Fructosyltransferases

The present invention is in the field of enzymatic production of biomolecules. The invention is particularly concerned with two novel fructosyltransferases derived from lactobacilli and with a process for recombinant production of the enzymes and for the production of useful levans, inulins and fructo-oligosaccharides from sucrose.

BACKGROUND OF THE INVENTION

Lactic acid bacteria (LAB) play an important role in the fermentative production of food and feed. Traditionally, these bacteria have been used for the production of for instance wine, beer, bread, cheese and yoghurt, and for the preservation of food and feed, e.g. olives, pickles, sausages, sauerkraut and silage. Because of these traditional applications, lactic acid bacteria are food-grade micro-organisms that posses the Generally Recognised As Safe (GRAS) status. Due to the different products which are formed during fermentation with lactic acid bacteria, these bacteria contribute positively to the taste, smell and preservation of the final product. The group of lactic acid bacteria encloses several genera such as Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, etc.

In recent years also the health promoting properties of lactic acid bacteria have received much attention. They produce an abundant variety of exopolysaccharides (EPS's). These polysaccharides are thought to contribute to human health by acting as prebiotic substrates, nutraceuticals, cholesterol lowering agents or immunomodulants.

To date high molecular weight polysaccharides produced by plants (such as cellulose, starch and pectin), seaweeds (such as alginate and carrageenan) and bacteria (such as alginate, gellan and xanthan) are used in several industrial applications as viscosifying, stabilising, emulsifying, gelling or water binding agents. Although all these polysaccharides are used as food additives, they originate from organisms not having the GRAS status. Thus they are less desirable than the exopolysaccharides of microorganisms, such as lactic acid bacteria, which have the GRAS status.

The exopolysaccharides produced by LAB can be divided in two groups, heteropolysaccharides and homopolysaccharides; these are synthesized by totally different mechanisms. The former consist of repeating units in which residues of different types of sugars are present and the latter consist of one type of monosaccharide. The synthesis of heteropolysaccharides by lactic acid bacteria, including lactobacilli, has been studied extensively in recent years. Considerably less information is available on the synthesis of homopolysaccharides from lactobacilli, although some studies have been performed. Homopolysaccharides with fructose as the constituent sugar can be divided into two groups, inulins and levans. Inulins consist of 2,1-linked β-fructofuranoside residues, whereas levans consist of 2,6-linked β-fructofuranoside residues. Both can be linear or branched. The size of bacterial levans can vary from 20 kDa up to several MDa. There is limited information on the synthesis of levans. In most detail this synthesis has been studied in

Zymomonas mobilis

and in Bacillus species. Within lactic acid bacteria, fructosyltransferases have only been studied in streptococci. So far no fructosyltransferases have been reported in lactobacilli.

In a recent report the

Lactobacillus reuteri

strain LB 121 was found to produce both a glucan and a fructan when grown on sucrose, but only a fructan when grown on raffinose (van Geel-Schutten, G. H. et al., Appl. Microbiol. Biotechnol. (1998) 50, 697-703). In another report the glucan and fructan were characterised by their molecular weights (of 3,500 and 150 kDa respectively) and the glucan was reported to be highly branched with a unique structure consisting of a terminal, 4-substituted, 6-substituted, and 4,6-disubstituted α-glucose in a molar ratio 1.1: 2.7:1.5:1.0 (van Geel-Schutten, G. H. et al., Appl. Environ. Microbiol. (1999) 65, 3008-3014). The fructan was identified as a linear (2→6)-β-D-fructofuranan (also called a levan). This was the first example of fructan synthesis by a Lactobacillus species.

SUMMARY OF THE INVENTION

Two novel genes encoding enzymes having fructosyltransferase activity have now been found in

Lactobacillus reuteri

, and their amino acid sequences have been determined. These are the first two enzymes identified in a Lactobacillus species capable of producing a fructan. One of the enzymes is an inulosucrase which produces a high molecular weight (>10

7

Da) fructan containing β(2-1) linked fructosyl units and fructo-oligosaccharides, while the other is a levansucrase which produces a fructan containing β(2-6) linked fructosyl units. The invention thus pertains to the enzymes, to DNA encoding them, to recombinant cells containing such DNA and to their use in producing carbohydrates, as defined in the appending claims.

DESCRIPTION OF THE INVENTION

It was found according to the invention that one of the novel fructosyltransferases (FTFA; an inulosucrase) produces a high molecular weight inulin with β(2-1) linked fructosyl units and fructo-oligosaccharides. The fructo-oligosaccharides synthesis was also observed in certain Lactobacillus strains, in particular in certain strains of

Lactobacillus reuteri

. However, the inulin has not been found in

Lactobacillus reuteri

culture supernatants, but only in extracts of

E. coli

cells expressing the above-mentioned fructosyltransferase. This inulosucrase consists of either 798 amino acids (2394 nucleotides) or 789 amino acids (2367 nucleotides) depending on the potential start codon used. The molecular weight (MW) deduced of the amino acid sequence of the latter form is 86 kDa and its isoelectric point is 4.51, at pH 7.

The amino acid sequence of the inulosucrase is shown in SEQ ID No. 1 (

FIG. 1

, amino acid residues 1-789). As mentioned above, the nucleotide sequence contains two putative start codons leading to either a 2394 (see SEQ ID No. 3) or 2367 (see SEQ ID No. 2) nucleotide form of the inulinsucrase. Both putative start codons are preceded by a putative ribosome binding site, GGGG (located 12 base pairs upstream its start codon) or AGGA (located 14 base pairs upstream its start codon), respectively (see FIG.

1

and SEQ ID No. 4).

The present invention covers a protein having inulosucrase activity with an amino acid identity of at least 65%, preferably at least 75%, and more preferably at least 85%, compared to the amino acid sequence of SEQ ID No. 1. The invention also covers a part of a protein with at least 15 contiguous amino acids which are identical to the corresponding part of the amino acid sequence of SEQ ID No. 1.

Fructosyltransferases have been found in several bacteria such as

Zymomonas mobilis, Erwinia amylovora, Acetobacter amylovora, Bacillus polymyxa, Bacillus amyloliquefaciens, Bacillus stearothermophilus

, and

Bacillus subtilis

. In lactic acid bacteria this type of enzyme previously has only been found in some streptococci. Most bacterial fructosyltransferases have a molecular mass of 50-100 kDa (with the exception of the fructosyltransferase found in

Streptococcus salivarius

which has a molecular mass of 140 kDa). Amino acid sequence alignment revealed that the novel inulosucrase of lactobacilli has high homology with fructosyltransferases originating from Gram positive bacteria, in particular with Streptococcus enzymes. The highest homology (

FIG. 2

) was found with the SacB enzyme of

Streptococcus mutans

Ingbritt A (62% identity within 539 amino acids).

Certain putative functions based on the alignment and site-directed mutagenesis studies can be ascribed to several amino acids of the novel inulosucrase. Asp-263, Glu-330, Asp-415, Glu-431, Asp-511, Glu-514, Arg-532 and/or Asp-551 of the amino acid sequence of SEQ ID No. 1 are identified as putative catalytic residues. Noteworthy, a hydrophobicity plot according to Kyte and Doolittle (1982) J. Mol. Biol. 157, 105-132 suggests that the novel inulosucrase contains a putative signal sequence according to the Von Heijne rule. The putative signal peptidase site is located between Gly at position 21 and Ala at position 22. Furthermore, it is striking that the C-terminal amino acid sequence of the novel inulosucrase contains a putative cell wall anchor amino acid signal LPXTG (SEQ ID No. 5) and a 20-fold repeat of the motif PXX (residues 690-749 of SEQ ID NO: 1) (see figure

1

.), where P is proline and X is any other amino acid. In 15 out of 20 repeats, however, the motif is PXT. This motif has so far not been reported in proteins of prokaryotic and eukaryotic origin.

A nucleotide sequence encoding any of the above mentioned proteins, mutants, variants or parts thereof is also a subject of the invention. Furthermore, the nucleic acid sequences corresponding to expression-regulating regions (promoters, enhancers, terminators) of at least 30 contiguous nucleic acids contained in the nucleic acid sequence (-67)-(-1) or 2367-2525 of SEQ ID No. 4 (see also

FIG. 1

) can be used for homologous or heterologous expression of genes. Such expression-regulating sequences are operationally linked to a polypeptide-encoding nucleic acid sequence such as the genes of the fructosyltransferase according to the invention. A nucleic acid construct comprising the nucleotide sequence operationally linked to an expression-regulating nucleic acid sequence is also covered by the invention.

A recombinant host cell, such as a mammalian (with the exception of human), plant, animal, fungal or bacterial cell, containing one or more copies of the nucleic acid construct mentioned above is an additional subject of the invention. The inulosucrase gene (starting at nucleotide 41) has been cloned in an

E. coli

expression vector under the control of an ara promoter in

E. coli

Top10.

E. coli

Top10 cells expressing the recombinant inulosucrase hydrolysed sucrose and synthesized fructan material. SDS-PAGE of arabinose induced

E. coli

Top10 cell extracts suggested that the recombinant inulosucrase has a molecular weight of 80-100 kDa, which is in the range of other known fructosyltransferases and in line with the molecular weight of 86 kDa deduced of the amino acid sequence depicted in FIG.

1

.

The invention further covers an inulosucrase according to the invention which, in the presence of sucrose, produces a inulin having β(2-1)-linked D-fructosyl units and fructo-oligosaccharides. Two different types of fructans, inulins and levans, exist in nature. Surprisingly, the novel inulosucrase expressed in

E. coli

Top10 cell synthesizes a high molecular weight (>10

7

Da) inulin and fructo-oligosaccharides, while in

Lactobacillus reuteri

culture supernatants, in addition to the fructo-oligosaccharides, a levan and not an inulin is found. This discrepancy can have several explanations: the inulosucrase gene may be silent in

Lactobacillus reuteri

, or may not be expressed in

Lactobacillus reuteri

under the conditions tested, or the inulosucrase may only synthesize fructo-oligosaccharides in its natural host, or the inulin polymer may be degraded shortly after synthesis, or may not be secreted and remains cell-associated, or the inulosucrase may have different activities in

Lactobacillus reuteri

and

E. coli

Top10 cells.

It was furthermore found according to the invention that certain lactobacilli, in particular

Lactobacillus reuteri

, possess another fructosyltransferase, a levansucrase (FTFB), in addition to the inulosucrase described above. The N-terminal amino acid sequence of the fructosyltransferase purified from Lactobacillus reuteri supernatant was found to be QVESNNYNGVAEVNTERQANGQI (residues 2-24 of SEQ ID No. 6). Furthermore, three internal sequences were identified, namely (M)(A)HLDVWDSWPVQDP(V) (SEQ ID No. 7), NAGSIFGT(K) (SEQ ID No. 8), V(E) (E) VYSPKVSTLMASDEVE (SEQ ID No. 9). The N-terminal amino acid sequence could not be identified in the deduced inulosucrase sequence. Also the amino acid sequences of the three internal peptide fragments of the purified fructosyltransferase were not present in the putative inulosucrase sequence. Evidently, the inulosucrase gene does not encode the purified fructosyltransferase synthesizing the levan. The complete amino acid sequence of the levansucrase is shown in SEQ ID No. 11 and the nucleotide sequence is shown in SEQ ID No. 10. The levansucrase comprises a putative membrane anchor (see amino acids 761-765 in SEQ ID No. 11) and a putative membrane spanning domain (see amino acids 766-787 in SEQ ID No. 11). The fructan produced by the levansucrase was identified in the Lactobacillus reuteri culture supernatant as a linear (2→6)-β-D-fructofuranan with a molecular weight of 150 kDa. The purified enzyme also produces this fructan.

Additionally, the invention thus covers a protein having levansucrase activity with an amino acid identity of at least 65%, preferably at least 75%, and more preferably at least 85%, compared to the amino acid sequence of SEQ ID NO. 11. The second novel fructosyltransferase produces a high molecular weight fructan with β(2-6) linked fructosyl units with sucrose or raffinose as substrate. The invention also covers a part of a protein with least 15 contiguous amino acids, which are identical to the corresponding part of the amino acid sequence of SEQ ID No. 11. A nucleotide sequence encoding any of the above-mentioned proteins, mutants, variants or parts thereof is a subject of the invention as well as a nucleic acid construct comprising the nucleotide sequence mentioned above operationally linked to an expression-regulating nucleic acid sequence. A recombinant host cell, such as a mammalian (with the exception of human), plant, animal, fungal or bacterial cell, containing one or more copies of the nucleic acid construct mentioned above is an additional subject of the invention. The invention further covers a protein according to the invention which, in the presence of sucrose, produces a fructan having β(2-6)-linked D-fructosyl units.

The invention also pertains to a process of producing an inulin-type and/or a levan-type of fructan as described above using fructosyltransferases according to the invention and a suitable fructose source such as sucrose, stachyose or raffinose. The fructans may either be produced by Lactobacillus strains or recombinant host cells according to the invention containing one or both fructosyl transferases or by a fuctosyltransferase enzyme isolated by conventional means from the culture of fructosyltransferase-positive lactobacilli, especially a

Lactobacillus reuteri

, or from a recombinant organism containing the fructosyltransferase gene or genes.

Additionally, the invention concerns a process of producing fructo-oligosaccharides containing the characteristic structure of the fructans described above using a Lactobacillus strain or a recombinant host cell according to the invention containing one or both fructosyltransferases or an isolated fructosyltransferase according to the invention. There is a growing interest in oligosaccharides derived from homopolysaccharides, for instance for prebiotic purposes. Several fructo- and gluco-oligosaccharides are known to stimulate the growth of bifidobacteria in the human colon. Fructo-oligosaccharides produced by the fructosyltransferase described above are also part of the invention. Another way of producing fructo-oligosaccharides is by hydrolysis of the fructans described above. This hydrolysis can be performed by known hydrolysis methods such as enzymatic hydrolysis with enzymes such as levanase or inulinase or by acid hydrolysis. The fructo-oligosaccharides can also be produced in the presence of a fructosyltransferase according to the invention and an acceptor molecule such as lactose or maltose. The fructo-oligosaccharides to be produced according to the invention prefarably contain at least 2, more preferably at least 3, up to about 20 anhydrofructose units, optionally in addition to one or more other (glucose, galactose, etc.) units. These fructo-oligosaccharides are useful as prebiotics, and can be administered to a mammal in need of improving the bacterial status of the colon.

The invention also concerns chemically modified fructans and fructo-oligosaccharides based on the fructans described above. Chemical modification can be achieved by oxidation, such as hypochlorite oxidation resulting in ring-opened 2,3-dicarboxy-anhydrofructose units (see e.g. EP-A-427349), periodate oxidation resulting in ring-opened 3,4-dialdehyde-anhydrofructose units (see e.g. WO 95/12619), which can be further oxidised to (partly) carboxylated units (see e.g. WO 00/26257), TEMPO-mediated oxidation resulting in 1- or 6-carboxy-anhydrofructose units (see e.g. WO 95/07303). The oxidised fructans have improved water-solubility, altered viscosity and a retarded fermentability and can be used as metal-complexing agents, detergent additives, strengthening additives, bioactive carbohydrates, emulsifiers and water binding agents. They can also be used as starting materials for further derivatisation such as cross-linking and the introduction of hydrophobes. Oxidised fructans coupled to amino compounds such as proteins, or fatty acids can be used as emulsifiers and stabilizers. (Partial) hydrolysis of fructans according to the invention and modified fructans according to the invention results in fructo-oligosaccharides, which can be used as bioactive carbohydrates or prebiotics. The oxidised fructans of the invention preferably contain 0.05-1.0 carboxyl groups per anhydrofructose unit, e.g. as 6- or 1-carboxyl units.

Another type of chemical modification is phosphorylation, as described in O.B. Wurzburg (1986) Modified Starches: properties and uses. CRC Press Inc., Boca Raton, 97-112. One way to achieve this modification is by dry heating fructans with a mixture of monosodium and disodium hydrogen phosphate or with tripolyphosphate. The phosphorylated fructans are suitable as wet-end additives in papermaking, as binders in paper coating compositions, as warp sizing-agents, and as core binders for sand molds for metal casting. A further type of derivatisation of the fructans is acylation, especially acetylation using acetic or propionic anhydride, resulting in products suitable as bleaching assistants and for the use in foils. Acylation with e.g. alkenyl succinic anhydrides or (activated) fatty acids results in surface-active products suitable as e.g. surfactants, emulsifiers, and stabilizers.

Hydroxyalkylation, carboxymethylation, and aminoalkylation are other methods of chemical derivatisation of the fructans. Hydroxyalkylation is commonly performed by base-catalysed reaction with alkylene oxides, such as ethylene oxide, propylene oxide or epichlorohydrine; the hydroxyalkylated products have improved solubility and viscosity characteristics. Carboxymethylation is achieved by reaction of the fructans with mono-chloroacetic acid or its alkali metal salts and results in anionic polymers suitable for various purposes including crystallisation inhibitors, and metal complexants. Amino-alkylation can be achieved by reaction of the fructans with alkylene imines, haloalkyl amines or amino-alkylene oxides, or by reaction of epichlorohydrine adducts of the fructans with suitable amines. These products can be used as cationic polymers in a variety of applications, especially as a wet-end additive in paper making to increase strength, for filler and fines retention, and to improve the drainage rate of paper pulp. Other potential applications include textile sizing and wastewater purification. The above mentioned modifications can be used either separately or in combination depending on the desired product. Furthermore, the degree of chemical modification is variable and depends on the intended use. If necessary 100% modification, i.e. modification of all anhydrofructose units can be performed. However, partial modification, e.g. from 1 modified anhydrofructose unit per 100 up to higher levels, will often be sufficient in order to obtain the desired effect. The modified fructans have a DP (degree of polymerisation) of at least 100, preferably at least 1000 units.

Use of a Lactobacillus strain capable of producing a levan, inulin or fructo-oligosaccharides or a mixture thereof, as a probiotic, is also covered by the invention. Preferably, the Lactobacillus strain is also capable of producing a glucan, especially an 1,4/1,6-α-glucan as referred to above. The efficacy of some

Lactobacillus reuteri

strains as a probiotics has been demonstrated in various animals such as for instance poultry and humans. The administration of some

Lactobacillus reuteri

strains to pigs resulted in significantly lower serum total and LDL-cholesterol levels, while in children

Lactobacillus reuteri

is used as a therapeutic agent against acute diarrhea. For this and other reasons

Lactobacillus reuteri

strains, which were not reported to produce the glucans or fructans described herein, have been supplemented to commercially available probiotic products. The mode of action of

Lactobacillus reuteri

as a probiotic is still unclear. Preliminary studies indicated that gut colonization by

Lactobacillus reuteri

may be of importance. According to the invention, it was found that the mode of action of

Lactobacillus reuteri

as a probiotic may reside partly in the ability to produce polysaccharides. Lactobacillus strains, preferably

Lactobacillus reuteri

strains, and more preferably

Lactobacillus reuteri

strain LB 121 and other strains containing one or more fructosyltransferase genes encoding proteins capable of producing inulins, levans and/or fructo-oligosaccharides can thus advantageously be used as a probiotic. They can also, together with these polysaccharides, be used as a symbiotic (instead of the term symbiotic, the term synbiotic can also be used). In that respect another part of the invention concerns a probiotic or symbiotic composition containing a Lactobacillus strain capable of producing an inulin, a levan or fructo-oligosaccharides and/or a glucan or a mixture thereof, said production being performed according to the process according to the invention. The probiotic or symbiotic compositions of the invention may be directly ingested with or without a suitable vehicle or used as an additive in conjunction with foods. They can be incorporated into a variety of foods and beverages including, but not limited to, yoghurts, ice creams, cheeses, baked products such as bread, biscuits and cakes, dairy and dairy substitute foods, confectionery products, edible oil compositions, spreads, breakfast cereals, juices and the like.

Furthermore, the invention pertains to a process of improving the microbial status in the mammalian colon comprising administering an effective amount of a Lactobacillus strain capable of producing an oligosaccharide or polysaccharide according ot the invention and to a process of improving the microbial status of the mammalian colon comprising administering an effective amount of an oligosaccharide or polysaccharide produced according to the process according ot the invention.

EXAMPLES

EXAMPLE 1

Isolation of DNA from

Lactobacillus reuteri

Nucleotide Sequence Analysis of the Inulosucrase (ftfA) Gene, Construction of Plasmids for Expression of the Inulosucrase Gene in

E. coli

Top10 Expression of the Inulosucrase gene in

E. coli

Top10 and Identification of the Produced Polysaccharides Produced by the Recombinant Enzyme.

General procedures for cloning, DNA manipulations and agarose gel electrophoresis were essentially as described by Sambrook et al. (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. Restriction endonuclease digestions and ligations with T4 DNA ligase were performed as recommended by the suppliers. DNA was amplified by PCR techniques using ampliTAQ DNA polymerase (Perkin Elmer) or Pwo DNA polymerase. DNA fragments were isolated from agarose gels using the Qiagen extraction kit (Qiagen GMBH), following the instructions of the suppliers.

Lactobacillus reuteri

strain 121 (LMG 18388) was grown at 37° C. in MRS medium (DIFCO) or in MRS-s medium (MRS medium containing 100 g/l sucrose instead of 20 g/l glucose). When fructo-oligosaccharides production was investigated phosphate was omitted and ammonium citrate was replaced by ammonium nitrate in the MRS-s medium.

E. coli

strains were grown aerobically at 37° C. in LB medium, where appropriate supplemented with 50 μg/ml ampicillin (for selection of recombinant plasmids) or with 0.02% (w/v) arabinose (for induction of the inulosucrase gene).

Total DNA of

Lactobacillus reuteri

was isolated according to Verhasselt et al. (1989) FEMS Microbiol. Lett. 59, 135-140 as modified by Nagy et al. (1995) J. Bacteriol. 177, 676-687.

The inulosucrase gene was identified by amplification of chromosomal DNA of

Lactobacillus reuteri

with PCR using degenerated primers (5 ftf, 6 ftfi, and 12 ftfi, see table 1) based on conserved amino acid sequences deduced from different bacterial fructosyltranferase genes (SacB of

Bacillus amyloliquefaciens

, SacB of

Bacillus subtilis, Streptococcus mutans

fructosyltransferase and

Streptococcus salivarius

fructosyltransferase, see

FIG. 4

) and

Lactobacillus reuteri

DNA as template. Using primers 5 ftf and 6 ftfi, an amplification product with the predicted size of about 234 bp was obtained (FIG.

5

A). This 234 bp fragment was cloned in

E. coli

JM109 using the pCR2.1 vector and sequenced. Transformations were performed by electroporation using the BioRad gene pulser apparatus at 2.5 kV, 25 μF and 200 Ω, following the instructions of the manufacturer. Sequencing was performed according to the method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. Analysis of the obtained sequence data confirmed that part of a fructosyltransferase (ftf gene had been isolated. The 234 bp amplified fragment was used to design primers 7 ftf and 8 ftfi (see table 1). PCR with the primers 7 ftf and 12 ftfi gave a product of the predicted size of 948 bp (see FIG.

5

B); its sequence showed clear similarity with previously characterized fructosyltransferase genes. The 948 bp amplified fragment was used to design the primers ftfAC1(i) and ftfAC2(i) (see table 1) for inverse PCR. Using inverse PCR techniques a 1438 bp fragment of the inulosucrase gene was generated, including the 3′ end of the inulosucrase gene (see FIG.

5

C). The remaining 5′ fragment of the inulosucrase gene was isloated with a combination of standard and inverse PCR techniques. Briefly,

Lactobacillus reuteri

DNA was cut with restriction enzyme XhoI and ligated. PCR with the primers 7 ftf and 8 ftfi, using the ligation product as a template, yielded a 290 bp PCR product which was cloned into pCR2.1 and sequenced. This revealed that primer 8 ftfi had annealed aspecifically as well as specifically yielding the 290 bp product (see FIG.

5

D).

At this time, the N-terminal amino acid sequence of a fructosyltransferase enzyme (FTFB) purified from the Lactobacillus reuteri strain 121 was obtained. This sequence consisted of the following 23 amino acids: QVESNNYNGVAEVNTERQANGQI (residues 2-24 of SEQ ID No. 6). The degenerated primer 19 ftf (YNGVAEV) (residues 8-14 of SEQ ID NO: 6) was designed on the basis of a part of this N-terminal peptide sequence and primer 20 ftfi was designed on the 290 bp PCR product. PCR with primers 19 ftf and 20 ftfi gave a 754 bp PCR product (see FIG.

5

E), which was cloned into pCR2.1 and sequenced. Both DNA strands of the entire fructosyltransferase gene were double sequenced. In this way the sequence of a 2.6 kb region of the

Lactobacillus reuteri

DNA, containing the inulosucrase gene and its surroundings were obtained.

The plasmids for expression of the inulosucrase gene in

E. coli

Top10 were constructed as described hereafter. A 2414 bp fragment, containing the inulosucrase gene starting at the first putative start codon at position 41, was generated by PCR, using primers ftfA1 and ftfA2i. Both primers contained suitable restriction enzyme recognition sites (a NcoI site at the 5′end of ftfA1 and a BglII site at the 3′end of ftfA2i). PCR with

Lactobacillus reuteri

DNA, Pwo DNA polymerase and primers ftfA1 and ftfA2i yielded the complete inulosucrase gene flanked by NcoI and BglII restriction sites. The PCR product with blunt ends was ligated directly into pCRbluntII-Topo. Using the NcoI and BglII restriction sites, the putative ftfA gene was cloned into the expression vector pBAD, downstream of the inducible arabinose promoter and in frame upstream of the Myc epitope and the His tag. The pBAD vector containing the inulosucrase gene (pSVH101) was transformed to

E. coli

Top10 and used to study inulosucrase expression. Correct construction of plasmid containing the complete inulosucrase gene was confirmed by restriction enzyme digestion analysis and by sequence analysis, showing an in frame cloning of the inulosucrase gene using the ribosomal binding site provided by the pBAD vector and the first putative start codon (at position 41) of inulosucrase (see FIG.

1

).

Plasmid DNA of

E. coli

was isolated using the alkaline lysis method of Birnboim and Doly (1979) Nucleic Acids Res. 7, 1513-1523 or with a Qiagen plasmid kit following the instructions of the supplier. Cells of

E. coli

Top10 with pSVH101 were grown overnight in LB medium containing 0.02% (w/v) arabinose and were harvested by centrifugation. The pellet was washed with 25 mM sodium acetate buffer pH 5.4 and the suspension was centrifuged again. Pelleted cells were resuspended in 25 mM sodium acetate buffer pH 5.4. Cells were broken by sonication. Cell debris and intact cells were removed by centrifugation for 30 min at 4° C. at 10,000×g and the resulting cell free extract was used in the enzyme assays.

The fructosyltranferase activities were determined at 37° C. in reaction buffer (25 mM sodium acetate, pH 5.4, 1 mM CaCl

2

, 100 g/l sucrose) by monitoring the release of glucose from sucrose, by detecting fructo-oligosaccharides or by determining the amount of fructan polymer produced using

E. coli

cell free extracts or

Lactobacillus reuteri

culture supernatant as enzyme source. Sucrose, glucose and fructose were determined enzymatically using commercially available kits.

Fructan production by

Lactobacillus reuteri

was studied with cells grown in MRS-s medium. Product formation was also studied with cell-free extracts of

E. coli

containing the novel inulosucrase incubated in reaction buffer (1 mg protein/10 ml buffer, incubated overnight at 37° C.). Fructans were collected by precipitation with ethanol.

1

H-NMR spectroscopy and methylation analysis were performed as described by van Geel-Schutten et al. (1999) Appl. Environ. Microbiol. 65, 3008-3014. The molecular weights of the fructans were determined by high performance size exclusion chromatography coupled on-line with a multi angle laser light scattering and a differential refractive index detector. Fructo-oligosaccharide synthesis was studied in

Lactobacillus reuteri

culture supernatants and in extracts of

E. coli

cells containing the novel inulosucrase incubated in reaction buffer (1 mg protein/10 ml buffer, incubated overnight at 37° C.). Glucose and fructose were determined enzymatically as described above and fructo-oligosaccharides produced were analyzed using a Dionex column. The incubation mixtures were centrifuged for 30 min at 10,000×g and diluted 1:5 in a 100% DMSO solution prior to injection on a Dionex column. A digest of inulin (DP1-20) was used as a standard. Separation of compounds was achieved with anion-exchange chromatography on a CarboPac Pa1 column (Dionex) coupled to a CarboPac PA1 guard column (Dionex). Using a Dionex GP50 pump the following gradient was generated: % eluent B is 5% (0 min); 35% (10 min); 45% (20 min); 65% (50 min); 100% (54-60 min); 5% (61-65 min). Eluent A was 0.1 M NaOH and eluent B was 0.6 M NaAc in a 0.1 M NaOH solution. Compounds were detected using a Dionex ED40 electrochemical detector with an AU working electrode and a Ag/AgCl reference-electrode with a sensitivity of 300 nC. The pulse program used was: +0.1 Volt (0-0.4 s); +0.7 Volt (0.41-0.60 s); −0.1 Volt (0.61-1.00 s). Data were integrated using a Perkin Elmer Turbochrom data integration system. A different separation of compounds was done on a cation exchange column in the calcium form (Benson BCX4). As mobile phase Ca-EDTA in water (100 ppm) was used. The elution speed was 0.4 ml/min at a column temperature of 85° C. Detection of compounds was done by a refractive index (Jasco 830-RI) at 40° C. Quantification of compounds was achieved by using the software program Turbochrom (Perkin Elmer).

SDS-PAGE was performed according to Laemmli (1970) Nature 227, 680-685 using 7.5% polyacrylamide gels. After electrophoresis gels were stained with Coomassie Briljant Blue or an activity staining (Periodic Acid Schiff, PAS) was carried out as described by Van Geel-Schutten et al. (1999) Appl. Environ. Microbiol. 65, 3008-3014.

TABLE 1

Nucleotide sequence of primers used in PCR reactions to identify the

inulosucrase gene.

Primer

Location

name

(bp)

Nucleotide sequence (and SEQ ID No)

ftfAC1

1176

CTG-ATA-ATA-ATG-GAA-ATG-TAT-CAC

(SEQ ID No. 12)

ftfAC2i

1243

CAT-GAT-CAT-AAG-TTT-GGT-AGT-AAT-AG

(SEQ ID No. 13)

ftfac1

1176

GTG-ATA-CAT-TTC-CAT-TAT-TAT-CAG

(SEQ ID No. 14)

ftfAC2

1243

CTA-TTA-CTA-CCA-AAC-TTA-TGA-TCA-TG

(SEQ ID No. 15)

ftfA1

CCA-TGG-CCA-TGG-TAG-AAC-GCA-AGG-

AAC-ATA-AAA-AAA-TG

(SEQ ID No. 16)

ftfA2i

AGA-TCT-AGA-TCT-GTT-AAA-TCG-ACG-TTT-

GTT-AAT-TTC-TG

(SEQ ID No. 17)

5ftf

845

GAY-GTN-TGG-GAY-WSN-TGG-GCC

(SEQ ID No. 18)

6ftfi

1052

GTN-GCN-SWN-CCN-SWC-CAY-TSY-TG

(SEQ ID No. 19)

7ftf

1009

GAA-TGT-AGG-TCC-AAT-TTT-TGG-C

(SEQ ID No. 20)

8ftfi

864

CCT-GTC-CGA-ACA-TCT-TGA-ACT-G

(SEQ ID No. 21)

12ftfi

1934

ARR-AAN-SWN-GGN-GCV-MAN-GTN-SW

(SEQ ID No. 22)

19ftf

1

TAY-AAY-GGN-GTN-GCN-GAR-GTN-AA

(SEQ ID No. 23)

20ftfi

733

CCG-ACC-ATC-TTG-TTT-GAT-TAA-C

(SEQ ID No. 24)

Listed from left to right are: primer name (i, inverse primer), location (in bp) in ftfA and the sequence from 5' to 3' according to IUB group codes (N = any base; M = A or C; R = A or G; W = A or T; S = C or G; Y = C or T; K = G or T; B = not A; D = not C; H = not G; and V = not T).

EXAMPLE 2

Purification and Amino Acid Sequencing of the Levansucrase (FTFB).

Protein Purification

Samples were taken between each step of the purification process to determine the enzyme activity (by glucose GOD-Perid method) and protein content (by Bradford analysis and acrylamide gel electrophoresis). Collected chromatography fractions were screened for glucose liberating activity (GOD-Perid method) to determine the enzyme activity.

One liter of an overnight culture of LB121 cells grown on MRS medium containing 50 grams per liter maltose was centrifuged for 15 min. at 10,000×g. The supernatant was precipitated with 1.5 liter of a saturated ammonium sulphate solution. The ammonium sulphate solution was added at a rate of 50 ml/min. under continuous stirring. The resulting 60% (w/v) ammonium sulphate solution was centrifuged for 15 min. at 10,000×g. The precipitate was resuspended in 10 ml of a sodium phosphate solution (10 mM, pH 6.0) and dialysed overnight against 10 mM sodium phosphate, pH 6.0.

A hydroxylapatite column was washed with a 10 mM sodium phosphate solution pH 6.0; the dialysed sample was loaded on the column. After eluting the column with 200 mM sodium phosphate, pH 6.0 the eluted fractions were screened for glucose releasing activity and fractions were pooled for phenyl superose (a hydrophobic interactions column) chromatography. The pooled fractions were diluted 1:1 (v:v) with 25 mM sodium acetate, 2 M ammonium sulphate, pH 5.4 and loaded on a phenyl superose column (washed with 25 mM sodium acetate, 1 M ammonium sulphate, pH 5.4). In a gradient from 25 mM sodium acetate, 1 M ammonium sulphate, pH 5.4 (A) to 25 mM sodium acetate, pH 5.4 (B) fractions were collected from 35% B to 50% B.

Pooled fractions from the phenyl superose column were loaded on a gel filtration (superdex) column and eluted by a 25 mM acetate, 0.1 M sodium chloride, pH 5.4 buffer. The superdex fractions were loaded on a washed (with 25 mM sodium acetate, pH 5.4) Mono Q column and eluted with 25 mM sodium acetate, 1 M sodium chloride, pH 5.4. The fractions containing glucose liberating activity were pooled, dialysed against 25 mM sodium acetate, pH 5.4, and stored at −20° C.

A levansucrase enzyme was purified from LB121 cultures grown on media containing maltose using ammonium sulfate precipitation and several chromatography column steps (table 2). Maltose (glucose—glucose) was chosen because both glucansucrase and levansucrase can not use maltose as substrate. LB121 will grow on media containing maltose but will not produce polysaccharide. From earlier experiments it was clear that even with harsh methods the levansucrase enzyme could not be separated from its product levan. These harsh methods included boiling the levan in a SDS solution and treating the levan with HCl and TFA. No levanase enzyme was commercially available for the enzymatic breakdown of levan. Only a single levansucrase was detected in maltose culture supernatants. In order to prove that the enzyme purified from maltose culture supernatant is the same enzyme which is responsible for the levan production during growth on raffinose, biochemical and biophysical tests were performed.

TABLE 2

Purification of the

Lactobacillus reuteri

LB 121

levansucrase (FTFB) enzyme

Total

Specific

Protein

Activity

Activity

Purification

Yield

Step

(mg)

(U)

(U/mg)

(fold)

(%)

Supernatant

128

64

0.5

1

100

Ammonium

35.2

42

1.2

2.4

65.6

sulfate

precipitation

(65%)

Hydroxyl

1.5

30.6

20.4

40.8

47.8

apatite

Phenyl

0.27

23

85

170

36

superose

Gel

0.055

10

182

360

16

Filtration

MonoQ

0.0255

4

176

352

6

Amino Acid Sequencing of FTFB

A 5% SDS-PAA gel was allowed to “age” overnight in order to reduce the amount of reacting chemical groups in the gel. Reaction of chemicals in the PAA gel (TEMED and ammonium persulphate) with proteins can cause some undesired effects, such as N-terminal blocking of the protein, making it more difficult to determine the protein amino acid composition. 0.1 mM thioglycolic acid (scavenger to reduce the amount of reactive groups in the PAA gel material) was added to the running buffer during electrophoresis.

In order to determine the amino acid sequence of internal peptides of protein bands running in a SDS-PAA gel, protein containing bands were cut out of the PAA gel. After fractionating the protein by digestion with chymotrypsin the N-terminal amino acid sequences of the digested proteins were determined (below).

N-terminal sequencing was performed by Western blotting of the proteins from the PAA gel to an Immobilon PVDF membrane (Millipore/Waters Inc.) at 0.8 mA/cm

2

for 1 h. After staining the PVDF membrane with Coomassie Brilliant Blue without adding acetic acid (to reduce N-terminal blocking) and destaining with 50% methanol, the corresponding bands were cut out of the PVDF membrane for N-terminal amino acid sequence determination.

Amino acid sequence determination was performed by automated Edman degradation as described by Koningsberg and Steinman (1977) The proteins (third edition) volume 3, 1-178 (Neurath and Hill, eds.). The automated equipment for Edman degradation was an Applied Biosystems model 477A pulse-liquid sequenator described by Hewick et al. (1981), J. Biol. Chem. 15, 7990-7997 connected to a RP-HPLC unit (model 120A, Applied Biosystems) for amino acid identification.

The N-terminal sequence of the purified FTFB was determined and found to be: (A) Q V E S N N Y N G V A E V N T E R Q A N G Q I (G) (V) (D) (SEQ ID No. 6). Three internal peptide sequences of the purified FTFB were determined: (M) (A) H L D V W D S W P V Q D P (V) (SEQ ID No. 7); N A G S I F G T (K) (SEQ ID No. 8); and V (E) (E) V Y S P K V S T L M A S D E V E (SEQ ID No. 9).

The following primers were designed on the basis of the N-terminal and internal peptide fragments of FTFB. Listed from left to right are: primer name, source peptide fragment and sequence (from 5′ to 3′). FTFB1+FTFB3i yields approximately a 1400 bp product in a PCR reaction. FTFB1 forward (N-terminal): AA T/C-TAT-AA T/C-GG T/C-GTT-GC G/A-T/C GA-AGT (SEQ ID No. 25); and FTFB3i reverse (Internal 3): TAC-CGN-A/T C/G N-CTA-CTT-CAA-CTT (SEQ ID No. 26). The FTFB gene was partly isolated by PCR with primers FTFB1 and FTFB3i. PCR with these primers yielded a 1385 bp amplicon, which after sequencing showed high homology to ftfA and SacB from

Streptococcus mutans.

EXAMPLE 3

Oxidation of Levans

For TEMPO-mediated oxidation, a levan according to the invention prepared as described above (dry weight 1 g, 6.15 mmol) was resuspended in 100 ml water. Next, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO; 1% by weight compared to the polysaccharide (0.01 g, 0.065 mmol)) was added and resuspended in 20 min. Sodium bromide (0.75 g, 7.3 mmol) was added and the suspension was cooled down to 0° C. This reaction also proceded without bromide. A solution of hypochlorite (6 ml, 15% solution, 12.6 mmol) was adjusted to pH 10.0 with 3M HCl and cooled to 0° C. This solution was added to the suspension of the polysaccharide and TEMPO. The course of the reaction was followed by monitoring the consumption of sodium hydroxide solution, which is equivalent to the formation of uronic acid. After 30 min, 60 ml 0.1M NaOH was consumed. This amount corresponds to the formation of 97% uronic acid. Thereafter, the solution was poured out in 96% ethanol (comprising 70% of the volume of the solution) causing the product to precipitate. The white precipitate was centrifuged, resuspended in ethanol/water (70/30 v/v) and centrifuged again. Next, the precipitate was resuspended in 96% ethanol and centrifuged. The obtained product was dried at reduced pressure. The uronic acid content was determined by means of the uronic acid assay according to Blumenkrantz and Abdoe-Hansen (Anal. Biochem., 54 (1973), 484). A calibration curve was generated using polygalacturonic acid (5, 10, 15 and 20 μg). With this calibration curve the uronic acid content in a sample of 20 μg of the product was determined. The obtained result was a content of 95% uronic acid with a yield of 96%.

Partial Oxidation

For partial oxidation, a levan according to the invention (dry weight 2 g, 12.3 mmol) was resuspended in 25 ml water. Next, TEMPO (1% by weight compared to the polysaccharide (0.02 g, 0.13 mmol)) was added, resuspended in 20 min and cooled to 0° C. A solution of hypochlorite (1 ml, 15% solution, 2.1 mmol) was adjusted to pH 9.0 with 3M HCl and cooled down to 0° C. This solution was added to the suspension of the polysaccharide and TEMPO. Within 5 min the mixture became a solid gel.

EXAMPLE 4

Adhesion of

Lactobacillus reuteri

Strains to Caco-2 Cell Lines

The adhesion of

Lactobacillus reuteri

strains to Caco-2 cell lines was determined as described below. Firstly, a bacterial suspension was prepared as follows.

Lactobacillus reuteri

strains LB 121, 35-5, K24 and DSM20016 and

L. rhamnosus

LGG (a well known probiotic strain with good adhering properties) were cultured in MRS broth supplemented with 5 μl/ml of methyl-1,2-[

3

H]-thymidine at 37° C. for 18-20 h before the adhesion assays. The cultures were harvested by centrifugation, washed with phosphate buffered saline (PBS) and resuspended in PBS or PBS supplemented with 30 g/l sucrose (see Table 3) to a final density of about 2×10

9

cfu/ml. Prior to the adhesion assay, the cell suspensions in PBS with 30 g/l sucrose were incubated for 1 hour at 37° C., whereas the cell suspensions in PBS were kept on ice for 1 hour. After incubation at 37° C., the suspensions in PBS with sucrose were centrifuged and the cells were washed with and resuspended in PBS to a final density of about 2×10

9

cfu/ml.

Caco-2 cells were cultured as follows. Subcultures of Caco-2 cells (ATCC, code HTB 37, human colon adenocarcinoma), stored as frozen stock cultures in liquid nitrogen were used for the adhesion tests. The Caco-2 cells were grown in culture medium consisting of Dulbecco's modified Eagle medium (DMEM), supplemented with heat-inactivated foetal calf serum (10% v/v), non-essential amino acids (1% v/v), L-glutamine (2 mM) and gentamicin (50 μg/ml). About 2,000,000 cells were seeded in 75 cm

2

tissue culture flasks containing culture medium and cultured in a humidified incubator at 37° C. in air containing 5% CO

2

. Near confluent Caco-2 cell cultures were harvested by trypsinisation and resuspended in culture medium. The number of cells was established using a Bürker-Türk counting chamber.

TABLE 3

Incubation of the different Lactobacillus strains prior to the

adhesion assays.

Lactobacillus

Polysaccharide

strain

Extra incubation

produced

Group

reuteri 121

PBS sucrose, 37° C. for

glucan and fructan

As

1 hr

reuteri 35-5

PBS sucrose, 37° C. for

glucan

Bs

1 hr

reuteri K24

PBS sucrose, 37° C. for

none

Cs

1 hr

reuteri 121

PBS on ice

none

D

reuteri

PBS on ice

none

E

DSM20016*

rhamnosus GG

PBS on ice

none

F

*Type strain of L. reuteri

For the following experiments a Caco-2 monolayer transport system was used. Caco-2 cells cultured in a two-compartment transport system are commonly used to study the intestinal, epithelial permeability. In this system the Caco-2 cell differentiates into polarized columnar cells after reaching confluency. The Caco-2 system has been shown to simulate the passive and active transcellular tranport of electrolytes, sugars, amino acids and lipophilic compounds (Hillgren et al. 1995, Dulfer et al., 1996, Duizer et al., 1997). Also, a clear correlation between the in vivo absorption and the permeability across the monolayers of Caco-2 cells has been reported (Artursson and Karlsson, 1990). For the present transport studies, Caco-2 cells were seeded on semi-permeable filter inserts (12 wells Transwell plates, Costar) at ca. 100,000 cells per filter (growth area ±1 cm

2

containing 2.5 ml culture medium). The cells on the insert were cultured for 17 to 24 days at 37° C. in a humidified incubator containing 5% CO

2

in air. During this culture period the cells have been subjected to an enterocyte-like differentiation. Gentamycin was eliminated from the culture medium two days prior to the adhesion assays.

The adhesion assay was performed as follows. PBS was used as exposure medium. 25 μl of a bacterial suspension (2×10

9

cfu/ml ) were added to 0.5 ml medium. The apical side of the Caco-2 monolayers was incubated with the bacterial suspensions for 1 hour at 37° C. After incubation, remaining fluid was removed and the cells were washed three times with 1 ml PBS. Subsequently, the Caco-2 monolayers were digested overnight with 1 ml 0.1M NaOH, 1% SDS. The lysate was mixed with 10 ml Hionic Fluor scintillation liquid and the radioactivity was measured by liquid scintillation counting using a LKB/Wallac scintillation counter. As a control, the radioactivity of the bacterial suspensions was measured. For each test group, the percentage of bacteria attached to the monolayers was calculated. All adhesion tests were performed in quadruple. In Table 4 the results of the bacterial adhesion test to Caco-2 cellines are given. From the results can be concluded that the glucans and the fructans contribute to the adherence of

Lactobacillus reuteri

to Caco-2 cellines. This could indicate that

Lactobacillus reuteri

strains producing EPS possess improved probiotic characteristics or that

Lactobacillus reuteri

and its polysaccharides could function as an exellent symbiotic.

TABLE 4

The results of the bacterial adhesion test to Caco-2 cellines.

Group

0% of bacteria

(see Table

bound to the

1)

monolayer

As

6.5

Bs

5.7

Cs

1.8

D

2.3

E

0.9

F

1.3

DESCRIPTION OF THE FIGURES

FIG.

1

: The nucleic acid (SEQ ID NO: 4) and deduced amino acid sequences (SEQ ID NOS 27 and 1) of the novel inulosucrase of

Lactobacillus reuteri

. Also encompassed within the figure is the comparison peptide (SEQ ID NO: 28). Furthermore, the designations and orientation (< for 3′ to 5′ and > for 5′ to 3′) of the primers and the restriction enzymes used for (inverse) PCR, are shown at the right hand side. Putative start codons (ATG, at positions 41 and 68) and stop codon (TAA, at position 2435) are shown in bold. The positions of the primers used for PCR are shown in bold/underlined. The NheI restriction sites (at positions 1154 and 2592) used for inverse PCR are underlined. The primers used and their exact positions in the inulosucrase sequence are shown in table 1. Starting at amino acid 690, the 20 PXX (residues 690-749 of SEQ ID NO: 1) repeats are underlined. At amino acid 755 the LPXTG (SEQ ID NO: 5) motif is underlined.

FIG.

2

: Dendrogram of bacterial and plant fructosyltransferases. The horizontal distances are a measure for the difference at the amino acid sequence level. 10% difference is indicated by the upper bar. Bootstrap values (in percentages) are given at the root of each tree. Fructosyltransferases of Gram positive bacteria are indicated in the lower half of the figure (

B. staerothermophilus

SurB;

B. amyloliquefaciens

SacB; B. subtilis SacB;

S. mutans

SacB;

L. reuteri

FtfA (inulosucrase);

S. salivarius

Ftf). Plant fructosyltransferases are indicated in the middle part of the figure (

Cynara scolymus

Ss-1 ft;

Allium cepa

F-6 gft;

Hordeum vulgare

Sf-6 ft). Fructosyltransferases of Gram negative bacteria are shown in the upper part of the figure (

Z. mobilis

LevU;

Z. mobilis

SucE2;

Z mobilis

SacB;

E. amylovora

Lcs;

A. diazotrophicus

LsdA).

FIG.

3

: The N-terminal (SEQ ID NO: 6) and three internal amino acid sequences (SEQ ID NOS 7-9) of the novel levansucrase of

Lactobacillus reuteri.

FIG.

4

: Parts of an alignment of the deduced amino acid sequences of some bacterial fructosyltransferase genes (SEQ ID NOS 29-40). Sequences in bold indicate the consensus sequences used to construct the degenerated primers 5 ftf, 6 ftfi and 12 ftfi. (*) indicates a position with a fully conserved amino acid residue. (:) indicates a position with a fully conserved ‘strong’ group: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. (.) indicates a position with a fully conserved ‘weaker’ group: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY. Goups are according to the Pam250 residue weight matrix described by Altschul et al. (1990) J. Mol. Biol. 215, 403-410.

FIG.

5

: The strategy used for the isolation of the inulosucrase gene from

Lactobacillus reuteri

121 chromosomal DNA.

# SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 40

<210> SEQ ID NO 1

<211> LENGTH: 789

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 1

Met Tyr Lys Ser Gly Lys Asn Trp Ala Val Va

#l Thr Leu Ser Thr Ala

1 5

# 10

# 15

Ala Leu Val Phe Gly Ala Thr Thr Val Asn Al

#a Ser Ala Asp Thr Asn

20

# 25

# 30

Ile Glu Asn Asn Asp Ser Ser Thr Val Gln Va

#l Thr Thr Gly Asp Asn

35

# 40

# 45

Asp Ile Ala Val Lys Ser Val Thr Leu Gly Se

#r Gly Gln Val Ser Ala

50

# 55

# 60

Ala Ser Asp Thr Thr Ile Arg Thr Ser Ala As

#n Ala Asn Ser Ala Ser

65

# 70

# 75

# 80

Ser Ala Ala Asn Thr Gln Asn Ser Asn Ser Gl

#n Val Ala Ser Ser Ala

85

# 90

# 95

Ala Ile Thr Ser Ser Thr Ser Ser Ala Ala Se

#r Leu Asn Asn Thr Asp

100

# 105

# 110

Ser Lys Ala Ala Gln Glu Asn Thr Asn Thr Al

#a Lys Asn Asp Asp Thr

115

# 120

# 125

Gln Lys Ala Ala Pro Ala Asn Glu Ser Ser Gl

#u Ala Lys Asn Glu Pro

130

# 135

# 140

Ala Val Asn Val Asn Asp Ser Ser Ala Ala Ly

#s Asn Asp Asp Gln Gln

145 1

#50 1

#55 1

#60

Ser Ser Lys Lys Asn Thr Thr Ala Lys Leu As

#n Lys Asp Ala Glu Asn

165

# 170

# 175

Val Val Lys Lys Ala Gly Ile Asp Pro Asn Se

#r Leu Thr Asp Asp Gln

180

# 185

# 190

Ile Lys Ala Leu Asn Lys Met Asn Phe Ser Ly

#s Ala Ala Lys Ser Gly

195

# 200

# 205

Thr Gln Met Thr Tyr Asn Asp Phe Gln Lys Il

#e Ala Asp Thr Leu Ile

210

# 215

# 220

Lys Gln Asp Gly Arg Tyr Thr Val Pro Phe Ph

#e Lys Ala Ser Glu Ile

225 2

#30 2

#35 2

#40

Lys Asn Met Pro Ala Ala Thr Thr Lys Asp Al

#a Gln Thr Asn Thr Ile

245

# 250

# 255

Glu Pro Leu Asp Val Trp Asp Ser Trp Pro Va

#l Gln Asp Val Arg Thr

260

# 265

# 270

Gly Gln Val Ala Asn Trp Asn Gly Tyr Gln Le

#u Val Ile Ala Met Met

275

# 280

# 285

Gly Ile Pro Asn Gln Asn Asp Asn His Ile Ty

#r Leu Leu Tyr Asn Lys

290

# 295

# 300

Tyr Gly Asp Asn Glu Leu Ser His Trp Lys As

#n Val Gly Pro Ile Phe

305 3

#10 3

#15 3

#20

Gly Tyr Asn Ser Thr Ala Val Ser Gln Glu Tr

#p Ser Gly Ser Ala Val

325

# 330

# 335

Leu Asn Ser Asp Asn Ser Ile Gln Leu Phe Ty

#r Thr Arg Val Asp Thr

340

# 345

# 350

Ser Asp Asn Asn Thr Asn His Gln Lys Ile Al

#a Ser Ala Thr Leu Tyr

355

# 360

# 365

Leu Thr Asp Asn Asn Gly Asn Val Ser Leu Al

#a Gln Val Arg Asn Asp

370

# 375

# 380

Tyr Ile Val Phe Glu Gly Asp Gly Tyr Tyr Ty

#r Gln Thr Tyr Asp Gln

385 3

#90 3

#95 4

#00

Trp Lys Ala Thr Asn Lys Gly Ala Asp Asn Il

#e Ala Met Arg Asp Ala

405

# 410

# 415

His Val Ile Glu Asp Gly Asn Gly Asp Arg Ty

#r Leu Val Phe Glu Ala

420

# 425

# 430

Ser Thr Gly Leu Glu Asn Tyr Gln Gly Glu As

#p Gln Ile Tyr Asn Trp

435

# 440

# 445

Leu Asn Tyr Gly Gly Asp Asp Ala Phe Asn Il

#e Lys Ser Leu Phe Arg

450

# 455

# 460

Ile Leu Ser Asn Asp Asp Ile Lys Ser Arg Al

#a Thr Trp Ala Asn Ala

465 4

#70 4

#75 4

#80

Ala Ile Gly Ile Leu Lys Leu Asn Lys Asp Gl

#u Lys Asn Pro Lys Val

485

# 490

# 495

Ala Glu Leu Tyr Ser Pro Leu Ile Ser Ala Pr

#o Met Val Ser Asp Glu

500

# 505

# 510

Ile Glu Arg Pro Asn Val Val Lys Leu Gly As

#n Lys Tyr Tyr Leu Phe

515

# 520

# 525

Ala Ala Thr Arg Leu Asn Arg Gly Ser Asn As

#p Asp Ala Trp Met Asn

530

# 535

# 540

Ala Asn Tyr Ala Val Gly Asp Asn Val Ala Me

#t Val Gly Tyr Val Ala

545 5

#50 5

#55 5

#60

Asp Ser Leu Thr Gly Ser Tyr Lys Pro Leu As

#n Asp Ser Gly Val Val

565

# 570

# 575

Leu Thr Ala Ser Val Pro Ala Asn Trp Arg Th

#r Ala Thr Tyr Ser Tyr

580

# 585

# 590

Tyr Ala Val Pro Val Ala Gly Lys Asp Asp Gl

#n Val Leu Val Thr Ser

595

# 600

# 605

Tyr Met Thr Asn Arg Asn Gly Val Ala Gly Ly

#s Gly Met Asp Ser Thr

610

# 615

# 620

Trp Ala Pro Ser Phe Leu Leu Gln Ile Asn Pr

#o Asp Asn Thr Thr Thr

625 6

#30 6

#35 6

#40

Val Leu Ala Lys Met Thr Asn Gln Gly Asp Tr

#p Ile Trp Asp Asp Ser

645

# 650

# 655

Ser Glu Asn Leu Asp Met Ile Gly Asp Leu As

#p Ser Ala Ala Leu Pro

660

# 665

# 670

Gly Glu Arg Asp Lys Pro Val Asp Trp Asp Le

#u Ile Gly Tyr Gly Leu

675

# 680

# 685

Lys Pro His Asp Pro Ala Thr Pro Asn Asp Pr

#o Glu Thr Pro Thr Thr

690

# 695

# 700

Pro Glu Thr Pro Glu Thr Pro Asn Thr Pro Ly

#s Thr Pro Lys Thr Pro

705 7

#10 7

#15 7

#20

Glu Asn Pro Gly Thr Pro Gln Thr Pro Asn Th

#r Pro Asn Thr Pro Glu

725

# 730

# 735

Ile Pro Leu Thr Pro Glu Thr Pro Lys Gln Pr

#o Glu Thr Gln Thr Asn

740

# 745

# 750

Asn Arg Leu Pro Gln Thr Gly Asn Asn Ala As

#n Lys Ala Met Ile Gly

755

# 760

# 765

Leu Gly Met Gly Thr Leu Leu Ser Met Phe Gl

#y Leu Ala Glu Ile Asn

770

# 775

# 780

Lys Arg Arg Phe Asn

785

<210> SEQ ID NO 2

<211> LENGTH: 2367

<212> TYPE: DNA

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 2

atgtataaaa gcggtaaaaa ttgggcagtc gttacactct cgactgctgc gc

#tggtattt 60

ggtgcaacaa ctgtaaatgc atccgcggac acaaatattg aaaacaatga tt

#cttctact 120

gtacaagtta caacaggtga taatgatatt gctgttaaaa gtgtgacact tg

#gtagtggt 180

caagttagtg cagctagtga tacgactatt agaacttctg ctaatgcaaa ta

#gtgcttct 240

tctgccgcta atacacaaaa ttctaacagt caagtagcaa gttctgctgc aa

#taacatca 300

tctacaagtt ccgcagcttc attaaataac acagatagta aagcggctca ag

#aaaatact 360

aatacagcca aaaatgatga cacgcaaaaa gctgcaccag ctaacgaatc tt

#ctgaagct 420

aaaaatgaac cagctgtaaa cgttaatgat tcttcagctg caaaaaatga tg

#atcaacaa 480

tccagtaaaa agaatactac cgctaagtta aacaaggatg ctgaaaacgt tg

#taaaaaag 540

gcgggaattg atcctaacag tttaactgat gaccagatta aagcattaaa ta

#agatgaac 600

ttctcgaaag ctgcaaagtc tggtacacaa atgacttata atgatttcca aa

#agattgct 660

gatacgttaa tcaaacaaga tggtcggtac acagttccat tctttaaagc aa

#gtgaaatc 720

aaaaatatgc ctgccgctac aactaaagat gcacaaacta atactattga ac

#ctttagat 780

gtatgggatt catggccagt tcaagatgtt cggacaggac aagttgctaa tt

#ggaatggc 840

tatcaacttg tcatcgcaat gatgggaatt ccaaaccaaa atgataatca ta

#tctatctc 900

ttatataata agtatggtga taatgaatta agtcattgga agaatgtagg tc

#caattttt 960

ggctataatt ctaccgcggt ttcacaagaa tggtcaggat cagctgtttt ga

#acagtgat 1020

aactctatcc aattatttta tacaagggta gacacgtctg ataacaatac ca

#atcatcaa 1080

aaaattgcta gcgctactct ttatttaact gataataatg gaaatgtatc ac

#tcgctcag 1140

gtacgaaatg actatattgt atttgaaggt gatggctatt actaccaaac tt

#atgatcaa 1200

tggaaagcta ctaacaaagg tgccgataat attgcaatgc gtgatgctca tg

#taattgaa 1260

gatggtaatg gtgatcggta ccttgttttt gaagcaagta ctggtttgga aa

#attatcaa 1320

ggcgaggacc aaatttataa ctggttaaat tatggcggag atgacgcatt ta

#atatcaag 1380

agcttattta gaattctttc caatgatgat attaagagtc gggcaacttg gg

#ctaatgca 1440

gctatcggta tcctcaaact aaataaggac gaaaagaatc ctaaggtggc ag

#agttatac 1500

tcaccattaa tttctgcacc aatggtaagc gatgaaattg agcgaccaaa tg

#tagttaaa 1560

ttaggtaata aatattactt atttgccgct acccgtttaa atcgaggaag ta

#atgatgat 1620

gcttggatga atgctaatta tgccgttggt gataatgttg caatggtcgg at

#atgttgct 1680

gatagtctaa ctggatctta taagccatta aatgattctg gagtagtctt ga

#ctgcttct 1740

gttcctgcaa actggcggac agcaacttat tcatattatg ctgtccccgt tg

#ccggaaaa 1800

gatgaccaag tattagttac ttcatatatg actaatagaa atggagtagc gg

#gtaaagga 1860

atggattcaa cttgggcacc gagtttctta ctacaaatta acccggataa ca

#caactact 1920

gttttagcta aaatgactaa tcaaggggat tggatttggg atgattcaag cg

#aaaatctt 1980

gatatgattg gtgatttaga ctccgctgct ttacctggcg aacgtgataa ac

#ctgttgat 2040

tgggacttaa ttggttatgg attaaaaccg catgatcctg ctacaccaaa tg

#atcctgaa 2100

acgccaacta caccagaaac ccctgagaca cctaatactc ccaaaacacc aa

#agactcct 2160

gaaaatcctg ggacacctca aactcctaat acacctaata ctccggaaat tc

#ctttaact 2220

ccagaaacgc ctaagcaacc tgaaacccaa actaataatc gtttgccaca aa

#ctggaaat 2280

aatgccaata aagccatgat tggcctaggt atgggaacat tgcttagtat gt

#ttggtctt 2340

gcagaaatta acaaacgtcg atttaac

#

# 2367

<210> SEQ ID NO 3

<211> LENGTH: 2394

<212> TYPE: DNA

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 3

atgctagaac gcaaggaaca taaaaaaatg tataaaagcg gtaaaaattg gg

#cagtcgtt 60

acactctcga ctgctgcgct ggtatttggt gcaacaactg taaatgcatc cg

#cggacaca 120

aatattgaaa acaatgattc ttctactgta caagttacaa caggtgataa tg

#atattgct 180

gttaaaagtg tgacacttgg tagtggtcaa gttagtgcag ctagtgatac ga

#ctattaga 240

acttctgcta atgcaaatag tgcttcttct gccgctaata cacaaaattc ta

#acagtcaa 300

gtagcaagtt ctgctgcaat aacatcatct acaagttccg cagcttcatt aa

#ataacaca 360

gatagtaaag cggctcaaga aaatactaat acagccaaaa atgatgacac gc

#aaaaagct 420

gcaccagcta acgaatcttc tgaagctaaa aatgaaccag ctgtaaacgt ta

#atgattct 480

tcagctgcaa aaaatgatga tcaacaatcc agtaaaaaga atactaccgc ta

#agttaaac 540

aaggatgctg aaaacgttgt aaaaaaggcg ggaattgatc ctaacagttt aa

#ctgatgac 600

cagattaaag cattaaataa gatgaacttc tcgaaagctg caaagtctgg ta

#cacaaatg 660

acttataatg atttccaaaa gattgctgat acgttaatca aacaagatgg tc

#ggtacaca 720

gttccattct ttaaagcaag tgaaatcaaa aatatgcctg ccgctacaac ta

#aagatgca 780

caaactaata ctattgaacc tttagatgta tgggattcat ggccagttca ag

#atgttcgg 840

acaggacaag ttgctaattg gaatggctat caacttgtca tcgcaatgat gg

#gaattcca 900

aaccaaaatg ataatcatat ctatctctta tataataagt atggtgataa tg

#aattaagt 960

cattggaaga atgtaggtcc aatttttggc tataattcta ccgcggtttc ac

#aagaatgg 1020

tcaggatcag ctgttttgaa cagtgataac tctatccaat tattttatac aa

#gggtagac 1080

acgtctgata acaataccaa tcatcaaaaa attgctagcg ctactcttta tt

#taactgat 1140

aataatggaa atgtatcact cgctcaggta cgaaatgact atattgtatt tg

#aaggtgat 1200

ggctattact accaaactta tgatcaatgg aaagctacta acaaaggtgc cg

#ataatatt 1260

gcaatgcgtg atgctcatgt aattgaagat ggtaatggtg atcggtacct tg

#tttttgaa 1320

gcaagtactg gtttggaaaa ttatcaaggc gaggaccaaa tttataactg gt

#taaattat 1380

ggcggagatg acgcatttaa tatcaagagc ttatttagaa ttctttccaa tg

#atgatatt 1440

aagagtcggg caacttgggc taatgcagct atcggtatcc tcaaactaaa ta

#aggacgaa 1500

aagaatccta aggtggcaga gttatactca ccattaattt ctgcaccaat gg

#taagcgat 1560

gaaattgagc gaccaaatgt agttaaatta ggtaataaat attacttatt tg

#ccgctacc 1620

cgtttaaatc gaggaagtaa tgatgatgct tggatgaatg ctaattatgc cg

#ttggtgat 1680

aatgttgcaa tggtcggata tgttgctgat agtctaactg gatcttataa gc

#cattaaat 1740

gattctggag tagtcttgac tgcttctgtt cctgcaaact ggcggacagc aa

#cttattca 1800

tattatgctg tccccgttgc cggaaaagat gaccaagtat tagttacttc at

#atatgact 1860

aatagaaatg gagtagcggg taaaggaatg gattcaactt gggcaccgag tt

#tcttacta 1920

caaattaacc cggataacac aactactgtt ttagctaaaa tgactaatca ag

#gggattgg 1980

atttgggatg attcaagcga aaatcttgat atgattggtg atttagactc cg

#ctgcttta 2040

cctggcgaac gtgataaacc tgttgattgg gacttaattg gttatggatt aa

#aaccgcat 2100

gatcctgcta caccaaatga tcctgaaacg ccaactacac cagaaacccc tg

#agacacct 2160

aatactccca aaacaccaaa gactcctgaa aatcctggga cacctcaaac tc

#ctaataca 2220

cctaatactc cggaaattcc tttaactcca gaaacgccta agcaacctga aa

#cccaaact 2280

aataatcgtt tgccacaaac tggaaataat gccaataaag ccatgattgg cc

#taggtatg 2340

ggaacattgc ttagtatgtt tggtcttgca gaaattaaca aacgtcgatt ta

#ac 2394

<210> SEQ ID NO 4

<211> LENGTH: 2592

<212> TYPE: DNA

<213> ORGANISM: Lactobacillus reuteri

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)..(51)

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (68)..(2434)

<400> SEQUENCE: 4

tac aat ggg gtg gcg gag gtg aag aaa cgg gg

#t tac ttc tat gct aga 48

Tyr Asn Gly Val Ala Glu Val Lys Lys Arg Gl

#y Tyr Phe Tyr Ala Arg

1 5

# 10

# 15

acg caaggaacat aaaaaa atg tat aaa agc ggt aaa

#aat tgg gca gtc gtt 100

Thr

# Met Tyr Lys Ser Gly Lys Asn Trp Ala Val

#Val

# 20

# 25

aca ctc tcg act gct gcg ctg gta ttt ggt gc

#a aca act gta aat gca 148

Thr Leu Ser Thr Ala Ala Leu Val Phe Gly Al

#a Thr Thr Val Asn Ala

30

# 35

# 40

tcc gcg gac aca aat att gaa aac aat gat tc

#t tct act gta caa gtt 196

Ser Ala Asp Thr Asn Ile Glu Asn Asn Asp Se

#r Ser Thr Val Gln Val

45

# 50

# 55

# 60

aca aca ggt gat aat gat att gct gtt aaa ag

#t gtg aca ctt ggt agt 244

Thr Thr Gly Asp Asn Asp Ile Ala Val Lys Se

#r Val Thr Leu Gly Ser

65

# 70

# 75

ggt caa gtt agt gca gct agt gat acg act at

#t aga act tct gct aat 292

Gly Gln Val Ser Ala Ala Ser Asp Thr Thr Il

#e Arg Thr Ser Ala Asn

80

# 85

# 90

gca aat agt gct tct tct gcc gct aat aca ca

#a aat tct aac agt caa 340

Ala Asn Ser Ala Ser Ser Ala Ala Asn Thr Gl

#n Asn Ser Asn Ser Gln

95

# 100

# 105

gta gca agt tct gct gca ata aca tca tct ac

#a agt tcc gca gct tca 388

Val Ala Ser Ser Ala Ala Ile Thr Ser Ser Th

#r Ser Ser Ala Ala Ser

110

# 115

# 120

tta aat aac aca gat agt aaa gcg gct caa ga

#a aat act aat aca gcc 436

Leu Asn Asn Thr Asp Ser Lys Ala Ala Gln Gl

#u Asn Thr Asn Thr Ala

125 1

#30 1

#35 1

#40

aaa aat gat gac acg caa aaa gct gca cca gc

#t aac gaa tct tct gaa 484

Lys Asn Asp Asp Thr Gln Lys Ala Ala Pro Al

#a Asn Glu Ser Ser Glu

145

# 150

# 155

gct aaa aat gaa cca gct gta aac gtt aat ga

#t tct tca gct gca aaa 532

Ala Lys Asn Glu Pro Ala Val Asn Val Asn As

#p Ser Ser Ala Ala Lys

160

# 165

# 170

aat gat gat caa caa tcc agt aaa aag aat ac

#t acc gct aag tta aac 580

Asn Asp Asp Gln Gln Ser Ser Lys Lys Asn Th

#r Thr Ala Lys Leu Asn

175

# 180

# 185

aag gat gct gaa aac gtt gta aaa aag gcg gg

#a att gat cct aac agt 628

Lys Asp Ala Glu Asn Val Val Lys Lys Ala Gl

#y Ile Asp Pro Asn Ser

190

# 195

# 200

tta act gat gac cag att aaa gca tta aat aa

#g atg aac ttc tcg aaa 676

Leu Thr Asp Asp Gln Ile Lys Ala Leu Asn Ly

#s Met Asn Phe Ser Lys

205 2

#10 2

#15 2

#20

gct gca aag tct ggt aca caa atg act tat aa

#t gat ttc caa aag att 724

Ala Ala Lys Ser Gly Thr Gln Met Thr Tyr As

#n Asp Phe Gln Lys Ile

225

# 230

# 235

gct gat acg tta atc aaa caa gat ggt cgg ta

#c aca gtt cca ttc ttt 772

Ala Asp Thr Leu Ile Lys Gln Asp Gly Arg Ty

#r Thr Val Pro Phe Phe

240

# 245

# 250

aaa gca agt gaa atc aaa aat atg cct gcc gc

#t aca act aaa gat gca 820

Lys Ala Ser Glu Ile Lys Asn Met Pro Ala Al

#a Thr Thr Lys Asp Ala

255

# 260

# 265

caa act aat act att gaa cct tta gat gta tg

#g gat tca tgg cca gtt 868

Gln Thr Asn Thr Ile Glu Pro Leu Asp Val Tr

#p Asp Ser Trp Pro Val

270

# 275

# 280

caa gat gtt cgg aca gga caa gtt gct aat tg

#g aat ggc tat caa ctt 916

Gln Asp Val Arg Thr Gly Gln Val Ala Asn Tr

#p Asn Gly Tyr Gln Leu

285 2

#90 2

#95 3

#00

gtc atc gca atg atg gga att cca aac caa aa

#t gat aat cat atc tat 964

Val Ile Ala Met Met Gly Ile Pro Asn Gln As

#n Asp Asn His Ile Tyr

305

# 310

# 315

ctc tta tat aat aag tat ggt gat aat gaa tt

#a agt cat tgg aag aat 1012

Leu Leu Tyr Asn Lys Tyr Gly Asp Asn Glu Le

#u Ser His Trp Lys Asn

320

# 325

# 330

gta ggt cca att ttt ggc tat aat tct acc gc

#g gtt tca caa gaa tgg 1060

Val Gly Pro Ile Phe Gly Tyr Asn Ser Thr Al

#a Val Ser Gln Glu Trp

335

# 340

# 345

tca gga tca gct gtt ttg aac agt gat aac tc

#t atc caa tta ttt tat 1108

Ser Gly Ser Ala Val Leu Asn Ser Asp Asn Se

#r Ile Gln Leu Phe Tyr

350

# 355

# 360

aca agg gta gac acg tct gat aac aat acc aa

#t cat caa aaa att gct 1156

Thr Arg Val Asp Thr Ser Asp Asn Asn Thr As

#n His Gln Lys Ile Ala

365 3

#70 3

#75 3

#80

agc gct act ctt tat tta act gat aat aat gg

#a aat gta tca ctc gct 1204

Ser Ala Thr Leu Tyr Leu Thr Asp Asn Asn Gl

#y Asn Val Ser Leu Ala

385

# 390

# 395

cag gta cga aat gac tat att gta ttt gaa gg

#t gat ggc tat tac tac 1252

Gln Val Arg Asn Asp Tyr Ile Val Phe Glu Gl

#y Asp Gly Tyr Tyr Tyr

400

# 405

# 410

caa act tat gat caa tgg aaa gct act aac aa

#a ggt gcc gat aat att 1300

Gln Thr Tyr Asp Gln Trp Lys Ala Thr Asn Ly

#s Gly Ala Asp Asn Ile

415

# 420

# 425

gca atg cgt gat gct cat gta att gaa gat gg

#t aat ggt gat cgg tac 1348

Ala Met Arg Asp Ala His Val Ile Glu Asp Gl

#y Asn Gly Asp Arg Tyr

430

# 435

# 440

ctt gtt ttt gaa gca agt act ggt ttg gaa aa

#t tat caa ggc gag gac 1396

Leu Val Phe Glu Ala Ser Thr Gly Leu Glu As

#n Tyr Gln Gly Glu Asp

445 4

#50 4

#55 4

#60

caa att tat aac tgg tta aat tat ggc gga ga

#t gac gca ttt aat atc 1444

Gln Ile Tyr Asn Trp Leu Asn Tyr Gly Gly As

#p Asp Ala Phe Asn Ile

465

# 470

# 475

aag agc tta ttt aga att ctt tcc aat gat ga

#t att aag agt cgg gca 1492

Lys Ser Leu Phe Arg Ile Leu Ser Asn Asp As

#p Ile Lys Ser Arg Ala

480

# 485

# 490

act tgg gct aat gca gct atc ggt atc ctc aa

#a cta aat aag gac gaa 1540

Thr Trp Ala Asn Ala Ala Ile Gly Ile Leu Ly

#s Leu Asn Lys Asp Glu

495

# 500

# 505

aag aat cct aag gtg gca gag tta tac tca cc

#a tta att tct gca cca 1588

Lys Asn Pro Lys Val Ala Glu Leu Tyr Ser Pr

#o Leu Ile Ser Ala Pro

510

# 515

# 520

atg gta agc gat gaa att gag cga cca aat gt

#a gtt aaa tta ggt aat 1636

Met Val Ser Asp Glu Ile Glu Arg Pro Asn Va

#l Val Lys Leu Gly Asn

525 5

#30 5

#35 5

#40

aaa tat tac tta ttt gcc gct acc cgt tta aa

#t cga gga agt aat gat 1684

Lys Tyr Tyr Leu Phe Ala Ala Thr Arg Leu As

#n Arg Gly Ser Asn Asp

545

# 550

# 555

gat gct tgg atg aat gct aat tat gcc gtt gg

#t gat aat gtt gca atg 1732

Asp Ala Trp Met Asn Ala Asn Tyr Ala Val Gl

#y Asp Asn Val Ala Met

560

# 565

# 570

gtc gga tat gtt gct gat agt cta act gga tc

#t tat aag cca tta aat 1780

Val Gly Tyr Val Ala Asp Ser Leu Thr Gly Se

#r Tyr Lys Pro Leu Asn

575

# 580

# 585

gat tct gga gta gtc ttg act gct tct gtt cc

#t gca aac tgg cgg aca 1828

Asp Ser Gly Val Val Leu Thr Ala Ser Val Pr

#o Ala Asn Trp Arg Thr

590

# 595

# 600

gca act tat tca tat tat gct gtc ccc gtt gc

#c gga aaa gat gac caa 1876

Ala Thr Tyr Ser Tyr Tyr Ala Val Pro Val Al

#a Gly Lys Asp Asp Gln

605 6

#10 6

#15 6

#20

gta tta gtt act tca tat atg act aat aga aa

#t gga gta gcg ggt aaa 1924

Val Leu Val Thr Ser Tyr Met Thr Asn Arg As

#n Gly Val Ala Gly Lys

625

# 630

# 635

gga atg gat tca act tgg gca ccg agt ttc tt

#a cta caa att aac ccg 1972

Gly Met Asp Ser Thr Trp Ala Pro Ser Phe Le

#u Leu Gln Ile Asn Pro

640

# 645

# 650

gat aac aca act act gtt tta gct aaa atg ac

#t aat caa ggg gat tgg 2020

Asp Asn Thr Thr Thr Val Leu Ala Lys Met Th

#r Asn Gln Gly Asp Trp

655

# 660

# 665

att tgg gat gat tca agc gaa aat ctt gat at

#g att ggt gat tta gac 2068

Ile Trp Asp Asp Ser Ser Glu Asn Leu Asp Me

#t Ile Gly Asp Leu Asp

670

# 675

# 680

tcc gct gct tta cct ggc gaa cgt gat aaa cc

#t gtt gat tgg gac tta 2116

Ser Ala Ala Leu Pro Gly Glu Arg Asp Lys Pr

#o Val Asp Trp Asp Leu

685 6

#90 6

#95 7

#00

att ggt tat gga tta aaa ccg cat gat cct gc

#t aca cca aat gat cct 2164

Ile Gly Tyr Gly Leu Lys Pro His Asp Pro Al

#a Thr Pro Asn Asp Pro

705

# 710

# 715

gaa acg cca act aca cca gaa acc cct gag ac

#a cct aat act ccc aaa 2212

Glu Thr Pro Thr Thr Pro Glu Thr Pro Glu Th

#r Pro Asn Thr Pro Lys

720

# 725

# 730

aca cca aag act cct gaa aat cct ggg aca cc

#t caa act cct aat aca 2260

Thr Pro Lys Thr Pro Glu Asn Pro Gly Thr Pr

#o Gln Thr Pro Asn Thr

735

# 740

# 745

cct aat act ccg gaa att cct tta act cca ga

#a acg cct aag caa cct 2308

Pro Asn Thr Pro Glu Ile Pro Leu Thr Pro Gl

#u Thr Pro Lys Gln Pro

750

# 755

# 760

gaa acc caa act aat aat cgt ttg cca caa ac

#t gga aat aat gcc aat 2356

Glu Thr Gln Thr Asn Asn Arg Leu Pro Gln Th

#r Gly Asn Asn Ala Asn

765 7

#70 7

#75 7

#80

aaa gcc atg att ggc cta ggt atg gga aca tt

#g ctt agt atg ttt ggt 2404

Lys Ala Met Ile Gly Leu Gly Met Gly Thr Le

#u Leu Ser Met Phe Gly

785

# 790

# 795

ctt gca gaa att aac aaa cgt cga ttt aac ta

#aatacttt aaaataaaac 2454

Leu Ala Glu Ile Asn Lys Arg Arg Phe Asn

800

# 805

cgctaagcct taaattcagc ttaacggttt tttattttaa aagtttttat tg

#taaaaaag 2514

cgaattatca ttaatactaa tgcaattgtt gtaagacctt acgacagtag ta

#acaatgaa 2574

tttgcccatc tttgtcgg

#

#

#2592

<210> SEQ ID NO 5

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<220> FEATURE:

<221> NAME/KEY: MOD_RES

<222> LOCATION: (3)

<223> OTHER INFORMATION: Any amino acid

<400> SEQUENCE: 5

Leu Pro Xaa Thr Gly

1 5

<210> SEQ ID NO 6

<211> LENGTH: 27

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 6

Ala Gln Val Glu Ser Asn Asn Tyr Asn Gly Va

#l Ala Glu Val Asn Thr

1 5

# 10

# 15

Glu Arg Gln Ala Asn Gly Gln Ile Gly Val As

#p

20

# 25

<210> SEQ ID NO 7

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 7

Met Ala His Leu Asp Val Trp Asp Ser Trp Pr

#o Val Gln Asp Pro Val

1 5

# 10

# 15

<210> SEQ ID NO 8

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 8

Asn Ala Gly Ser Ile Phe Gly Thr Lys

1 5

<210> SEQ ID NO 9

<211> LENGTH: 19

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 9

Val Glu Glu Val Tyr Ser Pro Lys Val Ser Th

#r Leu Met Ala Ser Asp

1 5

# 10

# 15

Glu Val Glu

<210> SEQ ID NO 10

<211> LENGTH: 4634

<212> TYPE: DNA

<213> ORGANISM: Lactobacillus reuteri

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1220)..(3598)

<220> FEATURE:

<221> NAME/KEY: RBS

<222> LOCATION: (1205)..(1210)

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (2702)..(2707)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (3686)..(3698)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<400> SEQUENCE: 10

gttaacaaag acaaaatttt atataattct tcaaattaaa tttcccactg ta

#agaacata 60

aatgggtacc tgtttgatgg gaataatata tttgtaacta accggccggc ac

#ctctttct 120

aatgtgccta ggatgcataa tggatgtaaa ttactagatg gcggttttta ta

#cattaacc 180

tcgcaggaga gaaaagaagc aattagtaag gatccatatg cagataaatt ta

#ttaggcct 240

tatttaggtg ctaaaaattt cattcatgga actgctaggt actgtatttg gt

#taaaggac 300

gcaaacccga aagatatcca tcaatcgcca tttatactgg atagaatcaa ta

#aagtagcg 360

gaattcagat cgcagcaaaa aagtaaagat acacaaaaat atgcaaaacg gc

#ccatgcta 420

acaacacgac ttgcctatta tagccacgat gtacatacgg atatgctgat ag

#tacctgca 480

acatcatcgc aacgtagaga atatcttcca attggatatg tttcagaaaa ga

#atattgtg 540

tcttattcac taatgctaat ccccaatgct agtaatttta atttcggtat tc

#tagaatct 600

aaagttcact atatttggtt aaaaaacttt tgcggtcggt tgaagtccga tt

#atcgttat 660

tcaaacacta ttatttataa taatttccct tggccgactg ttggtgacaa gc

#caggamca 720

acaccatctc tgacactcgc tcaaggtata ttaaatactc gcaagctcta tc

#cagacagc 780

tcactggctg atctttatga tccactaaca atgccragtt gaactcgtaa ag

#ctcatgaa 840

gccaatgata aagctgttct taaagcatat ggattgagcc ctaaagctac tg

#agcaagaa 900

atcgtagaac atctatttaa gatgtatgaa aaactgacta aaggtgaaag at

#aactttgt 960

aaaaccaata ttttataaag acagtaaatg ttaatttgat aaaaacatat at

#ttaataaa 1020

caaaagtgat ataatcaagt agttctttgt attacaaaat acatttaata tc

#tctcagca 1080

ttttgcatac tgggagattt tttattgaca aattgtttga aagtgcttat ga

#tgaaaccg 1140

tgtagaaact aattcaattt gataaacgtt agacatttct gaggaggaag tc

#attttgga 1200

gtacaaagaa cataagaaa atg tat aaa gtc ggc aag aat

# tgg gcc gtt gct 1252

# Met Tyr Lys Val Gly Lys Asn Trp Ala

# Val Ala

# 1

# 5

# 10

aca ttg gta tca gct tca att tta atg gga gg

#g gtt gta acc gct cat 1300

Thr Leu Val Ser Ala Ser Ile Leu Met Gly Gl

#y Val Val Thr Ala His

15

# 20

# 25

gct gat caa gta gaa agt aac aat tac aac gg

#t gtt gct gaa gtt aat 1348

Ala Asp Gln Val Glu Ser Asn Asn Tyr Asn Gl

#y Val Ala Glu Val Asn

30

# 35

# 40

act gaa cgt caa gct aat ggt caa att ggc gt

#a gat gga aaa att att 1396

Thr Glu Arg Gln Ala Asn Gly Gln Ile Gly Va

#l Asp Gly Lys Ile Ile

45

# 50

# 55

agt gct aac agt aat aca acc agt ggc tcg ac

#a aat caa gaa tca tct 1444

Ser Ala Asn Ser Asn Thr Thr Ser Gly Ser Th

#r Asn Gln Glu Ser Ser

60

# 65

# 70

# 75

gct act aac aat act gaa aat gct gtt gtt aa

#t gaa agc aaa aat act 1492

Ala Thr Asn Asn Thr Glu Asn Ala Val Val As

#n Glu Ser Lys Asn Thr

80

# 85

# 90

aac aat act gaa aat gct gtt gtt aat gaa aa

#c aaa aat act aac aat 1540

Asn Asn Thr Glu Asn Ala Val Val Asn Glu As

#n Lys Asn Thr Asn Asn

95

# 100

# 105

act gaa aat gct gtt gtt aat gaa aac aaa aa

#t act aac aac aca gaa 1588

Thr Glu Asn Ala Val Val Asn Glu Asn Lys As

#n Thr Asn Asn Thr Glu

110

# 115

# 120

aac gat aat agt caa tta aag tta act aat aa

#t gaa caa cca tca gcc 1636

Asn Asp Asn Ser Gln Leu Lys Leu Thr Asn As

#n Glu Gln Pro Ser Ala

125

# 130

# 135

gct act caa gca aac ttg aag aag cta aat cc

#t caa gct gct aag gct 1684

Ala Thr Gln Ala Asn Leu Lys Lys Leu Asn Pr

#o Gln Ala Ala Lys Ala

140 1

#45 1

#50 1

#55

gtt caa aat gcc aag att gat gcc ggt agt tt

#a aca gat gat caa att 1732

Val Gln Asn Ala Lys Ile Asp Ala Gly Ser Le

#u Thr Asp Asp Gln Ile

160

# 165

# 170

aat gaa tta aat aag att aac ttc tct aag tc

#t gct gaa aag ggt gca 1780

Asn Glu Leu Asn Lys Ile Asn Phe Ser Lys Se

#r Ala Glu Lys Gly Ala

175

# 180

# 185

aaa ttg acc ttt aag gac tta gag ggg att gg

#t aat gct att gtt aag 1828

Lys Leu Thr Phe Lys Asp Leu Glu Gly Ile Gl

#y Asn Ala Ile Val Lys

190

# 195

# 200

caa gat cca caa tat gct att cct tat tct aa

#t gct aag gaa atc aag 1876

Gln Asp Pro Gln Tyr Ala Ile Pro Tyr Ser As

#n Ala Lys Glu Ile Lys

205

# 210

# 215

aat atg cct gca aca tac act gta gat gcc ca

#a aca ggt aag atg gct 1924

Asn Met Pro Ala Thr Tyr Thr Val Asp Ala Gl

#n Thr Gly Lys Met Ala

220 2

#25 2

#30 2

#35

cat ctt gat gtc tgg gac tct tgg cca gta ca

#a gat cct gtc aca ggt 1972

His Leu Asp Val Trp Asp Ser Trp Pro Val Gl

#n Asp Pro Val Thr Gly

240

# 245

# 250

tat gta tct aat tac atg ggt tat caa cta gt

#t att gct atg atg ggt 2020

Tyr Val Ser Asn Tyr Met Gly Tyr Gln Leu Va

#l Ile Ala Met Met Gly

255

# 260

# 265

att cca aat tcg cca act gga gat aat cat at

#c tat ctt ctt tac aac 2068

Ile Pro Asn Ser Pro Thr Gly Asp Asn His Il

#e Tyr Leu Leu Tyr Asn

270

# 275

# 280

aag tat ggt gat aat gac ttt tct cat tgg cg

#c aat gca ggt tca atc 2116

Lys Tyr Gly Asp Asn Asp Phe Ser His Trp Ar

#g Asn Ala Gly Ser Ile

285

# 290

# 295

ttt gga act aaa gaa aca aat gtg ttc caa ga

#a tgg tca ggt tca gct 2164

Phe Gly Thr Lys Glu Thr Asn Val Phe Gln Gl

#u Trp Ser Gly Ser Ala

300 3

#05 3

#10 3

#15

att gta aat gat gat ggt aca att caa cta tt

#t ttc acc tca aat gat 2212

Ile Val Asn Asp Asp Gly Thr Ile Gln Leu Ph

#e Phe Thr Ser Asn Asp

320

# 325

# 330

acg tct gat tac aag ttg aat gat caa cgc ct

#t gct acc gca aca tta 2260

Thr Ser Asp Tyr Lys Leu Asn Asp Gln Arg Le

#u Ala Thr Ala Thr Leu

335

# 340

# 345

aac ctt aat gtt gat gat aac ggt gtt tca at

#c aag agt gtt gat aat 2308

Asn Leu Asn Val Asp Asp Asn Gly Val Ser Il

#e Lys Ser Val Asp Asn

350

# 355

# 360

tat caa gtt ttg ttt gaa ggt gat gga ttt ca

#c tac caa act tat gaa 2356

Tyr Gln Val Leu Phe Glu Gly Asp Gly Phe Hi

#s Tyr Gln Thr Tyr Glu

365

# 370

# 375

caa ttc gca aac ggc aaa gat cgt gaa aat ga

#t gat tac tgc tta cgt 2404

Gln Phe Ala Asn Gly Lys Asp Arg Glu Asn As

#p Asp Tyr Cys Leu Arg

380 3

#85 3

#90 3

#95

gac cca cac gtt gtt caa tta gaa aat ggt ga

#t cgt tat ctt gta ttc 2452

Asp Pro His Val Val Gln Leu Glu Asn Gly As

#p Arg Tyr Leu Val Phe

400

# 405

# 410

gaa gct aat act ggg aca gaa gat tac caa ag

#t gac gac caa att tat 2500

Glu Ala Asn Thr Gly Thr Glu Asp Tyr Gln Se

#r Asp Asp Gln Ile Tyr

415

# 420

# 425

aat tgg gct aac tat ggt ggc gat gat gcc tt

#c aat att aag agt tcc 2548

Asn Trp Ala Asn Tyr Gly Gly Asp Asp Ala Ph

#e Asn Ile Lys Ser Ser

430

# 435

# 440

ttc aag ctt ttg aat aat aag aag gat cgt ga

#a ttg gct ggt tta gct 2596

Phe Lys Leu Leu Asn Asn Lys Lys Asp Arg Gl

#u Leu Ala Gly Leu Ala

445

# 450

# 455

aat ggt gca ctt ggt atc tta aag ctc act aa

#c aat caa agt aag cca 2644

Asn Gly Ala Leu Gly Ile Leu Lys Leu Thr As

#n Asn Gln Ser Lys Pro

460 4

#65 4

#70 4

#75

aag gtt gaa gaa gta tac tca cca ttg gta tc

#t act ttg atg gct tgc 2692

Lys Val Glu Glu Val Tyr Ser Pro Leu Val Se

#r Thr Leu Met Ala Cys

480

# 485

# 490

gat gag gta nnn nnn aag ctt ggt gat aag ta

#t tat ctc ttc tcc gta 2740

Asp Glu Val Xaa Xaa Lys Leu Gly Asp Lys Ty

#r Tyr Leu Phe Ser Val

495

# 500

# 505

act cgt gta agt cgt ggt tcc gat cgt gaa tt

#a acc gct aag gat aac 2788

Thr Arg Val Ser Arg Gly Ser Asp Arg Glu Le

#u Thr Ala Lys Asp Asn

510

# 515

# 520

aca atc gtt ggt gat aac gtt gct atg att gg

#t tac gtt tcc gat agc 2836

Thr Ile Val Gly Asp Asn Val Ala Met Ile Gl

#y Tyr Val Ser Asp Ser

525

# 530

# 535

tta atg ggt aag tac aag cca tta aat aac tc

#a ggt gtc gta tta act 2884

Leu Met Gly Lys Tyr Lys Pro Leu Asn Asn Se

#r Gly Val Val Leu Thr

540 5

#45 5

#50 5

#55

gca tca gta cct gca aac tgg cgt act gct ac

#t tat tcc tac tat gca 2932

Ala Ser Val Pro Ala Asn Trp Arg Thr Ala Th

#r Tyr Ser Tyr Tyr Ala

560

# 565

# 570

gta cct gta gct ggt cat cct gat caa gta tt

#a att act tct tac atg 2980

Val Pro Val Ala Gly His Pro Asp Gln Val Le

#u Ile Thr Ser Tyr Met

575

# 580

# 585

agt aac aag gac ttt gct tca ggt gaa gga aa

#c tat gca act tgg gca 3028

Ser Asn Lys Asp Phe Ala Ser Gly Glu Gly As

#n Tyr Ala Thr Trp Ala

590

# 595

# 600

cca agt ttc tta gta caa atc aat cca gat ga

#c acg aca act gta tta 3076

Pro Ser Phe Leu Val Gln Ile Asn Pro Asp As

#p Thr Thr Thr Val Leu

605

# 610

# 615

gca cgt gca act aac caa ggt gac tgg gtg tg

#g gac gac tct agt cgg 3124

Ala Arg Ala Thr Asn Gln Gly Asp Trp Val Tr

#p Asp Asp Ser Ser Arg

620 6

#25 6

#30 6

#35

aac gat aat atg ctc ggt gtt ctt aaa gaa gg

#t gca gct aac agt gcc 3172

Asn Asp Asn Met Leu Gly Val Leu Lys Glu Gl

#y Ala Ala Asn Ser Ala

640

# 645

# 650

gcc tta cca ggt gaa tgg ggt aag cca gtt ga

#c tgg agt ttg att aac 3220

Ala Leu Pro Gly Glu Trp Gly Lys Pro Val As

#p Trp Ser Leu Ile Asn

655

# 660

# 665

aga agt cct ggc tta ggc tta aag cct cat ca

#a cca gtt caa cca aag 3268

Arg Ser Pro Gly Leu Gly Leu Lys Pro His Gl

#n Pro Val Gln Pro Lys

670

# 675

# 680

att gat caa cct gat caa caa cct tct ggt ca

#a aac act aag aat gtc 3316

Ile Asp Gln Pro Asp Gln Gln Pro Ser Gly Gl

#n Asn Thr Lys Asn Val

685

# 690

# 695

aca cca ggt aat ggt gat aag cct gct ggt aa

#g gca act cct gat aac 3364

Thr Pro Gly Asn Gly Asp Lys Pro Ala Gly Ly

#s Ala Thr Pro Asp Asn

700 7

#05 7

#10 7

#15

act aat att gat cca agt gca caa cct tct gg

#t caa aac act aat att 3412

Thr Asn Ile Asp Pro Ser Ala Gln Pro Ser Gl

#y Gln Asn Thr Asn Ile

720

# 725

# 730

gat cca agt gca caa mct tct ggt caa aac ac

#t aag aat gtc aca cca 3460

Asp Pro Ser Ala Gln Xaa Ser Gly Gln Asn Th

#r Lys Asn Val Thr Pro

735

# 740

# 745

ggt aat gag aaa caa ggt aag aat acc gat gc

#a aaa caa tta cca caa 3508

Gly Asn Glu Lys Gln Gly Lys Asn Thr Asp Al

#a Lys Gln Leu Pro Gln

750

# 755

# 760

aca ggt aat aag tct ggt tta gca gga ctt ta

#c gct ggt tca tta ctt 3556

Thr Gly Asn Lys Ser Gly Leu Ala Gly Leu Ty

#r Ala Gly Ser Leu Leu

765

# 770

# 775

gcc ttg ttt gga ttg gca gca att gaa aag cg

#t cac gct taa

#3598

Ala Leu Phe Gly Leu Ala Ala Ile Glu Lys Ar

#g His Ala

780 7

#85 7

#90

tagagtaaaa aaacatcctc cactcaagtt acaagtagga taatatgtat ta

#tttctacg 3658

cytagtcaag aggrattact ggacatannn nnnnnnnnnn tccagttacc aa

#gtggaata 3718

tagtattatt ccacgctagt caggaggatt actgacatta ttggctacat gg

#ccggtagt 3778

cctcttttct tttgtgacga attgtcaaac caagtgcaac ggtttctcaa aa

#aacacctc 3838

atatggggtt tcataattta acacttttcg aggacggcgg ttcagctgat gt

#tggcagaa 3898

actgacgtcc ttatctgtat aatcatcaat attagccctt ttaggaaagt at

#tccctaat 3958

tagsccattg gtattttcat tgggtcctct ttcctctggt gaatagggat ct

#ggccaata 4018

gatagctact cctaaacgtc ctcgaatatc attcaagcca agaaattcac gc

#ccatgatc 4078

tggagtcaat gaatggacaa attctttagg aatagaccct aagagatcaa tt

#aagccctg 4138

atatttgaat tcggagaagg ggagttgtcc aacaattgcc gttataatac ca

#gggttaat 4198

acggccctgg gcctctacgg taatattgta tttttggctc agatcagtga ta

#gaaaccca 4258

cagatttagc ttgccggtgg agtgctgctt gaagtcttca attacttcgt ta

#ccatgttt 4318

gattgctaat ctgatgtgtc gttgttgtgg tgtagtaggc atcataccac ct

#cctcataa 4378

aataaggtat aacaggaatt tcttgtacta tatgatcctt ccaatataat aa

#tattaggc 4438

cgataagaaa tgaccagcta ccatttcttg atgcttagtg aatataatcg ga

#tgatacgt 4498

cacccctcaa caatccaatt tcacggaggt gagtaatcat gccgagagct ag

#gaatgatt 4558

ggaggaacga acacggtcca tgcggcagtg gctatttgga ttttagccaa ag

#cagcgtta 4618

ctgcttgcaa aagctt

#

#

# 4634

<210> SEQ ID NO 11

<211> LENGTH: 792

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<220> FEATURE:

<221> NAME/KEY: MOD_RES

<222> LOCATION: (495)..(496)

<223> OTHER INFORMATION: Any amino acid

<220> FEATURE:

<221> NAME/KEY: MOD_RES

<222> LOCATION: (737)

<223> OTHER INFORMATION: Thr or Pro

<400> SEQUENCE: 11

Met Tyr Lys Val Gly Lys Asn Trp Ala Val Al

#a Thr Leu Val Ser Ala

1 5

# 10

# 15

Ser Ile Leu Met Gly Gly Val Val Thr Ala Hi

#s Ala Asp Gln Val Glu

20

# 25

# 30

Ser Asn Asn Tyr Asn Gly Val Ala Glu Val As

#n Thr Glu Arg Gln Ala

35

# 40

# 45

Asn Gly Gln Ile Gly Val Asp Gly Lys Ile Il

#e Ser Ala Asn Ser Asn

50

# 55

# 60

Thr Thr Ser Gly Ser Thr Asn Gln Glu Ser Se

#r Ala Thr Asn Asn Thr

65

# 70

# 75

# 80

Glu Asn Ala Val Val Asn Glu Ser Lys Asn Th

#r Asn Asn Thr Glu Asn

85

# 90

# 95

Ala Val Val Asn Glu Asn Lys Asn Thr Asn As

#n Thr Glu Asn Ala Val

100

# 105

# 110

Val Asn Glu Asn Lys Asn Thr Asn Asn Thr Gl

#u Asn Asp Asn Ser Gln

115

# 120

# 125

Leu Lys Leu Thr Asn Asn Glu Gln Pro Ser Al

#a Ala Thr Gln Ala Asn

130

# 135

# 140

Leu Lys Lys Leu Asn Pro Gln Ala Ala Lys Al

#a Val Gln Asn Ala Lys

145 1

#50 1

#55 1

#60

Ile Asp Ala Gly Ser Leu Thr Asp Asp Gln Il

#e Asn Glu Leu Asn Lys

165

# 170

# 175

Ile Asn Phe Ser Lys Ser Ala Glu Lys Gly Al

#a Lys Leu Thr Phe Lys

180

# 185

# 190

Asp Leu Glu Gly Ile Gly Asn Ala Ile Val Ly

#s Gln Asp Pro Gln Tyr

195

# 200

# 205

Ala Ile Pro Tyr Ser Asn Ala Lys Glu Ile Ly

#s Asn Met Pro Ala Thr

210

# 215

# 220

Tyr Thr Val Asp Ala Gln Thr Gly Lys Met Al

#a His Leu Asp Val Trp

225 2

#30 2

#35 2

#40

Asp Ser Trp Pro Val Gln Asp Pro Val Thr Gl

#y Tyr Val Ser Asn Tyr

245

# 250

# 255

Met Gly Tyr Gln Leu Val Ile Ala Met Met Gl

#y Ile Pro Asn Ser Pro

260

# 265

# 270

Thr Gly Asp Asn His Ile Tyr Leu Leu Tyr As

#n Lys Tyr Gly Asp Asn

275

# 280

# 285

Asp Phe Ser His Trp Arg Asn Ala Gly Ser Il

#e Phe Gly Thr Lys Glu

290

# 295

# 300

Thr Asn Val Phe Gln Glu Trp Ser Gly Ser Al

#a Ile Val Asn Asp Asp

305 3

#10 3

#15 3

#20

Gly Thr Ile Gln Leu Phe Phe Thr Ser Asn As

#p Thr Ser Asp Tyr Lys

325

# 330

# 335

Leu Asn Asp Gln Arg Leu Ala Thr Ala Thr Le

#u Asn Leu Asn Val Asp

340

# 345

# 350

Asp Asn Gly Val Ser Ile Lys Ser Val Asp As

#n Tyr Gln Val Leu Phe

355

# 360

# 365

Glu Gly Asp Gly Phe His Tyr Gln Thr Tyr Gl

#u Gln Phe Ala Asn Gly

370

# 375

# 380

Lys Asp Arg Glu Asn Asp Asp Tyr Cys Leu Ar

#g Asp Pro His Val Val

385 3

#90 3

#95 4

#00

Gln Leu Glu Asn Gly Asp Arg Tyr Leu Val Ph

#e Glu Ala Asn Thr Gly

405

# 410

# 415

Thr Glu Asp Tyr Gln Ser Asp Asp Gln Ile Ty

#r Asn Trp Ala Asn Tyr

420

# 425

# 430

Gly Gly Asp Asp Ala Phe Asn Ile Lys Ser Se

#r Phe Lys Leu Leu Asn

435

# 440

# 445

Asn Lys Lys Asp Arg Glu Leu Ala Gly Leu Al

#a Asn Gly Ala Leu Gly

450

# 455

# 460

Ile Leu Lys Leu Thr Asn Asn Gln Ser Lys Pr

#o Lys Val Glu Glu Val

465 4

#70 4

#75 4

#80

Tyr Ser Pro Leu Val Ser Thr Leu Met Ala Cy

#s Asp Glu Val Xaa Xaa

485

# 490

# 495

Lys Leu Gly Asp Lys Tyr Tyr Leu Phe Ser Va

#l Thr Arg Val Ser Arg

500

# 505

# 510

Gly Ser Asp Arg Glu Leu Thr Ala Lys Asp As

#n Thr Ile Val Gly Asp

515

# 520

# 525

Asn Val Ala Met Ile Gly Tyr Val Ser Asp Se

#r Leu Met Gly Lys Tyr

530

# 535

# 540

Lys Pro Leu Asn Asn Ser Gly Val Val Leu Th

#r Ala Ser Val Pro Ala

545 5

#50 5

#55 5

#60

Asn Trp Arg Thr Ala Thr Tyr Ser Tyr Tyr Al

#a Val Pro Val Ala Gly

565

# 570

# 575

His Pro Asp Gln Val Leu Ile Thr Ser Tyr Me

#t Ser Asn Lys Asp Phe

580

# 585

# 590

Ala Ser Gly Glu Gly Asn Tyr Ala Thr Trp Al

#a Pro Ser Phe Leu Val

595

# 600

# 605

Gln Ile Asn Pro Asp Asp Thr Thr Thr Val Le

#u Ala Arg Ala Thr Asn

610

# 615

# 620

Gln Gly Asp Trp Val Trp Asp Asp Ser Ser Ar

#g Asn Asp Asn Met Leu

625 6

#30 6

#35 6

#40

Gly Val Leu Lys Glu Gly Ala Ala Asn Ser Al

#a Ala Leu Pro Gly Glu

645

# 650

# 655

Trp Gly Lys Pro Val Asp Trp Ser Leu Ile As

#n Arg Ser Pro Gly Leu

660

# 665

# 670

Gly Leu Lys Pro His Gln Pro Val Gln Pro Ly

#s Ile Asp Gln Pro Asp

675

# 680

# 685

Gln Gln Pro Ser Gly Gln Asn Thr Lys Asn Va

#l Thr Pro Gly Asn Gly

690

# 695

# 700

Asp Lys Pro Ala Gly Lys Ala Thr Pro Asp As

#n Thr Asn Ile Asp Pro

705 7

#10 7

#15 7

#20

Ser Ala Gln Pro Ser Gly Gln Asn Thr Asn Il

#e Asp Pro Ser Ala Gln

725

# 730

# 735

Xaa Ser Gly Gln Asn Thr Lys Asn Val Thr Pr

#o Gly Asn Glu Lys Gln

740

# 745

# 750

Gly Lys Asn Thr Asp Ala Lys Gln Leu Pro Gl

#n Thr Gly Asn Lys Ser

755

# 760

# 765

Gly Leu Ala Gly Leu Tyr Ala Gly Ser Leu Le

#u Ala Leu Phe Gly Leu

770

# 775

# 780

Ala Ala Ile Glu Lys Arg His Ala

785 7

#90

<210> SEQ ID NO 12

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 12

ctgataataa tggaaatgta tcac

#

# 24

<210> SEQ ID NO 13

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 13

catgatcata agtttggtag taatag

#

# 26

<210> SEQ ID NO 14

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 14

gtgatacatt tccattatta tcag

#

# 24

<210> SEQ ID NO 15

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 15

ctattactac caaacttatg atcatg

#

# 26

<210> SEQ ID NO 16

<211> LENGTH: 38

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 16

ccatggccat ggtagaacgc aaggaacata aaaaaatg

#

# 38

<210> SEQ ID NO 17

<211> LENGTH: 38

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 17

agatctagat ctgttaaatc gacgtttgtt aatttctg

#

# 38

<210> SEQ ID NO 18

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (6)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (15)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<400> SEQUENCE: 18

gaygtntggg aywsntgggc c

#

#

#21

<210> SEQ ID NO 19

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (3)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (6)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (9)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (12)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 19

gtngcnswnc cnswccayts ytg

#

# 23

<210> SEQ ID NO 20

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 20

gaatgtaggt ccaatttttg gc

#

# 22

<210> SEQ ID NO 21

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 21

cctgtccgaa catcttgaac tg

#

# 22

<210> SEQ ID NO 22

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (6)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (9)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (12)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (18)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (21)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<400> SEQUENCE: 22

arraanswng gngcvmangt nsw

#

# 23

<210> SEQ ID NO 23

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (9)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (11)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (15)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (21)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<400> SEQUENCE: 23

tayaayggng tngcngargt naa

#

# 23

<210> SEQ ID NO 24

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 24

ccgaccatct tgtttgatta ac

#

# 22

<210> SEQ ID NO 25

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<400> SEQUENCE: 25

aaytataayg gygttgcryg aagt

#

# 24

<210> SEQ ID NO 26

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial

#Sequence: Primer

<220> FEATURE:

<221> NAME/KEY: modified_base

<222> LOCATION: (9)

<223> OTHER INFORMATION: a, c, t, g, other o

#r unknown

<400> SEQUENCE: 26

taccgnwsnc tacttcaact t

#

#

#21

<210> SEQ ID NO 27

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 27

Tyr Asn Gly Val Ala Glu Val Lys Lys Arg Gl

#y Tyr Phe Tyr Ala Arg

1 5

# 10

# 15

Thr

<210> SEQ ID NO 28

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Lactobacillus reuteri

<400> SEQUENCE: 28

Tyr Asn Gly Val Ala Glu Val Asn Thr Glu Ar

#g Gln Ala Asn Gly Gly

1 5

# 10

# 15

Ile

<210> SEQ ID NO 29

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Bacillus amyloliquefaciens

<400> SEQUENCE: 29

Gly Leu Asp Val Trp Asp Ser Trp Pro Leu Gl

#n Asn Ala Asp

1 5

# 10

<210> SEQ ID NO 30

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Bacillus subtilis

<400> SEQUENCE: 30

Gly Leu Asp Val Trp Asp Ser Trp Pro Leu Gl

#n Asn Ala Asp

1 5

# 10

<210> SEQ ID NO 31

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Streptococcus mutans

<400> SEQUENCE: 31

Asp Leu Asp Val Trp Asp Ser Trp Pro Val Gl

#n Asp Ala Lys

1 5

# 10

<210> SEQ ID NO 32

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Streptococcus salivarius

<400> SEQUENCE: 32

Glu Ile Asp Val Trp Asp Ser Trp Pro Val Gl

#n Asp Ala Lys

1 5

# 10

<210> SEQ ID NO 33

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Bacillus amyloliquefaciens

<400> SEQUENCE: 33

Gln Thr Gln Glu Trp Ser Gly Ser Ala Thr Ph

#e Thr Ser Asp Gly Lys

1 5

# 10

# 15

<210> SEQ ID NO 34

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Bacillus subtilis

<400> SEQUENCE: 34

Gln Thr Gln Glu Trp Ser Gly Ser Ala Thr Ph

#e Thr Ser Asp Gly Lys

1 5

# 10

# 15

<210> SEQ ID NO 35

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Streptococcus mutans

<400> SEQUENCE: 35

Leu Thr Gln Glu Trp Ser Gly Ser Ala Thr Va

#l Asn Glu Asp Gly Ser

1 5

# 10

# 15

<210> SEQ ID NO 36

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Streptococcus salivarius

<400> SEQUENCE: 36

Asp Asp Gln Gln Trp Ser Gly Ser Ala Thr Va

#l Asn Ser Asp Gly Ser

1 5

# 10

# 15

<210> SEQ ID NO 37

<211> LENGTH: 11

<212> TYPE: PRT

<213> ORGANISM: Bacillus amyloliquefaciens

<400> SEQUENCE: 37

Lys Ala Thr Phe Gly Pro Ser Phe Leu Met As

#n

1 5

# 10

<210> SEQ ID NO 38

<211> LENGTH: 11

<212> TYPE: PRT

<213> ORGANISM: Bacillus subtilis

<400> SEQUENCE: 38

Gln Ser Thr Phe Ala Pro Ser Phe Leu Leu As

#n

1 5

# 10

<210> SEQ ID NO 39

<211> LENGTH: 11

<212> TYPE: PRT

<213> ORGANISM: Streptococcus mutans

<400> SEQUENCE: 39

Asn Ser Thr Trp Ala Pro Ser Phe Leu Ile Gl

#n

1 5

# 10

<210> SEQ ID NO 40

<211> LENGTH: 11

<212> TYPE: PRT

<213> ORGANISM: Streptococcus salivarius

<400> SEQUENCE: 40

Lys Ser Thr Trp Ala Pro Ser Phe Leu Ile Ly

#s

1 5

# 10

	Number	Date	Country
Parent	09/604958	Jun 2000	US
Child	09/995587		US

Fructosyltransferases

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (1)

CROSS-REFERENCE TO RELATED APPLICATION

Non-Patent Literature Citations (2)

Continuation in Parts (1)

Entry
G.H. van Geel-Schutten et al. “Biochemical and Structural Characterization of the Glucan and Fructan Exoplysaccharides Synthesized by the Lactobacillus reuteri Wild-Type Strain and by Mutant Strains”, Applied Environ. Microbiol. 65(7); 3008-3014. (Jul. 1).*
G.H. van Geel-Schutten et al. “Screening and Charcterization of Lactobacillus Strains Producing Large Amounts of Exopolysaccharides”, Appl. Microbiol. Biotechnol. 50: 697-703. (1998).