Microorganisms producing d-allulose from allitol and method for producing d-allulose using same

Abstract

[Problem] To provide a novel microorganism capable of producing D-allulose from allitol. Also, to provide a method for oxidizing / converting allitol into D-allulose using the microorganism. [Solution] Bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella and Lelliottia and capable of producing D-allulose from allitol employed as the starting material were identified. By using these bacteria, D-allulose is produced at a high yield from allitol.

Description

Microorganism for producing D-allulose from allitol and method for producing D-allulose using the same

[0001] The present invention relates to a microorganism capable of producing D-allulose from allitol, and a method for producing D-allulose from allitol using the microorganism.

[0002] Known examples of carbohydrate sweeteners classified as sugar alcohols include reduced starch syrup and reduced palatinose. Reduced palatinose does not cause dental caries and is a low-calorie sweetener. Sugar alcohols have a variety of uses, and sugar alcohols with six carbon atoms, such as sorbitol and mannitol, are used in a variety of applications. For example, D-sorbitol, produced by hydrogenation of D-glucose, is used in a wide range of applications, including cosmetics and as a sweetener. Furthermore, talitol, allitol, iditol, and the like are sugar alcohols (rare sugar alcohols) that are rarely found in nature, but are expected to be used in foods, cosmetics, pharmaceuticals, chemicals, pesticides, plant growth regulators, and more. These examples suggest that six-carbon sugar alcohols are expected to be used in many applications, and research has begun.

[0003] However, compared to D-allulose, for which various functionalities have been reported, the useful functions of allitol remain unclear, and allitol produced as a by-product in the rare sugar production process is currently treated as industrial waste. The issue of carbohydrate loss is a key issue that must be resolved both domestically and internationally, not only from the perspective of reducing environmental impact but also ensuring sustainable production and consumption patterns for food and other resources. Recently, there has been a demand not only for recycling unnecessary materials but also for promoting reuse activities for the effective circulation of resources. Allitol is a monosaccharide classified as a sugar alcohol and is a rare sugar alcohol that is rarely found in nature. In principle, its production can be achieved by reducing monosaccharides such as D-allulose and L-allulose using organic chemistry techniques under high temperature and high pressure with hydrogen in the presence of a metal catalyst. Furthermore, polyols such as allitol can be produced by enzymatic reactions using ketose 3-epimerase, as described in Patent Documents 1 to 4. Compared to D-allulose, for which various functionalities have been reported, the useful functions of allitol remain unclear, and as mentioned above, allitol produced in the rare sugar production process is currently treated as industrial waste.

[0004] Therefore, as a method for effectively utilizing allitol as a resource, a biological method has been reported in which allitol is oxidized and converted to D-allulose using microorganisms (Non-Patent Documents 1 and 2). However, only two strains of microorganisms capable of producing D-allulose from allitol have been reported.

[0005] JP 6-125776, JP 8-154696, JP 11-056383, International Publication 2014 / 109254

[0006] J. Biosci. Bioeng. (2007) Vol.103, No.3, p.282-285Biosci. Biotechnol. Biochem. (2007) Vol.71, No.12, p.3048-3054

[0007] An object of the present invention is to provide a novel microorganism that produces D-allulose from allitol. Another object of the present invention is to provide a biological method using the microorganism to oxidize and convert allitol into D-allulose, which has been reported to have various useful functions, as a method for effectively utilizing allitol as a resource.

[0008] In order to solve the above problems, the present inventors extensively screened for microorganisms capable of producing D-allulose from allitol by searching for microorganisms with high oxidizing activity for allitol from a soil library. From among these, they discovered and identified a microorganism that produces D-allulose in high yield, thereby completing the present invention.

[0009] Specifically, microorganisms capable of producing D-allulose from allitol upon contact with an aqueous solution containing allitol were selected. As a result, bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, and Leliotia that are capable of producing D-allulose from allitol were identified, and it was confirmed that these bacteria can produce D-allulose in high yields.

[0010] The present invention relates to a novel microorganism capable of producing D-allulose from allitol, and to a method for producing D-allulose from allitol using the microorganism. More specifically, the present invention relates to a method using bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, and Leliotia that are capable of producing D-allulose from allitol. Furthermore, the microorganism preferably does not have the ability to convert D-talitol, which may be contaminated during the process of producing allitol by reducing D-allulose with hydrogen under high temperature and pressure in the presence of a metal catalyst.

[0011] The present invention relates to the following methods for producing D-allulose from allitol (1) to (6): (1) A method for producing D-allulose from the raw material allitol using a microorganism selected from the group consisting of bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, and Lelliottia, which is capable of producing D-allulose from allitol; (2) The method described in (1) above, which is carried out by adding the raw material allitol to a bacterial cell culture system of the microorganism and culturing the microorganism; (3) The method described in (1) or (2) above, in which the raw material allitol is an aqueous solution containing allitol; and (4) The method described in any of (1) to (3) above, in which the microorganism does not have the ability to convert D-talitol. (5) The microorganism is selected from the group consisting of Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp. The method according to any one of (1) to (4) above, wherein the bacterial species is selected from the group consisting of Buttiauxella sp. and Leliotia jeotgalli.(6) The microorganism is selected from the group consisting of Burkholderia lata Y534-1 = 3 strains, Burkholderia lata U459-1-1 = 1 strain, Burkholderia multivorans Y488-4 = 4 strains, Burkholderia multivorans S332-4 = 1 strain, Burkholderia multivorans Y555-2d = 2 strains, and Burkholderia diffusa. diffusa Y452-1=3 strains, Enterobacter hormaechei BCr11-1 strain, Enterobacter hormaechei BCr11-2 strain, Enterobacter soli BDr27-1 strain, Agrobacterium pusense BCr15-1 strain, Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. (6) The method according to any one of (1) to (5), wherein the microorganism is selected from the group consisting of bacteria of Buttiauxella sp. BCr16-1-1, Lelliottia jeotgalli NH309-4, Lelliottia jeotgalli Ou92-1-1, and Lelliottia jeotgalli T33-1.

[0012] The present invention also relates to the following microorganisms (7) and (8) capable of producing D-allulose from allitol, and (9) a culture capable of producing D-allulose from allitol. (7) Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp. A microorganism capable of producing D-allulose from allitol, selected from the group consisting of bacterial species Buttiauxella sp. and Lelliottia jeotgalli.(8) The microorganism is selected from the group consisting of Burkholderia lata Y534-1 = 3 strains, Burkholderia lata U459-1-1 = 1 strain, Burkholderia multivorans Y488-4 = 4 strains, Burkholderia multivorans S332-4 = 1 strain, Burkholderia multivorans Y555-2d = 2 strains, and Burkholderia diffusa. diffusa Y452-1=3 strains, Enterobacter hormaechei BCr11-1 strain, Enterobacter hormaechei BCr11-2 strain, Enterobacter soli BDr27-1 strain, Agrobacterium pusense BCr15-1 strain, Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. The microorganism according to (7) above, which is selected from the group consisting of Buttiauxella sp. BCr16-1-1 strain, Lelliottia jeotgalli NH309-4 strain, Lelliottia jeotgalli Ou92-1-1 strain, and Lelliottia jeotgalli T33-1 strain. (9) A culture capable of producing D-allulose from allitol, comprising the microorganism according to (7) or (8) above and a medium.

[0013] According to the present invention, a novel microorganism capable of producing D-allulose from allitol can be provided, which not only enables the recycling of unnecessary allitol but also promotes the reuse of resources through effective circulation. Furthermore, D-allulose, which has been reported to have various useful functions, can be easily and inexpensively produced by a biological method using allitol, whose useful functions have not yet been elucidated, as a raw material. The method of the present invention for producing D-allulose using a bacterial oxidation reaction is not only environmentally friendly, but also more economical than enzymatic reaction methods because it can be produced by an oxidation reaction using bacterial cofactors without the addition of expensive reaction cofactors such as NAD(P)+. It is also a method for efficiently producing D-allulose from allitol, which has not been possible using organic chemistry techniques.

[0014] 1 shows the relative amounts of allitol converted to D-allulose by each microorganism under secondary screening conditions A to D. FIG. 2 shows the conversion rates of allitol to D-allulose by three highly active strains.

[0015] The present inventors used a soil library to search for microorganisms capable of producing D-allulose from allitol upon contact with an aqueous solution containing allitol. The microorganism capable of producing D-allulose from allitol of the present invention may be any microorganism capable of producing D-allulose upon contact with an aqueous solution containing allitol. Specifically, preferred microorganisms capable of producing D-allulose from allitol are selected from the group consisting of bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, and Leliotia, or mutants thereof.

[0016] More specifically, any Burkholderia bacterium having the ability to produce D-allulose from allitol can be used as long as it is a Burkholderia bacterium having that ability. Examples of Burkholderia bacterial species having that ability include Burkholderia lata, Burkholderia multivorans, and Burkholderia diffusa.

[0017] The Burkholderia rata Y534-1 (3 strains), Burkholderia rata U459-1-1 (1 strain), Burkholderia marteivorans Y488-4 (4 strains), Burkholderia marteivorans S332-4 (1 strain), Burkholderia marteivorans Y555-2d (2 strains), and Burkholderia diffusa Y452-1 (3 strains), which were confirmed to be capable of producing D-allulose from allitol, were deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on February 16, 2021, and are assigned accession numbers NITE AP-03413, NITE AP-03410, NITE AP-03412, and NITE AP-03413, respectively. The strains were received under accession numbers NITE AP-03408, NITE AP-03414, and NITE AP-03411. Subsequently, the strains were deposited on April 30, 2021, under accession numbers NITE P-03413, NITE P-03410, NITE P-03412, NITE P-03408, NITE P-03414, and NITE P-03411, respectively. Furthermore, each strain was deposited internationally on February 1, 2022 under the accession numbers NITE BP-03413, NITE BP-03410, NITE BP-03412, NITE BP-03408, NITE BP-03414, and NITE BP-03411, respectively.

[0018] Furthermore, any Enterobacter bacterium having the ability to produce D-allulose from allitol can be used, as long as it is a bacterium of the genus Enterobacter that has this ability. Examples of Enterobacter species that have this ability include Enterobacter hormaechei and Enterobacter soli. The Enterobacter hormaekei BCr11-1 strain, Enterobacter hormaekei BCr11-2 strain, and Enterobacter sori BDr27-1 strain, which were confirmed to be capable of producing D-allulose from allitol, were deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NIET), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on February 16, 2021, under the accession numbers NITE AP-03400, NITE AP-03401, and NITE AP-03405, respectively. Subsequently, each strain was deposited on April 30, 2021, under the accession numbers NITE P-03400, NITE P-03401, and NITE P-03405, respectively. Furthermore, each strain was deposited internationally on February 1, 2022 under the accession numbers NITE BP-03400, NITE BP-03401, and NITE BP-03405, respectively.

[0019] Furthermore, bacteria belonging to the genera Agrobacterium, Buttiauxella, and Leliotteia that are capable of producing D-allulose from allitol can be used as long as they belong to the genera Agrobacterium, Buttiauxella, and Leliotteia. Examples of bacterial species of the genera Agrobacterium, Buttiauxella, and Leliotteia that have this ability include Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp. (Buttiauxella sp.) and Leliotia jeotgalli.

[0020] The Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. BCr16-1-1 strain, Leiotheia geogalli NH309-4 strain, Leiotheia geogalli Ou92-1-1 strain, and Leiotheia geogalli T33-1 strain, which have been confirmed to have the ability to produce D-allulose from allitol, were deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NIET), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on February 16, 2021, and received under the accession numbers NITE AP-03404, NITE AP-03403, NITE AP-03406, NITE AP-03407, and NITE AP-03409, respectively. Subsequently, each strain was deposited on April 30, 2021 under the accession numbers NITE P-03404, NITE P-03403, NITE P-03406, NITE P-03407, and NITE P-03409, respectively. Furthermore, each strain was deposited internationally on February 1, 2022 under the accession numbers NITE BP-03404, NITE BP-03403, NITE BP-03406, NITE BP-03407, and NITE BP-03409, respectively.

[0021] In the production method of the present invention, these bacteria are first cultured in a standard nutrient medium, preferably under aerobic conditions such as shaking or aeration / agitation. During the culture, or using the obtained live cells, allitol in the aqueous solution is converted to D-allulose, and the resulting D-allulose is collected. In this way, the bacteria used for oxidizing D-allulose can be utilized in the culture medium. Specifically, Burkholderia lata Y534-1 = 3 strains, Burkholderia lata U459-1-1 = 1 strain, Burkholderia multivorans Y488-4 = 4 strains, Burkholderia multivorans S332-4 = 1 strain, Burkholderia multivorans Y555-2d = 2 strains, Burkholderia diffusa Y452-1 = 3 strains, Enterobacter Enterobacter hormaechei BCr11-1 strain, Enterobacter hormaechei BCr11-2 strain, Enterobacter soli BDr27-1 strain, Agrobacterium pusense BCr15-1 strain, Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. A culture capable of producing D-allulose from allitol can be used, which comprises a microorganism that selectively produces D-allulose from allitol and a medium, and is selected from Buttiauxella sp. BCr16-1-1 strain, Lelliottia jeotgalli NH309-4 strain, Lelliottia jeotgalli Ou92-1-1 strain, and Lelliottia jeotgalli T33-1 strain. Also usable are live cells isolated from the culture medium and dried cells.

[0022] The culture method involves inoculating bacteria capable of producing D-allulose from allitol into a nutrient medium, preferably a liquid medium, containing nutrients required by these bacteria, such as a carbon source, a nitrogen source, inorganic salts, and yeast extract, and culturing the bacteria at a temperature of 20 to 40°C under aerobic conditions for 1 to 10 days.

[0023] The viable bacterial cells obtained by such a culture method are contacted with an aqueous solution containing allitol, preferably under specified conditions such as shaking, aeration, and oxygen injection, to convert allitol to D-allulose. When the reaction is carried out at 30°C with shaking, up to 20 wt% of allitol is converted to D-allulose after two days. These bacteria can also be used in an immobilized form, and highly active immobilized bacteria can be obtained by various immobilization methods, such as carrier binding, crosslinking, gel entrapment, and microencapsulation.

[0024] The aqueous solution containing D-allulose produced and accumulated by the various methods described above is separated from insoluble matter such as bacterial cells by an appropriate separation method, such as centrifugation or filtration, and then collected. If necessary, the resulting aqueous D-allulose solution can be purified by, for example, activated carbon treatment or treatment with an ion exchange resin, and then concentrated to collect a syrupy or crystalline D-allulose product.

[0025] According to the method of the present invention, D-allulose can be obtained at a yield of up to 20% w / w based on the raw material allitol, making it suitable for industrial production. This allows for the inexpensive supply of D-allulose in large quantities as a raw material or intermediate in various industries, including the pharmaceutical and food industries. This not only enables the recycling of unnecessary allitol, but also promotes the reuse of resources. Since ketoses are converted into the corresponding two sugar alcohols, D-allulose, a monosaccharide classified as a ketose, can be converted to allitol and D-talitol by hydrogenation. The reactivity of the microorganism of the present invention to D-talitol, which may be present in the product, was examined, and it was found that D-talitol is not used as a substrate. Further research into methods for utilizing this microorganism is anticipated. The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0026] [Screening of D-allulose-producing bacteria] As a primary screening from the soil library, microorganisms were cultured in an inorganic salt medium containing 5% of various polyols (allitol, D-talitol, D-sorbitol), and the ketose produced in the culture supernatant was detected by the cysteine ​​carbazole method. From the group of microorganisms that showed oxidation activity only for allitol in the primary screening, secondary microbial screening was performed under four conditions to select microorganisms with high oxidation activity for allitol.

[0027] Conditions A and C: Using bacterial cells cultured on TSB agar medium at 30°C, the cells were suspended in a 0.5% allitol solution for condition A and in a 10% allitol solution for condition C, and after shaking at 30°C for 24 hours, the conversion of allitol to D-allulose was evaluated using HPLC. Conditions B and D: Each microorganism was inoculated into a yeast extract liquid medium containing 10% allitol for condition B and 5% allitol for condition D, and cultured with shaking at 30°C for 24 hours, after which the conversion of allitol to psicose associated with the growth was evaluated using HPLC.

[0028] [Results] In the secondary screening, the allitol-to-D-allulose conversion activity of the microorganisms isolated from soil was qualitatively evaluated using four screening methods under conditions A to D. As a result, 15 species of bacteria were identified as highly active strains (Table 1). Microbial identification by the Japan Food Research Laboratories classified the strains into the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, and Leliotia. The serial numbers of the 15 highly active strains are A06, A12, A17, A29, A39, A54, A90, A173, A201, A324, A325, A335, A354, A355, and A358, and the serial numbers of the control low activity strains are A44, A119, A162, A219, A322, A363, and A419.

[0029]

[0030] 1 shows the results of qualitative evaluation of these strains obtained as high activity strains and control strains with low activity, with the activity of the strain that showed the highest activity in each evaluation method under conditions A to D taken as 100% as a relative activity value. In the screening under condition A, the strain that showed the highest activity was Burkholderia lata U459-1-1=1 (A12), in the screening under conditions B and C, the strain that showed the highest activity was Agrobacterium pusense BCr15-1 (A335), and in the screening under condition D, the strain that showed the highest activity was Lelliottia jeotgalli Ou92-1-1 (A201).

[0031] [Time-course evaluation of the conversion of allitol to D-allulose in highly active strains selected under condition D] Using the highly active strains A90 (Laeriotheia geogalli T33-1 strain), A201 (Laeriotheia geogalli Ou92-1-1 strain), and A358 (Enterobacter sori BDr27-1 strain) selected under condition D in the secondary screening, the time-course evaluation of the conversion of allitol to D-allulose was carried out as follows.

[0032] Each strain was cultured in a yeast extract liquid medium at 30°C, and the resulting cells were washed with phosphate buffer and suspended in a solution consisting of 10% allitol and phosphate buffer (pH 7.0) to a turbidity of 20. The cells were then shaken at 30°C, and aliquots of the reaction solution were collected at 5, 9, 15, 25, 40, and 50 hours. Each reaction solution was centrifuged to remove the cells, and after desalting and filtration, the product was confirmed by HPLC to evaluate the conversion of allitol to D-allulose. In both assays, HPLC separation and quantification of allitol and D-allulose were performed using a GL-C611 column.

[0033] The results are shown in Figure 2. A358 (Enterobacter soli BDr27-1 strain) had the highest conversion efficiency, converting 18% of allitol to D-allulose in 50 hours. A time-dependent evaluation of the conversion of allitol to D-allulose by washed cell reaction was conducted for three strains belonging to the genus Laerliotheia or Enterobacter. As a result, the conversion of allitol to D-allulose over time was observed in all strains.

[0034] The present invention establishes a method for easily producing D-allulose from allitol using a biological method employing the novel microorganism of the present invention, which has been difficult to achieve in a predetermined ratio using conventional organic chemical techniques. Therefore, the method of the present invention is suitable as a biological method for producing D-allulose, and the novel microorganism of the present invention is useful as a microorganism for use in that method.

Claims

1. The present invention relates to a method for producing D-allulose from a raw material allitol using a microorganism selected from the group consisting of bacteria belonging to the genera Burkholderia, Agrobacterium, Buttiauxella, and Lelliottia, the microorganism having the ability to produce D-allulose from allitol.

2. 2. The method according to claim 1, wherein the method is carried out by adding allitol as a raw material to a culture system of the microorganism and culturing the microorganism.

3. The method according to claim 1 or 2, wherein the raw material allitol is an aqueous solution containing allitol.

4. The method according to any one of claims 1 to 3, wherein the microorganism does not have the ability to convert D-talitol.

5. 5. The method according to any one of claims 1 to 4, wherein the microorganism is selected from the group consisting of the bacterial species Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp. and Lelliottia jeotgalli.

6. The microorganism is selected from the group consisting of Burkholderia lata Y534-1 = 3 strains, Burkholderia lata U459-1-1 = 1 strain, Burkholderia multivorans Y488-4 = 4 strains, Burkholderia multivorans S332-4 = 1 strain, Burkholderia multivorans Y555-2d = 2 strains, Burkholderia diffusa diffusa Y452-1=3 strain, Agrobacterium pusense BCr15-1 strain, Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. The method according to any one of claims 1 to 5, wherein the host cell is selected from the group consisting of Buttiauxella sp. BCr16-1-1 strain, Lelliottia jeotgali NH309-4 strain, Lelliottia jeotgali Ou92-1-1 strain, and Lelliottia jeotgali T33-1 strain.

7. A microorganism capable of producing D-allulose from allitol, said microorganism being selected from the group consisting of bacterial species of Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp., and Lelliottia jeotgalli.

8. The microorganism is selected from the group consisting of Burkholderia lata Y534-1=3 strains, Burkholderia lata U459-1-1=1 strain, Burkholderia multivorans Y488-4=4 strains, Burkholderia multivorans S332-4=1 strain, Burkholderia multivorans Y555-2d-2 strain, Burkholderia diffusa diffusa Y452-1=3 strain, Agrobacterium pusense BCr15-1 strain, Buttiauxella brennerae BCr16-1-3 strain, Buttiauxella sp. The microorganism according to claim 7, which is selected from the group consisting of Buttiauxella sp. BCr16-1-1 strain, Lelliottia jeotgali NH309-4 strain, Lelliottia jeotgali Ou92-1-1 strain, and Lelliottia jeotgali T33-1 strain.

9. A culture comprising the microorganism according to claim 7 or 8 and a medium, capable of producing D-allulose from allitol.