RU2816764C1 - Production of sialylated oligosaccharides in bacillus cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a non-spore-forming Bacillus cell for producing a sialylated oligosaccharide, wherein said Bacillus cell is genetically engineered to contain lactose permease, a CMP-NeuNAc biosynthesis pathway and sialyltransferase, wherein the Bacillus cell sporulation capacity is impaired as a result of deletion or functional inactivation of one or more genes coding Spo0A, sigma E and sigma F. Invention also relates to a method of producing a sialylated oligosaccharide using said Bacillus cell.

EFFECT: disclosed is a non-spore-forming Bacillus cell for producing a sialylated oligosaccharide.

13 cl, 1 dwg, 3 tbl, 6 ex

Description

The present invention relates to the field of genetic engineering, in particular to the genetic engineering of Bacillus cells to produce sialylated oligosaccharides in said Bacillus cells, to the enzymatic production of sialylated oligosaccharides using said Bacillus cells and to the use of the sialylated oligosaccharides thus obtained.

Prior Art

Human breast milk provides infants with all the nutrients they need for healthy growth and development. Sugars, which are present in human breast milk, represent its main component, surpassing fats and proteins. In addition to lactose, which serves as a source of energy, human breast milk contains molecules of more complex saccharides, namely oligosaccharides. To date, approximately 200 structurally distinct oligosaccharides have been identified in human breast milk. These oligosaccharides are found in significant concentrations only in human milk, and they are collectively known as human milk oligosaccharides (HMOs). 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), sialic acid/N- acetylneuraminic acid (NeuNAc) and/or 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.

A significant number of OGMs carry one NeuNAc group. Among these sialylated human milk oligosaccharides (HOMS), 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c and disialyllacto-N-tetraose are the most common members in human breast milk.

Sialic acids (Sia) are a family of negatively charged monosaccharides with a backbone of nine carbon atoms. More than 50 forms of these α-keto acids have been found in nature. The most common sialic acid appears to be N-acetylneuraminic acid (NANA - N-acetylneuraminic acid, NeuNAc, Neu5Ac).

Sialic acids are found as terminal monosaccharide groups of glycans that are found in glycoconjugates (glycoproteins and glycolipids) on the surface of vertebrate and higher invertebrate cells. Sialic acids are components of lipopolysaccharides and capsular polysaccharides of pathogenic bacteria, including Escherichia coli K1, Haemophilus influenzae, Haemophilus ducreyi, Pateurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, Campylobacter jejuni and Streptococcus agalactiae.

It has been observed that sialylated HGMs support the infant's resistance to enteropathogenic bacteria and viruses. Interestingly, recent studies have also demonstrated a protective effect of long-chain SOGM against necrotizing enterocolitis, which is one of the most common and fatal diseases in preterm infants. In addition, sialylated oligosaccharides have been shown to neutralize enterotoxins from a variety of pathogenic microbes, including Escherichia coli, Vibrio cholerae and Salmonella. In addition, sialylated oligosaccharides have been found to inhibit colonization of the intestine by Helicobacter pylori and consequently prevent or inhibit gastric and duodenal ulcers. In addition, sialylated oligosaccharides are believed to support infant brain development and cognitive abilities.

Due to the known benefits of HMOs, primarily sialylated HMOs, an economically viable method for preparing them is desirable such that these sialylated oligosaccharides, or at least some of these sialylated oligosaccharides, become available as an additive to infant formulas.

The limited availability of human milk for purposes other than infant breastfeeding and the difficulty of obtaining pure fractions of individual human milk oligosaccharides from natural sources have led to the development of chemical routes for their synthesis. However, both chemical synthesis and in-vitro 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.

As an alternative to chemical and biocatalytic synthesis in-vitro, the enzymatic production of OGM has been developed. Today, recombinant Escherichia coli cells are used for the microbial production of certain HMOs on an industrial scale.

However, the genus Escherichia coli includes pathogenic members as well as non-pathogenic members. Although non-pathogenic E. coli strains are used for HGM microbial production, such non-pathogenic E. coli are not considered safe for the manufacture of products intended for human consumption in a variety of applications. This prevents regulatory approval of HGMs produced by modern biotechnological methods for human consumption in these areas. Thus, microbial cells of genera that would be considered safe for human consumption or would be considered safe when used in the preparation of compounds for human consumption in such areas are needed for the manufacture of saccharides that are intended for human consumption, for example, human milk oligosaccharides, in particularly for consumption by infants. The use of productive strains considered safe will, at least presumably, reduce concerns regarding the likelihood of human health risks posed by the saccharide and will facilitate their regulatory approval in most areas.

The problem is solved by using bacterial cells of the genus Bacillus to produce sialylated oligosaccharides, in particular sialylated OGM. Bacterial cells of some species of the genus Bacillus are already considered safe for human consumption or for the production of compounds/food for human consumption. Accordingly, Bacillus cells of species and/or strains are provided which are generally regarded as safe for the production of sialylated oligosaccharides, in particular for the production of sialylated oligosaccharides in breast milk.

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 the biotechnological production of various proteins and compounds used in food and pharmaceutical industry.

Bacillus 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.

Bacillus amyloliquefaciens is a root colonizing bacterium that is used to control some plant root pathogens in agriculture, aquaculture and hydroponics as it has antibacterial and fungal pathogens and can prevent infection through competitive exclusion or displacement of 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®.

Bacillus 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 are often consumed in Japan and contain up to 10 ⁸ viable B. subtilis cells per gram. Natto is recognized for its 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 in 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), improved growth abilities, 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 SpoOA. 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 through ectopic integration of an expression cassette containing a regulated promoter (e.g. mannitol inducible promoter ) and the comK 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).

Bacillus cells can be genetically engineered in a variety of ways to produce sialylated oligosaccharides.

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

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 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 via stop codons, and point mutations. No prior genome modifications are required for seamless genome editing via CRISPR-Cas9.

Random chromosomal gene integration and insertional mutagenesis can be carried out using a modified mariner transposon. This system does not tend to hotspot in B. subtilis while demonstrating high efficiency in random ectopic integration.

Although Bacillus species are used for the industrial production of enzymes, to date Bacillus cells have not been introduced into the industrial production of oligosaccharides, in particular the industrial production of sialylated oligosaccharides.

Chinese patent application CN 108410787 A discloses recombinant Bacillus subtilis cells that synthesize lactyl-N-neotetraose. These recombinant B. subtilis cells have a lactose permease gene, which is integrated into the cell genome. In addition, said Bacillus cell which carries a plasmid containing a β-1,3-N-glucosamine transferase gene and a β-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.

Chinese patent application CN 109735479 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 fucosyltransferase. The yield of 2'-fucosyllactose in the fermentation medium was reported to range from 0.424 g/L to 1.042 g/L.

Although numerous patent applications mention Bacillus as a genus believed to be suitable for the production of neutral oligosaccharides such as lacto-N-neotetraose or 2'-fucosyllactose, there is no application of Bacillus for the production of sialylated oligosaccharides, in particular sialylated human milk oligosaccharides has not yet been realized, presumably due to the significant metabolic modification efforts required to implement the necessary biosynthetic pathways for HGM 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 sialylated oligosaccharide in Bacillus requires the implementation of a heterologous metabolic pathway in the cell to provide the donor substrate, namely SMP (from the English Cytidine Monophosphatase - cytidine monophosphate)-NeuNAc, which is extremely important for the synthesis of sialylated oligosaccharides.

The goal was achieved by providing the Bacillus cell, which has a lactose permease to import exogenous lactose into the cell, the CMP-NeuNAc biosynthetic pathway for the intracellular production of nucleotide-activated sialic acid, namely cytidine monophosphate-N-acetylneuraminic acid (CMP-NeuNAc), as a donor substrate for the sialic acid moiety, and a sialyltransferase to transfer the sialic acid moiety from CMP-NeuNAc to the acceptor substrate. Cultivation of such Bacillus cells in the presence of exogenous lactose allows the production of the desired sialylated oligosaccharide.

Summary of the invention

According to the first aspect, a non-spore-forming bacterial cell of the genus Bacillus is proposed for the production of sialylated oligosaccharide, wherein the Bacillus cell has a lactose permease, a CMP-NeuNAc biosynthetic pathway and a sialyltransferase.

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 sialylated oligosaccharide.

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

- providing a non-spore-forming bacterial cell of the genus Bacillus, wherein said Bacillus cell contains lactose permease, the CMP-NeuNAc biosynthetic pathway and sialyltransferase;

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

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

According to a fourth aspect, sialylated oligosaccharides are provided that have been produced by a Bacillus cell that contains lactose permease, the CMP-NeuNAc biosynthetic pathway, and sialyltransferase.

According to a fifth aspect, there is proposed the use of a sialylated oligosaccharide that has been produced by a Bacillus cell that has lactose permease, the CMP-NeuNAc biosynthetic pathway and sialyltransferase, for the manufacture of a nutritional composition.

According to a sixth aspect, nutritional compositions are provided comprising at least one sialylated oligosaccharide that is produced by a Bacillus cell that contains lactose permease, the CMP-NeuNAc biosynthetic pathway and sialyltransferase.

Description of graphic materials

In fig. 1 illustrates various embodiments of the CMP-NeuNAc biosynthetic pathway that occurs in a Bacillus cell to produce a sialylated oligosaccharide.

Detailed description

According to a first aspect, a non-spore-forming bacterial cell of the genus Bacillus is provided for the production of sialylated oligosaccharide. In order to be able to produce a sialylated oligosaccharide, the Bacillus cell must provide the sialyltransferase with a donor substrate containing a sialic acid moiety and an acceptor substrate that is a disaccharide or oligosaccharide, such that the sialyltransferase can transfer the sialic acid moiety from the donor substrate to said substrate-acceptor, resulting in the formation of a sialylated oligosaccharide.

It should be understood that the sialylated oligosaccharide that the specified Bacillus cell is intended to produce is the desired sialylated oligosaccharide, while other sialylated oligosaccharides that may be formed due to sialyltransferase heterogeneity during the production of the desired sialylated oligosaccharide in the Bacillus cell are considered undesired sialylated oligosaccharides or by-products. products.

The Bacillus cell for producing the sialylated oligosaccharide is a non-sporulating Bacillus cell. Wild-type Bacillus cells can form spores. It is believed that sporulation, namely the process of spore formation, in bacteria is 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 the production of two genetically identical cells with different morphologies and developmental directions.

However, during industrial production, it is not desirable if the producing bacterial strain forms spores during the enzymatic production of the target product. Thus, it is preferable to use Bacillus cells to produce sialylated oligosaccharides that are unable to form spores. Such Bacillus cells are called "non-spore-forming".

In one embodiment, the non-sporulating Bacillus cell to produce the sialylated oligosaccharide was from one of the B. subtilis strains listed in Table 1.

In some embodiments, the Bacillus cell is genetically engineered to become nonspore-forming, for example, through deletion or functional inactivation of SpoOA. SpoOA is a DNA-binding protein that influences, directly or indirectly, the expression of more than 500 genes during early sporulation. Suitable functional inactivation of SpoOA involves deletion of the phosphorylation site where the phosphotransferases SpoOF and SpoOB phosphorylate SpoOA. Deletion or functional inactivation of the phosphorylation site of the phosphotransferases SpoOF and SpoOB results in functional inactivation of SpoOA and consequently impairs the ability of the Bacillus cell to form spores. Alternatively, the gene or nucleotide sequence encoding SpoOA, or part of the gene or nucleotide sequence encoding SpoOA, can be deleted from the genome of the Bacillus cell.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered by deletion or functional inactivation of the gene(s) encoding the sigma factor SigE (sigE) and/or the sigma factor SigF (sigF). The sigma factors SigE and SigF are transcription factors that are involved in the expression of genes that encode proteins that are involved in the early phase of sporulation.

Deletion or functional inactivation of SpoOA, SigE and/or SigF impairs the ability of the Bacillus cell to form spores. Such a non-sporulating Bacillus cell can be used as a precursor to form a Bacillus cell that is capable of producing sialylated oligosaccharide.

Wild-type Bacillus cells neither synthesize lactose intracellularly nor absorb exogenous lactose. However, lactose is a substrate acceptor for sialic acid moiety via lactose-accepting sialyltransferase to form some sialylated oligosaccharides such as 3'-sialylactose (3-SL) or 6'-sialylactose (3-SL). Therefore, in order to have the ability to produce such sialylated oligosaccharides, the Bacillus cell must have the ability to provide lactose to lactose-accepting sialyltransferase, either by producing lactose intracellularly and/or by uptake of lactose externally. In addition, lactose is typically a disaccharide moiety that carries most, if not all, sialylated HMOs. Therefore, lactose uptake is also required for the biosynthesis of sialylated OGMs other than trisaccharides, namely when the acceptor substrate for the NeuNAc moiety is an oligosaccharide rather than the disaccharide lactose.

In one embodiment, a lactose permease is present in the Bacillus cell to produce the sialylated oligosaccharide for the uptake of exogenous lactose. Therefore, the Bacillus cell can take up exogenous lactose. Alternatively, the Bacillus cell could be genetically engineered to synthesize lactose intracellularly from glucose and galactose such that exogenous supply of lactose would not be necessary to produce the sialylated oligosaccharide.

The term "exogenous" with respect to lactose, as used herein, refers to lactose that does not originate from a Bacillus cell to produce the sialylated oligosaccharide, namely, being intracellularly synthesized by said Bacillus cell, but that originates outside of that Bacillus cells, and which is added to the culture medium in which the Bacillus cell is grown to obtain the sialylated oligosaccharide.

In some embodiments, the Bacillus cell is genetically engineered to have the ability to take up exogenous lactose, namely, genetically engineered to contain lactose permease. Thus, the Bacillus cell has a heterologous lactose permease to produce sialylated oligosaccharide. A suitable heterologous 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, nucleotide sequences and/or genes, refers to a molecule that is not native in relation to the type of Bacillus cell that contains the specified molecule. The term "non-native" indicates that the molecule is not present in a naturally occurring or wild-type Bacillus progenitor cell, namely a Bacillus cell of the same species that is most commonly found in nature. Thus, a “heterologous sequence” or “heterologous nucleic acid” or “heterologous polypeptide”, as used herein, is a sequence or nucleic acid or peptide that is derived from a source that is foreign to the cell Bacillus (eg from another species), or, if from the same source, modified from its original form. The heterologous sequence can be stably introduced, for example, by transfection, transformation, conjugation or transduction, into the genome of a microbial host cell, thereby representing a genetically engineered host cell. Techniques may be used that will depend on the host cell into which the sequence is to be introduced. Various techniques are known to one skilled in the art and, for example, are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Accordingly, a “heterologous polypeptide” is a polypeptide that does not naturally occur in a cell, and a “heterologous sialyltransferase” is a sialyltransferase that does not naturally occur in a microbial 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 reference enzyme or transporter molecule, but which has an amino acid sequence different from the amino acid sequence of the reference enzyme or transporter molecule. Thus, a typical protein/polypeptide variant differs in amino acid sequence from the reference protein/polypeptide. The variant and reference 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 reference protein/polypeptide that have the same activity as the reference 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, and 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 sequence that has greater than about 60% amino acid sequence identity, preferably 65%, 70%, 75% , 80%, 85%, 90%, more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97% -th, 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 compared to the activity of the reference protein/polypeptide.

In various embodiments, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes a lactose permease, preferably a nucleotide sequence that encodes E. coli LacY lactose permease (UniProtKB-P02920) 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 some embodiments, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence encoding a region encoding the E. coli LacY protein.

In some embodiments, the codon usage frequency of the nucleotide sequence encoding the heterologous lactose permease is adjusted to the codon usage 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%.

For expression of lactose permease, the Bacillus cell contains a recombinant lactose permease gene, wherein a nucleotide sequence that encodes lactose permease is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding lactose permease.

The term "operably linked", as used herein, will mean a functional relationship between a nucleotide sequence that encodes a polypeptide or protein (commonly referred to as a "protein coding region", "open reading frame" and sometimes even " genome") and a second nucleotide sequence (an expression control sequence such as a promoter, a signal sequence, or a series of transcription factor binding sites) that influences the transcription and/or translation of the nucleotide sequence that encodes the polypeptide or protein. Accordingly, the term “promoter” refers to the deoxyribonucleic acid (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 an additional 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 a sialic acid moiety to be transferred by lactose-accepting sialyltransferase (see later herein), for example, to form a 3'-SL or 6'-SL.

To produce a sialylated oligosaccharide, Bacillus must be able to provide a donor substrate for the transfer of the NeuNAc moiety to the acceptor substrate. A typical donor substrate for the NeuNAc moiety is CMP-NeuNAc. Therefore, the Bacillus cell must be able to produce CMP-NeuNAc intracellularly to produce sialylated oligosaccharides. In the case of intracellular biosynthesis of CMP-NeuNAc, the Bacillus cell has a CMP-NeuNAc biosynthesis pathway (Figure 1). Thus, the Bacillus cell is genetically engineered to have the CMP-NeuNAc biosynthetic pathway.

The CMP-NeuNAc biosynthesis pathway includes the NeuNAc recycling pathway or the sialic acid biosynthesis pathway for de novo intracellular biosynthesis of NeuNAc. Therefore, the Bacillus cell has a CMP-NeuNAc biosynthesis pathway, including a NeuNAc utilization pathway and/or a sialic acid biosynthesis pathway.

The NeuNAc utilization pathway involves the uptake of exogenous sialic acid by the Bacillus cell and the conversion of the ingested sialic acid to CMP-NeuNAc. For the uptake of exogenous NeuNAc, the Bacillus cell has a sialic acid transporter. In some embodiments, the genetically engineered Bacillus cell is genetically engineered to have sialic acid transport to take up exogenous NeuNAc. A suitable sialic acid transporter is E. coli NanT (UniProtKB P41036). E. coli NanT catalyzes the proton-dependent symport of sialic acid. NanT can transport NeuNAc as well as the related sialic acids N-glycolylneuraminic acid (NeuNGc) and 3-keto-3-deoxy-D-glycero-D-galactonic acid (KDN). In one embodiment, the genetically engineered Bacillus cell contains E. coli NanT or a functional variant thereof.

In some embodiments, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes a sialic acid transporter for uptake of exogenous NeuNAc, preferably a nucleotide sequence that encodes E. coli NanT or a functional variant thereof.

E. coli NanT is encoded by the protein-coding region of the E. coli naT gene. Therefore, in some embodiments, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes the E. coli NanT sialic acid transporter or a functional variant thereof.

For expression of the sialic acid transporter, the Bacillus cell contains a recombinant sialic acid transporter gene, wherein a nucleotide sequence that encodes the sialic acid transporter is operably linked to expression control sequences that mediate expression of the nucleotide sequence encoding the sialic acid transporter.

The recombinant sialic acid transporter gene may be present integrated into the Bacillus chromosome, or present as an episomal version on an additional plasmid in the Bacillus cell.

In one embodiment, the codon frequency of the nucleotide sequence encoding the sialic acid transporter is adapted to the codon frequency of Bacillus.

In various embodiments, the CMP-NeuNAc biosynthetic pathway includes the sialic acid biosynthetic pathway. Thus, the Bacillus cell may have a sialic acid biosynthetic pathway for the intracellular biosynthesis of N-acetylneuraminic acid. The sialic acid biosynthetic pathway involves the enzymatic activities of glutamine:fructose-6-phosphate aminotransferase (Figure 1: ) and N-acetylneuraminic acid synthase (same as sialic acid synthase) (Figure 1: ). Therefore, the Bacillus cell requires glutamine:fructose-6-phosphate aminotransferase and N-acetylneuraminic acid synthase to produce sialylated oligosaccharide.

The enzyme glutamine:fructose-6-phosphate aminotransferase (EC 2.6.1.16) catalyzes the conversion of fructose-6-phosphate (Frc-6P) to glucosamine-6-phosphate (GlcN-6P) using glutamine (Figure 1: ). This enzymatic reaction is generally believed to be the first step in the hexosamine biosynthetic pathway. Alternative names for glutamine:fructose-6-phosphate aminotransferase are D-fructose-6-phosphate aminotransferase, GFAT, glucosamine-6-phosphate synthase, hexosephosphate aminotransferase, and L-glutamine-D-fructose-6-phosphate aminotransferase.

In an additional and/or alternative embodiment, the genetically engineered Bacillus cell contains a glutamine:fructose-6-phosphate aminotransferase, preferably a heterologous glutamine:fructose-6-phosphate aminotransferase. An example of a suitable heterologous glutamine:fructose-6-phosphate aminotransferase comes from E. coli. Glutamine: fructose-6-phosphate aminotransferase from E. coli (UniProtKB - P17169) is designated GlmS. Therefore, the Bacillus cell contains E. coli GlmS or a functional variant of E. coli GlmS. Preferably, the functional E. coli GlmS variant is a version that exhibits significantly reduced sensitivity to glucosamine 6-phosphate inhibition compared to the wild type enzyme. An example of a functional E. coli GlmS variant that exhibits significantly reduced sensitivity to glucosamine 6-phosphate inhibition. Examples of E. coli GlmS functional variants that exhibit significantly reduced sensitivity to glucosamine-6-phosphate inhibition are GlmS*54 and GlmS*, as described in International Application No. PCT/EP2019/063669 (incorporated herein by reference).

In a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a glutamine:fuctose-6-phosphate aminotransferase, preferably a glutamine:fructose-6-phosphate aminotransferase E. coli GlmS, or a nucleotide sequence encoding a functional variant E. coli GlmS, which showed significantly reduced sensitivity for glucosamine-6-phosphate inhibition compared to the wild-type enzyme.

For expression of glutamine:fructose-6-phosphate aminotransferase, the Bacillus cell contains a recombinant glutamine:fructose-6-phosphate aminotransferase gene, where the nucleotide sequence that encodes the glutamine:fructose-6-phosphate aminotransferase is operably linked to expression control sequences that mediate expression of the glutamine open reading frame : fructose-6-phosphate aminotransferase.

In an additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous glutamine:fructose-6-phosphate aminotransferase is adapted to the codon frequency of Bacillus.

The recombinant glutamine:fuctose-6-phosphate aminotransferase gene can be integrated into the Bacillus chromosome or carried as an episomal version on a plasmid in the Bacillus cell.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell contains sialic acid synthase. Sialic acid synthase catalyzes the condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) with N-acetylneuraminic acid (NeuNAc) (Figure 1: ). The enzymatic formation of NeuNAc represents the last step in the sialic acid biosynthetic pathway.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a sialic acid synthase or a functional variant thereof, preferably a heterologous sialic acid synthase. Examples of sialic acid synthases are known from many bacterial species, such as Campylobacter jejuni, Streptococcus agalactiae, Butyrivibrio proteoclasticus, Methanobrevibacter ruminatium, Acetobacterium woodii, Desulfobacula toluolica, Escherichia coli, Prevotella nigescens, Halorhabdus tiamatea, Desulfotignum phosphitoxidans or Candidatus Scalindua sp., Idomarina loihiensis, Fusobacterium nucleatum or Neisseria meningitidis. An example of a suitable sialic acid synthase is the C. jejuni N-acetylneuraminic acid synthase NeuB, as encoded by the C. jejuni neuB gene.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes N-acetylneuraminic acid synthase. Therefore, a nucleotide sequence that encodes N-acetylneuraminic acid synthase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes N-acetylneuraminic acid synthase in a genetically engineered Bacillus cell with ensuring intracellular activity of N-acetylneuraminic acid synthase.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous N-acetylneuraminic acid synthase is adapted to the codon frequency of Bacillus.

The recombinant N-acetylneuraminic acid synthase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In one embodiment, the sialic acid biosynthetic pathway includes uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as an intermediate. The Bacillus cell, which has a sialic acid biosynthetic pathway that includes UDP-GlcNAc as an intermediate, additionally contains phosphoglucosamine mutase (Figure 1: ), glucosamine-1-phosphate N-acetyltransferase (Fig. 1: ), N-acetylglucosamine-1-phosphate uridyl transferase (Fig. 1 ) and UDP-N-acetylglucosamine 2-epimerase (Fig. 1: ).

Phosphoglucosamine mutase converts glucosamine 6-phosphate (GlcN-6P) to glucosamine 1-phosphate (GlcN-1 P).

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to contain phosphoglucosamine mutase. A suitable phosphoglucosamine mutase is E. coli phosphoglucosamine mutase GlmM or a functional variant thereof. Said E. coli GlmM is encoded by the E. coli glmM gene.

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

E. coli phosphoglucosamine mutase GlmM is encoded by the protein-coding region of the E. coli glmM gene. Therefore, the Bacillus cell is genetically engineered to contain and express the nucleotide sequence that encodes E. coli GlmM.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes a phosphoglucosamine mutase. Therefore, a nucleotide sequence that encodes a phosphoglucosamine mutase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of the nucleotide sequence that encodes a phosphoglucosamine mutase in a genetically engineered Bacillus cell to provide intracellular phosphoglucosamine mutase activity.

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

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

Glucosamine-1-phosphate-N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-coenzyme A to glucosamine-1-phosphate (GlcN-1-P) to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P).

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to contain glucosamine 1-phosphate N-acetyltransferase.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes glucosamine 1-phosphate N-acetyltransferase.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes glucosamine-1-phosphate-N-acetyltransferase. Therefore, a nucleotide sequence that encodes glucosamine-1-phosphate-N-acetyltransferase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes glucosamine-1-phosphate-N -acetyltransferase in a genetically engineered Bacillus cell to provide intracellular glucosamine-1-phosphate-N-acetyltransferase activity.

In an additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous glucosamine-1-phosphate-N-acetyltransferase is adapted to the codon frequency of Bacillus.

The recombinant glucosamine-1-phosphate-N-acetyltransferase gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

N-acetylglucosamine-1-phosphate uridyltransferase converts N-acetylglucosamine-1-phosphate (GlcNAc-1-P) to UDP-GlcNAc by transferring uridine 5-monophosphate (from uridine 5-triphosphate).

In a further and/or alternative embodiment, the cell is genetically engineered to have N-acetylglucosamine-1-phosphate uridyl transferase.

In a further and/or alternative embodiment, the cell is genetically engineered to contain or express a nucleotide sequence that encodes N-acetylglucosamine-1-phosphate uridyl transferase.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes N-acetylglucosamine-1-phosphate uridyl transferase.

Therefore, a nucleotide sequence that encodes N-acetylglucosamine-1-phosphate uridyltransferase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes N-acetylglucosamine-1-phosphate uridyltransferase in genetically engineered Bacillus cell to ensure intracellular activity of N-acetylglucosamine-1-phosphate uridyl transferase.

In an additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous N-acetylglucosamine-1-phosphate uridyl transferase is adapted to the codon frequency of Bacillus.

The recombinant N-acetylglucosamine-1-phosphate uridyl transferase gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In an additional and/or alternative embodiment, the glucosamine-1-phosphate N-acetyltransferase activity and N-acetylglucosamine-1-phosphate uridyltransferase activity are provided by a bifunctional enzyme. An example of such bifunctional enzymes is E. coli GlmU. E. coli GlmU catalyzes at least two sequential reactions that convert glucosamine 1-phosphate to UDP-GlcNAc (Figure 1: ). The C-terminal domain catalyzes the transfer of an acetyl group from acetyl-coenzyme A to glucosamine-1-phosphate (GlcN-1-P) to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P), which is converted to UDP-GlcNAc by transfer of uridine 5-monophosphate (from uridine 5-triphosphate), a reaction catalyzed by the N-terminal domain.

Such a bifunctional enzyme is also known from Bacillus subtilis and Haemophilus influenzae.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to have a heterologous bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase. A suitable example is E. coli GlmU or a functional variant thereof (including H. influenzae GlmU). Said E. coli GlmU is encoded by the E. coli glmU gene.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express or overexpress a nucleotide sequence that encodes said bifunctional enzyme, preferably a nucleotide sequence that encodes E. coli GlmU or a functional variant thereof.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes a bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-osphaturidyltransferase. Therefore, a nucleotide sequence that encodes a bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence, which encodes a bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-phosphate-uridyltransferase in a genetically engineered Bacillus cell to provide intracellular glucosamine-1-phosphate-N-acetyltransferase activity and N-acetylglucosamine-1-phosphate-uridyltransferase activity.

In an additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-phosphate-uridyltransferase is adapted to the codon frequency of Bacillus.

The recombinant bifunctional glucosamine-1-phosphate-N-acetyltransferase/N-acetylglucosamine-1-phosphate-uridyltransferase gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In an additional and/or alternative embodiment, the sialic acid biosynthetic pathway involves the conversion of UDP-GlcNAc to N-acetylmannosamine (ManNAc). This conversion can be catalyzed by UDP-N-acetylglucosamine-2-eimease, which not only converts UDP-N-acetylglucosamine to UDP-N-acetylmannosamine, but simultaneously releases UDP (Figure 1: ).

Therefore, the Bacillus cell contains UDP-N-acetylglucosamine-2-epimerase, which simultaneously releases UDP.

A suitable UDP-N-acetylglucosamine-2-epimerase with concomitant release of UDP is Campylobacter jejuni UDP-N-acetylglucosamine-2-epimerase NeuC, encoded by the C. jejuni neuC gene.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered to have UDP-N-acetylglucosamine-2-epimerase with concomitant release of UDP.

In a further and/or alternative embodiment, a Bacillus cell is genetically engineered to contain and express a nucleotide sequence that encodes UDP-N-acetylglucosamine-2-epimerase that simultaneously releases UDP, preferably to contain and express a nucleotide sequence that encodes NeuC C , jejuni or a functional variant thereof.

C. jejuni UDP-N-acetylglucosamine-2-epimerase NeuC is encoded by the protein-coding region of the C. jejuni NeuC gene. Therefore, the Bacillus cell is genetically engineered to contain and express the nucleotide sequence that encodes C. jejuni NeuC.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence that encodes UDP-N-acetylglucosamine-2-epimerase with concomitant release of UDP during its enzymatic reaction to release ManNAc. Therefore, a nucleotide sequence that encodes UDP-N-acetylglucosamine-2-epimerase, which simultaneously releases UDP, is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes said UDP -N-acetylglucosamine-2-epimerase in a genetically engineered Bacillus cell to provide intracellular UDP-N-acetylglucosamine-2-epimerase activity.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous UDP-N-acetylglucosamine-2-epimerase is adapted to the codon frequency of Bacillus.

The recombinant UDP-N-acetylglucosamine-2-epimerase gene may be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In an alternative embodiment, the sialic acid biosynthetic pathway uses N-acetylglucosamine-6-phosphate (GlcNAc-6-P) as an intermediate, but does not use UDP-GlcNAc. The genetically engineered Bacillus cell has a sialic acid biosynthetic pathway for the intracellular biosynthesis of N-acetylneuraminic acid, which uses GlcNAc-6-P as an intermediate, has glucosamine-6-phosphate-N-acetyltransferase (Figure 1: ). The sialic acid biosynthetic pathway, which uses glucosamine-6-phosphate-N-acetyltransferase for the intracellular biosynthesis of N-acetylneuraminic acid, does not use UDP-GlcNAc for the biosynthesis of sialic acid.

The sialic acid biosynthetic pathway using GlcNAc-6P involves the activities of glutamine:fructose-6-phosphate aminotransferase enzymes (Figure 1: ) and N-acetylneuraminic acid synthase (Fig. 1: ). This pathway for the biosynthesis of sialic acid additionally includes a) the activity of the enzymes glucosamine-6-phosphate-N-acetyltransferase (Fig. 1: ), N-acetylglucosamine-6-phosphate phosphatase (Fig. 1: ) and N-acetylglucosamine-2-epimerase (Fig. 1: ) or b) the activity of glucosamine-6-phosphate-N-acetyltransferase enzymes (Figure 1: ), N-acetylglucosamine-6-phosphate epimerase (Fig. 1: ) and N-acetylmannosamine-6-phosphate phosphatase (Fig. 1: ). Thus, it is not necessary for a genetically engineered Bacillus cell to possess the enzymatic activities of phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate-uridyltransferase and UDP-N-acetylglucosamine 2-epimerase with concomitant release of UDP for intracellular sialic acid biosynthesis. Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell capable of synthesizing sialic acid does not contain one or more enzyme activities selected from the group consisting of phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridyl transferase, and UDP N-acetylglucosamine enzyme activities -2-epimerase with concomitant release of UDP.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell has glucosamine 6-phosphate N-acetyltransferase activity. This glucosamine 6-phosphate N-acetyltransferase activity converts GlcN-6P to N-acetylglucosamine 6-phosphate (GlcNAc-6P). An example of glucosamine 6-phosphate N-acetyltransferase is Saccharomyces cerevisiae Gna1 (UniProtKB - P43577).

In a further and/or alternative embodiment, the genetically engineered Bacillus cell contains a glucosamine 6-phosphate N-acetyltransferase, preferably a heterologous glucosamine 6-phosphate N-acetyltransferase, more preferably Gna1 from S. cerevisiae or a functional variant thereof.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence encoding glucosamine 6-phosphate N-acetyltransferase, preferably S. cerevisiae Gna1, wherein said nucleotide sequence is operably linked to at least one nucleic acid expression control sequence influencing the transcription and/or translation of the specified nucleotide sequence in a genetically engineered Bacillus cell to ensure intracellular glucosamine-1-phosphate-N-acetyltransferase activity.

In an additional and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous glucosamine-6-phosphate-N-acetyltransferase is adapted to the codon frequency of Bacillus.

The recombinant glucosamine-6-phosphate-N-acetyltransferase gene can be integrated into the Bacillus chromosome or carried as an episomal version on a plasmid in the Bacillus cell.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell has N-acetylglucosamine-6-phosphate phosphatase activity. This N-acetylglucosamine 6-phosphate phosphatase activity converts GlcNAc-6P to N-acetylglucosamine (GlcNAc). Examples of N-acetylglucosamine-6-phosphate phosphatases are sugar phosphatases of the HAD-like superfamily, which catalyze the conversion of GlcNAc6P to GlcNAc. The HAD-like superfamily of enzymes is named after the bacterial enzyme halide dehydrogenase and includes phosphatases. A suitable HAD-like superfamily phosphatase catalyzing the conversion of GlcNAc6P to GlcNAc may be selected from the group consisting of fructose-1-phosphate phosphatase (YqaB, UniProtKB - P77475) and alpha-D-glucose-1-phosphate phosphatase (YihX, UniProtKB - P0A8Y3) . The E. coli YqaB and E. coli YihX enzymes are also thought to act on GlcNAc6P (Lee, S.-W. and Oh, M.-K. (2015) Metabolic Engineering 28: 143-150).

In an additional and/or alternative embodiment, the HAD-like superfamily sugar phosphatase that catalyzes the conversion of GlcNAc-6P to GlcNAc is a heterologous enzyme in a genetically engineered Bacillus cell. In an additional and/or alternative embodiment, the HAD-like superfamily sugar phosphatase that catalyzes the conversion of GlcNAc6P to GlcNAc is selected from the group consisting of E. coli YqaB, E. coli YihX, and functional variants thereof.

In an additional and/or alternative embodiment, the genetically engineered Bacillus cell contains a nucleic acid molecule that contains and expresses a nucleotide sequence encoding a HAD-like superfamily sugar phosphatase that catalyzes the conversion of GlcNAc-6P to GlcNAc. In an additional and/or alternative embodiment, the nucleotide sequence encoding a HAD-like superfamily sugar phosphatase that catalyzes the conversion of GlcNAc-6P to GlcNAc is a heterologous nucleotide sequence. In an additional and/or alternative embodiment, the nucleotide sequence encoding a HAD-like superfamily sugar phosphatase that catalyzes the conversion of GlcNAc-6P to GlcNAc encodes E. coli YqaB or E. coli YihX or a functional variant of one or two of these enzymes.

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

The recombinant N-acetylglucosamine-6-phosphate phosphatase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell has N-acetylglucosamine-2-epimerase activity. N-acetylglucosamine-2-epimerase (EC 5.1.3.8) is an enzyme that catalyzes the conversion of N-acetylglucosamine (GlcNAc) to N-acetylmannosamine (ManNAc). The enzyme is a racemase that acts on carbohydrates and their derivatives. The systematic name for this class of enzymes is N-acyl-D-glucosamine 2-epimerase. This enzyme is involved in the metabolism of amino sugars and the metabolism of nucleotide sugars, preferably heterologous N-acetylglucosamine 2-epimerase.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell contains N-acetylglucosamine-2-epimerase, preferably a heterologous N-acetylglucosamine-2-epimerase. Examples of N-acetylglucosamine 2-epimerases are described in Anabena variabilis, Acaryochloris sp., Nostoc sp., Nostoc punctiforme, Bacteroides ovatus or Synechocystis sp. An example of a suitable N-acetylglucosamine 2-epimerase is B. ovatus ATCC N-cetylglucosamine 2-epimerase 8483 (UniProtKB - A7LVG6), as encoded by the BACOVA_01816 gene. Another example is N-acetylglucosamine 2-epimerase from Synechocystis sp. (strain PCC 6803) (UniProtKB - P74124), which is also known as renin-binding protein and is encoded by the slr1975 gene.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising a nucleotide sequence that encodes N-acetylglucosamine 2-eimerase, preferably N-acetylglucosamine 2-epimerase of B. ovatus ATCC 8483 or Synechocystis sp. (strain PCC 6803) or a functional variant thereof.

Therefore, a nucleotide sequence that encodes N-acetylglucosamine-2-epimerase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes N-acetylglucosamine-2-epimerase in genetically engineered Bacillus cell to ensure intracellular activity of N-acetylglucosamine-2-epimerase.

In a further and/or alternative embodiment, the codon frequency of the nucleotide sequence encoding the heterologous N-acetylglucosamine-2-epimerase is adapted to the codon frequency of Bacillus.

The recombinant N-acetylglucosamine-2-epimerase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell has N-acetylglucosamine-6-phosphate epimerase activity and N-acetylmannosamine-6-phosphate phosphatase activity. N-acetylglucosamine-6-phosphate epimerase converts N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylmonnosamine-6-phosphate (ManNAc-6P), whereas N-acetylmannosamine-6-phosphate phosphatase dephosphorylates ManNAc-6P to produce N -acetylmonnosamine (ManNAc). Possession of N-acetylglucosamine-6-phosphate epimerase activity and N-acetylglucosamine-6-phosphate phosphatase activity provides an additional or alternative pathway to provide ManNAc for Neu5Ac production.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell contains N-acetylglucosamine-6-phosphate epimerase. An example of a suitable N-acetylglucosamine-6-phosphate epimerase is E. coli NanE (UniprotKB P0A761), as encoded by the E. coli paPE gene.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence encoding N-acetylglucosamine-6-phosphate epimerase, preferably a nucleotide sequence encoding E. coli NanE or a functional variant thereof.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence encoding N-acetylglucosamine-6-phosphate epimerase, wherein said nucleotide sequence is operably linked to at least one nucleic acid expression control sequence , affecting the transcription and/or translation of the specified nucleotide sequence in a genetically engineered microbial cell to ensure the intracellular activity of N-acetylglucosamine-2-phosphate epimerase.

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

In a further and/or alternative embodiment, the genetically engineered Bacillus cell contains N-acetylglucosamine-6-phosphate phosphatase, which converts ManNAc-6P to ManNAc.

Thus, in a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence encoding N-acetylmannosamine 6-phosphate phosphatase. Therefore, a nucleotide sequence that encodes N-acetylglucosamine-6-phosphate phosphatase is operably linked to at least one nucleic acid expression control sequence affecting the transcription and/or translation of said nucleotide sequence that encodes N-acetylglucosamine-6-phosphate phosphatase, in genetically engineered Bacillus cell to provide intracellular N-acetylglucosamine-6-phosphate phosphatase activity.

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

The recombinant N-acetylmannosamine 6-phosphate phosphatase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

The genetically engineered Bacillus cell has cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase activity (Figure 1: ) to transfer cytidine 5'-monophosphate to N-acetylneuraminic acid to form CMP-activated N-acetylneuraminic acid (CMP-NeuNAc). Several 5'-monophospho-(CMP)-sialic acid synthetases are known in the art and have been described, for example, 5'-monophospho-(CMP)-sialic acid synthetases from E. coli, Neisseria meningitidis, Campylobacter jejuni, Streptococcus sp., etc. .d.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises cytidine 5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase, preferably a heterologous cytidine 5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase, more preferably N-acetylneuraminate cytidyltransferase NeuA from C. jejuni. C. jejuni NeuA is encoded by the C. jejuni neuA gene.

Thus, in a further and/or alternative embodiment, a genetically engineered Bacillus cell comprises a nucleic acid molecule comprising and expressing a nucleotide sequence encoding cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase, wherein said nucleotide sequence is operably linked to at least one nucleic acid expression control sequence influencing the transcription and/or translation of the specified nucleotide sequence in a genetically engineered microbial cell to ensure N-acetylneuraminate cytidyl transferase activity.

In an additional and/or alternative embodiment, the codon usage frequency of the nucleotide sequence encoding heterologous cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase is adapted to the Bacillus codon usage frequency.

The recombinant cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid synthetase gene can be integrated into the Bacillus chromosome or present as an episomal version on a plasmid in the Bacillus cell.

The genetically engineered Bacillus cell contains a sialyltransferase, preferably a heterologous sialyltransferase and more preferably a sialyltransferase activity selected from the group consisting of α-2,3-sialyltransferase activity, α-2,6-sialyltransferase activity and/or α-2,8-sialyltransferase activity. Sialyltransferase activity can transfer the N-acetylneuraminic acid moiety from CMP-NeuNAc to an acceptor molecule, where the acceptor molecule is a saccharide molecule, to produce a sialylated saccharide.

In a further and/or alternative embodiment, the genetically engineered microbial cell contains at least one sialyltransferase, preferably at least one heterologous sialyltransferase, wherein said sialyltransferase may have α-2,3-sialyltransferase activity and/or α-2,6-sialyltransferase activity and/or α-2,8-sialyltransferase activity to transfer the NeuNAc moiety from CMP-NeuNAc as a donor substrate to an acceptor saccharide. Exemplary sialyltransferases and their genes are identified in Table 2.

The term “sialyltransferase”, as used herein, refers to polypeptides capable of having sialyltransferase activity. "Sialyltransferase activity" refers to the transfer of a sialic acid residue, preferably an N-acetylneuraminic acid residue (Neu5Ac), from a donor substrate to an acceptor molecule. The term “sialyltransferase” includes functional fragments of the sialyltransferases described herein, functional variants of the sialyltransferases described herein, and functional fragments of functional variants. "Functional" in this regard means that the fragments and/or variants may have sialyltransferase activity. Functional sialyltransferase fragments comprise truncated versions of the sialyltransferase as encoded by their naturally occurring gene, the truncated version of which may have sialyltransferase activity. Examples of truncated versions are sialyltransferases, which do not contain the so-called leader sequence that usually directs the polypeptide to a specific subcellular location. Typically, such leader sequences are removed from the polypeptide during intracellular transport, and they are also absent from the mature sialyltransferase found in nature.

A heterologous sialyltransferase can transfer a sialic acid residue from a donor substrate to an acceptor molecule. The term “may” in relation to a heterologous sialyltransferase refers to the sialyltransferase activity of the heterologous sialyltransferase and the proviso that suitable reaction conditions are required for the heterologous sialyltransferase to have its enzymatic activity. In the absence of suitable reaction conditions, the heterologous sialyltransferase does not have its enzymatic activity, but retains its enzymatic activity and has its enzymatic activity when suitable reaction conditions are restored. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of suitable acceptor molecules, the presence of significant cofactors such as, for example, monovalent or divalent ions, a pH value in the appropriate range, a suitable temperature, and the like. It is not necessary that the optimum values for each factor influencing the enzymatic reaction of the heterologous sialyltransferase be satisfied, but the reaction conditions must be such that the heterologous sialyltransferase performs its enzymatic activity. Accordingly, the term “may” excludes any conditions under which the enzymatic activity of the heterologous sialyltransferase is irreversibly impaired, and also excludes the heterologous sialyltransferase being affected by any such condition. In contrast, the term “may” means that the sialyltransferase is enzymatically active, namely, possesses its sialyltransferase activity if permissive reaction conditions (where all the requirements necessary for the sialyltransferase to exercise its enzymatic activity) are provided to the sialyltransferase.

Sialyltransferases can be distinguished by the type of sugar bond they form. As used herein, the terms "α-2,3-sialyltransferase" and "α-2,3-sialyltransferase activity" refer to polypeptides and their enzymatic activities that add a sialic acid residue to an α-2 ,3-bond to galactose, N-acetylgalactosamine or galactose or the N-acetylgalactosamine residue of the acceptor molecule. Similarly, the terms "α-2,6-sialyltransferase" and "α-2,6-sialyltransferase activity" refer to polypeptides and their enzymatic activities that add an α-2,6-linked sialic acid residue to galactose, N-acetylgalactosamine or galactose or an N-acetylgalactosamine residue of an acceptor molecule. Likewise, the terms "α-2,8-sialyltransferase" and "α-2,8-sialyltransferase activity" refer to polypeptides and their enzymatic activities that add an α-2,8-linked sialic acid residue to galactose, N-acetylgalactosamine or galactose or an N-acetylgalactosamine residue of an acceptor molecule.

In addition, the genetically engineered Bacillus cell is genetically engineered to contain and express a nucleotide sequence encoding a heterologous sialyltransferase. To this end, a nucleotide sequence encoding a heterologous sialyltransferase is operably linked to at least one expression control sequence affecting the transcription and/or translation of said nucleotide sequence encoding a heterologous sialyltransferase in a genetically engineered Bacillus cell to produce intracellular sialyltransferase activity.

In yet another embodiment, the heterologous sialyltransferase may have α-2,6-sialyltransferase activity.

In an additional and/or alternative embodiment, a genetically engineered Bacillus cell contains and expresses a nucleic acid molecule that contains at least one nucleotide sequence encoding said heterologous sialyltransferase capable of having α-2,6-sialyltransferase activity.

In a further and/or alternative embodiment, the heterologous sialyltransferase may have α-2,8-sialyltransferase activity. An example of a heterologous sialyltransferase that is capable of α-2,8-sialyltransferase activity is the Campylobacter jejuni sialyltransferase Cstll OH4384.

A sialyltransferase can transfer a sialic acid residue, such as an N-acetylneuraminic acid residue (Neu5Ac), from a donor substrate, such as CMP-Neu5Ac, to an acceptor molecule. The acceptor molecule is a saccharide molecule, preferably a saccharide molecule set forth in Table 3.

In an additional and/or alternative embodiment, the genetically engineered Bacillus microbial cell contains a nucleic acid molecule that contains and expresses at least one nucleotide sequence encoding said heterologous sialyltransferase capable of having α-2,3-sialyltransferase activity.

In a further and/or alternative embodiment, the acceptor molecule is a monosaccharide, preferably a monosaccharide selected from the group consisting of N-acetylglucosamine, galactose and N-acetylgalactosamine.

In a further and/or alternative embodiment, the acceptor molecule is a disaccharide, preferably a disaccharide selected from the group consisting of lactose, lactulose, N-acetyllactosamine, lacto-N-biose, lactulose and melibiose.

In an additional and/or alternative embodiment, the acceptor molecule is a trisaccharide, preferably a trisaccharide selected from the group consisting of raffinose, lacto-N-triose II, 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-N-

acetyllactosamine, 6'-sialyl-N-acetyllactosamine, 3'-galactosyllactose and 6'-galactosyllactose.

In an additional and/or alternative embodiment, the acceptor molecule is a tetrasaccharide, preferably a tetrasaccharide selected from the group consisting of lacto-N-tetraose, lacto-N-neotetraose, 2'3-difucosyllactose, 3-fucosyl-3'-sialyllactose and 3 -fucosyl-6'-sialyllactose.

In an additional and/or alternative embodiment, the acceptor molecule is a pentasaccharide, preferably a pentasaccharide selected from the group consisting of sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose I and lacto-N-neofucopentaose V.

It should be understood that a Bacillus cell already carrying one or more genes encoding these enzymes in a manner sufficient to produce NeuNAc, CMP-NeuNAc and/or sialylated saccharide does not require genetic modification to perform sialic acid biosynthesis and to transfer the sialic acid moiety to saccharide acceptor, but may nevertheless be genetically engineered to alter the expression level of one or more of said genes to increase the intracellular level of products of said one or more genes, such as, for example, the amount of glutamine: fructose-6-phosphate aminotransferase, glucosamine -6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylglucosamine 2-epimerase and/or N-acetylneuraminic acid synthetase, thus increasing the rate of Neu5Ac biosynthesis, and, as a consequence, sialylated saccharide, in genetically engineered cell.

In some embodiments, a Bacillus cell is intended for use in the production of 3'-SL and is genetically engineered to have a lactose permease, a CMP-NeuNAc biosynthetic pathway that uses GlcN-1P as an intermediate, and an α-2,3-sialyltransferase.

In various embodiments, a Bacillus cell is intended for use in the production of 6'-SL and is genetically engineered to have a lactose permease, a CMP-NeuNAc biosynthetic pathway that uses GlcN-1P as an intermediate, and an α-2,6-sialyltransferase.

In a further and/or alternative embodiment, the genetically engineered Bacillus microbial cell synthesizes more PEP than the wild type cell. In a further and/or alternative embodiment, the genetically engineered microbial cell is genetically engineered to have an enhanced PEP biosynthetic pathway. Preferably, the genetically engineered microbial cell is genetically engineered to have increased phosphoenolpyruvate synthase activity, for example, in that the ppsA gene encoding phosphoenolpyruvate synthase is overexpressed, and/or in that the non-naturally occurring microorganisms contain at least one additional copy a nucleotide sequence allowing the expression of phosphoenolpyruvate synthase or a functional variant thereof. Overexpression of ppsA enhances intracellular PEP synthesis such that more PEP is available for sialic acid production. For example, a suitable phosphoenolpyruvate synthase is E. coli PpsA.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell comprises a nucleic acid molecule comprising a nucleotide sequence encoding E. coli PpsA or a functional variant thereof. Said nucleotide sequence encoding E. coli PpsA or a functional variant thereof has at least sequence identity 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% with the E. coli ppsA gene.

In a further and/or alternative embodiment, the genetically engineered Bacillus cell is further modified to have the ability to transport said single carbon source into the cell through a mechanism that does not consume PEP.

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

Intracellular biosynthesis of sialylated oligosaccharides requires the import of lactose as an acceptor substrate for lactose-accepting sialyltransferase. Any intracellular enzyme activity that hydrolyzes ingested lactose will affect the efficiency of sialyllactose formation because the pool of intracellular lactose will be reduced. Thus, it will be advantageous if the Bacillus cell used to produce the sialylated 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 β-galactosidase activity of the cell.

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

The Bacillus ganA gene is also called yvfN or lacA. This is the GanR regulon gene, which 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 abolishes GanA-mediated β-galactosidase activity in the Bacillus cell, whereas reducing the level of ganA expression reduces the amount of GanA in the Bacillus cell and, therefore, β-galactosidase activity, which would interfere with sialylated oligosaccharide biosynthesis.

In a further and/or alternative embodiment, the Bacillus cell is genetically engineered by deleting or functionally inactivating 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 compared to a wild-type Bacillus cell.

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

When B. subtilis enters the post-exponential growth phase, they (begin to) produce large amounts of extracellular proteases. Foreign proteins are often protease sensitive. 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 Bacillus cell is genetically engineered such 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 of the genes selected from the group consisting of nprE, aprE, epr, bpr, mpr, nprB, vpr and wprA are deleted or functionally inactivated.

B, subtiiis synthesizes pulcherrimic acid when grown in media containing a carbohydrate such as glucose or lactose. The released pulcherrimic acid forms the red pigment pulcherrimin, a salt of pulcherrimic acid (iron(III) chelate) when iron is in the growth medium. The formation of this undesirable by-product during fermentation processes can be avoided/abrogated by deleting/disrupting the yvmC and/or surX genes. The yvmC gene encodes cyclodipeptide synthase, and the surX gene encodes cytochrome P450 cyclo-I-leucyl-I-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 has genes for the synthesis of more than 20 antibiotics. Among them are peptide antibiotics, such as lantibiotics and peptides similar to lactibiotics of Bacillus subtilis (subtilin, ericin S, mersacidin, sublancin 168, subtilosin A) and antibiotics synthesized non-ribosomally (peptide) (surfactin, ituirn, bacillomycin, mycosubtilin, corinebactin/ bacillibactin, fengycin, plipastatin, mycobacillin, TL-119, bacilysin, bacilizocin, amikoumacin, 3,3'-neotrehalosadiamine, difficidin, rhizocticin).

To obtain the sialylated 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, merzacidin, sublancin 168, subtilosin A; non-ribosomally synthesized antibiotics (peptide), such as surfactin, iturin, bacillomycin, mycosubtilin, corynebactin/bacillibactin, fengycin, plipastatin, mycobacillin, TL-119, bacilysin, bacilizocin, amikoumacin, 3,3'-neotrehalosadiamine, difficidin and rhizocticin. The Bacillus cell may have been genetically engineered to disrupt or abrogate its ability to synthesize one or more of these antibiotics.

A genetically engineered Bacillus cell that can produce a sialylated oligosaccharide may optionally include additional traits and may be genetically engineered to have these additional traits. These additional traits are believed to improve the productivity of the microorganism, which does not occur naturally, resulting in higher yields of sialylated oligosaccharide.

The Bacillus cell of the invention can produce sialylated oligosaccharide when cultured in the presence of lactose in a medium and under conditions that are permissive to the Bacillus cell to produce sialylated oligosaccharide. Thus, the use of a genetically engineered Bacillus cell, as previously described herein, to produce a sialylated oligosaccharide is also provided.

In one aspect, a method for producing a sialylated oligosaccharide is provided. The method comprises (i) providing a Bacillus cell as described previously herein, (ii) cultivating said Bacillus cell in a culture medium or fermentation broth and under conditions that are permissive for the production of sialylated oligosaccharide. The method may further comprise recovering the sialylated oligosaccharide from the culture medium/fermentation broth and/or from the Bacillus cell.

The fermentation broth contains at least one carbon source for the metabolism of the Bacillus cell. In some embodiments, the at least one carbon source is selected from the group consisting of glucose, fructose, sucrose, glycerol, and combinations thereof. The carbon source may be taken up by the Bacillus cell and may be metabolized by the Bacillus cell to produce biomass and/or energy in the form of high-energy triphosphates.

In some embodiments, the fermentation broth contains lactose, particularly if the Bacillus cell cannot synthesize lactose on its own. Lactose can be supplied to the fermentation broth from outside. Lactose can serve as an acceptor substrate for the NeuNAc group in the production of some sialylated oligosaccharides, in particular, in the production of sialyllactose.

In various embodiments, the fermentation broth contains sialic acid, particularly if a Bacillus cell is used to produce the sialylated oligosaccharide, which lacks a sialic acid biosynthesis pathway but does have a sialic acid utilization pathway.

In some embodiments, preparation of the sialylated oligosaccharide does not require the addition of N-acetylglucosamine, N-acetylmannosamine and/or N-acetylneuraminic acid to the fermentation broth and/or culturing the genetically engineered microbial cell in the presence of N-acetylglucosamine, N-acetylmannosamine and/or N-acetylneuraminic acid for intracellular biosynthesis of sialylated oligosaccharide, since the Bacillus cell is used to produce sialylated oligosaccharide, which has a de novo sialic acid biosynthesis pathway for intracellular biosynthesis of NeuNAC.

In this method, at least one genetically engineered Bacillus cell is cultured in a fermentation broth and under conditions that are permissive for the production of a saccharide containing at least one N-acetylneuraminic acid moiety.

While the process utilizes the carbon source in the fermentation broth for the genetically engineered Bacillus cell, it is not necessary to add glucosamine and/or N-acetylneuraminic acid and/or N-acetylglucosamine and/or N-acetylmannosamine to the fermentation broth when the Bacillus cell can produce N-acetylneuraminic acid intracellularly. Thus, in various embodiments, to produce a sialylated oligosaccharide, a genetically engineered Bacillus cell is cultured in the absence and/or without the addition of one or more selected from the group consisting of glucosamine, N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminic acid. The genetically engineered Bacillus cell can be cultured in the absence and/or without the addition of galactose, since galactose is not supplied as an acceptor substrate for the sialyltransferase reaction.

In additional and/or alternative embodiments, the genetically engineered Bacillus cell is cultured in the presence of one or more monosaccharides (e.g., galactose), disaccharides (e.g., lactose), trisaccharides (e.g., lacto-N-triose II), tetrasaccharides (e.g., lacto-N -tetraose) and/or pentasaccharides (for example, sialyllacto-N-tetraose a).

In a further and/or alternative embodiment, the genetically engineered Bacillus cell is cultured in the presence of at least one acceptor substrate selected from the group consisting of galactose, N-acetylgalactosamine, N-acetylglucosamine, lactose, lactulose, N-acetyllactosamine, lacto-N- bioses, lacto-N-triose, 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-N-acetyllactosamine, 6'-sialyl-N-acetyllactosamine, 3'-galactosyllactose, 6'-galactosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, 2'3-difucosyllactose, 3-fucosyl-3'-sialyllactose and 3-fucosyl-6'-sialyllactose. These substrates are imported into the cell and used as acceptor molecules in the cell.

The method includes the optional step of isolating a sialylated oligosaccharide that is produced by a genetically engineered Bacillus cell during its cultivation and proliferation in a fermentation broth. The sialylated oligosaccharide may be isolated from the fermentation broth after removal of the genetically engineered Bacillus cells, for example, by centrifugation or filtration, and/or may be isolated from the cells, for example, in that the cells are collected from the fermentation broth by centrifugation and subjected to a cell lysis step. The sialylated oligosaccharides can then be further purified from the fermentation broth and/or cell lysates by suitable techniques known to one of ordinary skill in the art. Suitable techniques include microfiltration, ultrafiltration, diafiltration, pseudo-moving bed chromatography, electrodialysis, reverse osmosis, gel filtration, anion exchange chromatography, cation exchange chromatography, and the like.

The method and the genetically engineered microbial cell used in the method are used to produce sialylated oligosaccharide. The term "sialylated oligosaccharide" refers to an oligosaccharide molecule containing at least one N-acetylneuraminic acid moiety.

In a further and/or alternative embodiment, the sialylated saccharide is an oligosaccharide. The term "oligosaccharide" as used herein refers to polymers of monosaccharide residues, wherein said polymers contain at least two monosaccharide residues, but no more than 10 monosaccharide residues, preferably no more than 7 monosaccharide residues. Oligosaccharides are either a linear chain of monosaccharides or are branched. In addition, the monosaccharide residues of oligosaccharides can be characterized by a number of chemical modifications. Accordingly, oligosaccharides may contain one or more non-saccharide moieties. The term "sialylated oligosaccharide", as used herein, refers to oligosaccharides containing one or more N-acetylneuraminic acid moieties.

In a further and/or alternative embodiment, the sialylated oligosaccharide is selected from the group consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, fucosyl-sialyllacto- N-tetraose a, fucosyl-sialyllacto-N-tetraose b, fucosyl-sialyllacto-N-tetraose c, disialyllacto-N-tetraose, fucosyllacto-N-tetraose I, fucosyllacto-N-tetraose II, 3'-sialylgalactose, 6' -sialylgalactose, 3'-sialyl-N-acetyllactosamine and 6'-sialyl-N-acetyllactosamine.

Another aspect of the invention provides the use of a genetically engineered Bacillus cell, as described previously herein, to produce a sialylated oligosaccharide in a whole cell fermentation process, namely, the sialylated oligosaccharide is synthesized by and in a genetically engineered Bacillus cell.

In yet another aspect of the invention, there is provided a sialylated oligosaccharide that is produced by the method and/or use of a genetically engineered Bacillus cell as described previously herein.

Another aspect of the invention provides the use of a sialylated oligosaccharide that is prepared by a method as described previously herein and/or through the use of a genetically engineered Bacillus cell as described previously herein for the manufacture of a nutritional composition.

Thus, a nutritional composition is provided comprising at least one sialylated oligosaccharide which is produced by a method and/or through a genetically engineered Bacillus cell as previously described herein.

In an additional and/or alternative embodiment, the sialylated oligosaccharide is selected from the group consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, fucosyl-sialyllacto- N-tetraose a, fucosyl-sialyllacto-N-tetraose b, fucosyl-sialyllacto-N-tetraose c, disialyllacto-N-tetraose, fucosyldisiallyllacto-N-tetraose I, fucosyldisiallyllacto-N-tetraose II, 3'-sialylgalactose, 6' -sialylgalactose, 3'-sialyl-N-acetyllactosamine and 6'-sialyl-N-acetyllactosamine.

In some embodiments, the nutritional composition further comprises at least one neutral HMO, preferably at least one neutral HMO that is produced through the use of a genetically engineered Bacillus cell. At least one neutral HGM is isolated from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N -fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaoses II, lacto-N-neodifucohexaoses I, lacto-N-hexaoses, lacto-N-neohexaoses, para-lacto-N-hexaoses and para-lacto-N-neohexaoses.

In a further embodiment, the nutritional composition is selected from the group consisting of medical compositions, pharmaceutical compositions, nutraceutical compositions, infant formulas and dietary supplements.

The nutritional composition may be presented in liquid form or solid form, including, but not limited to, powders, granules, flakes and pellets.

The present invention will be described with respect to specific embodiments, but the invention is not limited to them, but only to 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 in order to avoid obstruction of understanding. 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 the following concentrations: 5 μg⋅ml ^-1 erythromycin, 5 μg⋅ml ^-1 chloramphenicol, 10 μg⋅ml ^-1 kanamycin. 100 µg⋅ml ^-1 spectinomycin.

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 until stationary growth phase (OD ₆₀₀ (from the English optical density - optical phase) is 1.7-2). Cells were then pelleted 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, which is 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 4x PAB and 2x 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 medium DM3 contained the following sterile solutions per liter: 200 ml of 4% agar, 100 ml of 5% casamino acids, 50 ml of 10% 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 2x YT culture was diluted 100-fold with fresh 2x YT 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 another 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 delivery of the pulse, 1 ml of 2x 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 2x 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 sedum 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 xg 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 2x YT.

Luria broth (LB) medium consisted of 1% tryptone, 0.5% yeast extract, and 0.5% NaCl (pH 7.2).

2x 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 1000x solution of microelements (possibly additionally supplemented biotin and/or L-tryptophan).

The trace element solution (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 BO 3} , 9 g⋅l ^-1 ZnSO4 × 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 3'-sialyllactose

To synthesize 3'-sialyllactose from the metabolic intermediate UDP-N-acetylglucosamine using the neuCBA pathway, a B. subtilis expression plasmid (SEQ ID NO: 1) was constructed. First, the neuA, neuB, neuC genes, as well as the α-2,3-sialyltransferase, were codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp. The Campylobacter jejuni neuA gene (UniProtKB accession number: Q93MP7) encodes the CMP-Neu5Ac synthetase . The neuB gene of Campylobacter jejuni (UniProtKB accession number: Q93MP9) encodes sialic acid synthase. The neuC gene of Campylobacter jejuni (UniProtKB accession number: Q93MP8) encodes N-acetylglucosamine 6-phosphate 2-epimerase. The Pasteurella multocida siaT gene (UniProtKB accession number: Q9CLP3) encodes an α-2,3-sialyltransferase. The open reading frame of the E. coli lacY gene (Gen Bank accession number: NP_414877.1), encoding lactose permease, was amplified by PCR (polymerase chain reaction) from chromosomal DNA. Then, an expression cassette <P _grac100 -neuBCA-siaT-lacY-terminator> was constructed, which contains all the necessary genes under the control of the inducible P _grac100 promoter. For this purpose, the B. subtilis expression vector pHT253 (MoBiTec GmbH, Göttingen, Germany) was used as a scaffold. Each gene within the expression cassette was linked to the B. subtilis RBS sequence. Additionally, a suitable B. subtilis termination sequence from part of the iGem repository (sequence ID: BBa_B0015) was placed downstream of the expression cassette. The resulting plasmid <pHT253-P _grac100 -neuBCA-2,3siaT-lacY-terminator> (SEQ ID NO: 1) was used to transform the spore-forming B. subtilis strain (Table 1) by exploiting 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 3'-sialyllactose in the presence of exogenous lactose.

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

To synthesize 3'-sialyllactose from the metabolic intermediate UDP-N-acetylglucosamine using the neuCBA pathway, a B. subtilis constitutive expression plasmid (SEQ ID NO: 2) was constructed. First, the neuA, neuB, neuC, and siaT genes were codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp. The Campylobacter jejuni neuA gene (UniProtKB accession number: Q93MP7) encodes the CMP-Neu5Ac synthetase. The neuB gene of Campylobacter jejuni (UniProtKB accession number: Q93MP9) encodes sialic acid synthase.

The neuC gene of Campylobacter jejuni (UniProtKB accession number: Q93MP8) encodes N-acetylglucosamine 6-phosphate 2-epimerase. The Pasteurella multocida siaT gene (UniProtKB accession number: Q9CLP3) encodes an α-2,3-sialyltransferase. 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. Then, an expression cassette <P43-neuBCA-P _lepA -siaT-lacY-terminator> was constructed, which contains all the necessary genes functionally associated with two strong constitutive promoters of B. subtilis and/or P43. The B. subtilis expression vector pHT253 (MoBiTec GmbH, Göttingen, Germany) was used as the plasmid backbone. Each gene within the expression cassette was linked to the B. subtilis RBS sequence. Additionally, a suitable B. subtilis termination sequence from part of the iGem repository (sequence ID: BBa_B0015) was placed downstream of the expression cassette.

The resulting plasmid <phT253-P43-neuBCA- -siaT-lacY-terminator> (SEQ ID NO: 2) was used to transform spore-forming and non-spore-forming strains of B. subtilis (Table 1) by exploiting their 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 3'-sialyllactose in the presence of exogenous lactose.

Example 4: Construction of a Bacillus subtilis producer strain for 6'-sialyllactose

α-2,6-sialyltransferase from Photobacterium leiognathi was used to produce 6'-sialyllactose in B. subtilis. The open reading frame of the siaT gene (UniProtKB accession number: D0VYB7) was codon optimized for expression in B. subtilis and produced synthetically by GenScript Corp.

The expression plasmid <pHT253-P _grac100 -neuBCA-2,6siaT-lacY-terminator> (SEQ ID NO: 3) was constructed as described in example 2. The resulting plasmid (SEQ ID NO: 3) was used to transform the spore-forming and non-spore-forming strains B subtilis (Table 1) by exploiting their 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 6'-sialyllactose in the presence of exogenous lactose.

Example 5: Construction of a Bacillus subtilis strain for the production of 3'-sialyllactose

Engineering the metabolism of B. subtilis (Table 1) was achieved by integrating the heterologous neuA genes of Campylobacter jejuni, nanT of E. coli, and siaT of Haemophilus parahaemolyticus and simultaneous deletion of the endogenous ganA gene through homologous recombination. The ganA gene of B. subtilis (yvfN, lacA), which is located within the galactose operon, encodes beta-galactosidase.

For the production of 3'-sialyllactose from exogenous sialic acid/N-acetylneuraminic acid and lactose, the open reading frames neuA, nanT and siaT were functionally linked to the constitutive B. subtilis P43 promoter (part of the iGem repository: Sequence ID: BBa_K143013) as an operon. To this end, firstly, the neuA, nanT and siaT genes were codon optimized for expression in B. subtilis and obtained synthetically by GenScript Corp. The neuA gene of Campylobacter jejuni (UniProtKB accession number: Q93MP7) encodes the CMP-Neu5Ac synthetase, the nanT E gene . coli (GenBank accession number: NP_417691.4) encodes the sialic acid transporter and the siaT gene H. parahaemolyticus (UniProtKB accession number: I3DHL4) encodes alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3-N-acetylgalactosaminide 6-alpha-sialyltransferase (alpha-2,3-sialyltransferase).

Each gene within the expression cassette was linked to the B. subtilis RBS sequence. In addition, a suitable B. subtilis termination sequence from part of the iGem repository (sequence ID: BBa_B0015) was located downstream of the expression cassette.

Cloning vector pBR322 (New England Biolabs GmbH, Frankfurt, Germany) was used as the plasmid backbone. The full integration cassette was assembled to create the suicide plasmid<pBR322 flank ganA up-P43-siaT-neuA-nanT-terminator-erm-flank ganA down>(SEQ ID NO: 4). 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-siaT-neuA-nanT-terminator> expression cassette into the ganA locus of the B. subtilis genome, resulting in strain A, was verified by colony PCR. Gene expression was confirmed by targeted proteomics and/or real-time PCR.

To import exogenous lactose into the Bacillus cell, the E. coli lacY gene was integrated into the endogenous amyE (amyA) locus of the B. subtilis genome (encoding alpha-amylase). For this purpose, 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-1ox71-aad9-lox66-P43-lacY-flank amyE down> integration cassette (SEQ ID NO: 5) was constructed and cloned into pBR322 (New England Biolabs GmbH, Fankfurt, Germany). Strain A was transformed with the resulting suicide plasmid due to natural competence. Integration of the <P43-lacY> expression cassette into the amyE locus of strain A, resulting in the final strain B, was verified by colony PCR.

Gene expression was confirmed by targeted proteomics and/or real-time PCR. Strain B was cultured under conditions that are permissive for B. subtilis to produce 3'-sialyllactose in the presence of exogenous lactose and sialic acid/N-acetylneuraminic acid.

Example 6: Production of sialyllactose using modified metabolism strains of Bacillus subtilis

The preculture was inoculated with a Bacillus subtilis strain with a modified metabolism suitable for the biosynthesis of sialyllactose (as described in examples 2-5).

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 accomplished by lactose (2 mM) or both lactose (2 mM) and IPTG (1 mM). For the utilization biosynthesis of CMP-N-acetylneuraminic acid, 2 mM sialic acid/N-acetylneuraminic acid was additionally added. Culture was stopped after approximately 24 h/48 h post-induction, and intracellular and extracellular sialyllactose were 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 sialyllactose (3'-sialyllactose/6'-sialyllactose) was detected.

--->

LIST OF SEQUENCES

<110> Jennewein Biotechnologie GmbH

<120> PRODUCTION OF SIALYLATED OLIGOSACCHARIDES IN BACILLUS CELLS

<130>P 1906 WO

<160> 5

<170> Patent In version 3.5

<210> 1

<211> 13121

<212> DNA

<213> Artificial sequence

<220>

<223> Expression plasmid

<400> 1

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

acatgaaaga aatcaaaatc caaaacatca tcatcagcga agaaaaagcg ccgctggttg

2820

tgccggaaat cggcattaat cataacggat ctttagaact ggctaaaatc atggttgatg

2880

cagcgttttc agctggagcc aaaatcatca aacatcaaac acatatcgtc gaagatgaaa

2940

tgagcaaagc tgcaaagaaa gttatcccgg gcaacgctaa aatctctatc tacgaaatca

3000

tgcagaaatg cgctttagat tacaaagatg aacttgccct gaaagaatac acagaaaaac

3060

tgggacttgt gtatcttagc acaccgtttt caagagcagg cgcgaatcgc ttagagata

3120

tgggagtctc tgcatttaaa atcggctcag gagaatgtaa taactatccg ctgatcaaac

3180

atattgcagc gtttaaaaaa ccgatgattg tgtctacagg catgaactca atcgaaagca

3240

ttaaaccgac agtgaaaatc ctgcttgata acgaaatccc gtttgtcctg atgcatacaa

3300

caaacctgta tccgacaccg cataatcttg ttagattaaa cgccatgctg gaactgaaga

3360

aagaatttag ctgcatggtg ggactttctg atcatacaac agataatctg gcatgccttg

3420

gcgcggtcgt tcttggagcg tgtgtcttag aaagacattt tacagattca atgcatcgca

3480

gcggaccgga tattgtttgt tctatggata caaaagcctt aaaagaactg atcattcaat

3540

cagaacagat ggcaatcatt cgcggcaata acgaatcaaa gaaagcagcc aaacaagaac

3600

aggttacaat cgattttgct tttgcctctg tggtctcaat caaagatatt aagaaaggcg

3660

aagttctgtc aatggataat atttgggtga aaagaccggg acttggcgga atcagcgcag

3720

cggaatttga aaatattctg ggcaagaaag cactgcgcga tattgaaaac gatgcgcagt

3780

taagctatga agattttgct taagctctta aggaggattt tagaatgaag aaaattctgt

3840

ttatcacagg ctcaagagcg gattactcta aaatcaaatc acttatgtac cgcgtccaaa

3900

attcaagcga atttgaactt tacatctttg ctacaggcat gcatctgagc aaaaactttg

3960

gatacacagt taaagaatta tataaaaatg gctttaaaaa catctacgaa tttatcaact

4020

acgataaata ttatcagaca gataaagccc tggcaacaac aattgatgga ttttcaagat

4080

atgcgaacga attaaaaccg gatctgattg ttgtgcatgg cgatcgcatc gaaccgcttg

4140

cagcggctat tgtcggagcc cttaacaaca tcctggttgc acatatcgaa ggcggagaaa

4200

ttagcggaac aatcgatgat tctttaagac atgcgatttc aaaacttgct catatccatc

4260

tggtgaacga tgaatttgca aaaagacgcc ttatgcaatt aggcgaagat gaaaaatcaa

4320

tctttatcat cggatctccg gatctggaac tgcttaacga taacaaaatc tcacttagcg

4380

aagccaaaaa atactacgat attaactacg aaaactatgc attactgatg tttcatccgg

4440

tcacaacaga aatcacatct atcaaaaacc aagccgataa cctggtgaaa gcacttatcc

4500

agtcaaacaa aaactacatc gtcatctatc cgaataacga tctgggcttt gaactgatcc

4560

ttcagagcta tgaagaattt aaaaacaacc cgagatttaa actgtttccg tctctgcgct

4620

ttgaatactt tatcacactt ctgaaaaacg ccgattttat tatcggaaac tcttcatgca

4680

tcctgaaaga agcgttatac ctgaaaacag ctggcattct ggttggatca agacaaaatg

4740

gccgcttagg aaatgaaaac acactgaaag tgaatgcgaa cagcgatgaa atccttaaag

4800

ctatcaacac aatccataaa aaacaggatt tattttctgc taaactggaa attcttgata

4860

gctctaaact gtttttcgaa tatcttcaat caggcgattt ctttaaactt agcacacaga

4920

aagtttttaa agatattaaa taaaaaggag gaactactat gtcacttgca attatcccgg

4980

cgagaggcgg aagcaaaggc atcaaaaaca aaaacctggt tctgcttaac aacaaaccgc

5040

ttatctacta cacaatcaaa gcagcgctga atgctaaatc tatttcaaaa gttgtggtct

5100

caagcgatag cgatgaaatc cttaactacg ccaaatctca aaacgtggat attctgaaaa

5160

gaccgatctc tcttgcacag gatgatacaa catcagataa agtcttactg catgcgctga

5220

aattttacaa agattacgaa gatgttgtgt ttttacaacc gacatctccg ctgcgcacaa

5280

acattcatat caacgaagca tttaacctgt acaaaaattc aaacgctaat gccctgatta

5340

gcgtctctga atgcgataac aaaatcctta aagcatttgt ttgcaacgat tgtggcgatt

5400

tagccggaat ttgtaatgat gaatatccgt ttatgccgcg ccagaaactg ccgaaaacat

5460

atatgagcaa cggagcgatc tacatcctta aaatcaaaga atttctgaac aacccgtcat

5520

ttctgcaaag caaaacaaaa cattttctta tggatgaatc atcatcactg gatattgatt

5580

gcctggaaga tttaaagaaa gttgaacaaa tttggaaaaa ataaagatta actaataagg

5640

aggacaaaca tggataaatt tgctgaacat gaaattccga aagctgttat cgtggccggc

5700

aacggagaat ctctttcaca aatcgattac agactgcttc cgaaaaacta tgatgtcttt

5760

agatgcaacc agttttactt tgaagaacgc tattttcttg gcaacaaaat caaagcagtt

5820

ttctttacac cgggagtgtt tttagaacaa tactacacac tgtaccatct taaacgcaac

5880

aacgaatact ttgtcgataa cgttatcctg tcatcattta accatccgac agttgatctg

5940

gaaaaatcac aaaaaatcca ggctctgttt attgatgtga tcaacggcta cgaaaaatac

6000

ctgtctaaac tgacagcctt tgatgtttat ctgagataca aagaacttta cgaaaaccaa

6060

cgcattacat caggagtgta tatgtgcgcc gtcgcaatcg cgatgggcta tacagatatt

6120

taccttacag gaatcgattt ttatcaggca agcgaagaaa actacgcgtt tgataacaaa

6180

aaaccgaaca tcatcagatt actgccggat tttcgcaaag aaaaaacatt attttcatat

6240

catagcaaag atattgatct tgaagcatta tcttttctgc aacagcatta ccatgttaac

6300

ttttacagca tttctccgat gtctccgctg tcaaaacatt ttccgatccc gacagtggaa

6360

gatgattgtg aaacaacatt tgtcgcgccg ctgaaagaaa actacatcaa cgatattctt

6420

ttaccgccgc attttgtcta tgaaaaactt ggcgttgata aacttgcagc ggctttagaa

6480

catcatcatc atcatcatta aagtgatagc ggtaccatta taggtaagag aggaatgtac

6540

acatgtacta tttaaaaaac acaaactttt ggatgttcgg tttattcttt ttctttttact

6600

tttttatcat gggagcctac ttcccgtttt tcccgatttg gctacatgac atcaaccata

6660

tcagcaaaag tgatacgggt attatttttg ccgctatttc tctgttctcg ctattattcc

6720

aaccgctgtt tggtctgctt tctgacaaac tcgggctgcg caaatacctg ctgtggatta

6780

ttaccggcat gttagtgatg tttgcgccgt tctttatttt tatcttcggg ccactgttac

6840

aatacaacat tttagtagga tcgattgttg gtggtattta tctaggcttt tgttttaacg

6900

ccggtgcgcc agcagtagag gcatttattg agaaagtcag ccgtcgcagt aatttcgaat

6960

ttggtcgcgc gcggatgttt ggctgtgttg gctgggcgct gtgtgcctcg attgtcggca

7020

tcatgttcac catcaataat cagtttgttt tctggctggg ctctggctgt gcactcatcc

7080

tcgccgtttt actctttttc gccaaaacgg atgcgccctc ttctgccacg gttgccaatg

7140

cggtaggtgc caaccattcg gcatttagcc ttaagctggc actggaactg ttcagacagc

7200

caaaactgtg gtttttgtca ctgtatgtta ttggcgtttc ctgcacctac gatgtttttg

7260

accaacagtt tgctaatttc tttacttcgt tctttgctac cggtgaacag ggtacgcggg

7320

tatttggcta cgtaacgaca atgggcgaat tacttaacgc ctcgattatg ttctttgcgc

7380

cactgatcat taatcgcatc ggtgggaaaa acgccctgct gctggctggc actattatgt

7440

ctgtacgtat tattggctca tcgttcgcca cctcagcgct ggaagtggtt attctgaaaa

7500

cgctgcatat gtttgaagta ccgttcctgc tggtgggctg ctttaaatat attaccagcc

7560

agtttgaagt gcgtttttca gcgacgattt atctggtctg tttctgcttc tttaagcaac

7620

tggcgatgat ttttatgtct gtactggcgg gcaatatgta tgaaagcatc ggtttccagg

7680

gcgcttatct ggtgctgggt ctggtggcgc tgggcttcac cttaatttcc gtgttcacgc

7740

ttagcggccc cggcccgctt tccctgctgc gtcgtcaggt gaatgaagtc gcttaaggat

7800

ccatgtctag agtcgacgtc cccggggcag cccgcctaat gagcgggctt ttttcacgtc

7860

ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt

7920

gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct

7980

gcgtttatac ccggggcagc ccgcctaatg agcgggcttt tttcacgtca cgcgtccatg

8040

gagatctttg tctgcaactg aaaagtttat accttacctg gaacaaatgg ttgaaacata

8100

cgaggctaat atcggcttat taggaatagt ccctgtacta ataaaatcag gtggatcagt

8160

tgatcagtat attttggacg aagctcggaa agaatttgga gatgacttgc ttaattccac

8220

aattaaatta agggaaagaa taaagcgatt tgatgttcaa ggaatcacgg aagaagatac

8280

tcatgataaa gaagctctaa aactattcaa taaccttaca atggaattga tcgaaagggt

8340

ggaaggttaa tggtacgaaa attaggggat ctacctagaa agccacaagg cgataggtca

8400

agcttaaaga acccttacat ggatcttaca gattctgaaa gtaaagaaac aacagaggtt

8460

aaacaaacag aaccaaaaag aaaaaaagca ttgttgaaaa caatgaaagt tgatgtttca

8520

atccataata agattaaatc gctgcacgaa attctggcag catccgaagg gaattcatat

8580

tacttagagg atactattga gagagctatt gataagatgg ttgagacatt acctgagagc

8640

caaaaaactt tttatgaata tgaattaaaa aaaagaacca acaaaggctg agacagactc

8700

caaacgagtc tgttttttta aaaaaaatat taggagcatt gaatatatat tagagaatta

8760

agaaagacat gggaataaaa atattttaaa tccagtaaaa atatgataag attatttcag

8820

aatatgaaga actctgtttg tttttgatga aaaaacaaac aaaaaaaatc cacctaacgg

8880

aatctcaatt taactaacag cggccaaact gagaagttaa atttgagaag gggaaaaggc

8940

ggatttatac ttgtatttaa ctatctccat tttaacattt tattaaaccc catacaagtg

9000

aaaatcctct tttacactgt tcctttaggt gatcgcggag ggacattatg agtgaagtaa

9060

acctaaaagg aaatacagat gaattagtgt attatcgaca gcaaaccact ggaaataaaa

9120

tcgccaggaa gagaatcaaa aaagggaaag aagaagttta ttatgttgct gaaacggaag

9180

agaagatatg gacagaagag caaataaaaa acttttcttt agacaaattt ggtacgcata

9240

taccttacat agaaggtcat tatacaatct taaataatta cttctttgat ttttggggct

9300

attttttagg tgctgaagga attgcgctct atgctcacct aactcgttat gcatacggca

9360

gcaaagactt ttgctttcct agtctacaaa caatcgctaa aaaaatggac aagactcctg

9420

ttacagttag aggctacttg aaactgcttg aaaggtacgg ttttatttgg aaggtaaacg

9480

tccgtaataa aaccaaggat aacacagagg aatccccgat ttttaagatt agacgtaagg

9540

ttcctttgct ttcagaagaa cttttaaatg gaaaccctaa tattgaaatt ccagatgacg

9600

aggaagcaca tgtaaagaag gctttaaaaa aggaaaaaga gggtcttcca aaggttttga

9660

aaaaagagca cgatgaattt gttaaaaaaa tgatggatga gtcagaaaca attaatattc

9720

cagaggcctt acaatatgac acaatgtatg aagatatact cagtaaagga gaaattcgaa

9780

aagaaatcaa aaaacaaata cctaatccta caacatcttt tgagagtata tcaatgacaa

9840

ctgaagagga aaaagtcgac agtactttaa aaagcgaaat gcaaaatcgt gtctctaagc

9900

cttcttttga tacctggttt aaaaacacta agatcaaaat tgaaaataaa aattgtttat

9960

tacttgtacc gagtgaattt gcatttgaat ggattaagaa aagatattta gaaacaatta

10020

aaacagtcct tgaagaagct ggatatgttt tcgaaaaaat cgaactaaga aaagtgcaat

10080

aaactgctga agtatttcag cagttttttt tatttagaaa tagtgaaaaa aatataatca

10140

gggaggtatc aatatttaat gagtactgat ttaaatttat ttagactgga attaataatt

10200

aacacgtaga ctaattaaaa tttaatgagg gataaagagg atacaaaaat attaatttca

10260

atccctatta aattttaaca agggggggat taaaatttaa ttagaggttt atccacaaga

10320

aaagacccta ataaaatttt tactagggtt ataacactga ttaatttctt aatgggggag

10380

ggattaaaat ttaatgacaa agaaaacaat cttttaagaa aagcttttaa aagataataa

10440

taaaaagagc tttgcgatta agcaaaactc tttacttttt cattgacatt atcaaattca

10500

tcgatttcaa attgttgttg tatcataaag ttaattctgt tttgcacaac cttttcagga

10560

atataaaaca catctgaggc ttgttttata aactcagggt cgctaaagtc aatgtaacgt

10620

agcatatgat atggtatagc ttccacccaa gttagccttt ctgcttcttc tgaatgtttt

10680

tcatatactt ccatgggtat ctctaaatga ttttcctcat gtagcaaggt atgagcaaaa

10740

agtttatgga attgatagtt cctctctttt tcttcaactt ttttatctaa aacaaacact

10800

ttaacatctg agtcaatgta agcataagat gtttttccag tcataatttc aatcccaaat

10860

cttttagaca gaaattctgg acgtaaatct tttggtgaaa gaattttttt atgtagcaat

10920

atatccgata cagcaccttc taaaagcgtt ggtgaatagg gcattttacc tatctcctct

10980

cattttgtgg aataaaaata gtcatattcg tccatctacc tatcctatta tcgaacagtt

11040

gaacttttta atcaaggatc agtccttttt ttcattattc ttaaactgtg ctcttaactt

11100

taacaactcg atttgttttt cgagatctcg agggtaacta gcctcgccga tcccgcaaga

11160

ggcccggcag tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt

11220

tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca

11280

ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt

11340

ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga

11400

tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa

11460

gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct

11520

gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat

11580

acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga

11640

tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc

11700

caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat

11760

gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa

11820

cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac

11880

tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa

11940

agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc

12000

tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc

12060

ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag

12120

acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta

12180

ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa

12240

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc

12300

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat

12360

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga

12420

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt

12480

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata

12540

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac

12600

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg

12660

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg

12720

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag

12780

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct

12840

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc

12900

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt

12960

ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg

13020

tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga

13080

gtcagtgagc gaggaagcgg aagagcgccc aatacgcatg c

13121

<210> 2

<211> 11125

<212> DNA

<213> Artificial sequence

<220>

<223> 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 gctagcattt tacattttta gaaatgggcg tgaaaaaaag cgcgcgatta

1140

tgtaaaatat aaagattaac taataaggag gacaaacatg aaagaaatca aaatccaaaa

1200

catcatcatc agcgaagaaa aagcgccgct ggttgtgccg gaaatcggca ttaatcataa

1260

cggatcttta gaactggcta aaatcatggt tgatgcagcg ttttcagctg gagccaaaat

1320

catcaaacat caaacacata tcgtcgaaga tgaaatgagc aaagctgcaa agaaagttat

1380

cccggggcaac gctaaaatct ctatctacga aatcatgcag aaatgcgctt tagattacaa

1440

agatgaactt gccctgaaag aatacacaga aaaactggga cttgtgtatc ttagcacacc

1500

gttttcaaga gcaggcgcga atcgcttaga agatatggga gtctctgcat ttaaaatcgg

1560

ctcaggagaa tgtaataact atccgctgat caaacatatt gcagcgttta aaaaaccgat

1620

gattgtgtct acaggcatga actcaatcga aagcattaaa ccgacagtga aaatcctgct

1680

tgataacgaa atcccgtttg tcctgatgca tacaacaaac ctgtatccga caccgcataa

1740

tcttgttaga ttaaacgcca tgctggaact gaagaaagaa tttagctgca tggtgggact

1800

ttctgatcat acaacagata atctggcatg ccttggcgcg gtcgttcttg gagcgtgtgt

1860

cttagaaaga cattttacag attcaatgca tcgcagcgga ccggatattg tttgttctat

1920

ggatacaaaa gccttaaaag aactgatcat tcaatcagaa cagatggcaa tcattcgcgg

1980

caataacgaa tcaaagaaag cagccaaaca agaacaggtt acaatcgatt ttgcttttgc

2040

ctctgtggtc tcaatcaaag atattaagaa aggcgaagtt ctgtcaatgg ataatatttg

2100

ggtgaaaaga ccgggacttg gcggaatcag cgcagcggaa tttgaaaata ttctgggcaa

2160

gaaagcactg cgcgatattg aaaacgatgc gcagttaagc tatgaagatt ttgcttaagc

2220

tcttaaggag gattttagaa tgaagaaaat tctgtttatc acaggctcaa gagcggatta

2280

ctctaaaatc aaatcactta tgtaccgcgt ccaaaattca agcgaatttg aactttacat

2340

ctttgctaca ggcatgcatc tgagcaaaaa ctttggatac acagttaaag aattatataa

2400

aaatggcttt aaaaacatct acgaatttat caactacgat aaatattatc agacagataa

2460

agccctggca acaacaattg atggattttc aagatatgcg aacgaattaa aaccggatct

2520

gattgttgtg catggcgatc gcatcgaacc gcttgcagcg gctattgtcg gagcccttaa

2580

caacatcctg gttgcacata tcgaaggcgg agaaattagc ggaacaatcg atgattcttt

2640

aagacatgcg atttcaaaac ttgctcatat ccatctggtg aacgatgaat ttgcaaaaag

2700

acgccttatg caattaggcg aagatgaaaa atcaatcttt atcatcggat ctccggatct

2760

ggaactgctt aacgataaca aaatctcact tagcgaagcc aaaaaatact acgatattaa

2820

ctacgaaaac tatgcattac tgatgtttca tccggtcaca acagaaatca catctatcaa

2880

aaaccaagcc gataacctgg tgaaagcact tatccagtca aacaaaaact acatcgtcat

2940

ctatccgaat aacgatctgg gctttgaact gatccttcag agctatgaag aatttaaaaa

3000

caacccgaga tttaaactgt ttccgtctct gcgctttgaa tactttatca cacttctgaa

3060

aaacgccgat tttattatcg gaaactcttc atgcatcctg aaagaagcgt tatacctgaa

3120

aacagctggc attctggttg gatcaagaca aaatggccgc ttaggaaatg aaaacacact

3180

gaaagtgaat gcgaacagcg atgaaatcct taaagctatc aacacaatcc ataaaaaaca

3240

ggatttattt tctgctaaac tggaaattct tgatagctct aaactgtttt tcgaatatct

3300

tcaatcaggc gatttcttta aacttagcac acagaaagtt tttaaagata ttaaataaaa

3360

aggaggaact actatgtcac ttgcaattat cccggcgaga ggcggaagca aaggcatcaa

3420

aaacaaaaac ctggttctgc ttaacaacaa accgcttatc tactacacaa tcaaagcagc

3480

gctgaatgct aaatctattt caaaagttgt ggtctcaagc gatagcgatg aaatccttaa

3540

ctacgccaaa tctcaaaacg tggatattct gaaaagaccg atctctcttg cacaggatga

3600

tacaacatca gataaagtct tactgcatgc gctgaaattt tacaaagatt acgaagatgt

3660

tgtgttttta caaccgacat ctccgctgcg cacaaacatt catatcaacg aagcatttaa

3720

cctgtacaaa aattcaaacg ctaatgccct gattagcgtc tctgaatgcg ataacaaaat

3780

ccttaaagca tttgtttgca acgattgtgg cgatttagcc ggaatttgta atgatgaata

3840

tccgtttatg ccgcgccaga aactgccgaa aacatatatg agcaacggag cgatctacat

3900

ccttaaaatc aaagaatttc tgaacaaccc gtcatttctg caaagcaaaa caaaacattt

3960

tcttatggat gaatcatcat cactggatat tgattgcctg gaagatttaa agaaagttga

4020

acaaatttgg aaaaaataaa gtcaatgtat gaatggatac gggatatgaa tcaataagta

4080

cgtgaaagag aaaagcaacc cagatatgat agggaacttt tctctttctt gttttacatt

4140

gaatctttac aatcctattg atataatcta agctagtgta ttttgcgttt aatagtggag

4200

aaaagctagc gattaactaa taaggaggac aaacatggat aaatttgctg aacatgaaat

4260

tccgaaagct gttatcgtgg ccggcaacgg agaatctctt tcacaaatcg attacagact

4320

gcttccgaaa aactatgatg tctttagatg caaccagttt tactttgaag aacgctattt

4380

tcttggcaac aaaatcaaag cagttttctt tacaccggga gtgtttttag aacaatacta

4440

cacactgtac catcttaaac gcaacaacga atactttgtc gataacgtta tcctgtcatc

4500

atttaaccat ccgacagttg atctggaaaa atcacaaaaa atccaggctc tgtttattga

4560

tgtgatcaac ggctacgaaa aatacctgtc taaactgaca gcctttgatg tttatctgag

4620

atacaaagaa ctttacgaaa accaacgcat tacatcagga gtgtatatgt gcgccgtcgc

4680

aatcgcgatg ggctatacag atatttacct tacaggaatc gatttttatc aggcaagcga

4740

agaaaactac gcgtttgata acaaaaaacc gaacatcatc agattactgc cggattttcg

4800

caaagaaaaa acattatttt catatcatag caaagatatt gatcttgaag cattatcttt

4860

tctgcaacag cattaccatg ttaactttta cagcatttct ccgatgtctc cgctgtcaaa

4920

acattttccg atcccgacag tggaagatga ttgtgaaaca acatttgtcg cgccgctgaa

4980

agaaaactac atcaacgata ttcttttacc gccgcatttt gtctatgaaa aacttggcgt

5040

tgataaactt gcagcggctt tagaacatca tcatcatcat cattaaagtg atagcggtac

5100

cattataggt aagagaggaa tgtacacatg tactatttaa aaaacacaaa cttttggatg

5160

ttcggtttat tctttttctt ttactttttt atcatgggag cctacttccc gtttttcccg

5220

atttggctac atgacatcaa ccatatcagc aaaagtgata cgggtattat ttttgccgct

5280

atttctctgt tctcgctatt attccaaccg ctgtttggtc tgctttctga caaactcggg

5340

ctgcgcaaat acctgctgtg gattattacc ggcatgttag tgatgtttgc gccgttcttt

5400

atttttatct tcggggccact gttacaatac aacattttag taggatcgat tgttggtggt

5460

atttatctag gcttttgttt taacgccggt gcgccagcag tagaggcatt tattgagaaa

5520

gtcagccgtc gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg tgttggctgg

5580

gcgctgtgtg cctcgattgt cggcatcatg ttcaccatca ataatcagtt tgttttctgg

5640

ctgggctctg gctgtgcact catcctcgcc gttttactct ttttcgccaa aacggatgcg

5700

ccctcttctg ccacggttgc caatgcggta ggtgccaacc attcggcatt tagccttaag

5760

ctggcactgg aactgttcag acagccaaaa ctgtggtttt tgtcactgta tgttattggc

5820

gtttcctgca cctacgatgt ttttgaccaa cagtttgcta atttctttac ttcgttcttt

5880

gctaccggtg aacagggtac gcgggtattt ggctacgtaa cgacaatggg cgaattactt

5940

aacgcctcga ttatgttctt tgcgccactg atcattaatc gcatcggtgg gaaaaacgcc

6000

ctgctgctgg ctggcactat tatgtctgta cgtattattg gctcatcgtt cgccacctca

6060

gcgctggaag tggttattct gaaaacgctg catatgtttg aagtaccgtt cctgctggtg

6120

ggctgcttta aatatattac cagccagttt gaagtgcgtt tttcagcgac gatttatctg

6180

gtctgtttct gcttctttaa gcaactggcg atgattttta tgtctgtact ggcgggcaat

6240

atgtatgaaa gcatcggttt ccagggcgct tatctggtgc tgggtctggt ggcgctgggc

6300

ttcaccttaa tttccgtgtt cacgcttagc ggccccggcc cgctttccct gctgcgtcgt

6360

caggtgaatg aagtcgctta aggatccatg tctagagtcg acgtccccgg ggcagcccgc

6420

ctaatgagcg ggcttttttc acgtcccagg catcaaataa aacgaaaggc tcagtcgaaa

6480

gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact

6540

ggctcacctt cgggtgggcc tttctgcgtt tatagaattc atattactta gaggatacta

6600

ttgagagagc tattgataag atggttgaga cattacctga gagccaaaaa actttttatg

6660

aatatgaatt aaaaaaaaga accaacaaag gctgagacag actccaaacg agtctgtttt

6720

tttaaaaaaa atattaggag cattgaatat atattagaga attaagaaag acatgggaat

6780

aaaaatattt taaatccagt aaaaatatga taagattatt tcagaatatg aagaactctg

6840

tttgtttttg atgaaaaaac aaacaaaaaa aatccaccta acggaatctc aatttaacta

6900

acagcggcca aactgagaag ttaaatttga gaaggggaaa aggcggattt atacttgtat

6960

ttaactatct ccattttaac attttattaa accccataca agtgaaaatc ctcttttaca

7020

ctgttccttt aggtgatcgc ggagggacat tatgagtgaa gtaaacctaa aaggaaatac

7080

agatgaatta gtgtattatc gacagcaaac cactggaaat aaaatcgcca ggaagagaat

7140

caaaaaaggg aaagaagaag tttattatgt tgctgaaacg gaagagaaga tatggacaga

7200

agagcaaata aaaaactttt ctttagacaa atttggtacg catatacctt acatagaagg

7260

tcattataca atcttaaata attacttctt tgatttttgg ggctattttt taggtgctga

7320

aggaattgcg ctctatgctc acctaactcg ttatgcatac ggcagcaaag acttttgctt

7380

tcctagtcta caaacaatcg ctaaaaaaat ggacaagact cctgttacag ttagaggcta

7440

cttgaaactg cttgaaaggt acggttttat ttggaaggta aacgtccgta ataaaaccaa

7500

ggataacaca gaggaatccc cgatttttaa gattagacgt aaggttcctt tgctttcaga

7560

agaactttta aatggaaacc ctaatattga aattccagat gacgaggaag cacatgtaaa

7620

gaaggcttta aaaaaggaaa aagagggtct tccaaaggtt ttgaaaaaag agcacgatga

7680

atttgttaaa aaaatgatgg atgagtcaga aacaattaat attccagagg ccttacaata

7740

tgacacaatg tatgaagata tactcagtaa aggagaaatt cgaaaagaaa tcaaaaaaca

7800

aatacctaat cctacaacat cttttgagag tatatcaatg acaactgaag aggaaaaagt

7860

cgacagtact ttaaaaagcg aaatgcaaaa tcgtgtctct aagccttctt ttgatacctg

7920

gtttaaaaac actaagatca aaattgaaaa taaaaattgt ttattacttg taccgagtga

7980

atttgcattt gaatggatta agaaaagata tttagaaaca attaaaacag tccttgaaga

8040

agctggatat gttttcgaaa aaatcgaact aagaaaagtg caataaactg ctgaagtatt

8100

tcagcagttt tttttattta gaaatagtga aaaaaatata atcagggagg tatcaatatt

8160

taatgagtac tgatttaaat ttatttagac tggaattaat aattaacacg tagactaatt

8220

aaaatttaat gagggataaa gaggatacaa aaatattaat ttcaatccct attaaatttt

8280

aacaaggggg ggattaaaat ttaattagag gtttatccac aagaaaagac cctaataaaa

8340

tttttactag ggttataaca ctgattaatt tcttaatggg ggagggatta aaatttaatg

8400

acaaagaaaa caatctttta agaaaagctt ttaaaagata ataataaaaa gagctttgcg

8460

attaagcaaa actctttact ttttcattga cattatcaaa ttcatcgatt tcaaattgtt

8520

gttgtatcat aaagttaatt ctgttttgca caaccttttc aggaatataa aacacatctg

8580

aggcttgttt tataaactca gggtcgctaa agtcaatgta acgtagcata tgatatggta

8640

tagcttccac ccaagttagc ctttctgctt cttctgaatg tttttcatat acttccatgg

8700

gtatctctaa atgattttcc tcatgtagca aggtatgagc aaaaagttta tggaattgat

8760

agttcctctc tttttcttca acttttttat ctaaaacaaa cactttaaca tctgagtcaa

8820

tgtaagcata agatgttttt ccagtcataa tttcaatccc aaatctttta gacagaaatt

8880

ctggacgtaa atcttttggt gaaagaattt ttttatgtag caatatatcc gatacagcac

8940

cttctaaaag cgttggtgaa tagggcattt tacctatctc ctctcatttt gtggaataaa

9000

aatagtcata ttcgtccatc tacctatcct attatcgaac agttgaactt tttaatcaag

9060

gatcagtcct ttttttcatt attcttaaac tgtgctctta actttaacaa ctcgatttgt

9120

ttttccagat ctcgagggta actagcctcg ccgatcccgc aagaggcccg gcagtcaggt

9180

ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca

9240

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg

9300

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc

9360

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg

9420

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt

9480

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta

9540

ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat

9600

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga

9660

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca

9720

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact

9780

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc

9840

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact

9900

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt

9960

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt

10020

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt

10080

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata

10140

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag

10200

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat

10260

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa

10320

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca

10380

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt

10440

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg

10500

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc

10560

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga

10620

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc

10680

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc

10740

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca

10800

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg

10860

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta

10920

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct

10980

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag

11040

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa

11100

gcggaagagc gcccaatacg catgc

11125

<210> 3

<211> 13805

<212> DNA

<213> Artificial sequence

<220>

<223> Expression plasmid

<400> 3

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

acatgaaaga aatcaaaatc caaaacatca tcatcagcga agaaaaagcg ccgctggttg

2820

tgccggaaat cggcattaat cataacggat ctttagaact ggctaaaatc atggttgatg

2880

cagcgttttc agctggagcc aaaatcatca aacatcaaac acatatcgtc gaagatgaaa

2940

tgagcaaagc tgcaaagaaa gttatcccgg gcaacgctaa aatctctatc tacgaaatca

3000

tgcagaaatg cgctttagat tacaaagatg aacttgccct gaaagaatac acagaaaaac

3060

tgggacttgt gtatcttagc acaccgtttt caagagcagg cgcgaatcgc ttagagata

3120

tgggagtctc tgcatttaaa atcggctcag gagaatgtaa taactatccg ctgatcaaac

3180

atattgcagc gtttaaaaaa ccgatgattg tgtctacagg catgaactca atcgaaagca

3240

ttaaaccgac agtgaaaatc ctgcttgata acgaaatccc gtttgtcctg atgcatacaa

3300

caaacctgta tccgacaccg cataatcttg ttagattaaa cgccatgctg gaactgaaga

3360

aagaatttag ctgcatggtg ggactttctg atcatacaac agataatctg gcatgccttg

3420

gcgcggtcgt tcttggagcg tgtgtcttag aaagacattt tacagattca atgcatcgca

3480

gcggaccgga tattgtttgt tctatggata caaaagcctt aaaagaactg atcattcaat

3540

cagaacagat ggcaatcatt cgcggcaata acgaatcaaa gaaagcagcc aaacaagaac

3600

aggttacaat cgattttgct tttgcctctg tggtctcaat caaagatatt aagaaaggcg

3660

aagttctgtc aatggataat atttgggtga aaagaccggg acttggcgga atcagcgcag

3720

cggaatttga aaatattctg ggcaagaaag cactgcgcga tattgaaaac gatgcgcagt

3780

taagctatga agattttgct taagctctta aggaggattt tagaatgaag aaaattctgt

3840

ttatcacagg ctcaagagcg gattactcta aaatcaaatc acttatgtac cgcgtccaaa

3900

attcaagcga atttgaactt tacatctttg ctacaggcat gcatctgagc aaaaactttg

3960

gatacacagt taaagaatta tataaaaatg gctttaaaaa catctacgaa tttatcaact

4020

acgataaata ttatcagaca gataaagccc tggcaacaac aattgatgga ttttcaagat

4080

atgcgaacga attaaaaccg gatctgattg ttgtgcatgg cgatcgcatc gaaccgcttg

4140

cagcggctat tgtcggagcc cttaacaaca tcctggttgc acatatcgaa ggcggagaaa

4200

ttagcggaac aatcgatgat tctttaagac atgcgatttc aaaacttgct catatccatc

4260

tggtgaacga tgaatttgca aaaagacgcc ttatgcaatt aggcgaagat gaaaaatcaa

4320

tctttatcat cggatctccg gatctggaac tgcttaacga taacaaaatc tcacttagcg

4380

aagccaaaaa atactacgat attaactacg aaaactatgc attactgatg tttcatccgg

4440

tcacaacaga aatcacatct atcaaaaacc aagccgataa cctggtgaaa gcacttatcc

4500

agtcaaacaa aaactacatc gtcatctatc cgaataacga tctgggcttt gaactgatcc

4560

ttcagagcta tgaagaattt aaaaacaacc cgagatttaa actgtttccg tctctgcgct

4620

ttgaatactt tatcacactt ctgaaaaacg ccgattttat tatcggaaac tcttcatgca

4680

tcctgaaaga agcgttatac ctgaaaacag ctggcattct ggttggatca agacaaaatg

4740

gccgcttagg aaatgaaaac acactgaaag tgaatgcgaa cagcgatgaa atccttaaag

4800

ctatcaacac aatccataaa aaacaggatt tattttctgc taaactggaa attcttgata

4860

gctctaaact gtttttcgaa tatcttcaat caggcgattt ctttaaactt agcacacaga

4920

aagtttttaa agatattaaa taaaaaggag gaactactat gtcacttgca attatcccgg

4980

cgagaggcgg aagcaaaggc atcaaaaaca aaaacctggt tctgcttaac aacaaaccgc

5040

ttatctacta cacaatcaaa gcagcgctga atgctaaatc tatttcaaaa gttgtggtct

5100

caagcgatag cgatgaaatc cttaactacg ccaaatctca aaacgtggat attctgaaaa

5160

gaccgatctc tcttgcacag gatgatacaa catcagataa agtcttactg catgcgctga

5220

aattttacaa agattacgaa gatgttgtgt ttttacaacc gacatctccg ctgcgcacaa

5280

acattcatat caacgaagca tttaacctgt acaaaaattc aaacgctaat gccctgatta

5340

gcgtctctga atgcgataac aaaatcctta aagcatttgt ttgcaacgat tgtggcgatt

5400

tagccggaat ttgtaatgat gaatatccgt ttatgccgcg ccagaaactg ccgaaaacat

5460

atatgagcaa cggagcgatc tacatcctta aaatcaaaga atttctgaac aacccgtcat

5520

ttctgcaaag caaaacaaaa cattttctta tggatgaatc atcatcactg gatattgatt

5580

gcctggaaga tttaaagaaa gttgaacaaa tttggaaaaa ataaagatta actaataagg

5640

aggacaaaca tgaaaagaat cttttgcctt gtgtctgcaa tcctgctttc agcgtgtaat

5700

gataaccaga atacagtcga tgttgtggtc tcaacagtga acgataacgt catcgaaaac

5760

aacacatacc aggtcaaacc gatcgataca ccgacaacat ttgatagcta ttcttggatt

5820

caaacatgcg gcacaccgat ccttaaagat gatgaaaaat actcactgtc atttgatttt

5880

gttgctccgg aattagatca ggatgaaaaa ttttgttttg aatttacagg cgatgttgat

5940

ggaaaacgct atgtgacaca aacaaatctg acagttgtgg caccgacact tgaagtgtat

6000

gtcgatcatg cgagcctgcc gtctcttcaa cagctgatga aaatcatcca acagaaaaac

6060

gaatactctc agaacgaaag atttatctca tggggccgca tcggacttac agaagataac

6120

gctgaaaaac tgaatgccca tatttatccg ctggctggaa ataacacatc tcaagaatta

6180

gtcgatgcag ttatcgatta cgcggattca aaaaacagac tgaatctgga acttaacaca

6240

aacacagctc atagctttcc gaacttagcc ccgattctgc gcattatctc aagcaaaagc

6300

aacatcctga tctctaacat caacctttac gatgatggct cagccgaata cgtgaacctg

6360

tacaactgga aagatacaga agataaaagc gtcaaactgt ctgattcatt tttagttctg

6420

aaagattact ttaatggaat ttcttcagaa aaaccgagcg gcatctatgg aagatataac

6480

tggcatcagc tgtacaacac atcttactat tttctgagaa aagattatct gacagtcgaa

6540

ccgcaactgc atgatcttcg cgaatatctt ggcggatcat taaaacagat gagctgggat

6600

ggcttttcac aactgagcaa aggagataaa gaactgtttc tgaacattgt tggctttgat

6660

caagaaaaac tgcaacagga atatcaacag agcgaacttc cgaattttgt gtttacagga

6720

acaacaacat gggcaggcgg agaaacaaaa gaatattatg cgcaacagca agttaacgtc

6780

gttaacaacg caatcaacga aacatctccg tactacctgg gcagagaaca tgatcttttc

6840

tttaaaggac atccgcgcgg cggaattatc aatgatatta tcctgggctc atttaataac

6900

atgattgata ttccggcgaa agttagcttt gaagtgctta tgatgacagg aatgttaccg

6960

gatacagtgg gcggaattgc atcatcactg tattttagca tcccggccga aaaagtctct

7020

tttatcgttt ttacatcaag cgatacaatc acagatcgcg aagatgctct gaaatctccg

7080

cttgttcagg tgatgatgac actgggcatt gtgaaagaaa aagatgtcct gttttggtca

7140

gatttaccgg attgctcttc aggagtttgt attgcccaat attaaagtga tagcggtacc

7200

attataggta agagaggaat gtacacatgt actatttaaa aaacacaaac ttttggatgt

7260

tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg tttttcccga

7320

tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt tttgccgcta

7380

tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac aaactcgggc

7440

tgcgcaaata cctgctgtgg attattaccg gcatgttagt gatgtttgcg ccgttcttta

7500

tttttatctt cggggccactg ttacaataca acattttagt aggatcgatt gttggtggta

7560

tttatctagg cttttgtttt aacgccggtg cgccagcagt agaggcattt attgagaaag

7620

tcagccgtcg cagtaatttc gaatttggtc gcgcgcggat gtttggctgt gttggctggg

7680

cgctgtgtgc ctcgattgtc ggcatcatgt tcaccatcaa taatcagttt gttttctggc

7740

tgggctctgg ctgtgcactc atcctcgccg ttttactctt tttcgccaaa acggatgcgc

7800

cctcttctgc cacggttgcc aatgcggtag gtgccaacca ttcggcattt agccttaagc

7860

tggcactgga actgttcaga cagccaaaac tgtggttttt gtcactgtat gttattggcg

7920

tttcctgcac ctacgatgtt tttgaccaac agtttgctaa tttctttact tcgttctttg

7980

ctaccggtga acagggtacg cgggtatttg gctacgtaac gacaatgggc gaattactta

8040

acgcctcgat tatgttcttt gcgccactga tcattaatcg catcggtggg aaaaacgccc

8100

tgctgctggc tggcactatt atgtctgtac gtattattgg ctcatcgttc gccacctcag

8160

cgctggaagt ggttattctg aaaacgctgc atatgtttga agtaccgttc ctgctggtgg

8220

gctgctttaa atatattacc agccagtttg aagtgcgttt ttcagcgacg atttatctgg

8280

tctgtttctg cttctttaag caactggcga tgatttttat gtctgtactg gcgggcaata

8340

tgtatgaaag catcggtttc cagggcgctt atctggtgct gggtctggtg gcgctgggct

8400

tcaccttaat ttccgtgttc acgcttagcg gccccggccc gctttccctg ctgcgtcgtc

8460

aggtgaatga agtcgcttaa ggatccatgt ctagagtcga cgtccccggg gcagcccgcc

8520

taatgagcgg gcttttttca cgtcccaggc atcaaataaa acgaaaggct cagtcgaaag

8580

actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg

8640

gctcaccttc gggtggggcct ttctgcgttt atacccgggg cagcccgcct aatgagcggg

8700

cttttttcac gtcacgcgtc catggagatc tttgtctgca actgaaaagt ttatacctta

8760

cctggaacaa atggttgaaa catacgaggc taatatcggc ttattaggaa tagtccctgt

8820

actaataaaa tcaggtggat cagttgatca gtatattttg gacgaagctc ggaaagaatt

8880

tggagatgac ttgcttaatt ccacaattaa attaagggaa agaataaagc gatttgatgt

8940

tcaaggaatc acggaagaag atactcatga taaagaagct ctaaaactat tcaataacct

9000

tacaatggaa ttgatcgaaa gggtggaagg ttaatggtac gaaaattagg ggatctacct

9060

agaaagccac aaggcgatag gtcaagctta aagaaccctt acatggatct tacagattct

9120

gaaagtaaag aaacaacaga ggttaaacaa acagaaccaa aaagaaaaaa agcattgttg

9180

aaaacaatga aagttgatgt ttcaatccat aataagatta aatcgctgca cgaaattctg

9240

gcagcatccg aagggaattc atattactta gaggatacta ttgagagagc tattgataag

9300

atggttgaga cattacctga gagccaaaaa actttttatg aatatgaatt aaaaaaaaga

9360

accaacaaag gctgagacag actccaaacg agtctgtttt tttaaaaaaa atattaggag

9420

cattgaatat atattagaga attaagaaag acatgggaat aaaaatattt taaatccagt

9480

aaaaatatga taagattatt tcagaatatg aagaactctg tttgtttttg atgaaaaaac

9540

aaacaaaaaa aatccaccta acggaatctc aatttaacta acagcggcca aactgagaag

9600

ttaaatttga gaaggggaaa aggcggattt atacttgtat ttaactatct ccattttaac

9660

attttattaa accccataca agtgaaaatc ctcttttaca ctgttccttt aggtgatcgc

9720

ggagggacat tatgagtgaa gtaaacctaa aaggaaatac agatgaatta gtgtattatc

9780

gacagcaaac cactggaaat aaaatcgcca ggaagagaat caaaaaaggg aaagaagaag

9840

tttattatgt tgctgaaacg gaagagaaga tatggacaga agagcaaata aaaaactttt

9900

ctttagacaa atttggtacg catatacctt acatagaagg tcattataca atcttaaata

9960

attacttctt tgatttttgg ggctattttt taggtgctga aggaattgcg ctctatgctc

10020

acctaactcg ttatgcatac ggcagcaaag acttttgctt tcctagtcta caaacaatcg

10080

ctaaaaaaat ggacaagact cctgttacag ttagaggcta cttgaaactg cttgaaaggt

10140

acggttttat ttggaaggta aacgtccgta ataaaaccaa ggataacaca gaggaatccc

10200

cgatttttaa gattagacgt aaggttcctt tgctttcaga agaactttta aatggaaacc

10260

ctaatattga aattccagat gacgaggaag cacatgtaaa gaaggcttta aaaaaggaaa

10320

aagagggtct tccaaaggtt ttgaaaaaag agcacgatga atttgttaaa aaaatgatgg

10380

atgagtcaga aacaattaat attccagagg ccttacaata tgacacaatg tatgaagata

10440

tactcagtaa aggagaaatt cgaaaagaaa tcaaaaaaca aatacctaat cctacaacat

10500

cttttgagag tatatcaatg acaactgaag aggaaaaagt cgacagtact ttaaaaagcg

10560

aaatgcaaaa tcgtgtctct aagccttctt ttgatacctg gtttaaaaac actaagatca

10620

aaattgaaaa taaaaattgt ttattacttg taccgagtga atttgcattt gaatggatta

10680

agaaaagata tttagaaaca attaaaacag tccttgaaga agctggatat gttttcgaaa

10740

aaatcgaact aagaaaagtg caataaactg ctgaagtatt tcagcagttt tttttattta

10800

gaaatagtga aaaaaatata atcagggagg tatcaatatt taatgagtac tgatttaaat

10860

ttatttagac tggaattaat aattaacacg tagactaatt aaaatttaat gagggataaa

10920

gaggatacaa aaatattaat ttcaatccct attaaatttt aacaaggggg ggattaaaat

10980

ttaattagag gtttatccac aagaaaagac cctaataaaa tttttactag ggttataaca

11040

ctgattaatt tcttaatggg ggagggatta aaatttaatg acaaagaaaa caatctttta

11100

agaaaagctt ttaaaagata ataataaaaa gagctttgcg attaagcaaa actcttttact

11160

ttttcattga cattatcaaa ttcatcgatt tcaaattgtt gttgtatcat aaagttaatt

11220

ctgttttgca caaccttttc aggaatataa aacacatctg aggcttgttt tataaactca

11280

gggtcgctaa agtcaatgta acgtagcata tgatatggta tagcttccac ccaagttagc

11340

ctttctgctt cttctgaatg tttttcatat acttccatgg gtatctctaa atgattttcc

11400

tcatgtagca aggtatgagc aaaaagttta tggaattgat agttcctctc tttttcttca

11460

acttttttat ctaaaacaaa cactttaaca tctgagtcaa tgtaagcata agatgttttt

11520

ccagtcataa tttcaatccc aaatctttta gacagaaatt ctggacgtaa atcttttggt

11580

gaaagaattt ttttatgtag caatatatcc gatacagcac cttctaaaag cgttggtgaa

11640

tagggcattt tacctatctc ctctcatttt gtggaataaa aatagtcata ttcgtccatc

11700

tacctatcct attatcgaac agttgaactt tttaatcaag gatcagtcct ttttttcatt

11760

attcttaaac tgtgctctta actttaacaa ctcgatttgt ttttccagat ctcgagggta

11820

actagcctcg ccgatcccgc aagaggcccg gcagtcaggt ggcacttttc ggggaaatgt

11880

gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag

11940

acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca

12000

tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc

12060

agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat

12120

cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc

12180

aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg

12240

gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc

12300

agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat

12360

aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga

12420

gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc

12480

ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc

12540

aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt

12600

aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc

12660

tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc

12720

agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca

12780

ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca

12840

ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt

12900

ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta

12960

acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg

13020

agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc

13080

ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag

13140

cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa

13200

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc

13260

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc

13320

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta

13380

caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag

13440

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct

13500

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga

13560

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc

13620

ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt

13680

atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg

13740

cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg

13800

catgc

13805

<210> 4

<211> 10838

<212> DNA

<213> Artificial sequence

<220>

<223> Suicide plasmid

<400> 4

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 aattttacat ttttagaaat gggcgtgaaa aaaagcgcgc gattatgtaa

3180

aatataaaga ttaactaata aggaggacaa acatgacaga acagtacatc aaaaacgttg

3240

aagtgtatct tgattatgca acaatcccga cactgaacta cttttaccat tttacagaaa

3300

acaaagatga tattgcgaca attagactgt ttggccttgg acgctttaac atctctaaat

3360

caatcatcga atcatatccg gaaggcatta tcagatattg cccgattatc tttgaagatc

3420

agacagcttt tcaacagctg tttatcacac tgcttacaga agattctttt tgtcaatacc

3480

gctttaactt tcatattaac ctgtttcatt catggaaaat gcttatcccg ctgctgcata

3540

ttatctggca atttaaacat aaagtcctgg atattaaact taacttttat gatgatggct

3600

ctgaaggact ggttacactg tcaaaaatcg aacaaaacta ttcaagcgaa attcttcaga

3660

aaatcatcga tattgatagc caatcttttt acgctgataa acttagcttt ttagatgaag

3720

atattgccag atatttatgg aatagcctgt ttgaatcaca ttactatctt ttaaacgatt

3780

ttctgcttaa aaacgaaaaa ctgagcttac tgaaaaactc tatcaaatac tgccatatca

3840

tggatcttga acgctattta caatttacac aggaagaaaa agatttcttt aacgaacttc

3900

tgggcattaa catccagagc cttgaagata aaatcaaaat ctttcaacaa aagaaaacat

3960

ttatttttac aggaacaaca atcttttctc tgccgaaaga agaagaagaa acactttaca

4020

gactgcatct gaacgctatc cttaactaca tccatccgaa cggcaaatac tttatcggcg

4080

atggatttac actggtgatc aaaggacatc cgcatcaaaa agaaatgaat agcagactgg

4140

aaaaatcatt tgaaaaagcc gtcatgctgc cggataacat cccgtttgaa attctttacc

4200

tgatcggctg caaaccggat aaaattggcg gatttgtttc aacaagctac ttttcatgtg

4260

ataagaaaaa tattgcggat ctgctgttta tcagcgcgag acaggaagaa gtgcgcaaaa

4320

acgattatct ttttaacatc caataccagc tgagagatat gatgatcaaa acaggattta

4380

tccaagaaga aaaaacacat ttttacagcg atattccgat ctttatttct taaaaaggag

4440

gaactactat gtcacttgca attatcccgg cgagaggcgg aagcaaaggc atcaaaaaca

4500

aaaacctggt tctgcttaac aacaaaccgc ttatctacta cacaatcaaa gcagcgctga

4560

atgctaaatc tatttcaaaa gttgtggtct caagcgatag cgatgaaatc cttaactacg

4620

ccaaatctca aaacgtggat attctgaaaa gaccgatctc tcttgcacag gatgatacaa

4680

catcagataa agtcttactg catgcgctga aattttacaa agattacgaa gatgttgtgt

4740

ttttacaacc gacatctccg ctgcgcacaa acattcatat caacgaagca tttaacctgt

4800

acaaaaattc aaacgctaat gccctgatta gcgtctctga atgcgataac aaaatcctta

4860

aagcatttgt ttgcaacgat tgtggcgatt tagccggaat ttgtaatgat gaatatccgt

4920

ttatgccgcg ccagaaactg ccgaaaacat atatgagcaa cggagcgatc tacatcctta

4980

aaatcaaaga atttctgaac aacccgtcat ttctgcaaag caaaacaaaa cattttctta

5040

tggatgaatc atcatcactg gatattgatt gcctggaaga tttaaagaaa gttgaacaaa

5100

tttggaaaaa ataagctctt aaggaggatt ttagaatgag cacaacaaca caaaatatcc

5160

cgtggtatcg ccatcttaac agagcccagt ggcgcgcatt ttctgcagcg tggttaggct

5220

atctgcttga tggatttgat tttgttctga ttgcactggt tcttacagaa gtgcaaggcg

5280

aatttggact tacaacagtg caggctgcct ctttaatttc agcagcgttt atcagcagat

5340

ggtttggcgg attaatgctg ggcgcaatgg gagatcgcta tggaagacgc ctggcgatgg

5400

tcacatctat tgttctgttt tcagcgggca cattagcttg cggctttgcc ccgggatata

5460

ttacaatgtt tatcgcgaga cttgtgattg gcatgggaat ggcaggcgaa tatggatcaa

5520

gcgcgacgta tgttatcgaa tcttggccga aacatctgcg caataaagca tcaggatttc

5580

ttattagcgg cttttctgtg ggagcggttg tggctgccca agtctattct cttgtcgttc

5640

cggtttgggg atggagagca ctgtttttca ttggcatcct tccgattatc tttgctcttt

5700

ggttacgcaa aaatatcccg gaagccgaag attggaaaga aaaacatgcc ggaaaagcac

5760

ctgtgagaac aatggtcgat attctgtata gaggcgaaca tcgcattgct aacatcgtta

5820

tgacattagc agcggctaca gctctgtggt tttgctttgc cggaaattta caaaacgccg

5880

caatcgttgc tgtgctgggc ttactgtgtg cggctatttt tatcagcttt atggttcagt

5940

ctgccggaaa acgctggccg acaggcgtga tgttaatggt ggtcgttctg tttgcttttc

6000

tttattcatg gccgattcaa gcccttttac cgacatacct gaaaacagat ttagcttaca

6060

atccgcatac agtggccaac gtcctgtttt tctcaggctt tggagccgca gttggctgct

6120

gtgtgggcgg atttcttggc gattggttag gaacaagaaa agcgtatgtt tgttcactgc

6180

ttgcgagcca gttactgatt atcccggttt ttgcaatcgg cggagcgaat gtctgggttc

6240

ttggactttt actgtttttc caacagatgt taggccaagg aattgctggc atcttaccga

6300

aactgattgg cggatatttt gatacagatc agagagcggc tggccttgga tttacatata

6360

atgttggagc actgggcgga gcccttgcac cgattatcgg agcgttaatt gctcaacgcc

6420

tggatcttgg cacagcgctt gcttctttat catttagcct gacatttgtg gtcattcttt

6480

taatcggcct tgatatgccg tcaagagttc agcgctggtt aagaccggaa gcactgagaa

6540

cacatgatgc gattgatggc aaaccgtttt caggagctgt gccgtttggc agcgccaaaa

6600

acgatctggt caaaacaaaa tcataaccag gcatcaaata aaacgaaagg ctcagtcgaa

6660

agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac

6720

tggctcacct tcgggtgggc ctttctgcgt ttatatacgc gttaacccgg gcccgcggat

6780

ggatatgatc agatccttta actctggcaa ccctcaaaat tgaatgagac atgctacacc

6840

tccggataat aaatatatat aaacgtatat agatttcata aagtctaaca cactagactt

6900

atttacttcg taattaagtc gttaaaccgt gtgctctacg accaaaacta taaaaccttt

6960

aagaactttc tttttttaca agaaaaaaga aattagataa atctctcata tcttttattc

7020

aataatcgca tccgattgca gtataaattt aacgatcact catcatgttc atatttatca

7080

gagctcgtgc tataattata ctaattttat aaggaggaaa aaatatgggc atttttagta

7140

tttttgtaat cagcacagtt cattatcaac caaacaaaaa ataagtggtt ataatgaatc

7200

gttaataagc aaaattcata taaccaaatt aaagagggtt ataatgaacg agaaaaatat

7260

aaaacacagt caaaacttta ttacttcaaa acataatata gataaaataa tgacaaatat

7320

aagattaaat gaacatgata atatctttga aatcggctca ggaaaaggcc attttaccct

7380

tgaattagta aagaggtgta atttcgtaac tgccattgaa atagaccata aattatgcaa

7440

aactacagaa aataaacttg ttgatcacga taatttccaa gttttaaaca aggatatatt

7500

gcagtttaaa tttcctaaaa accaatccta taaaatatat ggtaatatac cttataacat

7560

aagtacggat ataatacgca aaattgtttt tgatagtata gctaatgaga tttatttaat

7620

cgtggaatac gggtttgcta aaagattatt aaatacaaaa cgctcattgg cattactttt

7680

aatggcagaa gttgatattt ctatattaag tatggttcca agagaatatt ttcatcctaa

7740

acctaaagtg aatagctcac ttatcagatt aagtagaaaa aaatcaagaa tatcacacaa

7800

agataaacaa aagtataatt atttcgttat gaaatgggtt aacaaagaat acaagaaaat

7860

atttacaaaa aatcaattta acaattcctt aaaacatgca ggaattgacg atttaaacaa

7920

tattagcttt gaacaattct tatctctttt caatagctat aaattattta ataagtaagc

7980

gaggcaggat caggacaatg attatatttt tgtcatgaat ttcacggaag aaaaacagct

8040

ggtcacgttt gatcagagtg tgaaggacat aatgacagga gacatattgt caggcgacct

8100

gacgatggaa aagtatgaag tgagaattgt cgtaaacaca cattaggctg atgctccgct

8160

cgatatgggc ggattctttt ttctatagaa tgaaaacgct tgctaagtct tggggggatg

8220

aaatcatgaa aagcaaagtg aaaatgttct ttgcggctgc catcgtgtgg agtgcatgta

8280

gttcaacagg atatgccgct gccattgaga aggagaagca cgtgtcagag cttcgggcag

8340

aggatctttt tgttaaaaaa gtagagggga tgaacaagga ttttatcaaa ggggcagatg

8400

tatccagcgt tattgctttg gaaaacagcg gtgtcacctt ttacaataca aacggaaaac

8460

gccaggatat ctttacaact ttaaaacagg ctggggtcaa ctatgttcgc gtccgcatct

8520

ggaatcaccc gtatgattca aatggcaacg ggtatggcgg gggaaacaat gatgttcaaa

8580

aagccatcga aatcggaaaa agagcgacag cgaacggaat gaaggtgctg gccgactttc

8640

actactctga tttctgggcc gatccagcga aacaaaaggt gcccaaagcc tgggcgaatc

8700

tcagctttga agcaaaaaaa gcaaagctct atgagtatac gaaacaaagc ctgcaaaaga

8760

tgatcaagga aggcgtgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca

8820

tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc

8880

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg

8940

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt

9000

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa

9060

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct

9120

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc

9180

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg

9240

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct

9300

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag

9360

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga

9420

agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga

9480

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg

9540

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag

9600

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag

9660

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat

9720

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct

9780

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac

9840

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa

9900

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg

9960

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt

10020

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca

10080

ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt

10140

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct

10200

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg

10260

cagcactgca taattctctt actngtcatgc catccgtaag atgcttttct gtgactggtg

10320

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg

10380

cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa

10440

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt

10500

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt

10560

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt

10620

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca

10680

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat

10740

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata

10800

aaaataggcg tatcacgagg ccctttcgtc ttcaagaa

10838

<210> 5

<211> 4254

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 5

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

<---

Claims (16)

1. A non-sporulating Bacillus cell for producing a sialylated oligosaccharide, wherein said Bacillus cell is genetically engineered to contain a lactose permease, a CMP-NeuNAc biosynthetic pathway, and a sialyltransferase, wherein the sporulating ability of the Bacillus cell is impaired by deletion or functional inactivation of one or more genes encoding Spo0A , sigma E and sigma F. 2. The Bacillus cell according to claim 1, wherein the lactose permease is E. coli LacY or a functional variant thereof. 3. Bacillus cell according to any one of paragraphs. 1, 2, where the CMP-NeuNAc biosynthetic pathway uses GlcN-1P or GlcNAc-6P as an intermediate. 4. Bacillus cell according to any one of paragraphs. 1-3, where the CMP-NeuNAc biosynthetic pathway utilizes the sialic acid utilization pathway. 5. Bacillus cell according to any one of paragraphs. 1-4, wherein the sialyltransferase is a lactose-accepting sialyltransferase, preferably a sialyltransferase selected from the group consisting of α-2,3-sialyltransferases, α-2,6-sialyltransferases and α-2,8-sialyltransferases. 6. Bacillus cell according to any one of paragraphs. 1-5, 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. 7. The Bacillus cell of claim 6, wherein the Bacillus cell is genetically engineered by deletion or functional inactivation of at least one of the genes selected from the group consisting of yesZ and ganA. 8. Bacillus cell according to any one of paragraphs. 1-7, wherein said Bacillus cell is a Bacillus subtilis cell. 9. Bacillus cell according to any one of paragraphs. 1-8, for use in the production of 3'-sialyllactose, wherein the CMP-NeuNAc biosynthetic pathway uses GlcN-1P as an intermediate and where the sialyltransferase is an α-2,3-sialyltransferase. 10. Bacillus cell according to any one of paragraphs. 1-8, for use in the production of 6'-sialyllactose, wherein the CMP-NeuNAc biosynthetic pathway uses GlcN-1P as an intermediate and where the sialyltransferase is an α-2,6-sialyltransferase. 11. Use of a Bacillus cell according to any one of paragraphs. 1-10 to obtain sialylated oligosaccharide. 12. A method for producing sialylated oligosaccharide, including: - providing a non-sporulating Bacillus cell as defined in any one of paragraphs. 1-10; - culturing said Bacillus cell in a fermentation broth and under conditions that are permissive for the production of sialylated oligosaccharide; and, perhaps, - extraction of sialylated oligosaccharide from the medium and/or Bacillus cells. 13. The method according to claim 12, wherein the fermentation broth contains lactose.

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