacg

4560

cagaaaacga tgatcatggc ggatatagac tgaataacct tcaaatcccg gaagaatatc

4620

ttcagtacta cacaccgaaa atcaacaaca tctacaaact gcttgtcaga ggcagccgct

4680

tatatccgga tatttttctg tttcttggct tttgcaacga atttcatgcg tatggctatg

4740

attttgaata catcgcgcaa aaatggaaaa gcaaaaaata catcggatac tggcagtctg

4800

aacatttctt tcataaacat attctggatc tgaaagaatt tttcatcccg aaaaacgtta

4860

gcgaacaagc taacttactg gcagcgaaaa tcttagaatc tcagtcaagc ctgtcaattc

4920

atatcagacg cggcgattac atcaaaaaca aaacagccac actgacacat ggagtttgct

4980

ctcttgaata ctacaagaaa gcactgaaca aaatcagaga tttagccatg attcgcgatg

5040

tgtttatctt ttctgatgat attttctggt gtaaagaaaa catcgaaaca cttctgagca

5100

aaaaatacaa catctactac tcagaagatt taagccaaga agaagattta tggctgatgt

5160

ctctggctaa tcatcatatt atcgccaact cttcattttc atggtggggc gcatatcttg

5220

gaagctctgc gagccagatt gttatctatc cgacaccgtg gtatgatatt acaccgaaaa

5280

acacatacat tcctatcgtg aaccattgga tcaacgtgga caaacattcc tcctgctaaa

5340

gtgatagcgg taccattata ggtaagagag gaatgtacac atgtactatt taaaaaacac

5400

aaacttttgg atgttcggtt tattcttttt cttttacttt tttatcatgg gagcctactt

5460

cccgtttttc ccgatttggc tacatgacat caaccatatc agcaaaagtg atacgggtat

5520

tatttttgcc gctatttctc tgttctcgct attattccaa ccgctgtttg gtctgctttc

5580

tgacaaactc gggctgcgca aatacctgct gtggattatt accggcatgt tagtgatgtt

5640

tgcgccgttc tttattttta tcttcgggcc actgttacaa tacaacattt tagtaggatc

5700

gattgttggt ggtatttatc taggcttttg ttttaacgcc ggtgcgccag cagtagaggc

5760

atttattgag aaagtcagcc gtcgcagtaa tttcgaattt ggtcgcgcgc ggatgtttgg

5820

ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc atgttcacca tcaataatca

5880

gtttgttttc tggctgggct ctggctgtgc actcatcctc gccgttttac tctttttcgc

5940

caaaacggat gcgccctctt ctgccacggt tgccaatgcg gtaggtgcca accattcggc

6000

atttagcctt aagctggcac tggaactgtt cagacagcca aaactgtggt ttttgtcact

6060

gtatgttatt ggcgtttcct gcacctacga tgtttttgac caacagtttg ctaatttctt

6120

tacttcgttc tttgctaccg gtgaacaggg tacgcgggta tttggctacg taacgacaat

6180

gggcgaatta cttaacgcct cgattatgtt ctttgcgcca ctgatcatta atcgcatcgg

6240

tgggaaaaac gccctgctgc tggctggcac tattatgtct gtacgtatta ttggctcatc

6300

gttcgccacc tcagcgctgg aagtggttat tctgaaaacg ctgcatatgt ttgaagtacc

6360

gttcctgctg gtgggctgct ttaaatatat taccagccag tttgaagtgc gtttttcagc

6420

gacgatttat ctggtctgtt tctgcttctt taagcaactg gcgatgattt ttatgtctgt

6480

actggcgggc aatatgtatg aaagcatcgg tttccagggc gcttatctgg tgctgggtct

6540

ggtggcgctg ggcttcacct taatttccgt gttcacgctt agcggccccg gcccgctttc

6600

cctgctgcgt cgtcaggtga atgaagtcgc ttaaagtcaa tgtatgaatg gatacgggat

6660

atgaatcaat aagtacgtga aagagaaaag caacccagat atgataggga acttttctct

6720

ttcttgtttt acattgaatc tttacaatcc tattgatata atctaagcta gtgtattttg

6780

cgtttaatag tggagaaaag ctagcgatta actaataagg aggaactgaa atgtcaaaag

6840

tggcgcttat tacaggcgtc acaggacaag atggcagcta tttagctgaa tttctgcttg

6900

aaaaaggata tgaagtgcat ggcattaaaa gacgcgcatc atcatttaac acagaaagag

6960

tcgatcatat ctatcaagat ccgcatacat gcaacccgaa atttcatctg cattatggcg

7020

atctgagcga tacatctaac ctgacaagaa ttttacgcga agtccagccg gatgaagttt

7080

ataacctggg cgcgatgtct catgtcgctg tttcatttga aagcccggaa tatacagccg

7140

atgtcgatgc aatgggaaca cttagattac tggaagccat tcgcttttta ggcctggaaa

7200

agaaaacacg cttttatcag gcatctacat cagaactgta tggacttgtt caagaaatcc

7260

cgcagaaaga aacaacaccg ttttatccga gaagcccgta tgcggtcgct aaactttacg

7320

cgtactggat cacagttaat tatcgcgaat cttatggaat gtatgcttgc aatggcatct

7380

tatttaacca tgaatcaccg agacgcggag aaacatttgt tacaagaaaa atcacacgcg

7440

ccattgcaaa tatcgcccaa ggactggaaa gctgtcttta tttaggcaac atggattctc

7500

ttagagattg gggccatgca aaagattacg ttaaaatgca atggatgatg ttacaacagg

7560

aacagccgga agattttgtg atcgccacag gagtgcaata ttcagtcaga cagtttgttg

7620

aaatggcagc ggctcagctt ggcattaaac tgcgctttga aggcacagga gtggaagaaa

7680

aaggaatcgt tgtgagcgtt acaggccatg atgcgccggg agtgaaaccg ggcgatgtga

7740

ttatcgccgt cgatccgaga tattttcgcc cggcagaagt cgaaacactt ttaggagatc

7800

cgacaaaagc gcatgaaaaa ctgggctgga aaccggaaat tacactgaga gaaatggtta

7860

gcgaaatggt ggctaacgat ttagaagccg cgaaaaaaca ttctctgctt aaatcacatg

7920

gctatgatgt ggcgatcgct ctggaatctt aaaaaggagg aactactatg agcaaacaaa

7980

gagtttttat cgctggccat cgcggaatgg tgggctctgc cattagacgc caattagaac

8040

agagaggaga tgtggaactt gtcttaagaa cacgcgatga actgaatctg cttgattcaa

8100

gagctgtcca tgatttcttt gccagcgaac gcatcgatca ggtttatctt gcagcggcta

8160

aagttggcgg aattgtggca aataacacat atccggcgga ttttatctac caaaacatga

8220

tgatcgaatc taacattatc catgccgcac atcagaatga tgttaacaaa ctgctgtttc

8280

tgggatcaag ctgcatctat ccgaaacttg caaaacaacc gatggcggaa tcagaacttt

8340

tacagggcac actggaaccg acaaatgaac cgtatgcaat tgcgaaaatc gcgggaatca

8400

aactttgtga atcttacaac agacaatacg gcagagatta tcgctcagtg atgccgacaa

8460

acttatatgg accgcatgat aattttcatc cgtctaactc acatgtcatt ccggcactgc

8520

ttagacgctt tcatgaagct acagcccaga acgcgccgga tgttgtggtc tggggctcag

8580

gaacaccgat gcgcgaattt ctgcatgttg atgatatggc ggctgccagc atccatgtga

8640

tggaacttgc tcatgaagtc tggttagaaa atacacaacc gatgcttagc catattaacg

8700

tcggcacagg agttgattgc acaatcagag aattagctca gacaattgcc aaagttgtgg

8760

gatataaagg ccgcgtcgtt tttgatgcct ctaaaccgga tggcacaccg agaaaattac

8820

tggatgtgac acgcctgcat caacttggat ggtatcatga aattagctta gaagcaggcc

8880

tggcgtctac ataccaatgg tttttagaaa accaggatag atttcgcggc ggatcataag

8940

gatccatgtc tagagtcgac gtccccgggg cagcccgcct aatgagcggg cttttttcac

9000

gtcccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct

9060

gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt

9120

tctgcgttta tagaattcat attacttaga ggatactatt gagagagcta ttgataagat

9180

ggttgagaca ttacctgaga gccaaaaaac tttttatgaa tatgaattaa aaaaaagaac

9240

caacaaaggc tgagacagac tccgggcaag gctagacggg acttaccgaa agaaaccatc

9300

aatgatggtt tcttttttgt tcataaatca gacaaaactt ttctcttgca aaagtttgtg

9360

aagtgttgca caatataaat gtgaaatact tcacaaacaa aaagacatca aagagaaaca

9420

taccctggaa ggatgattaa tgatgaacaa acatgtaaat aaagtagctt taatcggagc

9480

gggttttgtt ggaagcagtt atgcatttgc gttaattaac caaggaatca cagatgagct

9540

tgtggtcatt gatgtaaata aagaaaaagc aatgggcgat gtgatggatt taaaccacgg

9600

aaaggcgttt gcgccacaac cggtcaaaac atcttacgga acatatgaag actgcaagga

9660

tgctgatatt gtctgcattt gcgccggagc aaaccaaaaa cctggtgaga cacgccttga

9720

attagtagaa aagaacttga agattttcaa aggcatcgtt agtgaagtca tggcgagcgg

9780

atttgacggc attttcttag tcgcgacaaa tccggttgat atcctgactt acgcaacatg

9840

gaaattcagc ggcctgccaa aagagcgggt gattggaagc ggcacaacac ttgattctgc

9900

gagattccgt ttcatgctga gcgaatactt tggcgcagcg cctcaaaacg tacacgcgca

9960

tattatcgga gagcacggcg acacagagct tcctgtttgg agccacgcga atgtcggcgg

10020

tgtgccggtc agtgaactcg ttgagaaaaa cgatgcgtac aaacaagagg agctggacca

10080

aattgtagat gatgtgaaaa acgcagctta ccatatcatt gagaaaaaag gcgcgactta

10140

ttatggggtt gcgatgagtc ttgctcgcat tacaaaagcc attcttcata atgaaaacag

10200

catattaact gtcagcacat atttggacgg gcaatacggt gcagatgacg tgtacatcgg

10260

tgtcatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc

10320

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta

10380

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag

10440

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg

10500

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg

10560

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg

10620

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga

10680

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc

10740

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt

10800

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact

10860

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg

10920

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt

10980

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt

11040

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct

11100

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg

11160

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt

11220

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt

11280

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc

11340

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg

11400

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc

11460

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg

11520

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca

11580

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga

11640

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct

11700

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg

11760

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca

11820

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca

11880

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct

11940

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact

12000

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa

12060

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc

12120

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga

12180

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga

12240

aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg

12300

cgtatcacga ggccctttcg tcttcaagaa

12330

<210> 4

<211> 4254

<212> DNA

<213> Artificial sequence

<220>

<223> integration cassette

<400> 4

gcaggctgtt attgtaacat gtaagccata agccattcgt aaaagtgcgg gaggaaggtc

60

atgaataatc tgcgtaatag actttcaggc gtgaatggga aaaataagag agtaaaagaa

120

aaagaacaaa aaatctggtc ggagattggg atgatagcgg gagcatttgc gctgcttgat

180

gtgatcatcc gcggcattat gtttgaattt ccgtttaaag aatgggctgc aagccttgtg

240

tttttgttca tcattatctt atattactgc atcagggctg cggcatccgg aatgctcatg

300

ccgagaatag acaccaaaga agaactgcaa aaacgggtga agcagcagcg aatagaatca

360

attgcggtcg cctttgcggt agtggtgctt acgatgtacg acagggggat tccccataca

420

ttcttcgctt ggctgaaaat gattcttctt tttatcgtct gcggcggcgt tctgtttctg

480

cttcggtatg tgattgtgaa gctggcttac agaagagcgg taaaagaaga aataaaaaag

540

aaatcatctt ttttgtttgg aaagcgaggg aagcgttcac agtttcgggc agcttttttt

600

ataggaacat tgatttgtat tcactctgcc aagttgtttt gatagagtga ttgtgataat

660

tttaaatgta agcgttaaca aaattctcca gtcttcacat cggtttgaaa ggaggaagcg

720

gaagaatgaa gtaagaggga tttttgactc cgaagtaagt cttcaaaaaa tcaaataagg

780

agtgtcaaga atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt

840

tttattgctg tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa

900

caaatcgaat gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggcc

960

agtgaattcg agctcggtac ctaccgttcg tataatgtat gctatacgaa gttatgataa

1020

aaaatttaga agccaatgaa atctataaat aaactaaatt aagtttattt aattaacaac

1080

tatggatata aaataggtac taatcaaaat agtgaggagg atatatttga atacatacga

1140

acaaattaat aaagtgaaaa aaatacttcg gaaacattta aaaaataacc ttattggtac

1200

ttacatgttt ggatcaggag ttgagagtgg actaaaacca aatagtgatc ttgacttttt

1260

agtcgtcgta tctgaaccat tgacagatca aagtaaagaa atacttatac aaaaaattag

1320

acctatttca aaaaaaatag gagataaaag caacttacga tatattgaat taacaattat

1380

tattcagcaa gaaatggtac cgtggaatca tcctcccaaa caagaattta tttatggaga

1440

atggttacaa gagctttatg aacaaggata cattcctcag aaggaattaa attcagattt

1500

aaccataatg ctttaccaag caaaacgaaa aaataaaaga atatacggaa attatgactt

1560

agaggaatta ctacctgata ttccatttc tgatgtgaga agagccatta tggattcgtc

1620

agaggaatta atagataatt atcaggatga tgaaaccaac tctatattaa ctttatgccg

1680

tatgatttta actatggaca cgggtaaaat cataccaaaa gatattgcgg gaaatgcagt

1740

ggctgaatct tctccattag aacataggga gagaattttg ttagcagttc gtagttatct

1800

tggagagaat attgaatgga ctaatgaaaa tgtaaattta actataaact atttaaataa

1860

cagattaaaa aaattataaa aaaattgaaa aaatggtgga aacacttttt tcaatttttt

1920

tgttttatta tttaatattt gggaaatatt cattctaata taacttcgta taatgtatgc

1980

tatacgaacg gtaggatcct ctagagtcga cctgcaggca ttttacattt ttagaaatgg

2040

gcgtgaaaaa aagcgcgcga ttatgtaaaa tataaagtga tagcggtacc attataggta

2100

agagaggaat gtacacatgt actatttaaa aaacacaaac ttttggatgt tcggtttatt

2160

ctttttcttt tactttttta tcatgggagc ctacttcccg tttttcccga tttggctaca

2220

tgacatcaac catatcagca aaagtgatac gggtattatt tttgccgcta tttctctgtt

2280

ctcgctatta ttccaaccgc tgtttggtct gctttctgac aaactcgggc tgcgcaaata

2340

cctgctgtgg attattaccg gcatgttagt gatgtttgcg ccgttcttta tttttatctt

2400

cgggccactg ttacaataca acattttagt aggatcgatt gttggtggta tttatctagg

2460

cttttgtttt aacgccggtg cgccagcagt agaggcattt attgagaaag tcagccgtcg

2520

cagtaatttc gaatttggtc gcgcgcggat gtttggctgt gttggctggg cgctgtgtgc

2580

ctcgattgtc ggcatcatgt tcaccatcaa taatcagttt gttttctggc tgggctctgg

2640

ctgtgcactc atcctcgccg ttttactctt tttcgccaaa acggatgcgc cctcttctgc

2700

cacggttgcc aatgcggtag gtgccaacca ttcggcattt agccttaagc tggcactgga

2760

actgttcaga cagccaaaac tgtggttttt gtcactgtat gttattggcg tttcctgcac

2820

ctacgatgtt tttgaccaac agtttgctaa tttctttact tcgttctttg ctaccggtga

2880

acagggtacg cgggtatttg gctacgtaac gacaatgggc gaattactta acgcctcgat

2940

tatgttcttt gcgccactga tcattaatcg catcggtggg aaaaacgccc tgctgctggc

3000

tggcactatt atgtctgtac gtattattgg ctcatcgttc gccacctcag cgctggaagt

3060

ggttattctg aaaacgctgc atatgtttga agtaccgttc ctgctggtgg gctgctttaa

3120

atatattacc agccagtttg aagtgcgttt ttcagcgacg atttatctgg tctgtttctg

3180

cttctttaag caactggcga tgatttttat gtctgtactg gcgggcaata tgtatgaaag

3240

catcggtttc cagggcgctt atctggtgct gggtctggtg gcgctgggct tcaccttaat

3300

ttccgtgttc acgcttagcg gccccggccc gctttccctg ctgcgtcgtc aggtgaatga

3360

agtcgcttaa gcaatcaatg tcggatgcca gcctggcttt gattacgtgc taaatggttt

3420

atataatgac tcgggcttaa gcggttctct tccccattga gggcaaggct agacgggact

3480

taccgaaaga aaccatcaat gatggtttct tttttgttca taaatcagac aaaacttttc

3540

tcttgcaaaa gtttgtgaag tgttgcacaa tataaatgtg aaatacttca caaacaaaaa

3600

gacatcaaag agaaacatac cctggaagga tgattaatga tgaacaaaca tgtaaataaa

3660

gtagctttaa tcggagcggg ttttgttgga agcagttatg catttgcgtt aattaaccaa

3720

ggaatcacag atgagcttgt ggtcattgat gtaaataaag aaaaagcaat gggcgatgtg

3780

atggatttaa accacggaaa ggcgtttgcg ccacaaccgg tcaaaacatc ttacggaaca

3840

tatgaagact gcaaggatgc tgatattgtc tgcatttgcg ccggagcaaa ccaaaaacct

3900

ggtgagacac gccttgaatt agtagaaaag aacttgaaga ttttcaaagg catcgttagt

3960

gaagtcatgg cgagcggatt tgacggcatt ttcttagtcg cgacaaatcc ggttgatatc

4020

ctgacttacg caacatggaa attcagcggc ctgccaaaag agcgggtgat tggaagcggc

4080

acaacacttg attctgcgag attccgtttc atgctgagcg aatactttgg cgcagcgcct

4140

caaaacgtac acgcgcatat tatcggagag cacggcgaca cagagcttcc tgtttggagc

4200

cacgcgaatg tcggcggtgt gccggtcagt gaactcgttg agaaaaacga tgcg

4254

<210> 5

<211> 10340

<212> DNA

<213> Artificial sequence

<220>

<223> expression plasmid

<400> 5

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca

60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc

180

accatatgcc ttcaatcctt ttaataacaa ttatagcatc taatcttcaa caaactggcc

240

cgtttgttga actactcttt aataaaataa tttttccgtt cccaattcca cattgcaata

300

atagaaaatc catcttcatc ggctttttcg tcatcatctg tatgaatcaa atcgccttct

360

tctgtgtcat caaggtttaa ttttttatgt atttctttta acaaaccacc ataggagatt

420

aaccttttac ggtgtaaacc ttcctccaaa tcagacaaac gtttcaaatt cttttcttca

480

tcatcggtca taaaatccgt atcctttaca ggatattttg cagtttcgtc aattgccgat

540

tgtatatccg atttatattt atttttcggt cgaatcattt gaacttttac atttggatca

600

tagtctaatt tcattgcctt tttccaaaat tgaatccatt gtttttgatt cacgtagttt

660

tctgtattct taaaataagt tggttccaca cataccaata catgcatgtg ctgattataa

720

gaattatctt tattatttat tgtcacttcc gttgcacgca taaaaccaac aagattttta

780

ttaatttttt tatattgcat cattcggcga aatccttgag ccatatctga caaactctta

840

tttaattctt cgccatcata aacattttta actgttaatg tgagaaacaa ccaacgaact

900

gttggctttt gtttaataac ttcagcaaca accttttgtg actgaatgcc atgtttcatt

960

gctctcctcc agttgcacat tggacaaagc ctggatttac aaaaccacac tcgatacaac

1020

tttctttcgc ctgtttcacg attttgttta tactctaata tttcagcaca atctttttact

1080

ctttcagcct ttttaaattc aagaatatgc agaagttcaa agtaatcaac attagcgatt

1140

ttcttttctc tccatggtct cacttttcca ctttttgtct tgtccactaa aacccttgat

1200

ttttcatctg aataaatgct actattagga cacataatat taaaagaaac ccccatctat

1260

ttagttattt gtttagtcac ttataacttt aacagatggg gtttttctgt gcaaccaatt

1320

ttaagggttt tcaatacttt aaaacacata cataccaaca cttcaacgca cctttcagca

1380

actaaaataa aaatgacgtt atttctatat gtatcaagat aagaaagaac aagttcaaaa

1440

ccatcaaaaa aagacacctt ttcaggtgct ttttttattt tataaactca ttccctgatc

1500

tcgacttcgt tcttttttta cctctcggtt atgagttagt tcaaattcgt tctttttagg

1560

ttctaaatcg tgtttttctt ggaattgtgc tgttttatcc tttaccttgt ctacaaaccc

1620

cttaaaaacg tttttaaagg cttttaagcc gtctgtacgt tccttaagat tttacatttt

1680

tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat ataaagatta actaataagg

1740

aggacaaaca tgcaaaaact gctttctctg ccgtcaaatc ttgtccagtc ttttcatgaa

1800

cttgaaagag ttaaccgcac agattggttt tgcacatcag atccggttgg caaaaaactg

1860

ggctcaggcg gaggcacaag ctggttactg gaagaatgtt acaacgaata cagcgatgga

1920

gcgacatttg gcgaatggct ggaaaaagaa aaacgcattc tgttacatgc tggaggccaa

1980

agcagacgcc ttccgggata tgccccgtct ggcaaaatct taacaccggt cccggttttt

2040

agatgggaaa gaggacaaca tttaggccag aacctgctta gcttacaact gccgctttat

2100

gaaaaaatta tgtctctggc tccggataaa ctgcatacac tgattgcctc tggcgatgtg

2160

tatatccgca gcgaaaaacc gcttcagtct atcccggaag ctgatgttgt gtgctatgga

2220

ttatgggtcg atccgtctct ggcaacacat catggcgttt ttgcgtcaga tagaaaacat

2280

ccggaacaac tggattttat gctgcagaaa ccgtcacttg cagaattaga aagcctgtct

2340

aaaacacatt tatttctgat ggatattggc atctggttac tgtcagatag agcggtcgaa

2400

attctgatga aacgcagcca taaagaatca agcgaagaac ttaaatacta cgatctgtac

2460

tcagattttg gacttgcttt aggcacacat ccgcgcattg aagatgaaga agtgaataca

2520

cttagcgtcg caatcctgcc tcttccggga ggcgaatttt accattacgg aacatcaaaa

2580

gaactgattt cttcaacact tagcgttcaa aacaaagtgt atgatcagag acgcatcatg

2640

catagaaaag tgaaaccgaa tccggcaatg tttgtccaaa acgcggtcgt tcgcattccg

2700

ctttgcgctg aaaatgccga tttatggatt gaaaacagcc atatcggacc gaaatggaaa

2760

atcgcatcaa gacatattat cacaggcgtg ccggaaaatg attggtcact tgcagtcccg

2820

gcgggagtgt gtgtcgatgt ggtcccgatg ggagataaag gctttgttgc gagaccgtat

2880

ggattagatg atgtgtttaa aggcgatctg cgcgatagca aaacaacact tacaggcatt

2940

ccgtttggcg aatggatgtc taaaagagga ttatcataca cagatttaaa aggccgcaca

3000

gatgatctgc aggctgtttc agtgtttccg atggtgaaca gcgtcgaaga attaggcctg

3060

gtccttagat ggatgctttc tgaaccggaa ttagaagaag gaaaaaatat ttggctgcgc

3120

tctgaacatt tttcagcaga tgaaatctca gctggcgcca accttaaaag actgtacgcg

3180

caaagagaag aatttcgcaa aggaaattgg aaagcacttg cggttaacca tgaaaaatca

3240

gtgttttatc agctggatct ggcagatgca gcggaagatt ttgttcgcct tggcttagat

3300

atgccggaac ttttaccgga agatgctctg caaatgtcac gcattcataa tcgcatgctt

3360

agagcccgca tcctgaaact tgatggaaaa gattatagac cggaagaaca ggctgccttt

3420

gatctgctta gagatggatt actggatggc attagcaacc gcaaatctac accgaaactg

3480

gatgtctatt ctgatcaaat tgtttggggc agatcaccgg tgcgcatcga tatggcaggc

3540

ggctggacag atacaccgcc gtatagcctt tattctggag gcaatgttgt gaaccttgcg

3600

attgaattaa atggccaacc gccgcttcag gtctacgtta aaccgtgcaa agattttcat

3660

atcgttctga gatcaatcga tatgggagct atggaaatcg tgagcacatt tgatgaactg

3720

caagattaca agaaaattgg ctcaccgttt agcatcccga aagcagcgtt aagcctggcc

3780

ggatttgctc cggccttttc tgctgtcagc tatgcatcac tggaagaaca gcttaaagat

3840

tttggcgcag gaattgaagt tacactttta gctgccatcc cggcgggctc tggattaggc

3900

acaagctcta ttctggcttc aacagtgctt ggagccatca atgatttttg cggcctggca

3960

tgggataaaa acgaaatttg tcaaagaaca cttgtcttag aacagctgct tacaacaggc

4020

ggcggctggc aagatcagta tggcggagtc ctgcaaggag ttaaactgct gcagacagaa

4080

gctggctttg cccagtcacc gttagttcgc tggctgccgg atcatctttt tacacatccg

4140

gaatacaaag attgtcatct tttatactac acaggaatta caagaacagc aaaaggcatt

4200

ttagcggaaa tcgtgtcaag catgtttctg aatagctcac tgcatcttaa cctgcttagc

4260

gaaatgaaag cacatgcgct tgatatgaat gaagctattc aaagaggctc ttttgtcgaa

4320

tttggacgct tagttggcaa aacatgggaa cagaataaag ccctggatag cggaacaaac

4380

ccgccggcag tggaagcgat catcgatctg atcaaagatt acacactggg atacaaactg

4440

cctggagcag gcggcggcgg ctatctgtat atggttgcga aagatccgca agcagctgtg

4500

agaattagaa aaatcctgac agaaaatgct ccgaacccga gagcccgctt tgtggaaatg

4560

acactttcag ataaaggatt tcaggtctca agaagctaag ctcttaagga ggattttaga

4620

atgggttcta tcatcagact tcagggtggt ttaggcaatc aactgtttca atttagcttt

4680

ggttatgctc tttcaaaaat caatggcaca ccgctgtact ttgatatttc acattacgca

4740

gaaaacgatg atcatggcgg atatagactg aataaccttc aaatcccgga agaatatctt

4800

cagtactaca caccgaaaat caacaacatc tacaaactgc ttgtcagagg cagccgctta

4860

tatccggata tttttctgtt tcttggcttt tgcaacgaat ttcatgcgta tggctatgat

4920

tttgaataca tcgcgcaaaa atggaaaagc aaaaaataca tcggatactg gcagtctgaa

4980

catttctttc ataaacatat tctggatctg aaagaatttt tcatcccgaa aaacgttagc

5040

gaacaagcta acttactggc agcgaaaatc ttagaatctc agtcaagcct gtcaattcat

5100

atcagacgcg gcgattacat caaaaacaaa acagccacac tgacacatgg agtttgctct

5160

cttgaatact acaagaaagc actgaacaaa atcagagatt tagccatgat tcgcgatgtg

5220

tttatctttt ctgatgatat tttctggtgt aaagaaaaca tcgaaacact tctgagcaaa

5280

aaatacaaca tctactactc agaagatta agccaagaag aagatttatg gctgatgtct

5340

ctggctaatc atcatattat cgccaactct tcattttcat ggtggggcgc atatcttgga

5400

agctctgcga gccagattgt tatctatccg acaccgtggt atgatattac accgaaaaac

5460

acatacattc ctatcgtgaa ccattggatc aacgtggaca aacattcctc ctgctaaaaa

5520

ggaggaacta ctatgggcaa tacatcaatt caaacacaga gctatagagc agttgataaa

5580

gatgcgggac aaagccgctc ttatattatc ccgtttgcac tgctttgctc tctgtttttc

5640

ctgtgggctg tggccaataa cctgaatgat attttactgc cgcagtttca acaggcgttt

5700

acacttacaa actttcaagc tggattaatt cagagcgcct tttactttgg ctactttatc

5760

atcccgatcc cggctggaat ccttatgaaa aaactgtctt acaaagctgg aattatcaca

5820

ggcttatttc tgtatgcact gggagcagct ctgttttggc ctgctgccga aatcatgaat

5880

tatacactgt ttctggtcgg actgtttatt atcgcagcgg gactgggctg tcttgaaaca

5940

gctgccaacc cgtttgtcac agttcttgga ccggaatcaa gcggccattt tagactgaac

6000

cttgcacaaa catttaactc atttggagca atcatcgcgg ttgtgtttgg ccagagctta

6060

attctgtcta atgtcccgca tcaaagccag gatgttctgg ataaaatgtc tccggaacaa

6120

ctttcagcgt ataaacattc acttgtgtta agcgtccaga caccgtatat gattatcgtt

6180

gcaatcgtgc ttttagtcgc gctgcttatt atgttaacaa aatttccggc actgcaatct

6240

gataaccatt cagatgcgaa acagggctca tttagcgctt ctttatcaag actggccaga

6300

attagacatt ggcgctgggc tgtgttagcc caattttgct atgtcggagc acagacagcg

6360

tgttggtcat atctgatccg ctatgctgtc gaagaaattc cgggaatgac agccggcttt

6420

gcagcgaatt atcttacagg cacaatggtt tgctttttca tcggaagatt tacaggcaca

6480

tggttaattt ctcgctttgc accgcataaa gtgcttgctg cctatgcgtt aatcgcaatg

6540

gcgctgtgcc ttatttcagc ttttgccggc ggacatgtcg gccttattgc attaacactg

6600

tgtagcgcgt ttatgtctat ccaatacccg acaatcttta gcctgggaat taaaaacctt

6660

ggccaggata caaaatacgg atcttcattt atcgttatga caattatcgg cggaggcatt

6720

gttacaccgg tgatgggatt tgtgagcgat gcagcgggca atatcccgac agctgaatta

6780

attccggctc tgtgttttgc cgttatcttt atctttgcta gatttcgctc tcaaacagcc

6840

acaaactaat ctagagtcga cgtccaggca tcaaataaaa cgaaaggctc agtcgaaaga

6900

ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg

6960

ctcaccttcg ggtgggcctt tctgcgttta tatacgcgtt aacccgggcc cgcggatgga

7020

tatgatcaga tcctttaact ctggcaaccc tcaaaattga atgagacatg ctacacctcc

7080

ggataataaa tatatataaa cgtatataga tttcataaag tctaacacac tagacttatt

7140

tacttcgtaa ttaagtcgtt aaaccgtgtg ctctacgacc aaaactataa aacctttaag

7200

aactttcttt ttttacaaga aaaaagaaat tagataaatc tctcatatct tttattcaat

7260

aatcgcatcc gattgcagta taaatttaac gatcactcat catgttcata tttatcagag

7320

ctcgtgctat aattatacta attttataag gaggaaaaaa tatgggcatt tttagtattt

7380

ttgtaatcag cacagttcat tatcaaccaa acaaaaaata agtggttata atgaatcgtt

7440

aataagcaaa attcatataa ccaaattaaa gagggttata atgaacgaga aaaatataaa

7500

acacagtcaa aactttatta cttcaaaaca taatatagat aaaataatga caaatataag

7560

attaaatgaa catgataata tctttgaaat cggctcagga aaaggccatt ttacccttga

7620

attagtaaag aggtgtaatt tcgtaactgc cattgaaata gaccataaat tatgcaaaac

7680

tacagaaaat aaacttgttg atcacgataa tttccaagtt ttaaacaagg atatattgca

7740

gtttaaattt cctaaaaacc aatcctataa aatatatggt aatatacctt ataacataag

7800

tacggatata atacgcaaaa ttgtttttga tagtatagct aatgagattt atttaatcgt

7860

ggaatacggg tttgctaaaa gattattaaa tacaaaacgc tcattggcat tacttttaat

7920

ggcagaagtt gatatttcta tattaagtat ggttccaaga gaatattttc atcctaaacc

7980

taaagtgaat agctcactta tcagattaag tagaaaaaaa tcaagaatat cacacaaaga

8040

taaacaaaag tataattatt tcgttatgaa atgggttaac aaagaataca agaaaatatt

8100

tacaaaaaat caatttaaca attccttaaa acatgcagga attgacgatt taaacaatat

8160

tagctttgaa caattcttat ctcttttcaa tagctataaa ttatttaata agtaagttaa

8220

gggatgcata aactgcatcc cttaacttgt ttttcgtgtg cctatttttt gtgaatcgat

8280

tcagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc

8340

ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc

8400

agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa

8460

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt

8520

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg

8580

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg

8640

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag

8700

cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc

8760

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa

8820

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg

8880

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc

8940

taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac

9000

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg

9060

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt

9120

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt

9180

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa

9240

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga

9300

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt

9360

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg

9420

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga

9480

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga

9540

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg

9600

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc

9660

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc

9720

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca

9780

taattctctt actngtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac

9840

caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg

9900

ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc

9960

ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg

10020

tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac

10080

aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat

10140

actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata

10200

catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa

10260

agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg

10320

tatcacgagg ccctttcgtc

10340

<---

Claims (7)

1. A non-sporulating Bacillus subtilis cell for producing 2'-fucosyllactose, wherein sporulation is impaired due to deletion or functional inactivation of one or more genes encoding Spo0A, sigma E and sigma F, wherein said cell is genetically engineered to have a lactose permease, wherein the lactose permease is E. coli LacY is genetically engineered to express de novo genes from the GDP-fucose biosynthetic pathway, which are the E. coli manA , E. coli manB , E. coli manC , E. coli gmd , and E. coli wcaG genes; genetically engineered to have the α-1,2-fucosyltransferase WbgL of E. coli , and wherein said Bacillus cell does not have any β-galactosidase activity or has reduced β-galactosidase activity compared to a wild-type Bacillus progenitor cell of the same species as a result of deletion or functional inactivation of gan A. 2. The non-spore-forming Bacillus subtilis cell according to claim 1, wherein the Bacillus subtilis cell is additionally genetically engineered as a result of deletion or functional inactivation of yesZ . 3. Use of Bacillus cells according to claim 1 or 2 to obtain 2'-fucosyllactose. 4. A method for producing 2'-fucosyllactose, including: - provision of a non-spore-forming Bacillus cell described in paragraph 1 or 2; - culturing said Bacillus cell in a medium that contains lactose and under conditions that are permissive for the production of fucosylated oligosaccharide; and, perhaps - extraction of 2'-fucosyllactose from the medium and/or Bacillus cells.