Linear inulin synthesizing bacterium and method for producing linear inulin

Abstract

[Problem] To provide: a linear inulin synthesizing bacterium of species closely related to the genus Bacillus, the bacterium being characterized in that a branched inulin synthase gene or a specific branched inulin synthase-like gene is deleted or inactivated and an inulosucrase gene or a specific inulosucrase-like gene is carried; a method for producing linear inulin using the linear inulin synthesizing bacterium; and others. [Solution] Only linear inulin can be produced by deleting or mutating the whole or a part of BI gene in an inulin synthesizing bacterium to produce a ΔBI inulin synthesizing strain in which BI enzyme has been deleted or inactivated, and then allowing a culture supernatant of the produced ΔBI inulin synthesizing strain to act on sucrose.

Description

Linear inulin-synthesizing bacteria and method for producing linear inulin

[0001] The present invention relates to linear inulin-synthesizing bacteria and a method for producing linear inulin, and more specifically to one or more linear inulin-synthesizing bacteria selected from the group consisting of linear inulin-synthesizing bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus and Amoniphilus, which are characterized by having a deleted or inactivated branched inulin synthase gene or a specified branched inulin synthase-like gene and having an inulosucrase gene or a specified inulosucrase-like gene, and linear inulin-synthesizing bacteria of closely related species of the genus Bacillus other than these linear inulin-synthesizing bacteria, and a method for producing linear inulin using at least one of these linear inulin-synthesizing bacteria.

[0002] Inulin is a type of polysaccharide (fructan) found in the roots of chicory and other Asteraceae plants. It has a terminal sucrose structure, with other fructose units polymerized at the fructose moiety, and is characterized by primarily β-2,1 bonds between the fructose units. In a broad sense, fructans consisting only of fructose without terminal glucose are also considered inulin. The degree of polymerization of inulin in plants ranges from approximately 8 to 60. Inulin is synthesized within plants through the action of two enzymes: sucrose:sucrose 1-fructosyltransferase (1-SST), which transfers fructosyl between sucrose units, and 2,1-fructan:2,1-fructan 1-fructosyltransferase (1-FFT), which transfers fructosyl between fructans with a degree of polymerization of 3 or higher. Linear inulin, in which the bonds between fructose sugars are β-2,1 bonds, is difficult to digest and absorb, and is utilized by bifidobacteria in the digestive tract, improving the intestinal flora and intestinal environment. Therefore, it is incorporated as a probiotic into health-conscious processed foods. Inulin cream, which is made by dissolving inulin in water to create a creamy gel, has a texture similar to that of fat, and is therefore used to improve the taste of reduced-fat processed foods.

[0003] Linear inulin is abundant in chicory roots, which are often used as a raw material for industrial production. The production process involves chicory cultivation, root harvesting, transportation to the factory, root cleaning and shredding, hot water extraction, filtration, purification, concentration, and spray drying. To produce linear inulin on an industrial scale, first, sufficient raw materials must be secured, which requires sufficient cultivated land, a long cultivation period from spring to autumn, adequate fertilization, and appropriate pest control measures such as chemicals to prevent disease. Second, since inulin contained in harvested roots is prone to decomposition and degradation during storage, and the roots themselves are prone to storage rot, raw roots must be processed as quickly as possible. Therefore, linear inulin production requires large-scale machinery and equipment capable of processing large quantities of roots in a short period of time. For these reasons, linear inulin production requires considerable labor and equipment, resulting in high production costs, and the industry has long sought affordable linear inulin.

[0004] On the other hand, it has been reported that inulin can be synthesized from sucrose using an enzyme called inulosucrase (hereinafter sometimes referred to as "IS") secreted by microorganisms. Inulosucrase catalyzes the β-2,1 transfer reaction of the fructofuranosyl group of sucrose to the terminal fructose moiety of another sucrose or its transglycosylation product, thereby synthesizing inulin from sucrose. Many examples of inulin synthesis using inulosucrase produced by genetic engineering technology or inulosucrase whose purity has been increased by protein purification have been reported for lactic acid bacteria. For example, Lactobacillus gasseri DSM20243 strain, L. gasseri DSM20604 strain, L. johnsonii NCC533 strain, L. Inulin synthesis by inulosucrasses derived from L. reuteri strain 121, L. reuteri strain TMW1.106, Leuconostoc citreum strain CW28, and Weissella confusa strain MBFCNC-2(1) has been reported. In addition to lactic acid bacteria, inulin synthesis by inulosucrasses derived from Bacillus sp. strain 217C-11 and Salipaludibacillus agaradhaerens strain WDG185 has also been reported (Non-Patent Document 1).

[0005] When attempting to industrially produce inulin using microbial enzymes, if the enzyme that catalyzes the reaction needs to be purified, costs increase, making it difficult to produce inulin inexpensively. All inulosucrase-producing bacteria reported to date secrete inulosucrase into the culture supernatant. In the present invention, a microorganism that secretes inulosucrase into a microbial culture solution and produces inulin when the culture supernatant is mixed with sucrose and maintained at a certain temperature for a certain period of time is referred to as an inulin-synthesizing bacterium. If the culture supernatant of an inulin-synthesizing bacterium could be used as an enzyme solution without purification, inulin could be produced inexpensively. An example of analysis of inulin produced from sucrose using a culture supernatant as an enzyme solution has been reported for Bacillus sp. 217C-11 (Non-Patent Document 2). Based on the inulin peak pattern and shape of HPLC by HPAEC-PAD, the product described in this report was considered to be a composition containing inulin with a bonding mode different in structure from linear inulin.

[0006] Biotechnology Advances, 37, 306-318 (2019) J. Agric. Food Chem. , 53, 1246-1253 (2005)

[0007] In the present invention, inulin with a partially different structure produced by an enzymatic reaction using a culture supernatant (crude enzyme) of a microorganism with sucrose as a substrate is called branched inulin. Branched inulin is a polymer containing bonds between fructose sugars other than β-2,1, and no microorganism has yet been reported in nature that produces a culture supernatant capable of producing only linear inulin.

[0008] In other words, when the culture supernatant of an inulin-synthesizing bacterium is reacted with sucrose as an enzyme solution, branched inulin is inevitably produced, making it impossible to produce only linear inulin. Conventional techniques for producing only linear inulin using bacterial enzymes require the use of inulosucrase protein partially or completely purified from the culture supernatant, which significantly increases industrial production costs. Furthermore, the reason why branched inulin is contained in the inulin produced when the culture supernatant is used as the enzyme solution is unknown, and no microorganism has yet been discovered that produces only linear inulin using a microbial culture supernatant as the enzyme solution. On the other hand, it is technically easy to use genetic engineering to introduce the inulosucrase gene into non-inulin-synthesizing bacteria, cause them to produce inulosucrase, and produce only linear inulin from sucrose. However, industrial use of genetically modified microorganisms requires strict management as an environmental safety measure, which creates new cost factors. In addition, the use of genetic engineering technology in the food industry requires careful confirmation of safety, and consumer concerns about foods produced using genetic engineering technology remain strong.

[0009] The present invention aims to provide one or more straight-chain inulin-synthesizing bacteria selected from the group consisting of straight-chain inulin-synthesizing bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus and Amoniphilus, and straight-chain inulin-synthesizing bacteria of closely related species of the genus Bacillus other than these straight-chain inulin-synthesizing bacteria, characterized in that a branched inulin synthase gene or a specified branched inulin synthase-like gene is deleted or inactivated and an inulosucrase gene or a specified inulosucrase-like gene is present; and a method for producing straight-chain inulin using at least one of these straight-chain inulin-synthesizing bacteria.

[0010] As a result of extensive research to solve the above-mentioned problems, the present inventors have discovered that when attempting to produce only linear inulin from sucrose using the culture supernatant of an inulin-synthesizing bacterium, branched inulin is produced due to not only inulosucrase, which synthesizes linear inulin from sucrose, but also an unknown enzyme, branched inulin synthase (hereinafter sometimes referred to as "BI"), which converts linear inulin to branched inulin, and that branched inulin is synthesized by the cooperative action of these two enzymes. Based on this discovery, we created a linear inulin-synthesizing bacterium (hereinafter sometimes referred to as a "ΔBI inulin-synthesizing strain") in which the BI enzyme was deleted or inactivated by deleting or mutating all or part of the BI gene of the inulin-synthesizing bacterium, and found that when the culture supernatant of the created ΔBI inulin-synthesizing strain was allowed to act on sucrose, only linear inulin was produced, and we completed the following inventions.

[0011] (1) One or more linear inulin-synthesizing bacteria selected from the group consisting of linear inulin-synthesizing bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus and Amoniphilus, and linear inulin-synthesizing bacteria of closely related species of the genus Bacillus other than these linear inulin-synthesizing bacteria, characterized in that at least one gene shown in (a) to (e) below is deleted or inactivated, and at least one gene shown in (h) to (m) below is present: (a) a gene encoding a protein consisting of a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 35; (b) a gene that hybridizes under stringent conditions to the complementary strand of a gene consisting of the nucleotide sequence shown in SEQ ID NO: 35; (c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 36; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 36 in which 1 to 10 amino acids have been deleted, substituted, added or inserted; (e) a gene encoding a protein consisting of an amino acid sequence having 67.9% or more identity with the amino acid sequence shown in SEQ ID NO: 36.(h) a gene having the nucleotide sequence shown in SEQ ID NO: 33; (i) a gene consisting of a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 33 and encoding a protein having the activity of catalyzing the reaction of β-2,1-transfer of the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product; (j) a gene that hybridizes under stringent conditions to the complementary strand of the gene consisting of the nucleotide sequence shown in SEQ ID NO: 33 and encodes a protein having the activity of catalyzing the reaction of β-2,1-transfer of the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product; (k) a gene consisting of the amino acid sequence shown in SEQ ID NO: 34 and encoding a protein that is secreted outside the bacterial cell; (l) A gene encoding an extracellularly secreted protein, which consists of an amino acid sequence obtained by deleting, substituting, adding, or inserting 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 34, and which has the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product; (m) A gene encoding an extracellularly secreted protein, which consists of an amino acid sequence having an identity of 67.3% or more with the amino acid sequence shown in SEQ ID NO: 34, and which has the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product.

[0012] (2) A linear inulin-synthesizing bacterium according to (1), wherein the linear inulin-synthesizing bacterium has the following characteristics (f) or (g); (f) A linear inulin-synthesizing bacterium that produces inulin in which the bonds between fructoses are solely β-2,1 bonds, by inoculating one loopful of bacterial cells into 3 mL of a medium having the following composition, culturing the medium at 30°C for 24 hours at 130 rpm, adding sucrose to the culture supernatant to give a Bx of 40, and then reacting the medium at 37°C for 24 hours. <Culture medium composition> 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH ２ P.O. ４and adjusted to pH 7.2 with NaOH; (g) a linear inulin-synthesizing bacterium, characterized in that, when one loopful of the bacterial cells is inoculated into 3 mL of a medium having the composition (I) below, and cultured under shaking at 30°C for 24 hours at 130 rpm, sucrose is added to the culture supernatant to give a Bx of 40, and the reaction is continued at 37°C for 24 hours, and the resulting reaction product is subjected to HPAEC-PAD analysis under the conditions (II) below, a peak is observed at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16) that is the same as that of inulin consisting only of β-2,1 bonds, and the area of ​​the peaks other than said peak accounts for less than 1% of the total peak area; <(I) Medium composition> 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH ２ P.O. ４ , adjusted to pH 7.2 with NaOH. <(II) Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: constant 100 mM NaOH, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry.

[0013] (3) The linear inulin-synthesizing bacterium according to (1), wherein the gene consisting of SEQ ID NO: 35 or a nucleotide sequence having 75% or more identity to said SEQ ID NO: has the following characteristics: When a protein consisting of an amino acid sequence encoded by the gene consisting of SEQ ID NO: 35 or a nucleotide sequence having 75% or more identity to said SEQ ID NO: and a protein consisting of the amino acid sequence shown in SEQ ID NO: 34 are each added to a sucrose solution of Bx40 so as to give 2 U / g sucrose, and reacted at 37°C for 24 hours, and the resulting reaction product is subjected to HPAEC-PAD analysis under the following conditions, the reaction of the protein consisting of the amino acid sequence shown in SEQ ID NO: 34 with the sucrose solution of Bx40 is characterized in that, from 20 minutes onwards (at a degree of polymerization of 9 or more), 10 or more peaks having the same elution time as linear inulin in which the bond between fructose sugars is only β-2,1 bond are obtained, and 10 or more peaks of branched inulin appearing between the linear inulin peaks and having a structure in which a fructofuranosyl group is bonded to any fructose site of linear inulin by a bond type other than β-2,1 bond are detected, and the branched inulin ratio (sum of peak areas of branched inulins / total peak area×100) is 10% or more at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16). <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH 100 mM constant, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry.

[0014] (4) A purified enzyme, crude enzyme, or enzyme-containing substance, culture medium, culture supernatant, or cultured cells obtained from the linear inulin-synthesizing bacterium described in any one of (1) to (3), or a processed product thereof.

[0015] (5) A method for producing linear inulin, characterized by comprising a step of culturing the linear inulin-synthesizing bacterium described in any one of (1) to (3).

[0016] (6) A method for producing linear inulin, characterized by comprising a step of reacting at least one of a purified enzyme, crude enzyme or enzyme-containing substance, culture medium, culture supernatant or cultured bacterial cells or a processed product thereof obtained from a linear inulin-synthesizing bacterium described in any one of (1) to (3) with sucrose to obtain a reaction product.

[0017] (7) The method for producing linear inulin according to (6), characterized in that, when the reaction product is subjected to HPAEC-PAD analysis under the following conditions, the proportion of the branched inulin (sum of peak areas of branched inulins / total peak area x 100) having a structure in which a fructofuranosyl group is bonded to any fructose site of linear inulin in which the bond between fructoses is β-2,1 bond, by a bond type other than β-2,1 bond, is less than 1% at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16); <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH Constant 100 mM, AcONa gradient (0-60 min, 0 → 600 mM); detection, pulsed amperometry.

[0018] (8) A method for producing DFAIII, characterized by reacting at least one of a purified enzyme, crude enzyme or enzyme-containing substance, culture medium, culture supernatant or cultured bacterial cells, or a processed product thereof, obtained from a linear inulin-synthesizing bacterium described in any one of (1) to (3), with sucrose.

[0019] (9) A method for producing DFAIII according to (8), characterized by using inulin fructotransferase.

[0020] The linear inulin-synthesizing bacterium of the present invention can synthesize only linear inulin from sucrose using its culture supernatant.Furthermore, the highly pure linear inulin free of branched inulin obtained by the present invention can be used to produce DFA III, a type of oligosaccharide, in high yield and at low cost.

[0021]

[0023] Figure 1 shows the peaks of a reaction product obtained by reacting the culture supernatant of an inulin-synthesizing bacterium selected from nature with Bx40 sucrose, and plant (chicory)-derived inulin (raftilin ST) in HPAEC-PAD analysis. Figure 2 shows a simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of identified inulin-synthesizing bacterium of the genus Bacillus, closely related to Bacillus drentensis. Figure 3 shows the peaks of a reaction product obtained by reacting two sucrose metabolism-related enzymes purified from the culture supernatant of the SD33 strain with Bx40 sucrose, and plant-derived inulin (raftilin ST) in HPAEC-PAD analysis.

[0024] Figure 4 shows the peaks of a levan preparation, an inulin preparation, the reaction product of peak A, and the reaction product of peak B. １３ This figure shows C-nuclear magnetic resonance (NMR) signals. This figure shows GC-MS peaks of methylation analysis of the reaction product of peak A, the reaction product of peak B, and peak 2 in the reaction product of peak B. This figure shows peaks by HPAEC-PAD analysis of the reaction product obtained by reacting the IS and BI of recombinant Escherichia coli SD33 strain with Bx40 sucrose. This figure shows a molecular phylogenetic tree created using the 16S rDNA partial base sequences of the type strain of a microorganism with a clear species name, Amoniphilus sp. YIM78166 strain, the type strain of Bacillus subtilis, and the SD33 strain, among a group of microorganisms having base sequences highly homologous to the IS and BI of the SD33 strain. This figure shows peaks by HPAEC-PAD analysis of the reaction products obtained by reacting the culture supernatants of the SD33 strain and the SD33_ΔBI strain with Bx40 sucrose. 1 shows peak diagrams by HPAEC-PAD analysis of reaction products obtained by mixing the culture supernatant of the SD33_ΔBI strain with various purified BI-like recombinant enzymes and reacting them with Bx40 sucrose. 1 shows peak diagrams by HPAEC-PAD analysis of reaction products obtained by reacting the culture supernatant of the SD33_ΔBI strain, OV191_IS (67.3% homology with SD33 strain IS), and LL01_IS (68.8% homology with SD33 strain IS) with Bx40 sucrose.

[0022] The linear inulin-synthesizing bacteria of the present invention, the purified enzyme, crude enzyme or enzyme-containing substance obtained from the linear inulin-synthesizing bacteria of the present invention, the culture medium, the culture supernatant or cultured cells or processed products thereof, the method for producing linear inulin, and the method for producing DFA III will be described in detail below. The linear inulin-synthesizing bacterium of the present invention is one or more linear inulin-synthesizing bacteria selected from the group consisting of linear inulin-synthesizing bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandconia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus and Amoniphilus, and linear inulin-synthesizing bacteria of closely related species of the genus Bacillus other than these linear inulin-synthesizing bacteria, characterized in that the branched inulin synthase (BI) gene or a specific branched inulin synthase-like (BI-like) gene is deleted or inactivated and the inulosucrase (IS) gene or a specific inulosucrase-like (IS-like) gene is present.

[0023] The inventors isolated and collected a large number of inulin-synthesizing bacteria from nature, and used their culture supernatants to intensively search for bacteria that produce only linear inulin from sucrose.They conducted detailed investigations into the inulin products produced using sucrose as a substrate in the culture supernatants of many inulin-synthesizing bacteria, but all of the inulin-synthesizing bacteria produced branched inulin, and no inulin-synthesizing bacteria that produced only linear inulin were found.

[0024] After further intensive investigation, we discovered that an unknown protein is the cause of branched inulin when using the culture supernatant of inulin-synthesizing bacteria. Specifically, we discovered that the culture supernatant of inulin-synthesizing bacteria contains not only IS, which synthesizes linear inulin from sucrose, but also an unknown enzyme that converts linear inulin to branched inulin. This unknown enzyme is BI. We then discovered that branched inulin is synthesized by the cooperative action of these two enzymes, IS and BI.

[0025] Therefore, a Basic Local Alignment Search Tool (BLAST) search was performed using Non-redundant protein sequences (nr) as a database to search for bacterial species that have a nucleotide sequence encoding an IS-like protein but do not have a nucleotide sequence encoding the newly discovered BI-like protein. However, no such bacterial species were found. Instead, it was found that all bacteria that have a nucleotide sequence encoding an amino acid sequence that is 67.3% or more identical to the amino acid sequence of IS shown in SEQ ID NO: 34 also have a nucleotide sequence encoding an amino acid sequence that is 66.9% or more identical to the amino acid sequence of BI shown in SEQ ID NO: 36, and that even a protein that is 67.9% identical to BI produces branched inulin when reacted with sucrose together with IS. Bacterial species that have both the base sequences encoding the IS-like protein and the BI-like protein and are capable of producing branched inulin include those of the genus Priestia, Bacillus, Neobacillus, Fredinandcohnia, Metabacillus, Litchfieldia, and the like. The genera identified were Bacillus genus, Bacillus eldia, Bacillus robertmuraya, Bacillus mycobacteroides, Bacillus alkalihalobacillus, Bacillus brevibacillus, Bacillus paenibacillus, and Bacillus ammoniphilus, i.e., closely related species of the genus Bacillus.

[0026] Next, the BI gene of an inulin-synthesizing bacterium was deleted or mutated in whole or in part to produce a linear inulin-synthesizing bacterium in which the BI enzyme was deleted or inactivated (hereinafter referred to as a "ΔBI inulin-synthesizing strain"). When the culture supernatant of the produced ΔBI inulin-synthesizing strain was allowed to act on sucrose, it was possible to successfully produce only linear inulin, thereby completing the present invention. Suitable bacterial species for producing ΔBI inulin-synthesizing strains include the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus, and Amoniphilus, as mentioned above. In other words, examples of the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus, and Amoniphilus, i.e., closely related species of the genus Bacillus, can be mentioned, and it can be said that it is impossible to produce linear inulin using the culture supernatant of these bacterial species without breeding and improving them.

[0027] In the present invention, the term "IS-like gene" refers to a gene corresponding to SEQ ID NO: 33 that encodes a protein having the activity of inulosucrase, which catalyzes the β-2,1-transfer of the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product. Specific examples include genes consisting of a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 33, preferably 75% identical, more preferably 80% identical, even more preferably 85% identical, particularly preferably 90% identical, and most preferably 95% identical. Furthermore, the gene may be a gene having the same function as the gene, but consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted.

[0028] Another example is a gene that hybridizes under stringent conditions to the complementary strand of a gene consisting of the nucleotide sequence represented by SEQ ID NO: 33 and encodes a protein that has the activity of catalyzing the β-2,1-transfer of a fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product. As used herein, "stringent conditions" refers to conditions under which a complementary strand of a gene strand that is homologous to a target sequence preferentially hybridizes to the target sequence, while a complementary strand of a gene strand that is not homologous does not substantially hybridize. Specifically, stringent conditions include hybridization performed overnight at 65°C, followed by washing to remove nonspecific reactions using 2xSSC at room temperature for 5 minutes, followed by two 30-minute washes using 0.2xSSC containing 0.1% SDS at 65°C. The composition of 2×SSC is 0.3 mol / L NaCl and 30 mmol / L sodium citrate (pH 7.0).

[0029] Linear inulin-synthesizing bacteria lacking or inactivating the activity of a BI-like protein can be obtained by deleting or inactivating the BI-like gene in the base sequence of a parent strain. In the present invention, the term "BI-like gene" refers to DNA consisting of a transcriptional region containing an ORF and a transcriptional regulatory region such as a promoter for the gene. Specifically, the term refers to a gene corresponding to SEQ ID NO: 35 that encodes an enzyme protein that catalyzes the reaction of transferring a fructofuranosyl group from sucrose to one of the fructose moieties of linear inulin via a linkage other than a β-2,1 bond to synthesize branched inulin. More specifically, the term refers to a gene encoding a protein having an amino acid sequence that is at least 70% identical to the base sequence of SEQ ID NO: 35, preferably 75% identical, more preferably 80% identical, even more preferably 85% identical, particularly preferably 90% identical, and most preferably 95% identical. Furthermore, the term may refer to a gene having the same function as the gene, but with a base sequence in which one to several bases have been deleted, substituted, added, or inserted. "One to several" can be, for example, 1 to 10, preferably 1 to 7, and more preferably 1 to 4. In the present invention, "linear inulin" refers to a fructan having a structure in which other fructose is polymerized at the fructose moiety of sucrose via a β-2,1 bond, and in which the bonds between fructose are composed exclusively of β-2,1 bonds. "Bonds other than β-2,1 bonds" literally means "bonds other than β-2,1 bonds," and refers to bonds other than β-2,1 bonds such as at least β-2,6 bonds, β-2,3 bonds, β-2,4 bonds, etc.

[0030] Furthermore, in the present invention, a BI-like gene includes a gene that hybridizes under stringent conditions with a gene consisting of the base sequence represented by SEQ ID NO: 35 and encodes an enzyme protein that catalyzes the reaction of synthesizing branched inulin by transferring the fructofuranosyl group of sucrose to any fructose site of linear inulin via a bond type other than a β-2,1 bond.

[0031] In the present invention, "deleting or inactivating a BI-like gene or BI gene" means introducing a mutation into one or more bases in the base sequence of the gene, i.e., deleting all or part of the base sequence of the gene, or substituting or inserting another base sequence into the base sequence, thereby reducing the activity of the BI-like protein or BI protein that is transcribed and translated, or making it impossible to produce the BI-like protein or BI protein, or introducing a mutation into the base sequence that encodes a signal peptide normally present on the 5' end of the BI-like gene or BI gene, thereby causing the loss of the ability to produce and secrete the BI-like protein or BI protein outside the bacterial cell.

[0032] In the present invention, the inulosucrase that catalyzes the β-2,1-transfer of the fructofuranosyl group of sucrose to the terminal fructose moiety of another sucrose or its transglycosylation product comprises the amino acid sequence represented by SEQ ID NO: 34. Proteins having an amino acid sequence with 67.3% or greater identity to the amino acid sequence can be used as long as they have the activity. Specifically, proteins having an amino acid sequence with an identity of at least 67.3% or greater, preferably 70% or greater, more preferably 75% or greater, even more preferably 80% or greater, even more preferably 85% or greater, particularly preferably 90% or greater, and most preferably 95% or greater, more specifically, proteins having an amino acid sequence with an identity of at least 67.3% or greater, preferably 68.0% or greater, 68.6% or greater, 68.8% or greater, 70.3% or greater, 70.9% or greater, 71.7% or greater, 73.4% or greater, 74.4% or greater, 75.6% or greater, 76.2% or greater, 77.2% or greater, 78.2% or greater, 79.2% or greater, 80% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 9 ... % or more, 76.8% or more, 78.7% or more, 78.9% or more, 79.1% or more, 79.4% or more, 79.8% or more, 80.4% or more, 80.7% or more, 81.1% or more, 81.3% or more, 81.7% or more, 81.9% or more, 83.4% or more, 84.9% or more, 85.8% or more, 96.2% or more, 96.8% or more, 97.3% or more, 98.2% or more, 98.4% or more, or 98.6% or more identity.

[0033] In the present invention, the enzyme that catalyzes the reaction of transferring a fructofuranosyl group of sucrose to any fructose site of linear inulin via a bond other than a β-2,1 bond to synthesize branched inulin consists of the amino acid sequence represented by SEQ ID NO: 36. However, any protein having an amino acid sequence that is 67.9% or more identical to the amino acid sequence can be used as long as it has the activity. Specifically, a protein having an amino acid sequence that is at least 67.9% or more, preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more identical to the amino acid sequence represented by SEQ ID NO: 36, more specifically, a protein having an amino acid sequence that is at least 67.9% or more, preferably 71.5% or more, 72.2% or more, 75.1% or more, 75.3% or more, 76.6% or more, 76.9% or more, 77.1% or more, 78.1% or more, 79.1% or more, 80% or more, 85 ... % or more, 77.3% or more, 77.5% or more, 78.3% or more, 78.5% or more, 78.7% or more, 79.0% or more, 80.8% or more, 81.0% or more, 84.8% or more, 85.3% or more, 85.4% or more, 85.8% or more, 86.0% or more, 88.6% or more, 89.7% or more, 93.1% or more, 93.7% or more, 96.6% or more, 98.7% or more, 98.9% or more, or 99.1% or more identity.

[0034] Furthermore, it may also be an amino acid sequence in which one to several amino acids are deleted, substituted, added, or inserted in the amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 36, and have the same function as the protein. Here, "one to several" may mean, for example, one to ten, preferably one to seven, more preferably one to four amino acids.

[0035] Here, "deletion or inactivation of the activity of a BI-like protein or BI protein" may mean that the BI-like gene or BI gene itself is removed from the genome, or that a mutation that inactivates or reduces the activity of BI is introduced by base substitution, deletion, insertion, etc. Alternatively, the nucleotide sequence encoding a signal peptide that is usually present at the 5' end of the BI-like gene or BI gene may be removed to cause the loss of the ability to produce and secrete BI extracellularly.

[0036] Procedures for "deleting or inactivating a BI-like protein or BI protein" include a method of deliberately deleting or inactivating a BI-like gene or BI gene, as well as a method of randomly mutating the genome and then obtaining a linear inulin-synthesizing bacterium in which BI activity has been eliminated or reduced by an appropriate method.

[0037] A method for deliberately deleting or inactivating a BI-like gene or BI gene may be, for example, a method using homologous recombination. Specifically, a circular recombinant plasmid obtained by inserting a DNA fragment containing the upstream and downstream sequences of the BI-like gene or BI gene into an appropriate plasmid vector can be incorporated into parent strain cells, and the BI-like gene in the genome of the parent strain can be deleted by double-crossover homologous recombination in a region outside the BI-like gene or BI gene. Alternatively, a linear DNA fragment in which an inactivating mutation has been introduced into the BI-like gene or BI gene by base substitution, base insertion, or the like can be constructed by PCR or other methods, and this can be incorporated into parent strain cells to cause double-crossover homologous recombination in two regions outside the mutation site in the BI-like gene or BI gene in the parent strain genome, thereby replacing the BI-like gene or BI gene in the genome with an inactivated gene fragment. Alternatively, deletion, substitution, or base insertion can be performed on the BI-like gene or BI gene using genome editing technology or the like.

[0038] On the other hand, methods for randomly deleting or inactivating genes include, for example, treating the parent strain with a mutagen such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), or irradiating it with UV or heavy ion beams.

[0039] By culturing a linear inulin-synthesizing bacterium in which the BI-like gene or BI gene has been deleted or inactivated by the above-mentioned method, a culture medium in which the BI-like protein or BI protein has been deleted or inactivated can be obtained.

[0040] When the culture supernatant of the culture medium is reacted with sucrose to obtain a reaction product, linear inulin accounts for the majority of the reaction product. When inulin is obtained from the reaction product and subjected to HPAEC-PAD analysis under the conditions described below, 10 or more linear inulin peaks are detected at elution times of 20 minutes or more. Assuming that branched inulin is produced in the same way as when inulin is produced from sucrose using the culture supernatant of the parent strain described in paragraph 0043 below, the peaks appear between the linear inulin peaks. At elution times of 20 to 30 minutes (degree of polymerization 9 to 16), the branched inulin ratio (sum of branched inulin peak areas / total peak area × 100) is less than 10%, preferably 7% or less, more preferably 5% or less, even more preferably 3% or less, particularly preferably 1% or less, and most preferably 0.3% or less. Here, in the present invention, "reaction product" refers to a product produced by conversion of a substrate by the activity of an IS or an IS and a BI, and the reaction product produced by conversion of a substrate by the activity of an IS includes not only reaction products containing linear inulin but also linear inulin itself, and the reaction product produced by conversion of a substrate by the activity of an IS and a BI includes not only reaction products containing linear inulin and branched inulin but also linear inulin and branched inulin themselves. The method for obtaining the linear inulin or branched inulin contained in the reaction product is not particularly limited, and any method selectable by one skilled in the art can be used.

[0041] <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH 100 mM constant, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry

[0042] The parent strain used to obtain the linear inulin-synthesizing bacterium of the present invention is not particularly limited, as long as it is an inulin-synthesizing bacterium having a gene with a base sequence that is recognized to have at least 60% identity to each of SEQ ID NO: 33 and SEQ ID NO: 35. Specifically, bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Lichfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus, and Amoniphilus, as well as other closely related species of the genus Bacillus, can be used.More specifically, Priestia megaterium, Bacillus sp., Neobacillus bataviensis, Metabacillus bambusae, Robertmuraya korlensis, Fredinandcohnia onubensis, Litchfieldia salsa, Mycobacteroides abscessus subsp. abscessus, subsp. abscessus, Brevibacillus sp., Brevibacillus formosus, Brevibacillus brevis, Brevibacillus dissolubilis, Brevibacillus migulae, Paenibacillus sp., Paenibacillus ergii, elgii), Paenibacillus prosopidis, Paenibacillus tyrfis, Ammoniphilus sp., Alkalihalobacillus krulwichiae, Salipaludibacillus agaradhaerens, and more specifically, Bacillus sp. SD33 strain (accession number: NITE BP-03890).According to molecular phylogenetic tree analysis based on the partial base sequences of 16S rDNA, the type strains of each of the above microbial species except for Mycobacteroides abscessus subsp. abscessus and the Amoniphilus sp. YIM78166 strain are all mapped to positions close to Bacillus subtilis (the most common Bacillus species). The genera Brevibacillus and Paenibacillus are conventionally treated as closely related species to the Bacillus genus, and it is appropriate to treat at least each of the above microbial species except for Mycobacteroides abscessus subsp. abscessus as closely related species to the Bacillus genus. The Bacillus sp. SD33 strain was deposited at the National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture) on May 22, 2023, and was assigned the accession number: NITE BP-03890.

[0043] When the culture supernatant of the parent strain is reacted with sucrose to obtain a reaction product, and inulin is obtained from the reaction product, and the obtained inulin is subjected to HPAEC-PAD analysis under the conditions described below, at elution times of 20 minutes and later, 10 or more branched inulin peaks and 10 or more linear inulin peaks are detected, and the branched inulin peaks appear between the linear inulin peaks, and the branched inulin ratio (sum of branched inulin peak areas / total peak area × 100) is 10% or more at elution times of 20 to 30 minutes (degree of polymerization 9 to 16).

[0044] <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH 100 mM constant, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry.

[0045] A medium suitable for culturing the linear inulin-synthesizing bacterium of the present invention contains, but is not limited to, a carbon source, a nitrogen source, inorganic salts, etc. Nutrients commonly used in conventional cultures, such as amino acids and vitamins, can also be used as needed. Carbon sources added to the medium include carbohydrates known in the art, such as sucrose, glucose, fructose, and maltose. However, when using Bacillus sp. SD33 as the microorganism, it is most preferable to use a liquid medium containing sucrose as the primary carbon source, as this improves the production of the inulin synthase of the present invention. These carbon sources can be used alone or in combination at an appropriate concentration (e.g., 5 to 50 g / L). These carbon sources may be isolated and purified, or may be contained in other substances. For example, when using sucrose as a carbon source, sucrose-containing substances such as raw sucrose or blackstrap molasses may be used instead of purified sucrose. Nitrogen sources to be added to the medium include, but are not limited to, organic nitrogen sources such as peptone, meat extract, yeast extract, and corn steep liquor, as well as inorganic nitrogen sources such as ammonium salts of sulfate, nitrate, and phosphate, which can be used alone or in combination. Inorganic salts include, but are not limited to, sulfates, hydrochlorides, carbonates, nitrates, and phosphates of potassium, sodium, calcium, magnesium, manganese, iron, and the like, which can be used alone or in combination.

[0046] The pH of the medium having the above composition is 6 to 9, preferably 7 to 8. The microorganism can be cultured by shaking culture or aeration and agitation culture. The culture temperature is preferably 25 to 35°C. The culture time may be any time long enough to allow the microorganism to grow, and is 5 to 96 hours, preferably 15 to 72 hours.

[0047] Next, the linear inulin-synthesizing bacterium of the present invention exocrinely secretes an IS and / or an IS-like protein, which may be in the form of a protein, i.e., an enzyme, or an enzyme-containing substance. The linear inulin-synthesizing bacterium exocrines an IS and / or an IS-like protein, which may be ...containing substance, and which may be an IS and / or an IS-like protein, which may be an IS and / or an IS-like protein, which may be an IS and / or an IS-like protein, which may be an IS and / or an IS-like protein, which may be an IS and / or an IS-like protein In the case of an enzyme, the form may be a purified enzyme or a crude enzyme, and in the case of an enzyme-containing substance, it may be an enzyme-containing substance, a processed product of an enzyme-containing substance, a culture solution, a culture supernatant, a cultured bacterial cell, or a processed product thereof. Furthermore, the purified enzyme, crude enzyme, enzyme-containing substance, culture solution, culture supernatant, cultured bacterial cell, or a processed product thereof are all substances obtained from the linear inulin-synthesizing bacterium according to the present invention, and the method of obtaining them may be any method selectable by a person skilled in the art, including extracellular secretion.

[0048] The method for producing inulin according to the present invention comprises the following steps (i) and / or (ii): (i) culturing the linear inulin-synthesizing bacterium according to the present invention, and (ii) reacting at least one of a purified enzyme, a crude enzyme, an enzyme-containing substance, a culture medium, a culture supernatant, or cultured cells obtained from the linear inulin-synthesizing bacterium according to the present invention, or a processed product thereof, with sucrose.

[0049] The above step (i), i.e., "the step of culturing the linear inulin-synthesizing bacterium of the present invention", is not particularly limited as long as it can culture the linear inulin-synthesizing bacterium of the present invention, and a culture method that can be appropriately selected by a person skilled in the art can be adopted for culturing the linear inulin-synthesizing bacterium of the present invention. For example, one such culture method may include inoculating the linear inulin-synthesizing bacterium of the present invention into a liquid medium containing sucrose as the main carbon source, and culturing it for 15 to 72 hours under conditions of pH 7 to 8 and 25 to 35°C.

[0050] The above-mentioned step (ii), i.e., "a step of reacting at least one of a purified enzyme, crude enzyme, or enzyme-containing material, culture medium, culture supernatant, or cultured bacterial cells, or a processed product thereof, obtained from the linear inulin-synthesizing bacterium of the present invention with sucrose," refers to a step of reacting a purified enzyme, crude enzyme, or enzyme-containing material, culture medium, culture supernatant, or cultured bacterial cells, or a processed product thereof, obtained by the above-mentioned method of obtaining the linear inulin-synthesizing bacterium, with sucrose. The method of "reaction" is not particularly limited as long as it allows the production of linear inulin, and any reaction method that can be appropriately selected by a person skilled in the art can be adopted. For example, such a "reaction" method may include a method in which at least one of a purified enzyme, crude enzyme, or enzyme-containing material, culture medium, culture supernatant, or cultured bacterial cells, or a processed product thereof, obtained from the linear inulin-synthesizing bacterium of the present invention is added to a reaction solution containing sucrose, and then reacted.

[0051] The method for producing linear inulin, which comprises the above-mentioned step (ii), i.e., "a step of reacting at least one of a purified enzyme, crude enzyme or enzyme-containing material, culture medium, culture supernatant or cultured bacterial cells or a processed product thereof obtained from the linear inulin-synthesizing bacterium of the present invention with sucrose to obtain a reaction product," can further perform the HPAEC-PAD analysis (iii) below on the obtained reaction product. (iii) When the reaction product (obtained in the above step (ii)) is subjected to HPAEC-PAD analysis under the following conditions, the proportion of branched inulin (sum of peak areas of branched inulins / total peak area×100) having a structure in which a fructofuranosyl group is bonded to any fructose site of linear inulin in which the bond between fructoses is β-2,1 bond, by a bond type other than β-2,1 bond, is less than 1% within an elution time of 20 to 30 minutes (degree of polymerization 9 to 16); <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4×250 mm; guard column: CarboPac PA1 4×50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH Constant 100 mM, AcONa gradient (0-60 min, 0 → 600 mM); detection, pulsed amperometry.

[0052] Next, the linear inulin-synthesizing bacterium of the present invention enables the direct production of substances such as difructose dianhydride III (DFA III), inulooligosaccharides, and cycloinulooligosaccharides (cyclofructans) from sucrose by using the culture supernatant in combination with the linear inulin-synthesizing bacterium. For the production of DFA III in particular, inulin fructotransferase (hereinafter referred to as "IFT") can be used, as well as purified enzymes, crude enzymes, enzyme-containing substances, culture broths, culture supernatants, cultured cells, or processed products thereof (hereinafter referred to as "IFT, etc.") obtained from microorganisms that produce IFT. Furthermore, DFA III can be produced by combining at least one of the purified enzyme, crude enzyme, or enzyme-containing substance, culture medium, culture supernatant, or cultured cells, or a processed product thereof, obtained from the linear inulin-synthesizing bacterium according to the present invention with IFT or the like, and reacting them with sucrose. By selecting such a method for producing DFA III, it becomes possible to produce DFA III directly from sucrose. In the production of DFA III described above, when IFT or the like is allowed to act on linear inulin, DFA III can be produced in a higher yield than when IFT or the like is allowed to act on inulin containing branched inulin.

[0053] In addition, as microorganisms that produce IFT, microorganisms belonging to the genera Arthrobacter, Kluyveromyces, Streptomyces, Enterobacter, Bacillus, and Microbacterium, as well as various bacteria, yeasts, filamentous fungi, actinomycetes, and the like can be appropriately used, and examples thereof include Arthrobacter ureafaciens (IFO12140), (ATCC21124), A. pascens (IFO12139), A. pascens T13-2, A. globiformis (IFO12137), A. globiformis C11-1, A. nictinovorans GS-9, A. ilicis OKU 17B, Arthrobacter sp. , H65-7, AHU1753 (FERM BP-8296), MCI-2493; Kluyveromyces marxianus (ATCC12424), CBS6556, K. marxianus var. marxianus, ibid. (IFO1735); Streptomyces fumigatus, S. marxianus, ibid. (IFO1735); rochei, Streptomyces sp. E87, Streptomyces sp. , same MCI-2524; Pseudomonas fluorescens, same No. 949; Bacillus circulans, same OKUMZ. 31B, MCI-2554, Bacillus sp., Snu-7; Aureobacterium sp., MCI-2494; Microbacterium sp., AL-210; Enterobacter sp., S45; Aspergillus fumigatus; and Penicillium purpurogenum.

[0054] The linear inulin-synthesizing bacteria according to the present invention, the purified enzyme obtained from the linear inulin-synthesizing bacteria according to the present invention, the crude enzyme or enzyme-containing substance, the culture medium, the culture supernatant or cultured cells or processed products thereof, the method for producing linear inulin, and the method for producing DFA III will be described below based on examples. Note that the technical scope of the present invention is not limited to the embodiments shown in these examples.

[0055] Example 1: Search for natural bacteria capable of synthesizing linear inulin In this Example 1, a search was conducted for natural bacteria capable of synthesizing only linear inulin. The procedures and results are described in detail below.

[0056] Soil samples collected from various parts of Japan were suspended in sterilized water, spread on a basal agar medium, and cultured at 35°C for 24 hours. Colonies that appeared on the agar medium were inoculated into 300 μL of basal medium and cultured statically at 35°C for 48 hours. 40 μL of the culture supernatant was added to 160 μL of 10 mM Na phosphate buffer (pH 7.0) containing 10% sucrose and incubated at 35°C for 24 hours. This was designated Sample 1. 2 μL of Aspergillus niger exoinulinase (Sigma-Aldrich) was added to 40 μL of Sample 1 and incubated at 35°C for 24 hours. This was designated Sample 2. Samples 1 and 2 were analyzed by TLC, and colonies in which the polysaccharide spot at the origin observed in Sample 1 disappeared in Sample 2 were identified as inulin-synthesizing bacteria. A total of 100 strains were obtained. The TLC analysis conditions were: plate: TLC silica gel 60 (Merck); load: 50 μg; mobile phase: 2-propanol, 1-butanol, H ２ O = 2,2,1, development: 10 cm, color development: spraying reagent (p-anisaldehyde: acetic acid: concentrated sulfuric acid: ethanol = 13:5:18:478) and then heating at 105°C for 3 minutes.

[0057] Of the 100 strains obtained, the 20 strains with the highest secretion of enzymes into the culture supernatant were selected. Next, these 20 strains of inulin-synthesizing bacteria were inoculated into 3 mL of basal medium and cultured at 30°C for 24 hours with shaking at 130 rpm. The culture supernatant was recovered by centrifugation at 12,000 x g for 5 minutes. After measuring the enzyme activity of the culture supernatant, it was mixed with Bx40 sucrose solution to a concentration of 2 U / g sucrose and maintained at 37°C for 24 hours. This reaction solution was maintained at 100°C for 5 minutes to inactivate the enzyme and then subjected to HPAEC-PAD analysis. The peaks of the resulting reaction product and plant-derived inulin (raftilin ST) obtained by HPAEC-PAD analysis are shown in Figure 1. The enzyme activity was calculated based on the amount of reducing sugar produced, measured by the DNS method. That is, the composition of 100 μL of reaction solution was appropriately diluted enzyme solution (10 μL), 300 mM sucrose, 40 mM MES-NaOH buffer (pH 6.5), and after 10 minutes of reaction at 37 ° C, 100 μL of DNS reagent (16 g / L NaOH, 5 g / L 3,5-dinitrosalicylic acid, 300 g / L potassium sodium tartrate) was added, and the mixture was heated for 20 minutes at 105 ° C to allow color development, after which the absorbance at 535 nm was measured. The amount of reducing sugar in the reaction solution was determined from a standard curve prepared using 0 to 5 mM D-glucose solution, and the amount of enzyme that produces 1 μmol of reducing sugar under these conditions was defined as 1 U. The HPAEC-PAD analysis in the following examples was carried out using an HPAEC-PAD system (ICS-3000) manufactured by Dionex under the following conditions: column: CarboPac PA1 4 × 250 mm; guard column: CarboPac PA1 4 × 50 mm; injection volume: 5 μL of a 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH 100 mM constant, AcONa gradient (0 to 60 min, 0 → 600 mM).

[0058] As shown in Figure 1, the peak patterns of the reaction solutions were different for fructan peaks (HPLC elution time 20 to 30 minutes) with a degree of polymerization of 9 or more, and all reaction solutions showed polysaccharide peak patterns not seen in plant-derived inulin (raftilin ST).

[0059] Furthermore, one strain of the obtained inulin-synthesizing bacterium was identified as a Bacillus bacterium closely related to Bacillus drentensis by DNA sequence analysis and molecular phylogenetic analysis (16S rDNA-full, commissioned to Techno Suruga Lab Co., Ltd.). Bacillus drentensis was reclassified as Neobacillus drentensis in 2020 (Int. J. Syst. Evol. Microbiol., 70, 406-438 (2020)). A simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of the identified Bacillus genus inulin-synthesizing bacterium closely related to Bacillus drentensis is shown in Figure 2. As shown in Figure 2, this strain was designated Bacillus sp. SD33 strain (hereinafter referred to as SD33 strain) (Accession number: NITE BP-03890).

[0060] Example 2: Identification of the gene responsible for producing branched inulin In this Example 2, the reason why the inulin-synthesizing bacterium discovered in Example 1 produces branched inulin and is unable to produce only linear inulin was clarified.

[0061] The SD33 strain was spread on a basal agar medium and cultured overnight at 35°C. The resulting single colony was inoculated into 6 mL of basal medium and pre-cultured at 30°C for 24 hours at 130 rpm. This was inoculated into two 500 mL aliquots of the same medium in a 2 L Erlenmeyer flask (2.5 mL each). Cultured at 30°C for 24 hours at 130 rpm, the culture supernatant was collected by centrifugation at 12,000 x g for 20 minutes. Ammonium sulfate was added to the resulting culture supernatant to 70% saturation, and the mixture was kept overnight at 4°C with stirring. The ammonium sulfate precipitate from the culture supernatant was collected by centrifugation at 12,000 x g for 20 minutes, dissolved in 10 mM sodium phosphate buffer (pH 7.0), placed in a dialysis tube, and then thoroughly dialyzed against the same buffer to obtain a crude enzyme solution. The crude enzyme solution was loaded onto Toyopearl DEAE 650M (2.5 cm i.d. x 20 cm, Tosoh Corporation) equilibrated with 10 mM sodium phosphate buffer (pH 7.0) in advance, and the non-adsorbed fraction was eluted with the same buffer. Since the non-adsorbed fraction showed activity to release reducing sugars from sucrose, ammonium sulfate was added to the non-adsorbed fraction to 80 mM, and the non-adsorbed fraction was loaded onto Toyopearl butyl 650M (2.5 cm i.d. x 10 cm, Tosoh Corporation) equilibrated with 10 mM sodium phosphate buffer (pH 7.0) containing 80 mM ammonium sulfate in advance, and the non-adsorbed fraction was eluted with the same buffer. The adsorbed fraction was eluted with a linear gradient of ammonium sulfate concentration from 80 mM to 0 mM. This operation yielded two activity peaks A and B, which show the activity of releasing reducing sugars from sucrose. The fractions from each peak were added to Bx40 sucrose to give a concentration of 2 U / g sucrose, and reaction mixtures were prepared using the method described in Example 1. The enzyme was inactivated and the resulting products were analyzed using HPAEC-PAD. The peaks for the reaction product of Peak A, the reaction product of Peak B, and plant-derived inulin (raftilin ST) are shown in Figure 3.

[0062] As shown in Figure 3, the peak pattern characteristics of the reaction product in peak A were consistent with linear inulin, and the reaction product in peak B was consistent with inulin containing branched inulin. Next, the structure of each product was analyzed by nuclear magnetic resonance (NMR). First, 99.5% ethanol was added to 1 mL of each reaction solution to a final concentration of 50%, and polysaccharides were precipitated. The precipitate was then recovered by centrifugation at 12,000 x g for 2 minutes. 2 mL of 50% ethanol was added to the recovered precipitate, and the suspension was suspended. The precipitate was then recovered by centrifugation at 12,000 x g for 2 minutes. This procedure was repeated five times to remove low molecular weight compounds. The resulting precipitate was freeze-dried, and inulin (derived from chicory, raftilin HP) and levan (produced by Zymomonas mobilis, Sigma-Aldrich) were used as reference samples. １３ C-NMR was performed on the levan preparation, the inulin preparation, the reaction product of peak A, and the reaction product of peak B. １３ The C-NMR signal is shown in FIG.

[0063] As shown in Figure 4, both products were almost equivalent to the inulin standard. １３ The C-NMR signal indicated that both the reaction products of Peak A and Peak B have the same basic structure as inulin, and that the difference between linear and branched inulin is due to partial structural differences. Since the protein of Peak A is contained in both peaks, it was determined that the protein of Peak A is IS, and the protein contained only in Peak B is the enzyme BI responsible for producing branched inulin.

[0064] Next, all of the free hydroxyl groups in 50 mg of the reaction product of peak A and the reaction product of peak B were methylated by the method described in "Nature Protocols, 7, 1590-1607 (2012)". After hydrolysis of the methylated polysaccharides, the liberated partially methylated sugars were dehydrogenated with NaBD. ４The resulting PMAAs were subjected to GC-MS analysis. GC-MS analysis was performed using a Shimadzu GC-MS system (GCMS-QP2010 Ultra). The GC analysis was performed under the following conditions: capillary column, BPX70 (25 m × 0.22 mm inner diameter, film = 0.25 μm; manufactured by SGE); injection volume, 1 μL (split ratio 1 / 10); vaporizer temperature, 240°C; carrier gas, helium; flow rate, 1.0 mL / min; column temperature, multi-step (0 to 2 min, 170°C; 2 to 32 min, 170 → 260°C; 32 to 35 min, 260°C); and MS analysis was performed under the following conditions: ion source temperature, 230°C; interface temperature, 250°C; solvent elution time, 3 min; measurement mode, scan; m / z, 100 to 350. The GC-MS peaks of the methylation analysis of the reaction product of peak A, the reaction product of peak B, and peak 2 in the reaction product of peak B are shown in FIG.

[0065] As shown in Figure 5, the reaction product of peak B contained several GC-MS peaks that were not contained in the reaction product of peak A. Of these, peak 2 had major primary fragments at m / z 189 and 190, minor fragments at m / z 233 and 234, and the only secondary fragment was m / z 129, and was therefore considered to be 1,2,5,6-tetra-O-acetyl-(2-deuterio)-3,4-di-O-methyl glucitol. In other words, it was considered that branched inulin contains at least a branched structure with a β-2,6 bond.

[0066] Next, to obtain the genetic sequence information of IS and BI, the N-terminal amino acid sequences of the proteins contained in peaks A and B were determined according to the following procedure. First, SDS-polyacrylamide gel electrophoresis (10%) of each peak was performed according to standard methods, and then transferred to a PVDF membrane and stained with CBB. Next, the membrane of the target band portion (both 44 kDa) was excised and washed with MeOH, 50% MeOH, and ultrapure water, and then the N-terminal amino acid sequence was analyzed using Procise 492 (Applied Biosystems). As a result, it was estimated that peak A was IS, and peak B was a mixture of IS and BI. The N-terminal amino acid sequence of IS was determined to be Ala-Glu-Ile-Ser-Ser-Asp-Tyr-Thr-Ser-Ile-Trp, and the N-terminal amino acid sequence of BI was predicted to be Glu-Asp-Val-Gly-Gln-Thr-Thr-Asn-Trp.

[0067] Next, the IS gene and its surrounding base sequence were determined according to standard methods. Specifically, Peak A was digested with lysyl endopeptidase, and the resulting peptides were separated by reverse-phase HPLC to analyze the internal sequence. Based on the internal sequence, several sense and antisense primers were prepared, and PCR was performed using the genomic DNA of the SD33 strain as a template. PCR for DNA fragment amplification was performed using PrimeSTAR max polymerase and an iCycle thermal cycler (Bio-Rad). The PCR reaction mixture was prepared by adding 1 μL of appropriately diluted template DNA, 20 pmol each of sense and antisense primers, and 25 μL of PrimeSTAR max Premix (2x), bringing the total reaction volume to 50 μL. The PCR reaction conditions were 98°C for 30 seconds as one cycle, followed by 30 cycles of three temperature changes: 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 5 to 30 seconds (adjusted depending on the target amplification product, with a guideline of 5 seconds per 1 kb).

[0068] Outward primers were created based on the sequence of the resulting amplified fragment, and the upstream and downstream base sequences were determined by adapter PCR using the LA in vitro cloning kit (Takara Bio). Here, "upstream" and "downstream" of a gene do not refer to positions from the replication origin, but rather "upstream" refers to the region following the 5' side of the start codon of the gene of interest in each operation / step, while "downstream" refers to the region following the 3' side of the stop codon of the gene of interest in each operation / step, and the same applies hereinafter.

[0069] The results are shown in SEQ ID NO: 37. Searching for open reading frames (ORFs) in the obtained sequences surrounding the IS gene revealed that an ORF presumed to be BI (hereinafter referred to as ORF1) was present immediately downstream of the IS. It was also revealed that both sequences had a signal peptide sequence on the N-terminal side.

[0070] Next, an E. coli recombinant enzyme expression system was constructed based on the nucleotide sequences encoding the mature proteins excluding the signal peptide of the IS gene and ORF1. Specifically, using the SD33 strain genomic DNA as a template, PCR was performed with the primer pairs for extending the IS and ORF1 sequences shown in Table 1 below to amplify the nucleotide sequences of each mature protein, each with an initiation codon attached to the N-terminus and a His tag for affinity purification attached to the C-terminus.

[0071] [Table 1]

[0072] PCR for amplifying the DNA fragment was performed as described in Example 2. The resulting amplified fragment was ligated to the NdeI-His tag region of the plasmid pET-22b (Merck Millipore) using TaKaRa Infusion HD (Takara Bio). The plasmid was produced in an E. coli DH5α transformant and purified by alkaline SDS. The accuracy of the plasmid sequence was confirmed by sequence analysis using universal primers, the T7 promoter and T7 terminator. The resulting plasmid was then used to transform E. coli BL21 (λDE3), which was then cultured for 18 hours at 37°C on LB agar medium containing 200 μg / mL ampicillin. The resulting colonies were inoculated into 3 mL of LB liquid medium containing 200 μg / mL ampicillin and cultured overnight at 37 ° C. and 130 rpm. Then, 1 / 100 of the culture was inoculated into 50 mL of the same liquid medium and cultured at 37 ° C. and 160 rpm until the OD600 reached 0.5. The mixture was left on ice for 10 minutes, and 50 μL of 0.1 M IPTG was added (final concentration 0.1 mM). The mixture was then cultured for 18 hours at 18 ° C. and 120 rpm. Subsequently, the cells were collected by centrifugation at 5000 × g for 10 minutes, suspended in 10 mM sodium phosphate buffer (pH 7.5), and subjected to ultrasonic disruption using a Sonifier 450 (Branson) (duty cycle 50, output 5, 2 minutes × 3). This was centrifuged again at 12,000 × g for 10 minutes, and the supernatant was loaded onto a Ni-chelating Sepharose column (φ1.0 × 4 cm) previously equilibrated with 10 mM sodium phosphate buffer (pH 7.5) containing 50 mM imidazole. After washing the non-adsorbed fraction with the same buffer, the imidazole concentration was increased to 250 mM to elute the adsorbed proteins, which were used as the purified IS and ORF1 enzymes.

[0073] When the purified IS enzyme was reacted with Bx40 sucrose at 2 U / g sucrose, linear inulin was produced. On the other hand, when the purified IS and ORF1 enzymes were mixed at 2 U / g sucrose each and reacted with Bx40 sucrose, branched inulin was produced, as analyzed by the method described in Example 1. The peaks obtained by HPAEC-PAD analysis of the reaction product obtained by reacting the IS and BI from the SD33 strain with Bx40 sucrose are shown in Figure 6.

[0074] As shown in Figure 6, under these analytical conditions, the number of branched inulin peaks detected among the linear inulin peaks after 20 minutes of elution was 10 or more, and the branched inulin ratio (sum of branched inulin peak areas / total peak area × 100) at 20-30 minutes of elution (degree of polymerization 9-16) was 23.9%, higher than 10% (Figure 6). Furthermore, when the ORF1 purified enzyme was reacted alone with Bx40 sucrose, transglycosylation products with degrees of polymerization 3-5 were observed. From these results, ORF1 was identified as the gene encoding the BI protein. The amino acid sequence of IS (SEQ ID NO: 34) and the amino acid sequence of BI (SEQ ID NO: 36) shared 40.2% identity.

[0075] Example 3: Searching for inulin-synthesizing bacteria that do not possess a BI-like gene using BLAST search In this Example 3, a search was performed using BLAST search to find inulin-synthesizing bacteria that do not possess the gene encoding the causative protein (BI) identified in Example 2, and it was revealed that "all bacteria that possess a gene encoding a protein with a certain level of homology to IS also possess a gene encoding a protein with a certain level of homology to the causative protein (BI)."

[0076] To search for inulin-synthesizing bacteria that do not possess a BI-like gene, a BLAST search was performed using the non-redundant protein sequences (nr) as a database for the amino acid sequences of IS and BI of the SD33 strain shown in SEQ ID NOs: 34 and 36. The results are shown in Table 2 below. In Table 2, microbial strains (recombinantly produced proteins) having a nucleotide sequence encoding a protein with a homology of 67.3% or more to IS are shown in bold.

[0077] [Table 2]

[0078] First, we identified strains having a base sequence encoding a protein (IS-like gene; SEQ ID NO: 33) that is 67.3% or more identical (specifically, 67.3%, 68.0%, 68.6%, 68.8%, 70.3%, 70.9%, 71.7%, 73.4%, 76.8%, 78.7%, 78.9%, 79.1%, 79.4%, 79.8%, 80.4%, 80.7%, 81.1%, 81.3%, 81.7%, 81.9, 83.4%, 84.9%, 85.8%, 96.2%, 96.8%, 97.3, 98.2%, 98.4%, 98.6%) to the amino acid sequence of IS. It was revealed that these strains also have a base sequence (BI-like gene; SEQ ID NO: 35) encoding a protein that is 67.9% or more homologous to the amino acid sequence of BI (specifically, 67.9%, 71.5%, 72.2%, 75.1%, 75.3%, 76.6%, 76.9%, 77.1%, 77.3%, 77.5%, 78.3%, 78.5%, 78.7%, 79.0%, 80.8%, 81.0%, 84.8%, 85.3%, 85.4%, 85.8%, 86.0%, 88.6%, 89.7%, 93.1%, 93.7%, 96.6%, 98.7%, 98.9%, 99.1%). Furthermore, all of the strains listed in Table 2 above, like strain SD33, had a BI-like gene located immediately downstream of the IS-like gene. Regarding Paenibacillus prosopidis and Paenibacillus sp. OV191 strains, no stop codon was found in the IS-like gene, and no signal peptide-like sequence was found at the N-terminus of the BI-like gene, suggesting the possibility that the BI-like protein and the IS-like protein are linked and produced. As described above, it was revealed that all bacteria possessing IS-like genes with a certain level of homology also possess BI-like genes. The amino acid sequence homology between the IS and each BI-like protein, and between the BI and each IS-like protein, was 50% or less.

[0079] Among the microorganisms listed in Table 2 above, the 16S rDNA partial base sequences of the type strains of microorganisms with clear species names, Amoniphilus sp. YIM78,166 strain, the type strain of Bacillus subtilis, and the SD33 strain were compared multiple times using clustalW, and a molecular phylogenetic tree was created using FastTree v2.1.8 with default parameters. The molecular phylogenetic tree created is shown in FIG.

[0080] As shown in Figure 7, the type strains of each microbial species except for Mycobacteroides abscessus subsp. abscessus and the Amoniphilus sp. YIM78,166 strain are all mapped to positions close to Bacillus subtilis (the most common Bacillus species). Given that the genera Brevibacillus and Paenibacillus are conventionally treated as closely related species to the genus Bacillus, it has been shown that it is appropriate to treat at least the microbial species in Table 2 above, excluding Mycobacteroides abscessus subsp. abscessus, i.e., the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Lichfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus, and Amoniphilus, as closely related species to the genus Bacillus.

[0081] Example 4: Creation of a plasmid for deleting the BI gene and a strain lacking the BI gene, and analysis of the inulin produced. This Example 4 describes the procedure for creating a plasmid for deleting the BI gene of the SD33 strain and a strain lacking the BI gene as a method for systematically breeding the inulin-synthesizing fungus SD33 strain isolated from nature into a strain suitable for synthesizing linear inulin. It was also demonstrated that deleting this gene enables the synthesis of linear inulin using the culture supernatant.

[0082] First, an overview of the BI gene deletion plasmid used in this method is provided below. The BI gene deletion plasmid is a plasmid obtained by subcloning a BI gene deletion DNA fragment into a plasmid containing any drug resistance gene. The BI gene deletion DNA fragment is a fragment formed by joining an approximately 0.5 kb fragment (A) adjacent to the upstream side of the BI gene with an approximately 0.5 kb fragment (B) adjacent to the downstream side. First, the upstream fragment (A) and downstream fragment (B) of the BI-like gene are prepared by a first PCR, using primers designed to add 10 to 30 bases upstream of the downstream fragment (B) to the downstream end of the upstream fragment (A). The primers used in this Example 4 are shown in Table 3 below.

[0083] [Table 3]

[0084] Next, a second PCR was performed using the PCR fragments (A) and (B) prepared in the first round as templates, with the upstream primer of the upstream fragment and the downstream primer of the downstream fragment. Annealing occurred between the downstream end of the upstream fragment and the upstream end of the downstream fragment, resulting in a DNA fragment in which (A) and (B) were ligated. The DNA fragment obtained in the second PCR was then inserted into a plasmid of interest by any method to obtain a plasmid for BI gene deletion. Specifically, PCR was performed using the SD33 strain genomic DNA as a template and primers (1) and (2) and (3) and (4) shown in Table 3 above. The two resulting amplified fragments were used as templates for PCR using primers (1) and (4) shown in Table 3 above. The PCR for amplifying the DNA fragments was performed as described in Example 2. The resulting amplified fragment was double-digested with restriction enzymes SmaI and XbaI (Takara Bio) and subcloned into the SmaI-XbaI region of pHY300PLK (Takara Bio). The DNA Ligation Kit Mighty Mix (Takara Bio) was used for ligation. The plasmid was produced by a transformant of Escherichia coli DH5α and prepared by the alkaline SDS method. The resulting plasmid was used as a template for sequence analysis using primer (5) listed in Table 3 above, confirming that the desired base sequence had been incorporated. This plasmid was used as the BI gene deletion plasmid.

[0085] Next, the SD33 strain was transformed with the resulting BI gene deletion plasmid, and a strain lacking the BI gene was obtained by double-crossover homologous recombination. Specifically, 100 μL of the SD33 strain glycerol stock was inoculated into 5 mL of TM medium placed in a 300 mL Erlenmeyer flask and cultured with shaking at 30°C and 130 rpm for 5 hours. The cells were collected by centrifugation at 5000 × g for 2 minutes, suspended in 5 mL of 50 mM Tris-HCl buffer (pH 7.5) and washed, and then the suspension was transferred to a 15 mL tube and centrifuged at 5000 × g for 2 minutes. The cells were suspended in 5 mL of 50 mM Tris-HCl buffer (pH 8.5) and shaken at 37°C and 85 rpm for 60 minutes. 500 μL of the suspension was sampled and centrifuged at 5000 × g for 1 minute to collect the cells, and the supernatant was removed. 5 μL of 300 ng / μL BI gene deletion plasmid solution was mixed with 50 μL of 70 mM phosphate buffer (pH 6.3) containing 0.5 M sodium sulfate, then added to the tube containing the bacteria, suspended by vortexing, and allowed to stand for 5 minutes. 150 μL of 70 mM phosphate buffer (pH 6.3) containing 40% PEG was added to the solution in the tube and suspended by vortexing. The bacteria were collected by centrifugation at 3000 × g for 5 minutes and resuspended in 20 mM MgCl . ２ The cells were suspended in 1 mL of TM medium containing 10 μg / mL tetracycline, transferred to a 15 mL tube, and shaken at 37° C. and 85 rpm for 60 minutes. The cells were collected by centrifugation at 5000×g for 1 minute, and then inoculated into LB medium containing 10 μg / mL tetracycline and cultured at 37° C. for 3 days to obtain transformants.

[0086] One loopful of the resulting colony was inoculated into 3 mL of LB liquid medium containing 10 μg / mL tetracycline and cultured for 8 hours with shaking at 30°C and 130 rpm. 50 μL of the culture medium was spread on the same agar medium and cultured for 24 hours at 35°C. Using the resulting single colony as a template, colony PCR was performed using primer (5), a universal primer for pHY300PLK shown in Table 3 above, and primer (6), designed based on the sequence near the transcription start site of the BI gene.

[0087] Colony PCR was performed using Emerald Amp Max Master Mix (Takara Bio). The colony PCR reaction solution was prepared by adding 1 μL of appropriately diluted bacterial cell suspension, 2 pmol each of sense and antisense primers, and 5 μL of Emerald Amp Max PCR Master Mix (2x Premix), and adjusting the total reaction volume to 10 μL. The PCR reaction conditions were 98°C for 30 seconds (1 cycle), 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 to 5 minutes (adjusted depending on the target amplification product, approximately 1 minute per 1 kb), for a total of 30 cycles. The sequencing reaction was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific), and the sequence was analyzed using an Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific). The composition of the medium used is described below. Unless otherwise specified, commercially available, high-grade reagents were used.

[0088] Media used: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, (15 g / L agar powder) Base medium: 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH ２ P.O. ４ , (15 g / L agar powder), adjusted to pH 7.2 with NaOH TM medium: 10 g / L glucose, 10 g / L soybean peptone, 5 g / L bonito extract, 2 g / L yeast extract, 10 mg / L FeSO ４ ・7H ２ O, 10 mg / L MnSO ４ ・7H ２ O, 1 mg / mL ZnSO ４ ・7H ２ 0, adjusted to pH 7.0 with NaOH

[0089] Colonies that yielded an approximately 500 bp amplified fragment by the colony PCR described above were designated as single-crossover recombinant strains. A colony from a single-crossover recombinant strain was inoculated into 3 mL of basal medium and cultured with shaking at 30°C and 130 rpm for 8 to 12 hours, after which 1 / 100 of the culture was inoculated into 3 mL of the same medium. This process was repeated four times, and the culture medium was then applied to the same agar medium and cultured overnight at 35°C. The cultured agar plate was pressed onto an autoclave-sterilized velvet cloth fixed to a base to allow colonies to adhere, and then LB agar medium containing no antibiotics and 10 μg / mL tetracycline were pressed onto the plate sequentially to transfer the colonies, which were then cultured overnight at 35°C. In this test, colonies that grew on LB agar medium containing no antibiotics but did not grow on LB agar medium containing 10 μg / mL tetracycline were selected, and colony PCR was performed using these as templates with primers (1) and (4). Strains that gave amplified fragments with a base size of approximately 1,000 bp, shorter than that of the original SD33 strain, were designated as BI gene deletion strains (hereinafter referred to as SD33_ΔBI strains).

[0090] Next, one loopful of SD33 strain and SD33_ΔBI strain was inoculated into 3 mL of basal medium and cultured for 24 hours at 30 ° C and 130 rpm with shaking. The supernatant was collected by centrifugation at 12,000 × g for 5 minutes, and sucrose was added to each culture supernatant to make Bx40. The mixture was then kept at 37 ° C for 24 hours, and then kept at 100 ° C for 5 minutes to inactivate the enzyme. The sugar composition of the reaction solution was analyzed by HPAEC-PAD. The peaks obtained by HPAEC-PAD analysis of the reaction products obtained by reacting the culture supernatants of SD33 strain and SD33_ΔBI strain with Bx40 sucrose are shown in Figure 8.

[0091] As shown in Figure 8, in the reaction mixture using the culture supernatant of the SD33 strain, branched inulin was confirmed in addition to linear inulin. On the other hand, in the reaction mixture using the culture supernatant of the SD33_ΔBI strain, only linear inulin was confirmed, and the area of ​​the peaks other than linear inulin was less than 1% of the total peak area at an elution time of 20 to 30 minutes.

[0092] Example 5: Analysis of sucrose reaction products of E. coli recombinant enzymes of BIs and ISs with various homologies In this Example 5, it was revealed that even proteins with a fairly low homology of 67.9% to the causative protein (BI) identified in Example 3 are responsible for producing branched inulin, and the genera Priestia, Bacillus, Neobacillus, Fredinandcornia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus, and Amoniphilus were identified as examples of inulin-synthesizing bacteria that harbor the genera encoding the causative proteins.

[0093] Of the base sequences encoding each BI-like protein shown in bold in Table 2 above, base sequences encoding the amino acid sequences excluding the signal peptide and termination codon sequences predicted by SignalP-5.0 were artificially synthesized (SEQ ID NOs: 38 to 47, commissioned to Integrated DNA Technologies). Two unknown bases in the base sequence information encoding the BI-like protein derived from Bacillus sp. strain LL01 were deduced from the base sequences of related BI-like proteins and used as guanidine bases to artificially synthesize the gene. Using each artificially synthesized base sequence as a template, PCR was performed with the corresponding primer pair. An E. coli recombinant enzyme expression system was constructed in the same manner as in Example 2, and the recombinant enzyme was purified. PCR for amplifying the DNA fragment was performed as described in Example 2. The BI-like protein (ATN45517.1) derived from the Salipaldibacillus agaradherens strain WDG185 was not expressed normally in E. coli and was therefore excluded from the subsequent tests. The primers used in Example 5 are listed in Table 4 below.

[0094] [Table 4]

[0095] Next, each purified BI-like recombinant enzyme and culture supernatant of the SD33_ΔBI strain (containing the IS enzyme) were added to a Bx40 sucrose solution at 2 U / g sucrose, and the mixture was incubated at 37°C for 24 hours, followed by incubation at 100°C for 5 minutes to inactivate the enzyme. The sugar composition of the reaction mixture was analyzed by HPAEC-PAD. Figure 9 shows the peaks obtained by HPAEC-PAD analysis of the reaction products obtained by mixing the culture supernatant of the SD33_ΔBI strain with various purified BI-like recombinant enzymes and reacting them with Bx40 sucrose. The enzyme activity was calculated as described in Example 1.

[0096] As shown in Figure 9, the culture supernatant of the SD33_ΔBI strain alone produced linear inulin. Table 5 below shows the branched inulin ratio (sum of branched inulin peak areas / total peak area × 100) at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16) when each purified BI-like recombinant enzyme and the culture supernatant of the SD33_ΔBI strain were mixed and reacted with Bx40 sucrose.

[0097]

[0098] As shown in Table 5 above, the branched inulin ratio was 0.3% at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16). On the other hand, as shown in Figure 9 , by adding any of the BI-like recombinant enzymes with a homology of 67.9% or more to SEQ ID NO: 36, branched inulin peaks were detected among the linear inulin peaks at elution time of 20 minutes or later, and the number of branched inulin peaks was 10 or more. Furthermore, as shown in Table 5 above, the branched inulin ratio at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16) exceeded 10% regardless of the BI used. When each BI-like recombinant enzyme was reacted alone with Bx40 sucrose, transglycosylation products with degrees of polymerization 3 to 5 were produced, similar to the SD33 strain BI.

[0099] Example 6: Study on the relationship between IS gene homology and linear inulin synthesis activity In this Example 6, it was revealed that even though the protein has a fairly low homology of 67.3% with the IS protein revealed in Example 2, it has inulosucrase activity to produce linear inulin.

[0100] OV191 strain IS-like protein, which has 67.3% homology with the amino acid sequence of the IS of the SD33 strain (SEQ ID NO: 34), and Bacillus sp. LL01 strain IS-like protein, which has 68.8% homology with the amino acid sequence of the IS of the SD33 strain (hereinafter referred to as "OV191_IS" and "LL01_IS," respectively), were used to artificially synthesize the signal peptide predicted by SignalP-5.0 and the amino acid sequence excluding the termination codon (SEQ ID NOs: 48 and 49), as in Example 5. An E. coli recombinant enzyme expression system was constructed, and the recombinant enzyme was purified. The peaks obtained by HPAEC-PAD analysis of the reaction products obtained by reacting the culture supernatant of the SD33_ΔBI strain, OV191_IS, and LL01_IS with Bx sucrose are shown in Figure 10.

[0101] As shown in Figure 10, the inulin produced when the two recombinant enzymes from the production reactions were reacted with a Bx40 sucrose solution showed a peak pattern of linear inulin, similar to that observed when the SD33_ΔBI strain was used, although there were fewer peaks at higher degrees of polymerization. From the above, it was found that even proteins with low homology (67.3% and 68.8%) to the IS of the SD33 strain possess the inulosucrase activity to produce linear inulin.

[0102] Example 7: Measurement of DFAIII yield in one-pot synthesis In this Example 7, it was revealed that even a protein with a fairly low homology of 67.9% to the causative protein (BI) identified in Example 3 can cause branched inulin to be produced, and can cause a decrease in the yield of DFAIII production from sucrose using the culture supernatant of the BI gene-deficient strain SD33 of Example 4 and inulin fructotransferase.

[0103] When inulin fructotransferase (IFT) is applied to inulin, DFAIII is produced, but the yield of DFAIII production is lower when inulin containing branched inulin is used compared to when linear inulin is used. Even when IS and IFT are added to a sucrose solution to perform a one-pot reaction of DFAIII, if the culture supernatant of a wild-type strain such as the SD33 strain is used as the IS, this culture supernatant also contains BI, so the yield of DFAIII production is lower compared to when purified IS is used. First, one-pot reactions were compared using the culture supernatant of the SD33 strain and the culture supernatant of the SD33_ΔBI strain.

[0104] To a Bx40 sucrose solution containing Arthrobacter sp. H65-7 IFT (manufactured by Nippon Beet Sugar Co., Ltd.) at 4 U / g sucrose, either (1) SD33 culture supernatant (2 U / g sucrose) or (2) SD33_ΔBI culture supernatant (1 U / g sucrose) was added. The solution was then incubated at 50°C for 90 hours, followed by 100°C for 10 minutes to terminate the enzymatic reaction. The reaction mixture was then diluted 10-fold with ultrapure water to analyze its sugar composition. To acid-hydrolyze the unreacted sucrose in the reaction mixture, 100 μL of 1N HCl was added to 1 mL of the 10-fold diluted solution and incubated at 60°C for 2 hours. Then, 100 μL of 1N NaOH was added to restore the pH, followed by HPLC analysis. The HPLC conditions were as follows: column: Shodex SUGAR KS-801 (guard column: SUGAR KS-G), column temperature: 60°C, solvent: 1 / 20,000N NaOH, flow rate: 1.0 mL / min, detection: RI, injection volume: 5 μL of 1 w / v% aqueous solution. The sugar composition percentage of DFA III (corresponding to the DFA III synthesis yield relative to sucrose) was calculated from the obtained peak area using the following formula. The DFA III synthesis yield when each purified BI-like recombinant enzyme was reacted with sucrose is shown in Table 6 below.

[0105] [Calculation formula] Sugar composition % of DFA III = [Disaccharide peak area after degradation] / [Total sugar peak area before degradation] × 100

[0106] [Table 6]

[0107] The yield of DFAIII synthesis when the culture supernatant of the SD33 strain shown in Table 6 above was used was 27.0%, whereas when the culture supernatant of the SD33_ΔBI strain was used, it was significantly improved to 38.6%.

[0108] Next, to clarify the homology of BI that reduces the reaction yield of the one-pot reaction of DFAIII, the following experiment was carried out using the BI-like recombinant enzyme prepared in Example 5. A mixture of 4 U / g sucrose of the culture supernatant of the SD33_ΔBI strain and purified BI-like recombinant enzyme (1 U / g sucrose each) was added to a Bx40 sucrose solution to which IFT (manufactured by Nippon Beet Sugar Co., Ltd.) had been added at 4 U / g sucrose, and the enzymatic reaction was carried out under the same reaction conditions as above to measure the DFAIII synthesis yield. The DFAIII synthesis yield relative to sucrose was as shown in Table 6 above. Regardless of which BI-like recombinant enzyme was mixed, the DFAIII yield was reduced compared to when only the culture supernatant of the SD33_ΔBI strain was used. This confirms that all BI-like proteins with 67.9% or more homology to the BI of the SD33 strain have enzymatic activity equivalent to that of the BI of the SD33 strain and reduce the yield of DFAIII in the one-pot DFAIII synthesis reaction.

[0109] Finally, we investigated whether the combination of BI and IS from different strains reduces the reaction yield of the one-pot DFAIII reaction. To a Bx40 sucrose solution containing IFT (4 U / g sucrose), an IS (1 U / g sucrose) from Paenibacillus sp. OV191 strain was added, or both an IS and a BI (1 U / g sucrose each) were added. The reaction was allowed to proceed at 50°C for 90 hours, and then the enzymatic reaction was stopped by holding the reaction at 100°C for 10 minutes. The DFAIII synthesis yield relative to sucrose was measured. A similar enzymatic reaction was also carried out using a combination of IS and BI from Bacillus sp. LL01 strain, and the DFAIII synthesis yield was measured. The yields of DFAIII synthesis when the IS and BI recombinant enzymes derived from Paenibacillus sp. strain OV191 and Bacillus sp. strain LL01 were reacted with sucrose are shown in Table 7 below.

[0110] [Table 7]

[0111] The DFAIII synthesis yields were as shown in Table 7. From the above, it was confirmed that when the IS and BI derived from Paenibacillus sp. OV191 strain and Bacillus sp. LL01 strain were used, the DFAIII yield decreased, as in the case of the SD33 strain.

[0112] [Rule 26 Amendment 02.10.2023]

Claims

1. One or more linear inulin-synthesizing bacteria selected from the group consisting of linear inulin-synthesizing bacteria of the genera Priestia, Bacillus, Neobacillus, Fredinandconia, Metabacillus, Richfieldia, Robertmuraya, Mycobacteroides, Alkalihalobacillus, Brevibacillus, Paenibacillus and Amoniphilus, and linear inulin-synthesizing bacteria of closely related species of the genus Bacillus other than these linear inulin-synthesizing bacteria, characterized in that at least one gene shown in (a) to (e) below is deleted or inactivated, and at least one gene shown in (h) to (m) below is present; (a) a gene encoding a protein consisting of a base sequence having 70% or more identity with the base sequence shown in SEQ ID NO:35; (b) a gene that hybridizes under stringent conditions to the complementary strand of the gene consisting of the base sequence shown in SEQ ID NO:35; (c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:36; (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:36 in which 1 to 10 amino acids have been deleted, substituted, added or inserted; (e) a gene encoding a protein consisting of an amino acid sequence having an identity of 67.9% or more with the amino acid sequence shown in SEQ ID NO:36.(h) a gene having the base sequence shown in SEQ ID NO: 33; (i) a gene consisting of a base sequence having 70% or more identity with the base sequence shown in SEQ ID NO: 33 and encoding a protein having the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product; (j) a gene which hybridizes under stringent conditions to the complementary strand of the gene consisting of the base sequence shown in SEQ ID NO: 33 and encodes a protein having the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or its transglycosylation product; (k) a gene consisting of the amino acid sequence shown in SEQ ID NO: 34 and encoding a protein which is secreted outside the bacterial cell; (l) a gene encoding an extracellularly secreted protein, which consists of an amino acid sequence obtained by deleting, substituting, adding or inserting 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO:34 and has the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or a transglycosylation product thereof; (m) a gene encoding an extracellularly secreted protein, which consists of an amino acid sequence having an identity of 67.3% or more with the amino acid sequence shown in SEQ ID NO:34 and has the activity of catalyzing the reaction of β-2,1-transferring the fructofuranosyl group of sucrose to the terminal fructose site of another sucrose or a transglycosylation product thereof.

2. A linear inulin-synthesizing bacterium according to (1), wherein the linear inulin-synthesizing bacterium has the following characteristics (f) or (g); (f) a linear inulin-synthesizing bacterium which produces inulin in which the bonds between fructose are composed only of β-2,1 bonds, by inoculating one platinum loop of the bacterium into 3 mL of a medium having the following composition, shaking culture at 30°C for 24 hours at 130 rpm, adding sucrose to the culture supernatant to a Bx of 40, and reacting at 37°C for 24 hours; <Medium composition> 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH ２ P.O. ４ and adjusting the pH to 7.2 with NaOH; (g) a linear inulin synthesizing bacterium, characterized in that when one loopful of the bacterial cells is inoculated into 3 mL of a medium having the composition (I) below, and cultured under shaking at 30°C for 24 hours at 130 rpm, sucrose is added to the culture supernatant to give a Bx of 40, and the reaction is carried out at 37°C for 24 hours, and the reaction product is subjected to HPAEC-PAD analysis under the conditions of (II) below, a peak having the same elution time as inulin consisting only of β-2,1 bonds is observed at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16), and the area of ​​the peaks other than this peak is less than 1% of the total peak area; <(I) Medium composition> 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH ２ P.O. ４ , and adjusted to pH 7.2 with NaOH. <(II) Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 × 250 mm; guard column: CarboPac PA1 4 × 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: constant 100 mM NaOH, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry.

3. The linear inulin synthesizing bacterium according to claim 1, wherein the gene consisting of SEQ ID NO: 35 or a base sequence having 75% or more identity with said SEQ ID NO: has the following characteristics: When a protein consisting of an amino acid sequence encoded by the gene consisting of SEQ ID NO: 35 or a base sequence having 75% or more identity with said SEQ ID NO: and a protein consisting of the amino acid sequence shown in SEQ ID NO: 34 are each added to a sucrose solution of Bx40 at 2 U / g sucrose, reacted at 37°C for 24 hours, and the reaction product obtained is subjected to HPAEC-PAD analysis under the following conditions, the reaction of the protein consisting of the amino acid sequence shown in SEQ ID NO: 34 with the sucrose solution of Bx40 is 2 U / g sucrose at 20 minutes or more (degree of polymerization 9 or more). 10 or more peaks having the same elution time as linear inulin in which the bond between fructose is only β-2,1 bond are obtained, and 10 or more peaks of branched inulin appearing between the peaks of linear inulin and having a structure in which a fructofuranosyl group is bonded to any fructose site of linear inulin by a bond type other than β-2,1 bond are detected, and the branched inulin ratio (sum of peak areas of branched inulin / total peak area×100) is 10% or more at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16). <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH 100 mM constant, AcONa gradient (0 to 60 min, 0 → 600 mM); detection: pulsed amperometry.

4. A purified enzyme, crude enzyme or enzyme-containing substance, culture medium, culture supernatant or cultured cells obtained from the linear inulin synthesizing bacteria described in any one of claims 1 to 3, or a processed product thereof.

5. A method for producing linear inulin, comprising a step of culturing a linear inulin synthesizing bacterium described in any one of claims 1 to 3.

6. A method for producing linear inulin, characterized in that it comprises a step of reacting at least one of a purified enzyme, a crude enzyme or an enzyme-containing substance, a culture medium, a culture supernatant or a cultured bacterial cell or a processed product thereof obtained from a linear inulin synthesizing bacterium described in any one of claims 1 to 3 with sucrose to obtain a reaction product.

7. The method for producing linear inulin according to claim 6, characterized in that, when the reaction product is subjected to HPAEC-PAD analysis under the following conditions, the ratio of the branched inulin having a structure in which a fructofuranosyl group is bonded to any fructose site of linear inulin in which the bond between fructose is β-2,1 bond by a bond type other than β-2,1 bond (sum of peak areas of branched inulins / total peak area x 100) is less than 1% at an elution time of 20 to 30 minutes (degree of polymerization 9 to 16); <Conditions for HPAEC-PAD analysis> Column: CarboPac PA1 4 x 250 mm; guard column: CarboPac PA1 4 x 50 mm; injection volume: 5 μL of 0.1% aqueous sample solution; flow rate: 1.0 mL / min; elution: NaOH Constant 100 mM, AcONa gradient (0-60 min, 0→600 mM); detection, pulsed amperometry.

8. A method for producing DFAIII, characterized by reacting at least one of a purified enzyme, a crude enzyme or an enzyme-containing substance, a culture medium, a culture supernatant or a cultured bacterial cell, or a processed product thereof, obtained from a linear inulin synthesizing bacterium described in any one of claims 1 to 3, with sucrose.

9. A method for producing DFAIII according to claim 8, characterized in that inulin fructotransferase is used.

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