KR102237465B1 - Recombinant yeast secreting inulosucrase and a method of producing fructooligosaccharides - Google Patents

Abstract

The present invention relates to an inulosucrase secretion expression cassette including a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion translational fusion partner (TFP), a vector including the cassette, a recombinant microorganism including the cassette or the vector, and a method for producing fructo-oligosaccharide (FOS) through inulosucrase production and direct fermentation of yeast using the recombinant microorganism. According to the present invention, the recombinant microorganism can secrete and express recombinant inulosucrase with excellent efficiency by using the protein secretion TFP and FOS can be effectively produced by fermenting inulosucrase produced from the microorganism in a medium containing sucrose.

Description

Yeast introduced with inulosucrase activity and method for producing fructooligosaccharides using the same {Recombinant yeast secreting inulosucrase and a method of producing fructooligosaccharides}

The present invention is an inulosucrase secretion expression cassette comprising a nucleic acid encoding an inulosucrase and a nucleic acid encoding a protein secretion fusion factor (TFP), a vector comprising the cassette, The present invention relates to a recombinant microorganism comprising the cassette or vector, and a method for producing inulosucrase and/or fructooligosaccharides using the recombinant microorganism.

Fructooligosaccharides (FOS) are fructose polymers produced from sucrose and are functional sweeteners with prebiotic effects that help the growth of beneficial bacteria in the intestine (European journal of clinical nutrition 2009, 63(11): 1277-1289). . FOS is a fructan (inulin, levane) having a polymerization degree of DP 3-60 or higher, medium-chain FOS (medium-chain FOS) of DP 2-20, DP 2- depending on the degree of polymerization (DP) of fructose. It is classified as short-chain FOS (scFOS) of 4, and has differences in sugar content and prebiotic effects depending on the degree of polymerization.

FOS is produced in two main ways. First, by using plants such as dahlias, chicory, and pork potatoes to accumulate inulin as storage carbohydrates in their tubers, endoinulinase (EC 3.2) was added to inulin obtained through hot water extraction from plant tubers. You can get FOS by processing .17). FOS produced by this method is known to have a degree of polymerization of DP 2-10. Alternatively, it is known that treatment of sucrose with inulosucrase (EC 2.4.1.9) mainly produces the short-chain FOS of DP 2-4 (Annual review of food science and technology 2010, 1: 305). -339). Inulosucrase, which is used for the reaction, can be obtained from various microbial resources such as fungi and bacteria. Among them, bacteria-derived enzymes also produce more than 10% of inulin during the FOS production process, so inulosucrases derived from fungi are mainly used.

When FOS is produced enzymatically from sucrose, glucose equivalent to half of the used sucrose and unconverted fructose are accumulated in the reaction solution, which acts to inhibit the activity of the enzyme or reduce the prebiotic effect. Therefore, in order to remove monosaccharides after the enzyme conversion reaction, a method of increasing the purity of FOS by culturing black yeast (Aureobasidium pullulans ) or baker's yeast ( Saccharomyces cerevisiae) is applied. (Carbohydrate polymers 2016, 136: 274-281).

Because yeast Saccharomyces cerevisiae is a single-celled eukaryotic microorganism, it has similar transcription and translation systems to higher cells, and is a system for removing introns through splicing and secretory organs such as endoplasmic reticulum and Golgi apparatus like higher cells. It is equipped with. Therefore, it is possible to secrete and produce proteins derived from higher cells in an active form through post-translational modification, and when secreting and producing a recombinant protein into a culture medium, it is possible to easily separate pure enzymes without going through a complicated purification process of several steps. There is an advantage to be able to. However, due to the low expression and secretion rate, the expression and application of the enzyme inulosucrase using Saccharomyces cerevisiae have not yet been reported.

Accordingly, the present inventors have used protein secretion fusion factor technology (Korea Patent Registration No. 10-0975596) to develop a system for mass-secreting and producing inulosucrase from yeast to effectively convert recombinant inulosucrase into cells. Protein secretion fusion factors that can be secreted outside were selected. It was confirmed that a transformant was prepared by introducing the expression vector in which the protein-secreting fusion factor gene and the inulosucrase gene selected through the above process were linked to the yeast, and mass secretion production of inulosucrase was possible from this. . In addition, the present invention was completed by confirming that it was possible to efficiently produce heavy chain FOS (DP 2-20), which is difficult to obtain by conventional methods, as a result of applying the transformant to FOS production by direct fermentation culture in a sucrose-containing medium.

One object of the present invention is to provide an inulosucrase secretion expression cassette comprising a nucleic acid encoding an inulosucrase and a nucleic acid encoding a translational fusion partner (TFP).

Another object of the present invention is to provide a vector comprising the cassette.

Another object of the present invention is to provide a recombinant microorganism containing an inulosucrase secretion expression cassette comprising a nucleic acid encoding an inulosucrase and a nucleic acid encoding a protein secretion fusion factor.

Another object of the present invention is (i) culturing the recombinant microorganism; And (ii) it is to provide a method for producing inulosucrase, comprising the step of recovering inullo sucrase from the cultured microorganism or a culture solution thereof.

Another object of the present invention is to provide a method for producing FOS, comprising the step of reacting the recombinant microorganism or inulosucrase prepared by the method with sucrose.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.

In order to achieve the above object, the present invention is a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion fusion factor (Translational fusion partner, TFP) as one embodiment, including inulosucrase secretion expression Cassette is provided.

In the present invention, in order to produce FOS with high efficiency, inulosucrase was secreted and produced from microorganisms using a protein-secreting fusion factor, and for this purpose, a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion fusion factor are used. After preparing a secretion expression cassette containing inulosucrase, a recombinant vector containing the cassette, and a recombinant microorganism, it was intended to produce inulosucrase useful for FOS production.

In the present invention, the term "fructooligosaccharides (FOS)" is a non-digestible, non-fermentable fructose polymer that does not decompose in the intestine of animals, and can be used as a low-calorie sweetener, and can also be used as a growth promoting factor of beneficial bacteria in vivo. I can.

In the present invention, the term "inulosucrase" refers to an enzyme that produces FOS using sucrose as a substrate. In the present invention, it is also referred to as Inu. The inulosucrase may specifically be derived from Lactobacillus leuteri , and more specifically may be an inulosucrase having the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and has FOS-producing ability. Any enzyme can be included regardless of its origin or sequence.

Therefore, it contains the amino acid sequence of SEQ ID NO: 1, or contains the conserved sequence of the amino acid sequence of SEQ ID NO: 1, and one or more at one or more positions (depending on the position or type in the conformational structure of the amino acid residue of the protein Although different, specifically 2 to 20 amino acids, more specifically 2 to 10, even more specifically 2 to 5) amino acids may include a substituted, deleted, inserted, added or reversed amino acid sequence. , 80% or more, specifically 90% or more, more specifically 95% or more, particularly specifically, with respect to the amino acid sequence of SEQ ID NO: 1, as long as it can maintain or enhance the activity of the inulosucrase. It may include an amino acid sequence having a homology of 97% or more, and substitution, deletion, insertion, addition, or inversion of the amino acid may include a mutant sequence naturally occurring in a microorganism containing the activity of the inulosucrase or an artificial It may even contain a variant sequence.

Specifically, the inulosucrase of the present invention includes not only the amino acid sequence of SEQ ID NO: 1, but also any one or more of the carboxy-terminal, amino-terminal, or amino acid sequences truncated at both ends of the amino acid sequence of SEQ ID NO: 1. It may be, but is not limited thereto. More specifically, the inulosucrase of the present invention may be composed of any one of the amino acid sequences of SEQ ID NO: 1 and the amino acid sequence truncated at both ends of the amino acid sequence of SEQ ID NO: 1, The amino acid sequence with both ends truncated in the amino acid sequence may be the amino acid sequence of SEQ ID NO: 9, but is not limited thereto.

The term "homology" of the present invention is intended to indicate the degree of similarity to the amino acid sequence of a wild-type protein or a nucleotide sequence encoding the same, and the amino acid sequence or nucleotide sequence of the present invention and the same percent or more of the same sequence It includes a sequence having. Such homology can be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Automated alignment algorithms in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determinations for other useful arrangements are automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

In the present invention, the term "translational fusion partner (TFP)" refers to a factor useful for secretion of a protein of interest. The protein secretion fusion factor may be disclosed in Korean Patent Laid-Open Publication No. 10-2008-0042823, specifically, TFP 1 of SEQ ID NO: 11, TFP 2 of SEQ ID NO: 12, TFP 3 of SEQ ID NO: 13, and TFP of SEQ ID NO: 14. 4, TFP 5 of SEQ ID NO: 15, TFP 6 of SEQ ID NO: 16, TFP 7 of SEQ ID NO: 17, TFP 8 of SEQ ID NO: 18, TFP 9 of SEQ ID NO: 19, TFP 10 of SEQ ID NO: 20, TFP 11 of SEQ ID NO: 21 , TFP 12 of SEQ ID NO: 22, TFP 13 of SEQ ID NO: 23, TFP 14 of SEQ ID NO: 24, TFP 15 of SEQ ID NO: 25, TFP 16 of SEQ ID NO: 26, TFP 17 of SEQ ID NO: 27, TFP 18 of SEQ ID NO: 28, It may be TFP 19 of SEQ ID NO: 29 or TFP 20 of SEQ ID NO: 30, but any factor capable of improving the secreted expression of the target protein may be included without limitation.

Therefore, the TFP may include the amino acid sequence of any one of SEQ ID NOs: 11 to 30. Alternatively, the TFP includes one or more conserved sequences of any one of the amino acid sequences of SEQ ID NOs: 11 to 30, and one or more at one or more positions (different depending on the position and type in the conformational structure of the amino acid residue of the protein, Specifically, 2 to 20 amino acids, more specifically 2 to 10, even more specifically 2 to 5, but not limited thereto.) of amino acids substituted, deleted, inserted, added, or inverted amino acid sequence. It may include, as long as the secretion expression of the target protein can be improved, 80% or more, specifically 90% or more, and more specifically 95% or more with respect to any one amino acid sequence of SEQ ID NOs: 11 to 30 , In particular, it may include an amino acid sequence having a homology of 97% or more, and substitution, deletion, insertion, addition or inversion of the amino acid may include a mutant sequence naturally occurring in a microorganism containing the activity of the TFP or It may even contain artificial mutant sequences.

Further, the recombinant inulosucrase, in the case of Lactobacillus leuteri derived from Inulosucrase and TFP 6 of SEQ ID NO: 16, TFP 10 of SEQ ID NO: 20, or TFP 13 of SEQ ID NO: 23 are linked It may be, but is not limited thereto.

In the inulosucrase secretion expression cassette, a nucleic acid encoding an inulosucrase and a nucleic acid encoding a protein secretion fusion factor may be linked to each other by a linker.

The linker may include a protease recognition sequence or an affinity tag.

In addition, the nucleic acid sequence encoding inulosucrase used in the present invention may include the linear vector of the present invention and linker DNA used for recombination in cells. The addition of such a linker sequence to the nucleic acid sequence encoding inulosucrase can be performed by general DNA techniques, such as PCR and/or restriction enzyme cleavage and ligation.

The linker DNA of the present invention must be of sufficient length, and the nucleic acid sequence encoding inulosucrazase in the host cell must be of a sufficient length, so that recombination can occur in the linear vector containing the nucleic acid sequence encoding TFP. It may have sufficient homology with a portion of the nucleic acid sequence. Specifically, the linker DNA may have a length of 20 bp or more, such as 30 or 40 bp, but is not limited thereto. More specifically, the linker DNA has at least 80% homology with the linear vector, and may have 85%, 90%, 95% or 99% homology, but is not limited thereto.

As an example, the linker DNA can cause cleavage at the junction site between the TFP and the protein of interest by encoding a protease recognition sequence. For example, linker DNA is a yeast kex2p protease recognition sequence (amino acid sequence including Lys-Arg), mammalian purine recognition sequence (amino acid sequence including Arg-XX-Arg), Factor-Xa recognition sequence (Ile-Glu-Gly -Amino acid sequence including Arg), enterokinase-recognition sequence (amino acid sequence including Asp-Asp-Lys), subtilisin recognition sequence (amino acid sequence including Ala-Ala-His-Tyr), tobacco etch virus Recognition sequence (amino acid sequence including Glu-Asn-Leu-Tyr-Phe-Gln-Gly), ubiquitin hydrolase recognition sequence (amino acid sequence including Arg-Gly-Gly) or thrombin recognition sequence (Arg-Gly- Amino acid sequence including Pro-Arg) can be encoded.

In the present invention, the term "affinity tag" may be used for various purposes as a peptide or nucleic acid sequence that can be introduced into a recombinant fusion protein or a nucleic acid encoding the same, and may be, for example, to increase the purification efficiency of the target protein. The affinity tag may be GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, HIS, STREP, CBP, CBD, or S-tag, and specifically may be an HIS tag, but It is not limited.

The affinity tag may be linked to any one or more of the carboxy terminal, amino terminal, a truncated terminal of the carboxy moiety, and a truncated terminal of the amino moiety of inulosucrase, and in particular, the carboxy moiety or the amino moiety May be connected to the cut end, but is not limited thereto.

In the present invention, the term "nucleic acid" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs with modified sugar or base moieties. (analogue) is also included. The present invention provides an inulosucrase secretion expression cassette comprising a nucleic acid encoding an inulosucrase and a nucleic acid encoding a protein secretion fusion factor.

The term "expression cassette" of the present invention has a nucleic acid element capable of affecting the expression of a structural gene in a cell. As a naturally occurring or synthesized nucleic acid structure, an expression cassette generally includes a transcribed nucleic acid and a promoter. For the purposes of the present invention, the expression cassette may include a nucleic acid encoding a protein secretion fusion factor and a nucleic acid encoding inulosucrase, and in particular, "inulosucrase, which induces the secretion of inulosucrase. As a secretion expression cassette", inulosucrase can be expressed and secreted.

The nucleic acid encoding the inulosucrase is due to the degeneracy of the codon or in consideration of the preferred codon in an organism to express the inulosucrase, the amino acid sequence of the inulosucrase is determined. Various modifications may be made to the coding region within a range that does not change. Specifically, any polynucleotide sequence encoding an inulosucrase consisting of the amino acid sequence of SEQ ID NO: 1 or an inulosucrase having both ends of the amino acid sequence of SEQ ID NO: 9 may be included without limitation.

For example, the inulosucrase consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention is a nucleic acid sequence of SEQ ID NO: 2, and the inulosucrase consisting of the amino acid sequence of SEQ ID NO: 9 is truncated at both ends is SEQ ID NO: 10 It may be encoded by the nucleic acid sequence of, but is not limited thereto.

In addition, the nucleic acid encoding the protein secretion fusion factor of the present invention is the amino acid of the protein secretion fusion factor due to the degeneracy of the codon as described above or in consideration of the preferred codon in an organism to express the protein secretion fusion factor. Various modifications may be made to the coding region within a range that does not change the sequence. Specifically, any polynucleotide sequence encoding a protein secretion fusion factor may be included without limitation.

For example, as the protein secretion fusion factor of the present invention, TFP 1 is SEQ ID NO: 31, TFP 2 is SEQ ID NO: 32, TFP 3 is SEQ ID NO: 33, TFP 4 is SEQ ID NO: 34, TFP 5 is SEQ ID NO: 35, TFP 6 is SEQ ID NO: 36, TFP 7 is SEQ ID NO: 37, TFP 8 is SEQ ID NO: 38, TFP 9 is SEQ ID NO: 39, TFP 10 is SEQ ID NO: 40, TFP 11 is SEQ ID NO: 41, TFP 12 is SEQ ID NO: 42, TFP 13 is sequence Number 43, TFP 14 is SEQ ID NO: 44, TFP 15 is SEQ ID NO: 45, TFP 16 is SEQ ID NO: 46, TFP 17 is SEQ ID NO: 47, TFP 18 is SEQ ID NO: 48, TFP 19 is SEQ ID NO: 49, or TFP 20 is sequence It may be encoded by the nucleic acid sequence of number 50, but is not limited thereto.

In another aspect, the present invention provides a vector including the cassette.

The cassette is as described above.

In the present invention, the term "vector" refers to a DNA product containing a nucleotide sequence of a nucleic acid encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. In particular, the present invention In the target protein means a recombinant fusion protein comprising inulosucrase and a protein secretion fusion factor.

In the vector, the expression control fragment having various functions for suppressing, amplifying, or inducing the expression of a target gene, a marker for selection of a transformant, a resistance gene for antibiotics, and secreted expression of a poorly expressed protein It may further include suitable custom fusion factors and the like. In particular, as a customized fusion factor suitable for the poorly expressed protein, it may be preferable to use the protein secretion fusion factor.

In a specific embodiment of the present invention, YGaTFP13-LrInuΔNC-6H including TFP 13 and Lactobacillus luteri-derived inulosuclase gene as an expression vector for highly secreted production of Lactobacillus luteri-derived inulosucrase. Was produced (Fig. 7).

In another aspect, the present invention provides a recombinant microorganism containing an inulosucrase secretion expression cassette, comprising a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion fusion factor. to provide.

The inulosucrase, protein secretion fusion factor, nucleic acid, and inulosucrase secretion expression cassette are as described above.

The recombinant microorganism may be a microorganism transformed with the nucleic acid.

The term "transformation" of the present invention means that a nucleic acid is introduced into a host cell so that the nucleic acid becomes capable of replication as an extrachromosomal factor or by completion of chromosomal integration.

Any transformation method may be used for the transformation method of the present invention, and may be easily performed according to a conventional method in the art.

The host cell used for transformation according to the present invention may be any host cell well known in the art, but a host having high transduction and expression efficiency of the inulosucrase gene of the present invention may be used. Examples can include bacteria, fungi, and yeast. More specifically, the recombinant microorganism may be yeast, more particularly, the yeast is Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia), It may belong to the genus Schizosaccharomyces , Yarrowia , Saccharomyces , Schwanniomyces or Arxula , and more specifically Candida utility ( Candida utilis ), Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris or Komagataella phaffii , Pichia stipitis ), Schizosaccharomyces pombe , Saccharomyces cerevisiae , Hansenula polymorpha , Yarrowia lipolytica , Schwanniomyces oxy It may be dentalis (Schwanniomyces occidentalis ) or Arsula adeninivorans (Arxula adeninivorans), but is not limited thereto.

In a specific embodiment of the present invention, the optimal yeast secretion fusion factor capable of secreting and expressing Lactobacillus leuteri- derived inulosucrase is selected, and each expression vector and each vector produced therefrom are selected. The production of recombinant inulosucrase was confirmed using yeast transformed with (FIGS. 5 to 6 and Table 2 ).

As an example in the present invention, the GAL10 promoter used for inulosucrase expression is a promoter in which transcription is induced by galactose, but in a strain in which the GAL80 gene is inactivated, high gene expression can be maintained regardless of the presence or absence of galactose. For this reason, in the present invention, inulosucrase was expressed using a strain in which the GAL80 gene was inactivated.

As a specific example for producing the recombinant microorganism, the PCR product amplified with LNK39 (SEQ ID NO: 6) and HisGT50R primer (SEQ ID NO: 7) is a linker end and 40 bases of the vector, and the GAL7 transcription terminator end Since the 50 bases are the same, when introduced into a yeast cell together with a linearized vector, crossover occurs within the cell, so that the inulosucrase gene and the protein-secreting fusion factor vector can be linked in a certain direction. Furthermore, the PCR product and the yeast into which the vector was introduced were used as a selective medium lacking uracil, SD-Ura medium (a yeast substrate lacking 0.67% amino acid, a nutritional supplement lacking 0.77% uracil, 2% glucose). When selected in, it is possible to produce transformants into which each expression vector was introduced immediately without the process of making a vector using E. coli (FIG. 1).

In addition, the transformed yeast is fed-batch culture to purify inulosucrase, and then FOS is produced using the purified enzyme (FIG. 15), or the transformed yeast is added to a yeast medium containing sucrose. It was confirmed that FOS can be directly produced by fermentation (Fig. 11, Fig. 12, Fig. 13).

For the purposes of the present invention, the recombinant microorganism comprises a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion fusion factor, so that the activity or concentration of inulosucrase is in a wild-type or unmodified microorganism strain. Typically at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000% based on activity or concentration It may be increased, but is not limited thereto.

In addition, the recombinant microorganism may produce an excess of FOS compared to an unmodified wild-type strain or an unmodified mutant strain as the activity or concentration of the inulosucrase increases. In the present invention, the term "unmodified microorganism" may refer to a natural strain itself or a microorganism that does not contain the inulosucrase secretion expression cassette, but is not limited thereto.

In another aspect of the present invention, (i) culturing the recombinant microorganism; And it provides a method for producing inullo sucrase comprising the step of recovering the inullo sucrase from the cultured microorganism or a culture supernatant thereof.

Recombinant microorganisms and inulosucrase are as described above.

The medium and culture conditions for culturing the recombinant microorganism can be appropriately selected and used depending on the host cell. Specifically, it is cultured in YEPD medium (3% yeast extract, 2% glucose, 1.5% peptone) containing glucose as a carbon source, and 60 hours while adding the supply medium (30% glucose, 15% yeast extract) according to glucose consumption. It may be cultured, but is not limited thereto.

In a specific embodiment of the present invention, fed-batch culture was performed using the recombinant microorganism to obtain inulosucrase.

The term "fed-batch culture" of the present invention is a culture method having a high final concentration and productivity of a product, and is very often used for high-concentration cell culture, and after initially filling the medium once, a new culture medium or a growth limiting substrate solution or It may be a method of additionally supplying a precursor of the product, but allowing the reaction product to remain in the fermentor.

For example, but not limited thereto, the preferred method of fed-batch culture is a one-step seed culture in 50 ml of a minimal liquid medium (a yeast nitrogen source substrate lacking 0.67% amino acid, 0.5% casamino acid, 2% glucose). Then, after culturing again in 200 ml of YEPD liquid medium to activate, inoculate the main culture medium and incubate at 30° C. for 45 hours, adding fed-batch culture medium (15% yeast extract, 30% glucose) according to the cell growth rate. It may be to supply.

In another aspect, the present invention provides a method for producing FOS, comprising the step of reacting the recombinant microorganism or inulosucrase prepared by the method with sucrose.

Recombinant microorganisms, a method for producing inulosucrase, and FOS are as described above.

In another aspect, the present invention provides a method for producing FOS, comprising fermenting the recombinant microorganism in a medium containing sucrose.

Recombinant microorganisms, a method for producing inulosucrase, and FOS are as described above. Compared to the enzyme process that produces FOS by reacting with sucrose after producing inulosucrase, the transformed strain is directly fermented in a sucrose-containing medium to produce FOS, thereby eliminating the cost of enzyme production and purification. Since the transforming strain can use fructose and glucose generated by the action of sucrase as a carbon source, it is possible to produce high-purity FOS (Fig. 11, Fig. 13, Fig. 14).

In a specific embodiment of the present invention, a recombinant yeast strain secreting and producing inulosucrase derived from Lactobacillus luteri is cultured in a yeast medium to which 300, 400 g/L of sucrose is added to produce FOS at high yield. It was confirmed (Fig. 13).

The FOS produced according to the method of the present invention was confirmed to be the heavy chain FOS of DP2-20 (FIG. 14).

The recombinant microorganism of the present invention can secrete and express recombinant inulosucrase with excellent efficiency by a protein secretion fusion factor, and react with sucrose by reacting inulosucrase produced from the microorganism, or sucrose by reacting the microorganism with sucrose. Medium chain FOS (DP 2-20) can be effectively produced by fermentation in a medium containing.

1 is a schematic diagram of a domain analysis according to the amino acid sequence of the LrInu gene and a cleaved enzyme region accordingly.
2 is an SDS-PAGE result confirming the secretion and expression of the cleaved LrInu enzymes.
3 is a graph showing the relative activities of the cleaved LrInu enzymes.
Figure 4 is a schematic diagram showing the intracellular homologous recombination process for introduction into the Saccharomyces cerevisiae strain by linking the LrInu gene and 20 kinds of protein secretion fusion factors (TFPs) to the GAL 10 promoter.
5 is a result of SDS-PAGE and Western blot analysis of protein supernatant expressed in 20 strains of Saccharomyces cerevisiae and secreted out of cells.
6 is a graph showing the comparative activity of protein supernatant expressed in 20 strains of Saccharomyces cerevisiae and secreted out of cells.
7 is a schematic diagram of a map of the recombinant vector YGaTFP13-LrInuΔNC-6H.
FIG. 8 is a result of confirming cell growth and enzyme activity by fed-batch fermentation of the yeast Saccharomyces cerevisiae strain containing the recombinant vector YGaTFP13-LrInuΔNC-6H in a 5 L fermentor.
9 is a result of SDS-PAGE analysis of 10 µl of culture medium taken by time by fed-batch fermentation of the yeast Saccharomyces cerevisiae strain containing the recombinant vector YGaTFP13-LrInuΔNC-6H in a 5 L fermentor.
10 is a graph showing the growth of the strain by culturing the yeast Saccharomyces cerevisiae strain containing YGaTFP13-LrInuΔNC-6H in a medium containing various concentrations of sucrose.
11 is a graph showing the production of FOS by culturing the yeast Saccharomyces cerevisiae strain containing YGaTFP13-LrInuΔNC-6H in a medium containing various concentrations of sucrose.
12 is a graph showing the growth of the strain by culturing a strain in the yeast Saccharomyces cerevisiae containing YGaTFP13-LrInuΔNC-6H using a yeast medium containing sucrose in a fermentor.
13 is a graph showing the production of FOS by culturing a strain in the yeast Saccharomyces ceribage containing YGaTFP13-LrInuΔNC-6H using a yeast medium containing sucrose in a fermentor.
14 is a graph of DP analysis of FOS obtained by culturing a strain on the yeast Saccharomyces cerevisiae containing YGaTFP13-LrInuΔNC-6H in a sucrose-containing medium.
Fig. 15 is a graph showing the analysis of FOS produced using inulosucrase obtained from fed-batch culture of recombinant yeast.

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

Example 1. Secretion production of inulosucrase through protein cleavage

In order to construct an expression vector derived from Lactobacillus leuteri to secrete inulosucrase (LrInu), the gene (NCBI GenBank: AAN05575.1, SEQ ID NO: 1) was synthesized by requesting Bioneer. LrInu is known as a representative refractory protein, and has been produced as a recombinant protein in E. coli after cutting the carboxy terminus. Therefore, in order to confirm the effect of amino terminus cleavage as well as cleavage of the carboxy terminus of the gene, the research team has primer F1 (SEQ ID NO. Each fragment was prepared by PCR (95° C. 5 minutes once; 95° C. 30 seconds, 55° C. 30 seconds, 72° C. 1 minute cycle 25 times; 72° C. 7 minutes once) using PCR (FIG. 1 ). The produced fragment was amplified again with LNK39 (SEQ ID NO: 6) and HisGT50R (SEQ ID NO: 7) primers, and the sequence required for vector and intracellular recombination (in vivo recombination) was introduced, and a histidine tag at the carboxy terminus The introduced gene was secured. The four genes obtained were introduced into the yeast strain Y2805GS (Mat a pep4::HIS3 prb1 can1 his3-200 ura3-52, gal80, suc2) together with the YGaT1 vector containing the MFa secretion signal and transformed through intracellular recombination. The conversion was allowed to form. The four transformed yeasts produced were selected in a medium lacking uracil, cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose) for 40 hours, centrifuged to remove the cells, and culture supernatant 10 μl was analyzed by SDS-PAGE (Fig. 2). In addition, in order to confirm the activity of the produced inulosucrase, the amount of FOS produced by reacting the same amount of the culture supernatant with 1% sucrose substrate was compared relative to each other (FIG. 3). The amount of FOS in the sample was developed by flowing HPLC water added with 5 mM sulfuric acid to a column (Animex HPX-87H) at a flow rate of 0.6 mL using an HPLC 1100 series (Agilent). Analyzed using.

As a result of the analysis, it was confirmed that only two types of LrInu△C (white triangle) and LrInu△NC (black triangle) in the form of truncated carboxy terminus secrete recombinant inulosucrase extracellularly as shown in FIG.2. The FOS production ability of the culture supernatant was also consistent with the SDS-PAGE results. Therefore, the double-terminal truncated inulosucrase (LrInuΔNC, blunt inulosucrase, SEQ ID NO: 9), which showed higher activity among the two types of inulosucrase, was finally selected and used for later experiments.

Example 2. Yeast protein secretion fusion factor (TFP) selection for secretory production of inulosucrase

In order to maximize the secretion-producing ability of the selected gene (LrInu△NC), the amplified gene is a linear vector containing 20 kinds of protein secretion fusion factors (Korean Patent No. 0975596) that help protein secretion expression and Together, the yeast strain Y2805GS (Mat a pep4::HIS3 prb1 can1 his3-200 ura3-52, gal80, suc2) was introduced to form a transformant through intracellular recombination (FIG. 4). The amino acid sequence of the protein secretion fusion factor used in the present invention is SEQ ID NO: 11 to SEQ ID NO: 30 in Table 1, and the nucleotide sequence is SEQ ID NO: 31 to 50.

Sequence number TFP name Amino acid sequence 11 TFP1 amino acid MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR 12 TFP2 amino acid MFNRFNKFQAAVALALLSRGALGDSYTNSTSSADLSSITSVSSASASATASDSLSSSDGTVYLPSTTISGDLTVTGKVIATEAVEVAAGGKLTLLDGEKYVFSSEAASASAGLALDKR 13 TFP3 amino acid MTPYAVAITVALLIVTVSALQVNNSCVAFPPSNLRGKNGDGTNEQYATALLSIPWNGPPESSRDINLIELEPQVALYLLENYINHYYNTTRDNKCPNNHYLMGGQLGSSSDNRSLNEAASASAGLALDKR 14 TFP4 amino acid MQFKNVALAASVAALSATASAEGYTPGEPWSTLTPTGSISCGAAEYTTTFGIAVQAITSSKAKRDVISQIGDGQVQATSAATAQATDSQAQATTTATPTSSEKMAASASAGLA 15 TFP5 amino acid MLQSVVFFALLTFASSVSAIYSNNTVSTTTTLAPSYSLVPQETTISYADDLAASASAGLALDKR 16 TFP6 amino acid MKFSTAVTTLISSGAIVSALPHVDVHQEDAHQHKRAVAYKYVYETVVVDSDGHTVTPAASEVATAATSAIITTSVLAPTSSAAAADSSASIAVSSAALAKNEKISDAAASATASTSQGASSSSYLAASASAGLALDKR 17 TFP7 amino acid MNWLFLVSLVFFCGVSTHPALAMSSNRLLKLANKSPKKIIPLKDSSFENILAPPHENAYIVALFTATAPEIGCSLCLELESEYDTIVASWFDDHPDAKSSNSDTSIFFTKVNLEDPSKTIPKAFQFFQLNNVPRLFIFKLNSPSILDHSVISISTDTGSERMKASASLAKLDLPVGASASFSLAKLDQ 18 TFP8 amino acid MKFSSVTAITLATVATVATAKKGEHDFTTTLTLSSDGSLTTTTSTHTTHKYGKFNKTSKSKTPLAASASAGLALDKR 19 TFP9 amino acid MASFATKFVIACFLFFSASAHNVLLPAYGRRCFFEDLSKGDELSISFQFGDRNPQSSSQLTGDFIIYGPERHEVLKTVRELAASASAGLALDKR 20 TFP10 amino acid MQYKKTLVASALAATTLAAYAPSEPWSTLTPTATYSGGVTDYASTFGIAVQPISTTSSASSAATTASSKAKRAASQIGDGQVQAATTTASVSTKSTAAAVSQIGDGQIQATTKTTAAAVSQIGDGQIQATTKTTSAKTTAAAVSQISDGQIQATTTTLAPLA 21 TFP11 amino acid MQFKNALTATAILSASALAANSTTSIPSSCSIGTSATATAQATDSQAQATTTAPLAASASAGLALDKR 22 TFP12 amino acid MVSKTWICGFISIITVVQALSCEKHDVLKKYQVGKFSSLTSTERDTPPSTTIEKWWINVCEEHNVEPPEECKKNDMLCGLTDVILPGKDAITTQIIDFDKNIGFNVEETESALTLTLNGATWGANSFDAKLEFQCNDNMKQDELAASASAGLALDKR 23 TFP13 amino acid MKLSALLALSASTAVLAAPAVHHSDNHHHNDKRAVVTVTQYVNADGAVVIPAATTATSAAADGKVESVAAATTTLSSTAAAATTLAASASAGLALDKR 24 TFP14 amino acid MRFSTTLATAATALFFTASQVSAIGELAFNLGVKNNDGTCKSTSDYETELQALKSYTSTVKVYAASDCNTLQNLGPAAEAEGFTIFVGVWPLAASASAGLALDKR 25 TFP15 amino acid MRLSNLIASASLLSAATLAAPANHEHKDKRAVVTTTVQKQTTIIVNGAASTPVAALEENAVVNSAPAAATSTTSSAASVATAASSSENNSQVSAAASPASSSAATSTQSSLAASASAGLALDKR 26 TFP16 amino acid MQFSTVASIAAVAAVASAAANVTTATVSQESTTLVTITSCEDHVCSETVSPALVSTATVTVDDVITQYTTWCPLTTEAPKNGTSTAAPVTSTEAPKNTTSAAPTHSVTSYTGAAAKALPAAGALLAASASAGLALDKR 27 TFP17 amino acid MKFSSALVLSAVAATALAESITTTITATKNGHVYTKTVTQDATFVWGGEDSYASSTSAAESSAAETSAAETSAAATTSAAATTSAAETSSAAETSSADEGSGSSITTTITATKNGHVYTKTVTQDATFVWTGEGSSNTWSPSSTSTSSEAATSSASTTATTLLAASAGLDLA 28 TFP18 amino acid MKFQVVLSALLACSSAVVASPIENLFKYRAVKASHSKNINSTLPAWNGSNSSNVTYANGTNSTTNTTTAESSQLQIIVTGGQVPITNSSLTHTNYTRLFNSSSALNITELYNVARVVNETIQDNLAASASAGLALDKR 29 TFP19 amino acid MRAITLLSSVVSLALLSKEVLATPPACLLACVAQVGKSSSTCDSLNQVTCYCEHENSAVKKCLDSICPNNDADAAYSAFKSSCSEQNASLGDSSGSASSSVLAASASAGLALDKR 30 TFP20 amino acid MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTLAASASAGLALDKR

Inulosucrase is known to be a protein that is difficult to secrete production in yeast, but when using the protein secretion fusion factor technology, 12 of the 20 protein secretion fusion factors can secrete and produce LrInuΔNC as shown in FIG. 5. It was confirmed that it can be. Among them, TFP6, 10, 13 was able to confirm the improved secretion ability more than two times compared to MFα. Of these, it was confirmed that TFP 13 can secrete and produce inulosucrase most efficiently (FIG. 6).

According to the above results, YGaTFP13-LrInuΔNC-6H was constructed as an expression vector that produces highly secreted Lactobacillus luteri-derived inulosucrase (FIG. 7).

Example 3. Mass production of inulosucrase through fed-batch fermentation of recombinant yeast

In order to mass-produce inulosucrase, the recombinant yeast (YGaTFP13-LrInuΔNC-6H/Y2805GS) containing the YGaTFP13-LrInuΔNC-6H vector described above was fed-batch culture using a 5 L fermentor. After initial culture in 50 ml YNB (0.67% amino acid-deficient yeast substrate, 0.5% casamino acid, 2% glucose) medium before entering the main culture, 200 ml of YPD liquid medium (2% glucose, 3% yeast extract) , 1.5% peptone) and inoculated in 1.8 L of the main culture solution (2% glucose, 3% yeast extract, 1.5% peptone) and fermented at 30°C. When glucose was completely consumed during cultivation, the feed medium (30% glucose, 15% yeast extract) was fermented for 60 hours while gradually adding from 2 g/L to 20 g/L per hour according to the growth of the cells. Samples were taken for each time during incubation, and the degree of growth of cells was measured by measuring absorbance at a wavelength of 600 nm, and FOS production capability was analyzed by HPLC to measure the activity of the enzyme (FIG. 8). Inulosucrase secreted in the medium was confirmed by SDS-PAGE analysis of 10 µl of the culture supernatant (FIG. 9).

As a result of the analysis, the recombinant inulosucrase produced through fed-batch fermentation over 60 hours could be obtained from the cultured cells having an absorbance of 128 at an activity level of 220 U/mL.

Example 4. Verification of optimal sucrose concentration for FOS production using recombinant yeast

Existing FOS production processes extract and partially purify enzymes produced from wild-type or recombinant strains, and then convert the inulin substrate into FOS, but this method requires extraction of the enzyme and purification costs due to the removal of monosaccharides, which are by-products. However, if it is possible to produce FOS by directly culturing a yeast strain that secretes and produces inulosucrase in a sucrose-containing medium, the production cost can be reduced. In addition, since a large amount of glucose and fructose produced in the FOS production process from sucrose is used as a carbon source of yeast, the purity of the produced FOS can be increased.

Accordingly, the optimal sucrose concentration was verified using a 250 mL flask by pre-experiment of FOS production through direct fermentation of the prepared recombinant yeast (YGaTFP13-LrInuΔNC-6H/Y2805GS). Specifically, yeast (YGaTFP13-LrInu△NC-6H/Y2805GS) expressing inulosucrase was incubated for 39 hours in a YPS medium containing 10-40% sucrose, and the absorbance of the culture medium was increased at 600 nm at intervals of 3 hours. Cell growth was confirmed by measurement, and the amount of FOS generated in the supernatant after centrifugation was quantitatively analyzed.

As a result of the analysis, in the medium containing 10-30% sucrose, there was no significant difference in the initial growth rate of yeast cells, but in the case of 10%, cells having a maximum absorbance of 25 levels could be secured due to depletion of the carbon source. At a sucrose concentration of 20-30%, as a higher carbon source was supplied, cell growth of 35 levels of absorbance was observed. At a sucrose concentration of 40%, osmotic stress acts as a high concentration of sugar is contained, thereby inhibiting the initial cell growth rate, thereby confirming an absorbance value of 30 levels (FIG. 10). FOS production according to the sucrose concentration tended to increase as the sucrose concentration increased (FIG. 11). Accordingly, the maximum production and yield were calculated and scale-up experiments were performed under the conditions of 30% and 40%.

Example 5. FOS production using fermentation of recombinant yeast

FOS production using recombinant yeast fermentation was performed in a 5 L fermentor. In performing the fermentation of recombinant yeast in a medium containing 30% and 40% sucrose, which are the conditions of the maximum production amount and yield identified in Example 4, the pH of the culture solution was maintained at 5.5 using aqueous ammonia, and 900 rpm, Incubation was performed for 45 hours under the condition of 1 vvm. The culture solution was taken every 5 hours during the culture to check the cell growth rate and FOS production.

As a result of the analysis, the degree of cell growth was confirmed to be 50 levels of absorbance at 30% sucrose concentration, and at 40% sucrose concentration, the absorbance value was 40 levels, which was inhibited from 30% sucrose concentration according to the inhibition of cell growth by osmotic stress as in flask culture. It was confirmed that the cells were growing (FIG. 12). As summarized in Table 2 and FIG. 13 below, the FOS production was confirmed at a level of 128.3 g/L from 30% sucrose and 152.6 g/L from 40% sucrose. As for the production yield, 30% was calculated to be superior to 40%, but it was confirmed that the sucrose concentration of 40% was more excellent in production and productivity.

Sucrose concentration (%) Production (g/L) Production yield (%) Time(h) Productivity (g/L/h) 30 128.3 ± 2.3 85.6 36 3.56 40 152.6 ± 1.4 76.3 39 3.91

Example 6. Analysis of the degree of polymerization of FOS directly produced using recombinant yeast

In order to confirm the degree of polymerization of the FOS produced through the above example, ion chromatography was performed. For analysis, the samples were separated by increasing the concentrations of sodium acetate buffer A and buffer B in which 600 mM NaOH was added to buffer A using a Dionex ICS-3000 instrument.

As a result of the analysis, it was confirmed that the FOS produced by the culture had a wide range of DP 2-20 levels (FIG. 14).

From the above description, other persons skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or altered forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant yeast secreting inulosucrase and a method of producing fructooligosaccharides <130> KPA191023-KR <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 760 <212> PRT <213> Unknown <220> <223> inulosucrase amino acid <400> 1 Asp Thr Asn Ile Glu Asn Asn Asp Ser Ser Thr Val Gln Val Thr Thr 1 5 10 15 Gly Asp Asn Asp Ile Ala Val Lys Ser Val Thr Leu Gly Ser Gly Gln 20 25 30 Val Ser Ala Ala Ser Asp Thr Thr Ile Arg Thr Ser Ala Asn Ala Asn 35 40 45 Ser Ala Ser Ser Ala Ala Asn Thr Gln Asn Ser Asn Ser Gln Val Ala 50 55 60 Ser Ser Ala Ala Ile Thr Ser Ser Thr Ser Ser Ala Ala Ser Ser Asn 65 70 75 80 Asn Thr Asp Ser Lys Ala Ala Gln Glu Asn Thr Asn Thr Ala Lys Asn 85 90 95 Asp Asp Thr Gln Lys Ala Ala Pro Ala Asn Glu Ser Ser Glu Ala Lys 100 105 110 Asn Glu Pro Ala Val Asn Val Asn Asp Ser Ser Ala Ala Lys Asn Asp 115 120 125 Asp Gln Gln Ser Ser Lys Lys Asn Thr Thr Ala Lys Leu Asn Lys Asp 130 135 140 Ala Glu Asn Val Val Lys Lys Ala Gly Ile Asp Pro Asn Ser Leu Thr 145 150 155 160 Asp Asp Gln Ile Lys Ala Leu Asn Lys Met Asn Phe Ser Lys Ala Ala 165 170 175 Lys Ser Gly Thr Gln Met Thr Tyr Asn Asp Phe Gln Lys Ile Ala Asp 180 185 190 Thr Leu Ile Lys Gln Asp Gly Arg Tyr Thr Val Pro Phe Phe Lys Ala 195 200 205 Ser Glu Ile Lys Asn Met Pro Ala Ala Thr Thr Lys Asp Ala Gln Thr 210 215 220 Asn Thr Ile Glu Pro Leu Asp Val Trp Asp Ser Trp Pro Val Gln Asp 225 230 235 240 Val Arg Thr Gly Gln Val Ala Asn Trp Asn Gly Tyr Gln Leu Val Ile 245 250 255 Ala Met Met Gly Ile Pro Asn Gln Asn Asp Asn His Ile Tyr Leu Leu 260 265 270 Tyr Asn Lys Tyr Gly Asp Asn Glu Leu Ser His Trp Lys Asn Val Gly 275 280 285 Pro Ile Phe Gly Tyr Asn Ser Thr Ala Val Ser Gln Glu Trp Ser Gly 290 295 300 Ser Ala Val Leu Asn Ser Asp Asn Ser Ile Gln Leu Phe Tyr Thr Arg 305 310 315 320 Val Asp Thr Ser Asp Asn Asn Thr Asn His Gln Lys Ile Ala Ser Ala 325 330 335 Thr Leu Tyr Leu Thr Asp Asn Asn Gly Asn Val Ser Leu Ala Gln Val 340 345 350 Ala Asn Asp His Ile Val Phe Glu Gly Asp Gly Tyr Tyr Tyr Gln Thr 355 360 365 Tyr Asp Gln Trp Lys Ala Thr Asn Lys Gly Ala Asp Asn Ile Ala Met 370 375 380 Arg Asp Ala His Val Ile Glu Asp Asp Asn Gly Asp Arg Tyr Leu Val 385 390 395 400 Phe Glu Ala Ser Thr Gly Leu Glu Asn Tyr Gln Gly Glu Asp Gln Ile 405 410 415 Tyr Asn Trp Leu Asn Tyr Gly Gly Asp Asp Ala Phe Asn Ile Lys Ser 420 425 430 Leu Phe Arg Ile Leu Ser Asn Asp Asp Ile Lys Ser Arg Ala Thr Trp 435 440 445 Ala Asn Ala Ala Ile Gly Ile Leu Lys Leu Asn Lys Asp Glu Lys Asn 450 455 460 Pro Lys Val Ala Glu Leu Tyr Ser Pro Leu Ile Ser Ala Pro Met Val 465 470 475 480 Ser Asp Glu Ile Glu Arg Pro Asn Val Val Lys Leu Gly Asn Lys Tyr 485 490 495 Tyr Leu Phe Ala Ala Thr Arg Leu Asn Arg Gly Ser Asn Asp Asp Ala 500 505 510 Trp Met Asn Ala Asn Tyr Ala Val Gly Asp Asn Val Ala Met Val Gly 515 520 525 Tyr Val Ala Asp Ser Leu Thr Gly Ser Tyr Lys Pro Leu Asn Asp Ser 530 535 540 Gly Val Val Leu Thr Ala Ser Val Pro Ala Asn Trp Arg Thr Ala Thr 545 550 555 560 Tyr Ser Tyr Tyr Ala Val Pro Val Ala Gly Lys Asp Asp Gln Val Leu 565 570 575 Val Thr Ser Tyr Met Thr Asn Arg Asn Gly Val Ala Gly Lys Gly Met 580 585 590 Asp Ser Thr Trp Ala Pro Ser Phe Leu Leu Gln Ile Asn Pro Asp Asn 595 600 605 Thr Thr Thr Val Leu Ala Lys Met Thr Asn Gln Gly Asp Trp Ile Trp 610 615 620 Asp Asp Ser Ser Glu Asn Leu Asp Met Ile Gly Asp Leu Asp Ser Ala 625 630 635 640 Ala Leu Pro Gly Glu Arg Asp Lys Pro Val Asp Trp Asp Leu Ile Gly 645 650 655 Tyr Gly Leu Lys Pro His Asp Pro Ala Thr Pro Asn Asp Pro Glu Thr 660 665 670 Pro Thr Thr Pro Glu Thr Pro Glu Thr Pro Asn Thr Pro Lys Thr Pro 675 680 685 Lys Thr Pro Glu Asn Pro Gly Thr Pro Gln Thr Pro Asn Thr Pro Asn 690 695 700 Thr Pro Glu Ile Pro Leu Thr Pro Glu Thr Pro Lys Gln Pro Glu Thr 705 710 715 720 Gln Thr Asn Asn Arg Leu Pro Gln Thr Gly Asn Asn Ala Asn Lys Ala 725 730 735 Met Ile Gly Leu Gly Met Gly Thr Leu Leu Ser Met Phe Gly Leu Ala 740 745 750 Glu Ile Asn Lys Arg Arg Phe Asn 755 760 <210> 2 <211> 2280 <212> DNA <213> Unknown <220> <223> inulosucrase nucleotide <400> 2 gacactaata ttgagaataa tgactcgtct actgttcaag ttactactgg agataatgat 60 attgctgtta aatcggttac tttgggatcg ggtcaggttt ctgctgcttc tgatactact 120 attcgaactt ctgctaatgc taatagcgcg tcttcggctg caaatacgca aaattctaat 180 tctcaagttg cttcttctgc tgcaattacc tcttctacat cttccgcggc gtcttctaac 240 aacacggatt ctaaggctgc tcaagaaaat acgaataccg ctaaaaatga cgatactcag 300 aaagctgctc cagccaatga atcttctgaa gccaaaaatg aaccggctgt aaacgttaac 360 gactcttctg ctgcaaaaaa cgatgatcaa caatcctcta aaaaaaatac tactgctaaa 420 ttgaataagg atgcggagaa tgttgttaag aaagctggta ttgatccaaa ctctttgact 480 gatgatcaga ttaaagcttt gaataaaatg aatttctcta aggctgctaa atctggtacg 540 caaatgactt ataatgattt ccaaaagatt gctgatactc taattaaaca ggacggtcgc 600 tatactgtgc cgttctttaa ggcttctgaa attaagaata tgcccgctgc tactaccaag 660 gatgctcaaa ctaacactat tgaaccgttg gatgtttggg attcttggcc agttcaagat 720 gtaagaactg gccaggttgc caattggaat ggttatcaat tggtcatcgc tatgatgggt 780 attcctaatc agaatgataa ccatatctac ttgttgtaca ataaatatgg tgataatgaa 840 ttgtctcact ggaaaaatgt aggtccaatt tttggttaca atagtactgc agtttcgcag 900 gagtggtctg gtagcgctgt tttgaactct gataattcta tccaattgtt ttatacaaga 960 gttgatactt ctgataacaa tactaatcat cagaagatag cttctgctac tttgtatttg 1020 acggataata acggcaatgt gtctttggct caagttgcta atgaccatat tgttttcgag 1080 ggcgatggtt attactacca aacctacgac caatggaagg ccactaataa aggggctgat 1140 aatattgcga tgcgtgatgc tcatgttatt gaagatgaca atggcgatag atatcttgtc 1200 tttgaagcgt ctactggtct tgaaaattac caaggtgaag atcaaattta taactggttg 1260 aattacggtg gtgatgacgc tttcaacatt aaaagcttgt ttcgtatttt gtctaatgac 1320 gatattaagt ctagagctac ttgggctaac gctgctatcg gtattttgaa attaaataaa 1380 gacgaaaaaa atccaaaagt tgctgaatta tattctccac tgatctctgc tcctatggtt 1440 tctgacgaaa ttgagagacc aaatgttgtt aagttaggta ataaatatta tctctttgct 1500 gctactaggt tgaatagggg ttctaatgac gatgcttgga tgaatgccaa ctacgctgtt 1560 ggtgacaatg tcgctatggt tggatatgtc gctgactctt tgactggttc ctacaagcca 1620 ttgaatgatt ctggtgtcgt tttgactgct tctgtccccg ctaactggag aacagctact 1680 tattcttact acgctgttcc cgttgctggt aaagatgatc aggtcttggt tactagctat 1740 atgactaata gaaatggtgt tgctggtaaa ggtatggact ctacatgggc tccaagcttt 1800 ttgttgcaaa ttaacccaga taatactact actgtgttgg ctaaaatgac taatcaaggt 1860 gattggatat gggatgactc ttctgaaaat ttggatatga taggtgacct ggattctgct 1920 gctttgccag gtgaaaggga taagcccgtt gattgggact tgattggtta tggattgaag 1980 ccacacgacc cagctactcc aaatgatccc gaaactccaa ctactccaga aactccagag 2040 actccaaaca ctccaaagac accaaaaaca ccagaaaacc caggtactcc acaaacacca 2100 aatactccta atacccccga gattccgctg actccagaga cgccaaaaca accagaaact 2160 caaactaata acagattgcc gcagacaggg aacaacgcta acaaggctat gattggtttg 2220 ggtatgggta ctttgttgtc gatgtttggt ttggctgaga tcaataaaag aagattcaac 2280 2280 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer F1 <400> 3 ctcgccttag ataaaagaga cactaatatt gagaataatg actcg 45 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primer F2 <400> 4 ctgctggcct cgccttagat aaaagagatg atcaacaatc ctctaaaaaa 50 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer R1 <400> 5 gtgatggtga tggtgatggt tgaatcttct tttattgatc tcagc 45 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer R2 <400> 6 gtgatggtga tggtggtgtg gcttcaatcc ataacc 36 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer LNK39 <400> 7 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 8 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Primer HisGT50R <400> 8 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac ttagtgatgg 60 tgatggtgat g 71 <210> 9 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Truncated inulosucrase amino acid <400> 9 Asp Asp Gln Gln Ser Ser Lys Lys Asn Thr Thr Ala Lys Leu Asn Lys 1 5 10 15 Asp Ala Glu Asn Val Val Lys Lys Ala Gly Ile Asp Pro Asn Ser Leu 20 25 30 Thr Asp Asp Gln Ile Lys Ala Leu Asn Lys Met Asn Phe Ser Lys Ala 35 40 45 Ala Lys Ser Gly Thr Gln Met Thr Tyr Asn Asp Phe Gln Lys Ile Ala 50 55 60 Asp Thr Leu Ile Lys Gln Asp Gly Arg Tyr Thr Val Pro Phe Phe Lys 65 70 75 80 Ala Ser Glu Ile Lys Asn Met Pro Ala Ala Thr Thr Lys Asp Ala Gln 85 90 95 Thr Asn Thr Ile Glu Pro Leu Asp Val Trp Asp Ser Trp Pro Val Gln 100 105 110 Asp Val Arg Thr Gly Gln Val Ala Asn Trp Asn Gly Tyr Gln Leu Val 115 120 125 Ile Ala Met Met Gly Ile Pro Asn Gln Asn Asp Asn His Ile Tyr Leu 130 135 140 Leu Tyr Asn Lys Tyr Gly Asp Asn Glu Leu Ser His Trp Lys Asn Val 145 150 155 160 Gly Pro Ile Phe Gly Tyr Asn Ser Thr Ala Val Ser Gln Glu Trp Ser 165 170 175 Gly Ser Ala Val Leu Asn Ser Asp Asn Ser Ile Gln Leu Phe Tyr Thr 180 185 190 Arg Val Asp Thr Ser Asp Asn Asn Thr Asn His Gln Lys Ile Ala Ser 195 200 205 Ala Thr Leu Tyr Leu Thr Asp Asn Asn Gly Asn Val Ser Leu Ala Gln 210 215 220 Val Ala Asn Asp His Ile Val Phe Glu Gly Asp Gly Tyr Tyr Tyr Gln 225 230 235 240 Thr Tyr Asp Gln Trp Lys Ala Thr Asn Lys Gly Ala Asp Asn Ile Ala 245 250 255 Met Arg Asp Ala His Val Ile Glu Asp Asp Asn Gly Asp Arg Tyr Leu 260 265 270 Val Phe Glu Ala Ser Thr Gly Leu Glu Asn Tyr Gln Gly Glu Asp Gln 275 280 285 Ile Tyr Asn Trp Leu Asn Tyr Gly Gly Asp Asp Ala Phe Asn Ile Lys 290 295 300 Ser Leu Phe Arg Ile Leu Ser Asn Asp Asp Ile Lys Ser Arg Ala Thr 305 310 315 320 Trp Ala Asn Ala Ala Ile Gly Ile Leu Lys Leu Asn Lys Asp Glu Lys 325 330 335 Asn Pro Lys Val Ala Glu Leu Tyr Ser Pro Leu Ile Ser Ala Pro Met 340 345 350 Val Ser Asp Glu Ile Glu Arg Pro Asn Val Val Lys Leu Gly Asn Lys 355 360 365 Tyr Tyr Leu Phe Ala Ala Thr Arg Leu Asn Arg Gly Ser Asn Asp Asp 370 375 380 Ala Trp Met Asn Ala Asn Tyr Ala Val Gly Asp Asn Val Ala Met Val 385 390 395 400 Gly Tyr Val Ala Asp Ser Leu Thr Gly Ser Tyr Lys Pro Leu Asn Asp 405 410 415 Ser Gly Val Val Leu Thr Ala Ser Val Pro Ala Asn Trp Arg Thr Ala 420 425 430 Thr Tyr Ser Tyr Tyr Ala Val Pro Val Ala Gly Lys Asp Asp Gln Val 435 440 445 Leu Val Thr Ser Tyr Met Thr Asn Arg Asn Gly Val Ala Gly Lys Gly 450 455 460 Met Asp Ser Thr Trp Ala Pro Ser Phe Leu Leu Gln Ile Asn Pro Asp 465 470 475 480 Asn Thr Thr Thr Val Leu Ala Lys Met Thr Asn Gln Gly Asp Trp Ile 485 490 495 Trp Asp Asp Ser Ser Glu Asn Leu Asp Met Ile Gly Asp Leu Asp Ser 500 505 510 Ala Ala Leu Pro Gly Glu Arg Asp Lys Pro Val Asp Trp Asp Leu Ile 515 520 525 Gly Tyr Gly Leu Lys Pro 530 <210> 10 <211> 1602 <212> DNA <213> Artificial Sequence <220> <223> Truncated inulosucrase nucleotide <400> 10 gatgatcagc agagcagcaa aaaaaacacc accgcgaaac tgaacaaaga tgcggaaaac 60 gtggtgaaaa aagcgggcat tgatccgaac agcctgaccg atgatcagat taaagcgctg 120 aacaaaatga actttagcaa agcggcgaaa agcggcaccc agatgaccta taacgatttt 180 cagaaaattg cggataccct gattaaacag gatggccgct ataccgtgcc gttttttaaa 240 gcgagcgaaa ttaaaaacat gccggcggcg accaccaaag atgcgcagac caacaccatt 300 gaaccgctgg atgtgtggga tagctggccg gtgcaggatg tgcgcaccgg ccaggtggcg 360 aactggaacg gctatcagct ggtgattgcg atgatgggca ttccgaacca gaacgataac 420 catatttatc tgctgtataa caaatatggc gataacgaac tgagccattg gaaaaacgtg 480 ggcccgattt ttggctataa cagcaccgcg gtgagccagg aatggagcgg cagcgcggtg 540 ctgaacagcg ataacagcat tcagctgttt tatacccgcg tggataccag cgataacaac 600 accaaccatc agaaaattgc gagcgcgacc ctgtatctga ccgataacaa cggcaacgtg 660 agcctggcgc aggtggcgaa cgatcatatt gtgtttgaag gcgatggcta ttattatcag 720 acctatgatc agtggaaagc gaccaacaaa ggcgcggata acattgcgat gcgcgatgcg 780 catgtgattg aagatgataa cggcgatcgc tatctggtgt ttgaagcgag caccggcctg 840 gaaaactatc agggcgaaga tcagatttat aactggctga actatggcgg cgatgatgcg 900 tttaacatta aaagcctgtt tcgcattctg agcaacgatg atattaaaag ccgcgcgacc 960 tgggcgaacg cggcgattgg cattctgaaa ctgaacaaag atgaaaaaaa cccgaaagtg 1020 gcggaactgt atagcccgct gattagcgcg ccgatggtga gcgatgaaat tgaacgcccg 1080 aacgtggtga aactgggcaa caaatattat ctgtttgcgg cgacccgcct gaaccgcggc 1140 agcaacgatg atgcgtggat gaacgcgaac tatgcggtgg gcgataacgt ggcgatggtg 1200 ggctatgtgg cggatagcct gaccggcagc tataaaccgc tgaacgatag cggcgtggtg 1260 ctgaccgcga gcgtgccggc gaactggcgc accgcgacct atagctatta tgcggtgccg 1320 gtggcgggca aagatgatca ggtgctggtg accagctata tgaccaaccg caacggcgtg 1380 gcgggcaaag gcatggatag cacctgggcg ccgagctttc tgctgcagat taacccggat 1440 aacaccacca ccgtgctggc gaaaatgacc aaccagggcg attggatttg ggatgatagc 1500 agcgaaaacc tggatatgat tggcgatctg gatagcgcgg cgctgccggg cgaacgcgat 1560 aaaccggtgg attgggatct gattggctat ggcctgaaac cg 1602 <210> 11 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> TFP1 amino acid <400> 11 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 85 90 <210> 12 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> TFP2 amino acid <400> 12 Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu 1 5 10 15 Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ser Thr Ser Ser 20 25 30 Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala 35 40 45 Thr Ala Ser Asp Ser Leu Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro 50 55 60 Ser Thr Thr Ile Ser Gly Asp Leu Thr Val Thr Gly Lys Val Ile Ala 65 70 75 80 Thr Glu Ala Val Glu Val Ala Ala Gly Gly Lys Leu Thr Leu Leu Asp 85 90 95 Gly Glu Lys Tyr Val Phe Ser Ser Glu Ala Ala Ser Ala Ser Ala Gly 100 105 110 Leu Ala Leu Asp Lys Arg 115 <210> 13 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> TFP3 amino acid <400> 13 Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr 1 5 10 15 Val Ser Ala Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Pro Ser 20 25 30 Asn Leu Arg Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr 35 40 45 Ala Leu Leu Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Ser Arg Asp 50 55 60 Ile Asn Leu Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu 65 70 75 80 Asn Tyr Ile Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro 85 90 95 Asn Asn His Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Asp Asn 100 105 110 Arg Ser Leu Asn Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp 115 120 125 Lys Arg 130 <210> 14 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> TFP4 amino acid <400> 14 Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser 1 5 10 15 Ala Thr Ala Ser Ala Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr 20 25 30 Leu Thr Pro Thr Gly Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr 35 40 45 Thr Phe Gly Ile Ala Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg 50 55 60 Asp Val Ile Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala 65 70 75 80 Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Ala 85 90 95 Thr Pro Thr Ser Ser Glu Lys Met Ala Ala Ser Ala Ser Ala Gly Leu 100 105 110 Ala <210> 15 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> TFP5 amino acid <400> 15 Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser 1 5 10 15 Val Ser Ala Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu 20 25 30 Ala Pro Ser Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala 35 40 45 Asp Asp Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 50 55 60 <210> 16 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP6 amino acid <400> 16 Met Lys Phe Ser Thr Ala Val Thr Thr Leu Ile Ser Ser Gly Ala Ile 1 5 10 15 Val Ser Ala Leu Pro His Val Asp Val His Gln Glu Asp Ala His Gln 20 25 30 His Lys Arg Ala Val Ala Tyr Lys Tyr Val Tyr Glu Thr Val Val Val 35 40 45 Asp Ser Asp Gly His Thr Val Thr Pro Ala Ala Ser Glu Val Ala Thr 50 55 60 Ala Ala Thr Ser Ala Ile Ile Thr Thr Ser Val Leu Ala Pro Thr Ser 65 70 75 80 Ser Ala Ala Ala Ala Asp Ser Ser Ala Ser Ile Ala Val Ser Ser Ala 85 90 95 Ala Leu Ala Lys Asn Glu Lys Ile Ser Asp Ala Ala Ala Ser Ala Thr 100 105 110 Ala Ser Thr Ser Gln Gly Ala Ser Ser Ser Ser Tyr Leu Ala Ala Ser 115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 130 135 <210> 17 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> TFP7 amino acid <400> 17 Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser 1 5 10 15 Thr His Pro Ala Leu Ala Met Ser Ser Asn Arg Leu Leu Lys Leu Ala 20 25 30 Asn Lys Ser Pro Lys Lys Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu 35 40 45 Asn Ile Leu Ala Pro Pro His Glu Asn Ala Tyr Ile Val Ala Leu Phe 50 55 60 Thr Ala Thr Ala Pro Glu Ile Gly Cys Ser Leu Cys Leu Glu Leu Glu 65 70 75 80 Ser Glu Tyr Asp Thr Ile Val Ala Ser Trp Phe Asp Asp His Pro Asp 85 90 95 Ala Lys Ser Ser Asn Ser Asp Thr Ser Ile Phe Phe Thr Lys Val Asn 100 105 110 Leu Glu Asp Pro Ser Lys Thr Ile Pro Lys Ala Phe Gln Phe Phe Gln 115 120 125 Leu Asn Asn Val Pro Arg Leu Phe Ile Phe Lys Leu Asn Ser Pro Ser 130 135 140 Ile Leu Asp His Ser Val Ile Ser Ile Ser Thr Asp Thr Gly Ser Glu 145 150 155 160 Arg Met Lys Gln Ile Ile Gln Ala Ile Lys Gln Phe Ser Gln Val Asn 165 170 175 Asp Phe Ser Leu His Leu Pro Val Gly Leu Ala Ala Ser Ala Ser Ala 180 185 190 Gly Leu Ala Leu Asp Lys Arg 195 <210> 18 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> TFP8 amino acid <400> 18 Met Lys Phe Ser Ser Val Thr Ala Ile Thr Leu Ala Thr Val Ala Thr 1 5 10 15 Val Ala Thr Ala Lys Lys Gly Glu His Asp Phe Thr Thr Thr Leu Thr 20 25 30 Leu Ser Ser Asp Gly Ser Leu Thr Thr Thr Thr Ser Thr His Thr Thr 35 40 45 His Lys Tyr Gly Lys Phe Asn Lys Thr Ser Lys Ser Lys Thr Pro Leu 50 55 60 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 65 70 75 <210> 19 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> TFP9 amino acid <400> 19 Met Ala Ser Phe Ala Thr Lys Phe Val Ile Ala Cys Phe Leu Phe Phe 1 5 10 15 Ser Ala Ser Ala His Asn Val Leu Leu Pro Ala Tyr Gly Arg Arg Cys 20 25 30 Phe Phe Glu Asp Leu Ser Lys Gly Asp Glu Leu Ser Ile Ser Phe Gln 35 40 45 Phe Gly Asp Arg Asn Pro Gln Ser Ser Ser Gln Leu Thr Gly Asp Phe 50 55 60 Ile Ile Tyr Gly Pro Glu Arg His Glu Val Leu Lys Thr Val Arg Glu 65 70 75 80 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 85 90 <210> 20 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> TFP10 amino acid <400> 20 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr 1 5 10 15 Leu Ala Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr 20 25 30 Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile 35 40 45 Ala Val Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr 50 55 60 Thr Ala Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln Ile Gly Asp Gly 65 70 75 80 Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr 85 90 95 Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr 100 105 110 Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln 115 120 125 Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser 130 135 140 Gln Ile Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro 145 150 155 160 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 165 170 <210> 21 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> TFP11 amino acid <400> 21 Met Gln Phe Lys Asn Ala Leu Thr Ala Thr Ala Ile Leu Ser Ala Ser 1 5 10 15 Ala Leu Ala Ala Asn Ser Thr Thr Ser Ile Pro Ser Ser Cys Ser Ile 20 25 30 Gly Thr Ser Ala Thr Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln 35 40 45 Ala Thr Thr Thr Ala Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala 50 55 60 Leu Asp Lys Arg 65 <210> 22 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> TFP12 amino acid <400> 22 Met Val Ser Lys Thr Trp Ile Cys Gly Phe Ile Ser Ile Ile Thr Val 1 5 10 15 Val Gln Ala Leu Ser Cys Glu Lys His Asp Val Leu Lys Lys Tyr Gln 20 25 30 Val Gly Lys Phe Ser Ser Leu Thr Ser Thr Glu Arg Asp Thr Pro Pro 35 40 45 Ser Thr Thr Ile Glu Lys Trp Trp Ile Asn Val Cys Glu Glu His Asn 50 55 60 Val Glu Pro Pro Glu Glu Cys Lys Lys Asn Asp Met Leu Cys Gly Leu 65 70 75 80 Thr Asp Val Ile Leu Pro Gly Lys Asp Ala Ile Thr Thr Gln Ile Ile 85 90 95 Asp Phe Asp Lys Asn Ile Gly Phe Asn Val Glu Glu Thr Glu Ser Ala 100 105 110 Leu Thr Leu Thr Leu Asn Gly Ala Thr Trp Gly Ala Asn Ser Phe Asp 115 120 125 Ala Lys Leu Glu Phe Gln Cys Asn Asp Asn Met Lys Gln Asp Glu Leu 130 135 140 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 145 150 155 <210> 23 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> TFP13 amino acid <400> 23 Met Lys Leu Ser Ala Leu Leu Ala Leu Ser Ala Ser Thr Ala Val Leu 1 5 10 15 Ala Ala Pro Ala Val His His Ser Asp Asn His His His Asn Asp Lys 20 25 30 Arg Ala Val Val Thr Val Thr Gln Tyr Val Asn Ala Asp Gly Ala Val 35 40 45 Val Ile Pro Ala Ala Thr Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys 50 55 60 Val Glu Ser Val Ala Ala Ala Thr Thr Thr Leu Ser Ser Thr Ala Ala 65 70 75 80 Ala Ala Thr Thr Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp 85 90 95 Lys Arg <210> 24 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> TFP14 amino acid <400> 24 Met Arg Phe Ser Thr Thr Leu Ala Thr Ala Ala Thr Ala Leu Phe Phe 1 5 10 15 Thr Ala Ser Gln Val Ser Ala Ile Gly Glu Leu Ala Phe Asn Leu Gly 20 25 30 Val Lys Asn Asn Asp Gly Thr Cys Lys Ser Thr Ser Asp Tyr Glu Thr 35 40 45 Glu Leu Gln Ala Leu Lys Ser Tyr Thr Ser Thr Val Lys Val Tyr Ala 50 55 60 Ala Ser Asp Cys Asn Thr Leu Gln Asn Leu Gly Pro Ala Ala Glu Ala 65 70 75 80 Glu Gly Phe Thr Ile Phe Val Gly Val Trp Pro Leu Ala Ala Ser Ala 85 90 95 Ser Ala Gly Leu Ala Leu Asp Lys Arg 100 105 <210> 25 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> TFP15 amino acid <400> 25 Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala 1 5 10 15 Thr Leu Ala Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val 20 25 30 Val Thr Thr Thr Val Gln Lys Gln Thr Thr Ile Ile Val Asn Gly Ala 35 40 45 Ala Ser Thr Pro Val Ala Ala Leu Glu Glu Asn Ala Val Val Asn Ser 50 55 60 Ala Pro Ala Ala Ala Thr Ser Thr Thr Ser Ser Ala Ala Ser Val Ala 65 70 75 80 Thr Ala Ala Ser Ser Ser Glu Asn Asn Ser Gln Val Ser Ala Ala Ala 85 90 95 Ser Pro Ala Ser Ser Ala Ala Thr Ser Thr Gln Ser Ser Leu Ala 100 105 110 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 115 120 <210> 26 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP16 amino acid <400> 26 Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala 1 5 10 15 Ser Ala Ala Ala Asn Val Thr Thr Ala Thr Val Ser Gln Glu Ser Thr 20 25 30 Thr Leu Val Thr Ile Thr Ser Cys Glu Asp His Val Cys Ser Glu Thr 35 40 45 Val Ser Pro Ala Leu Val Ser Thr Ala Thr Val Thr Val Asp Asp Val 50 55 60 Ile Thr Gln Tyr Thr Thr Trp Cys Pro Leu Thr Thr Glu Ala Pro Lys 65 70 75 80 Asn Gly Thr Ser Thr Ala Ala Pro Val Thr Ser Thr Glu Ala Pro Lys 85 90 95 Asn Thr Thr Ser Ala Ala Pro Thr His Ser Val Thr Ser Tyr Thr Gly 100 105 110 Ala Ala Ala Lys Ala Leu Pro Ala Ala Gly Ala Leu Leu Ala Ala Ser 115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 130 135 <210> 27 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> TFP17 amino acid <400> 27 Met Lys Phe Ser Ser Ala Leu Val Leu Ser Ala Val Ala Ala Thr Ala 1 5 10 15 Leu Ala Glu Ser Ile Thr Thr Thr Ile Thr Ala Thr Lys Asn Gly His 20 25 30 Val Tyr Thr Lys Thr Val Thr Gln Asp Ala Thr Phe Val Trp Gly Gly 35 40 45 Glu Asp Ser Tyr Ala Ser Ser Thr Ser Ala Ala Glu Ser Ser Ala Ala 50 55 60 Glu Thr Ser Ala Ala Glu Thr Ser Ala Ala Ala Thr Thr Ser Ala Ala 65 70 75 80 Ala Thr Thr Ser Ala Ala Glu Thr Ser Ser Ala Ala Glu Thr Ser Ser 85 90 95 Ala Asp Glu Gly Ser Gly Ser Ser Ile Thr Thr Thr Ile Thr Ala Thr 100 105 110 Lys Asn Gly His Val Tyr Thr Lys Thr Val Thr Gln Asp Ala Thr Phe 115 120 125 Val Trp Thr Gly Glu Gly Ser Ser Asn Thr Trp Ser Pro Ser Ser Thr 130 135 140 Ser Thr Ser Ser Glu Ala Ala Thr Ser Ser Ala Ser Thr Thr Ala Thr 145 150 155 160 Thr Leu Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 165 170 175 <210> 28 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP18 amino acid <400> 28 Met Lys Phe Gln Val Val Leu Ser Ala Leu Leu Ala Cys Ser Ser Ala 1 5 10 15 Val Val Ala Ser Pro Ile Glu Asn Leu Phe Lys Tyr Arg Ala Val Lys 20 25 30 Ala Ser His Ser Lys Asn Ile Asn Ser Thr Leu Pro Ala Trp Asn Gly 35 40 45 Ser Asn Ser Ser Asn Val Thr Tyr Ala Asn Gly Thr Asn Ser Thr Thr 50 55 60 Asn Thr Thr Thr Ala Glu Ser Ser Gln Leu Gln Ile Ile Val Thr Gly 65 70 75 80 Gly Gln Val Pro Ile Thr Asn Ser Ser Leu Thr His Thr Asn Tyr Thr 85 90 95 Arg Leu Phe Asn Ser Ser Ser Ala Leu Asn Ile Thr Glu Leu Tyr Asn 100 105 110 Val Ala Arg Val Val Asn Glu Thr Ile Gln Asp Asn Leu Ala Ala Ser 115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 130 135 <210> 29 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> TFP19 amino acid <400> 29 Met Arg Ala Ile Thr Leu Leu Ser Ser Val Val Ser Leu Ala Leu Leu 1 5 10 15 Ser Lys Glu Val Leu Ala Thr Pro Pro Ala Cys Leu Leu Ala Cys Val 20 25 30 Ala Gln Val Gly Lys Ser Ser Ser Thr Cys Asp Ser Leu Asn Gln Val 35 40 45 Thr Cys Tyr Cys Glu His Glu Asn Ser Ala Val Lys Lys Cys Leu Asp 50 55 60 Ser Ile Cys Pro Asn Asn Asp Ala Asp Ala Ala Tyr Ser Ala Phe Lys 65 70 75 80 Ser Ser Cys Ser Glu Gln Asn Ala Ser Leu Gly Asp Ser Ser Gly Ser 85 90 95 Ala Ser Ser Ser Val Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu 100 105 110 Asp Lys Arg 115 <210> 30 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> TFP20 amino acid <400> 30 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr 1 5 10 15 Leu Ala Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr 20 25 30 Ser Ser Ser Ser Ile Ser Thr Ser Ser Gly Ser Val Thr Ile Thr Ser 35 40 45 Ser Glu Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr 50 55 60 Glu Thr Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr 65 70 75 80 Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr 85 90 95 Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro 100 105 110 Thr Asn Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala 115 120 125 Pro Thr Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro 130 135 140 Thr Thr Ser Leu Pro Pro Ser Asn Thr Thr Thr Thr Leu Ala Ala Ser 145 150 155 160 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 165 170 <210> 31 <211> 285 <212> DNA <213> Artificial Sequence <220> <223> TFP1 nucleotide <400> 31 aaaagaatga gatttccttc aatttttact gcagttttat tcgcagcatc ctccgcatta 60 gctgctccag tcaacactac aacagaagat gaaacggcac aaattccggc tgaagctgtc 120 atcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt ttccaacagc 180 acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc taaagaagaa 240 ggggtggccg cctcggcctc tgctggcctc gccttagata aaaga 285 <210> 32 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> TFP2 nucleotide <400> 32 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggtg actcttacac caatagcacc tcctccgcag acttgagttc tatcacttcc 120 gtctcgtcag ctagtgcaag tgccaccgct tccgactcac tttcttccag tgacggtacc 180 gtttatttgc catccacaac aattagcggt gatctcacag ttactggtaa agtaattgca 240 accgaggccg tggaagtcgc tgccggtggt aagttgactt tacttgacgg tgaaaaatac 300 gtcttctcat ctgaggccgc ctcggcctct gctggcctcg ccttagataa aaga 354 <210> 33 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> TFP3 nucleotide <400> 33 atgacgccct atgcagtagc aattaccgtg gccttactaa ttgtaacagt gagcgcactc 60 caggtcaaca attcatgtgt cgcttttccg ccatcaaatc tcaggggcaa gaatggagac 120 ggtactaatg aacagtatgc aactgcacta ctttctattc cctggaatgg gcctcctgag 180 tcatcgaggg atattaatct tatcgaactc gaaccgcaag ttgcactcta tttgctcgaa 240 aattatatta accattacta caacaccaca agagacaata agtgccctaa taaccactac 300 ctaatgggag ggcagttggg tagctcatcg gataatagga gtttgaacga ggccgcctcg 360 gcctctgctg gcctcgcctt agataaaaga 390 <210> 34 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> TFP4 nucleotide <400> 34 atgcaattca aaaacgtcgc cctagctgcc tccgttgctg ctctatccgc cactgcttct 60 gctgaaggtt acactccagg tgaaccatgg tccaccttaa ccccaaccgg ctccatctct 120 tgtggtgcag ccgaatacac taccaccttt ggtattgctg ttcaagctat tacctcttca 180 aaagctaaga gagacgttat ctctcaaatt ggtgacggtc aagtccaagc cacttctgct 240 gctactgctc aagccaccga tagtcaagcc caagctacta ctaccgctac cccaaccagc 300 tccgaaaaga tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 351 <210> 35 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> TFP5 nucleotide <400> 35 atgcttcaat ccgttgtctt tttcgctctt ttaaccttcg caagttctgt gtcagcgatt 60 tattcaaaca atactgtttc tacaactacc actttagcgc ccagctactc cttggtgccc 120 caagagacta ccatatcgta cgccgacgac ctggccgcct cggcctctgc tggcctcgcc 180 ttagataaaa ga 192 <210> 36 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP6 nucleotide <400> 36 atgaaattct caactgccgt tactacgttg attagttctg gtgccatcgt gtctgcttta 60 ccacacgtgg atgttcacca agaagatgcc caccaacata agagggccgt tgcgtacaaa 120 tacgtttacg aaactgttgt tgtcgattct gatggccaca ctgtaactcc tgctgcttca 180 gaagtcgcta ctgctgctac ctctgctatc attacaacat ctgtgttggc tccaacctcc 240 tccgcagccg ctgcggatag ctccgcttcc attgctgttt catctgctgc cttagccaag 300 aatgagaaaa tctctgatgc cgctgcatct gccactgcct caacatctca aggggcatcc 360 tcctcatcct acctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 37 <211> 597 <212> DNA <213> Artificial Sequence <220> <223> TFP7 nucleotide <400> 37 atgaattggc tgtttttggt ctcgctggtt ttcttctgcg gcgtgtcaac ccatcctgcc 60 ctggcaatgt ccagcaacag actactaaag ctggctaata aatctcccaa gaaaattata 120 cctctgaagg actcaagttt tgaaaacatc ttggcaccac ctcacgaaaa tgcctatata 180 gttgctctgt ttactgccac agcgcccgaa attggctgtt ctctgtgtct cgagctagaa 240 tccgaatacg acaccatagt ggcctcctgg tttgatgatc atccggatgc aaaatcgtcc 300 aattccgata catctatttt cttcacaaag gtcaatttgg aggacccttc taagaccatt 360 cctaaagcgt tccagttttt ccaactaaac aatgttccta gattgttcat cttcaaacta 420 aactctccct ctattctgga ccacagcgtg atcagtattt ccactgatac tggctcagaa 480 agaatgaagc aaatcataca agccattaag cagttctcgc aagtaaacga cttctcttta 540 cacttacctg tgggtctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 597 <210> 38 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> TFP8 nucleotide <400> 38 atgaagttct cttctgttac tgctattact ctagccaccg ttgccaccgt tgccactgct 60 aagaagggtg aacatgattt cactaccact ttaactttgt catcggacgg tagtttaact 120 actaccacct ctactcatac cactcacaag tatggtaagt tcaacaagac ttccaagtcc 180 aagacccccc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 231 <210> 39 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> TFP9 nucleotide <400> 39 atggcctcat ttgctactaa gtttgtcatt gcttgcttcc tgttcttctc ggcgtccgcc 60 cataatgtcc ttcttccagc ttatggccgt agatgcttct tcgaagactt gagtaagggt 120 gacgagctct ccatttcgtt ccagttcggt gatagaaacc ctcaatccag tagccagctg 180 actggtgact ttatcatcta cgggccggaa agacatgaag ttttgaaaac ggttagggaa 240 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 282 <210> 40 <211> 522 <212> DNA <213> Artificial Sequence <220> <223> TFP10 nucleotide <400> 40 atgcaataca aaaagacttt ggttgcctct gctttggccg ctactacatt ggccgcctat 60 gctccatctg agccttggtc cactttgact ccaacagcca cttacagcgg tggtgttacc 120 gactacgctt ccaccttcgg tattgccgtt caaccaatct ccactacatc cagcgcatca 180 tctgcagcca ccacagcctc atctaaggcc aagagagctg cttcccaaat tggtgatggt 240 caagtccaag ctgctaccac tactgcttct gtctctacca agagtaccgc tgccgccgtt 300 tctcagatcg gtgatggtca aatccaagct actaccaaga ctaccgctgc tgctgtctct 360 caaattggtg atggtcaaat tcaagctacc accaagacta cctctgctaa gactaccgcc 420 gctgccgttt ctcaaatcag tgatggtcaa atccaagcta ccaccactac tttagcccct 480 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 522 <210> 41 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> TFP11 nucleotide <400> 41 atgcaattca agaacgcttt gactgctact gctattctaa gtgcctccgc tctagctgct 60 aactcaacta cttctattcc atcttcatgt agtattggta cttctgccac tgctactgct 120 caagccaccg atagtcaagc ccaagctact actaccgcac ccctggccgc ctcggcctct 180 gctggcctcg ccttagataa aaga 204 <210> 42 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> TFP12 nucleotide <400> 42 atggtatcga agacttggat atgtggcttc atcagtataa ttacagtggt acaggccttg 60 tcctgcgaga agcatgatgt attgaaaaag tatcaggtgg gaaaatttag ctcactaact 120 tctacggaaa gggatactcc gccaagcaca actattgaaa agtggtggat aaacgtttgc 180 gaagagcata acgtagaacc tcctgaagaa tgtaaaaaaa atgacatgct atgtggttta 240 acagatgtca tcttgcccgg taaggatgct atcaccactc aaattataga ttttgacaaa 300 aacattggct tcaatgtcga ggaaactgag agtgcgctta cattgacact aaacggcgct 360 acgtggggcg ccaattcttt tgacgcaaaa ctagaatttc agtgtaatga caatatgaaa 420 caagacgaac tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 471 <210> 43 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> TFP13 nucleotide <400> 43 atgaaattat ccgctctatt agctttatca gcctccaccg ccgtcttggc cgctccagct 60 gtccaccata gtgacaacca ccaccacaac gacaagcgtg ccgttgtcac cgttactcag 120 tacgtcaacg cagacggcgc tgttgttatt ccagctgcca ccaccgctac ctcggcggct 180 gctgatggaa aggtcgagtc tgttgctgct gccaccacta ctttgtcctc gactgccgcc 240 gccgctacaa ccctggccgc ctcggcctct gctggcctcg ccttagataa aaga 294 <210> 44 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> TFP14 nucleotide <400> 44 atgcgtttct ctactacact cgctactgca gctactgcgc tatttttcac agcctcccaa 60 gtttcagcta ttggtgaact agcctttaac ttgggtgtca agaacaacga tggtacttgt 120 aagtccactt ccgactatga aaccgaatta caagctttga agagctacac ttccaccgtc 180 aaagtttacg ctgcctcaga ttgtaacact ttgcaaaact taggtcctgc tgctgaagct 240 gagggattta ctatctttgt cggtgtttgg ccactggccg cctcggcctc tgctggcctc 300 gccttagata aaaga 315 <210> 45 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> TFP15 nucleotide <400> 45 atgcgtctct ctaacctaat tgcttctgcc tctcttttat ctgctgctac tcttgctgct 60 cccgctaacc acgaacacaa ggacaagcgt gctgtggtca ctaccactgt tcaaaaacaa 120 accactatca ttgttaatgg tgccgcttca actccagttg ctgctttgga agaaaatgct 180 gttgtcaact ccgctccagc tgccgctacc agtacaacat cgtctgctgc ttctgtagct 240 accgctgctt cctcttctga gaacaactca caagtttctg ctgccgcatc tccagcctcc 300 agctctgctg ctacatctac tcaatcttct ctggccgcct cggcctctgc tggcctcgcc 360 ttagataaaa ga 372 <210> 46 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP16 nucleotide <400> 46 atgcaatttt ctactgtcgc ttctatcgcc gctgtcgccg ctgtcgcttc tgccgctgct 60 aacgttacca ctgctactgt cagccaagaa tctaccactt tggtcaccat cacttcttgt 120 gaagaccacg tctgttctga aactgtctcc ccagctttgg tttccaccgc taccgtcacc 180 gtcgatgacg ttatcactca atacaccacc tggtgcccat tgaccactga agccccaaag 240 aacggtactt ctactgctgc tccagttacc tctactgaag ctccaaagaa caccacctct 300 gctgctccaa ctcactctgt cacctcttac actggtgctg ctgctaaggc tttgccagct 360 gctggtgctt tgctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 47 <211> 528 <212> DNA <213> Artificial Sequence <220> <223> TFP17 nucleotide <400> 47 atgaaattct cttccgcttt ggttctatct gctgttgccg ctactgctct tgctgagagt 60 atcaccacca ccatcactgc caccaagaac ggtcatgtct acactaagac tgtcacccaa 120 gatgctactt ttgtttgggg tggtgaagac tcttacgcca gcagcacttc tgccgctgaa 180 tcttctgccg ccgaaacttc tgccgccgaa acctctgctg ccgctaccac ttctgctgcc 240 gctaccactt ctgctgctga gacttcttct gctgctgaga cttcttctgc tgatgaaggt 300 tctggttcta gtatcactac cactatcact gccaccaaga acggtcacgt ctacactaag 360 actgtcaccc aagatgctac ttttgtctgg actggtgaag gcagcagcaa cacctggtct 420 ccaagtagta cctctaccag ctcagaagct gctacctctt ctgcttcaac cactgcaacc 480 accctgctgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 528 <210> 48 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP18 nucleotide <400> 48 atgaagttcc aagttgtttt atctgccctt ttggcatgtt catctgccgt cgtcgcaagc 60 ccaatcgaaa acctattcaa atacagggca gttaaggcat ctcacagtaa gaatatcaac 120 tccactttgc cggcctggaa tgggtctaac tctagcaatg ttacctacgc taatggaaca 180 aacagtacta ccaatactac tactgccgaa agcagtcaat tacaaatcat tgtaacaggt 240 ggtcaagtac caatcaccaa cagttctttg acccacacaa actacaccag attattcaac 300 agttcttctg ctttgaacat taccgaattg tacaatgttg cccgtgttgt taacgaaacg 360 atccaagata acctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 49 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> TFP19 nucleotide <400> 49 atgcgtgcca tcactttatt atcttcagtc gtttctttgg cattgttgtc gaaggaagtc 60 ttagcaacac ctccagcttg tttattggcc tgtgttgcgc aagtcggcaa atcctcttcc 120 acatgtgact ctttgaatca agtcacctgt tactgtgaac acgaaaactc cgccgtcaag 180 aaatgtctag actccatctg cccaaacaat gacgctgatg ctgcttattc tgctttcaag 240 agttcttgtt ccgaacaaaa tgcttcattg ggcgattcca gcggcagtgc ctcctcatcc 300 gttctggccg cctcggcctc tgctggcctc gccttagata aaaga 345 <210> 50 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> TFP20 nucleotide <400> 50 atgaaattat caactgtcct attatctgcc ggtttggcct cgactacttt ggcccaattt 60 tccaacagta catctgcttc ttccaccgat gtcacttcct cctcttccat ctccacttcc 120 tctggctcag taactatcac atcttctgaa gctccagaat ccgacaacgg taccagcaca 180 gctgcaccaa ctgaaacctc aacagaggct ccaaccactg ctatcccaac taacggtacc 240 tctactgaag ctccaaccac tgctatccca actaacggta cctctactga agctccaact 300 gatactacta ctgaagctcc aaccaccgct cttccaacta acggtacttc tactgaagct 360 ccaactgata ctactactga agctccaacc accggtcttc caaccaacgg taccacttca 420 gctttcccac caactacatc tttgccacca agcaacacta ccaccactct ggccgcctcg 480 gcctctgctg gcctcgcctt agataaaaga 510

Claims (17)

An inulosucrase secretion expression cassette comprising a nucleic acid encoding inulosucrase and a nucleic acid encoding a translational fusion partner (TFP), wherein the protein secretion fusion factor is SEQ ID NO: A secretion expression cassette that is at least one selected from the group consisting of a protein secretion fusion factor 6 of 16, a protein secretion fusion factor 10 of SEQ ID NO: 20, and a protein secretion fusion factor 13 of SEQ ID NO: 23.
The secretion expression cassette of claim 1, wherein the inulosucrase is derived from Lactobacillus leuteri.
The secretory expression cassette according to claim 1, wherein the inulosucrase is composed of an amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 9.
delete The secretory expression cassette according to claim 1, wherein the protein secretion fusion factor is a protein secretion fusion factor 13 of SEQ ID NO: 23.
The secretory expression cassette according to claim 1, wherein the nucleic acid encoding the inulosucrase and the nucleic acid encoding the protein secretory fusion factor are linked to each other by a linker.
The secretory expression cassette according to claim 6, wherein the linker comprises a protease recognition sequence or an affinity tag.
The secretory expression cassette according to claim 1, wherein the inulosucrase further comprises an affinity tag.
The method of claim 8, wherein the affinity tag is selected from the group consisting of GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, HIS, STREP, CBP, CBD, and S-tag. , Secretion expression cassette.
A vector comprising the inulosucrase secretion expression cassette of any one of claims 1 to 3 and 5 to 9.
A recombinant microorganism containing an inulosucrase secretion expression cassette comprising a nucleic acid encoding inulosucrase and a nucleic acid encoding a protein secretion fusion factor, wherein the protein secretion fusion factor is a protein secretion fusion of SEQ ID NO: 16 Factor 6, the protein secretion fusion factor 10 of SEQ ID NO: 20 and the protein secretion fusion factor 13 of SEQ ID NO: 23 will be any one or more selected from the group consisting of, the recombinant microorganism.
The microorganism according to claim 11, wherein the recombinant microorganism is yeast.
The method of claim 12, wherein the yeast is Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia), ski irradiation Caro My process (Schizosaccharomyces) , Yarrowia (Yarrowia), Saccharomyces (Saccharomyces), Schwanniomyces (Schwanniomyces) and Arsula (Arxula) that is selected from the group consisting of the genus, the microorganism.
The method of claim 12, wherein the yeast is Candida utilis , Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris. pastoris ), Pichia stipitis , Schizosaccharomyces pombe , Saccharomyces cerevisiae , Hansenula polymorpha , Yarrowia repolitica ( Yarrowia lipolytica ), Schwanniomyces occidentalis (Schwanniomyces occidentalis) and Arsula adenini borans (Arxula adeninivorans) that is selected from the group consisting of, a microorganism.
(i) culturing the recombinant microorganism of claim 11; And
(ii) A method for producing inulosucrase comprising the step of recovering inulosucrase from the cultured microorganism or a culture supernatant thereof.
A method for producing fructo-oligosaccharide comprising the step of reacting the inulosucrase prepared by the method of claim 15 with sucrose.
A method for producing fructo-oligosaccharide, comprising fermenting the microorganism prepared by the method of claim 11 in a medium containing sucrose.

Images