WO2016013570A1

WO2016013570A1 - Method for producing putrescine by mixed bacterial culture

Info

Publication number: WO2016013570A1
Application number: PCT/JP2015/070812
Authority: WO
Inventors: 雄祐北田; 松本　光晴
Original assignee: 協同乳業株式会社
Priority date: 2014-07-22
Filing date: 2015-07-22
Publication date: 2016-01-28
Also published as: JP2017158434A

Abstract

A method for producing putrescine utilizing a cooperative action of two or more kinds of microorganisms. More specifically, a method for producing putrescine utilizing a microorganism that has an arginine-dependent acid resistance mechanism and a microorganism that has an agmatine deiminase (AgDI) metabolic pathway, and a method for producing putrescine utilizing a microorganism that produces ornithine and a microorganism that has inducible ornithine decarboxylase (iODC) capable of inducibly acting with an ornithine-putrescine antiporter.

Description

Method for producing putrescine by bacterial mixed culture

The present invention relates to a method for producing putrescine using a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway).

The present invention also relates to a method for producing putrescine using an ornithine-producing microorganism and a microorganism having an ornithine decarboxylase (iODC) that works inductively with an ornithine putrescine antiporter.

Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups, and putrescine (1,4-diaminobutane) is one of the simplest polyamines. Polyamines produced by intestinal bacteria are strongly related to the health of the host, such as having an intestinal barrier function enhancing action, antimutagenicity, and antioxidant action. Elucidation of the route is an important theme. Currently, research on the polyamine biosynthesis system of intestinal bacteria has been conducted on individual bacterial species, but the polyamine biosynthesis pathway has been specified only for some pathogenic bacteria. In addition, the mechanism of polyamine release to the outside of the fungus body (inside the intestinal lumen) and its physiological significance (why it is released outside the fungus body) have not been elucidated in those fungus species.

The polyamine-rich food material present in the current market is an extract from plants (Patent Document 1, Patent Document 2) and yeast (Patent Document 3). In obtaining a polyamine by extraction from a plant or yeast, there is a complexity including an extraction step and a concentration step. Considering such circumstances, fermentation production by microorganisms is simpler and is likely to lead to cost reduction. Examples of methods for producing putrescine using microorganisms include an example using one kind of microorganism (Patent Document 4) and an example using a mutant microorganism subjected to genetic recombination (Patent Document 5). However, in the example using one kind of microorganism, there is a problem that a sufficiently high production amount of putrescine has not been obtained, and a method using a genetically modified microorganism is not suitable for production of food materials.

JP 2010-26381 JP2012-236803 JP 2001-8663 A JP 2006-191808 A Special table 2010-535028

In view of the above background, there is still a need for a new method for producing putrescine that is highly safe, simple and sufficient in yield. The present invention provides a new method for producing putrescine satisfying such conditions.

In view of the above, the inventor of the present case noted that the in vivo study confirmed that the most abundant putrescine present in the human intestinal tract was induced in a concentration-dependent manner by oral administration of the precursor arginine. Started the research. As a result of intensive studies, we found that mixed culture produced more putrescine than single strains, and hypothesized that putrescine was biosynthesized and released in the intestinal tract by the joint action of multiple bacteria rather than a single bacterium. Stood up. They found a combination of bacteria that produced putrescine from arginine. Based on this finding, the present invention has been completed.

That is, in one aspect, the present invention may be as follows.
[1] A method for producing putrescine, wherein a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) are mixed and cultured in the presence of arginine under acidic conditions. Said method.
[2] The step of mixed culture includes
(A) culturing a microorganism having an arginine-dependent acid-resistant mechanism under acidic conditions in the presence of arginine; and
(B) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture of (A) and mixed and cultured;
The method according to [1], wherein
[3] The method according to [1] or [2], wherein the microorganism having an arginine-dependent acid resistance mechanism is a microorganism belonging to the family Enterobacteriaceae.
[4] The method according to [3], wherein the microorganism having an arginine-dependent acid resistance mechanism is Escherichia coli or Salmonella enterica.
[5] The microorganism having the AgDI pathway is a microorganism belonging to the family Enterococcuaceae, Streptococcaceae, Lactobacillaceae, Pseudomonadaceae, any one of [1] to [4] The method according to item.
[6] The method according to [5], wherein the microorganism having the AgDI pathway is Enterococcus faecalis.
[7] The method according to [1] or [2], wherein the microorganism having an arginine-dependent acid resistance mechanism is Escherichia coli, and the microorganism having an AgDI pathway is Enterococcus faecalis.
[8] The method according to any one of [1] to [7], wherein the acidic condition is pH 4.0 or higher and lower than pH 7.0.
[9] The method according to any one of [1] to [8], wherein the amount of arginine added in the mixed culture step is in the range of 0.1 mM to 500 mM.
[10] The method according to any one of [1] to [9], further comprising a step of separating putrescine from the culture solution of the mixed culture.
[11] A method for producing putrescine, comprising the following steps:
(1) culturing a microorganism having an arginine-dependent acid resistance mechanism under acidic conditions in the presence of arginine;
(2) obtaining a culture supernatant from the culture of step (1);
(3) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture supernatant of step (2) and cultured; and (4) putrescine is separated from the culture solution of step (3);
Said method.
[12] A method for producing putrescine, comprising the step of mixing and culturing a microorganism producing ornithine and a microorganism having iODC which is ornithine decarboxylase.
[13] The method according to [12], wherein the ornithine-producing microorganism is a microorganism having an ADI pathway (arginine deiminase metabolic pathway), and the mixed culture is performed in the presence of arginine.
[14] The method according to [12] or [13], wherein the microorganism having iODC is Escherichia coli having iODC.
[15] The method according to [14], wherein the Escherichia coli having iODC is Escherichia coli JCM5491 strain (ATCC25922) or Escherichia coli JCM1649 strain (ATCC11775).
[16] The method according to any one of [13] to [15], wherein the microorganism having an ADI pathway is Enterococcus faecalis.
[17] The conditions of the mixed culture are the following (a) to (c):
(A) pH 4.5 to pH 7.5;
(B) arginine is present in the range of 0.1 mM to 500 mM; and (c) aerobic conditions;
The method according to any one of [13] to [16], wherein the method has at least one of the following conditions.
[18] mixed culture in the presence of arginine,
Incubate in a medium supplemented with arginine; and / or add a microorganism that produces arginine and cultivate mixedly;
The method according to any one of [13] to [17], wherein
[19] In the mixed culture step, primary magnesium phosphate (Mg (H ₂ PO ₄ ) ₂ ), secondary magnesium phosphate (magnesium hydrogen phosphate (MgHPO ₄ )), and tertiary magnesium phosphate (Mg ₃ ( Any one of [13] to [18], wherein a medium containing an ammonia removing agent selected from the group consisting of PO ₄ ) ₂ ) and magnesium pyrophosphate (Mg ₂ P ₂ O ₇ ) is used. The method according to item.

By using the joint action of two or more types of microorganisms, putrescine can be produced more efficiently than when one type of microorganism is used. Further, since the method of the present invention does not use a gene recombinant, it is highly safe from the viewpoint of production of food materials.

FIG. 1 is a schematic view of a method for producing putrescine by two types of microorganisms, a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway. E. coli was exemplified as a microorganism having an arginine-dependent acid resistance mechanism, and En. Faecalis was exemplified as a microorganism having an AgDI pathway. FIG. 2 is a schematic view of a method for producing putrescine by a microorganism having an ADI pathway and a microorganism having iODC. En. Faecalis was exemplified as a microorganism having an ADI pathway, and E. coli was exemplified as a microorganism having iODC. FIG. 3 is a graph showing the difference between strains in the ability to produce putrescine from ornithine of E. coli. FIG. 4 is a graph showing the results of evaluating the amount of putrescine produced from arginine when combining E. coli and En. Faecalis strains. (A) The result when combining En. Faecalis JCM5803 and any of four strains of E. coli (JCM5491, JCM1649, JCM1246 or MG1655) is shown. (B) The results when En. Faecalis JCM8726 and any of the four strains of E. coli are combined are shown. (C) The results when combining En. Faecalis JCM7783 and any of the four strains of E. coli are shown. (D) The results when En.Enfaecalis V583 and any of the four strains of E. coli are combined are shown. FIG. 5 is a graph showing the effect of ammonia concentration on putrescine production from ornithine of E. coli. FIG. 6 is a graph showing the effect of magnesium hydrogen phosphate on the production of putrescine from arginine during E. coli and En. Faecalis mixed culture. FIG. 7 is a graph showing changes in the concentrations of arginine, ornithine and putrescine during E. coli and En. Faecalis mixed culture in a 1.2 M magnesium hydrogen phosphate-added medium.

The present invention will be specifically described below, but the present invention is not limited to these. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

<Method for producing putrescine>
The present invention relates to a method for producing putrescine using the joint action of two or more kinds of microorganisms. The present application provides a method for producing putrescine using the cooperative action of different microorganisms, wherein one of the different microorganisms has one of the metabolic pathways and the other has the other metabolic pathway.

<I. Method for producing putrescine using a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway>
In one aspect, the present invention relates to a method for producing putrescine using a microorganism having an arginine-dependent acid tolerance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway).

The arginine-dependent acid resistance mechanism is a mechanism possessed by some microorganisms, which absorbs arginine and releases agmatine under acidic conditions. The AgDI pathway is an agmatine metabolic pathway possessed by some microorganisms, and is a pathway that, after absorbing agmatine, decomposes agmatine using agmatine deiminase and produces putrescine via carbamoylputrescine.

The inventors of the present invention have found that a microorganism having an AgDI pathway biosynthesizes and releases putrescine using a metabolite produced by a microorganism having an arginine-dependent acid-resistant mechanism. That is, as illustrated in FIG. 1, it has been found that a microorganism having an arginine-dependent acid-resistant mechanism produces agmatine from arginine, and a microorganism having an AgDI pathway produces putrescine using the produced agmatine. It was.

In a first aspect, the present application provides a method for producing putrescine, comprising a step of mixing and culturing a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway under acidic conditions in the presence of arginine.

Microorganisms having an arginine-dependent acid resistance mechanism include, but are not limited to, microorganisms belonging to the family Enterobacteriaceae. In a preferred embodiment, the microorganism having an arginine-dependent acid resistance mechanism is a microorganism belonging to the genus Escherichia or Salmonella. Among each genus, Escherichia coli and Salmonella enterica are particularly preferable, and any one of these may be appropriately selected. Salmonella enterica is closely related to E. coli and is known to have a metabolic pathway similar to that of E. coli (Brenneman, K. E., et al., (2013) J. Bacteriol., 195 (13) : 3062-3072).

Microorganisms having the AgDI pathway (Agmatine deiminase metabolic pathway) include, but are not limited to, microorganisms belonging to the family Enterococcuaceae, Streptococcaceae, Lactobacillaceae, Pseudomonadaceae Not. These microorganisms are known to have metabolic pathways similar to Enterococcus faecalis (Ladero, V., et al., (2011) Appl. Envion. Microbiol., 77 (18): 6409-6418; Nakada, Y., et al., (2001) J. Bacteriol., 183 (22): 6517-6524). In a preferred embodiment, the microorganism having the AgDI pathway may be selected from Enterococcus faecalis, Lactococcus lactis, Lactobacillus brevis, and Pseudomonas aeruginosa. In another preferred embodiment, the microorganism having the AgDI pathway is a microorganism belonging to the family Enterococcus. In a further preferred embodiment, the microorganism having the AgDI pathway is Enterococcus faecalis.

In the mixed culture process, the microorganism is cultured in the presence of arginine. The arginine concentration in the culture solution is not particularly limited, but may be 0.1 mM or more, 0.5 以上 mM or more, or 1.0 mM or more. The upper limit of the arginine concentration is not particularly limited, but is 1.0 M or less, 800 mM or less, 600 mM or less, 400 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 75 mM or less, or 50 mM or less Good. The upper and lower limits of the arginine concentration can be appropriately selected from the above ranges. In a preferred embodiment, the concentration of arginine in the culture solution is 0.1 μm to 200 μm, 0.1 μm to 500 μm, 1.0 μm to 50 μm.

In the mixed culture process, the microorganism is cultured under acidic conditions. The acidic condition is not particularly limited as long as it is in the range of pH 4.0 or more and less than pH 7.0. In a preferred embodiment, the lower limit of the acidic condition is pH 4.0 or more, pH 4.5 or more, pH 5.0 or more, or pH 5.5 or more, and the upper limit is less than pH 7.0, pH 6.5 or less, or pH 6.0. It is as follows. The lower limit and the upper limit of the acidic condition can be appropriately selected from the above ranges. In a particularly preferred embodiment, the acidic conditions are pH 4.0 or higher and pH 6.0 or lower.

In the mixed culture process, the microorganism is preferably cultured under anaerobic conditions, but is not limited thereto.

In the mixed culture step, mixed culture may be performed using any medium available to those skilled in the art. In the method of the present invention, it has been observed that the amount of putrescine production increases as the number of bacteria in the culture increases. From this point of view, a medium in which a medium component sufficiently contains components necessary for the growth of microorganisms is preferable. In addition, the mixed culture may be performed by batch culture (that is, a method in which medium components are not added during culture) or fed-batch culture (that is, a method in which medium components are continuously added during culture). Good. Batch culture has the advantage that it can be industrialized relatively easily. On the other hand, when fed-batch culture is used, since the culture medium components are continuously added during the culture, the number of bacteria in the culture solution generally increases. For this reason, increase in putrescine production can be expected by using fed-batch culture. A person skilled in the art can appropriately select the type of medium, the amount of medium components, the culture method, and the like in consideration of various situations.

In the mixed culture step, the time for performing the mixed culture is not particularly limited, and those skilled in the art can appropriately set by sampling the mixed culture and measuring the amount of putrescine in the culture. The culture time of the mixed culture may be, for example, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours.

In the above mixed culture process, the mixed culture may be performed in any order as long as the microorganism having an arginine-dependent acid-resistant mechanism and the microorganism having the AgDI pathway are both present during the culture. Absent. In a preferred embodiment, a microorganism having an arginine-dependent acid resistance mechanism is first cultured, and a microorganism having an AgDI pathway is added later and mixed and cultured. That is, the mixed culture process is performed as follows:
(A) culturing a microorganism having an arginine-dependent acid-resistant mechanism under acidic conditions in the presence of arginine; and
(B) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture of (A) and mixed and cultured;
May be performed.

The time for culturing in the above step (A) is not particularly limited, and may be, for example, 6 hours, 12 hours, 24 hours, or 48 hours. Although the time which performs the mixed culture of the said process (B) is not specifically limited, For example, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours may be sufficient.

The method for producing putrescine of the present invention may further include a step of separating putrescine from the culture solution of the mixed culture. In the step of separating putrescine, putrescine is separated and / or purified from the culture solution obtained in the mixed culture step. Putrescine is contained in the culture solution, and the cells may be separated from the culture solution as a pretreatment for purification, and the separated cells may be reused. Separation of the bacterial cells from the culture solution may be performed by any method including known methods. For example, the separation may be performed by centrifugation, a filtration filter, a bacterial cell immobilization technique, or the like. Separation of putrescine from the culture solution may be performed by any method including known methods. For example, ultra high performance liquid chromatography (UPLC), high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis, ion chromatography, gel filtration, centrifugation, etc. may be used. For the separation and purification of putrescine, it is sufficient that the culture solution containing at least putrescine is separated from the cells. Therefore, purified putrescine may be obtained as a result of the step of separating putrescine, or a culture solution from which the cells have been removed may be obtained as a crude putrescine purified product.

In a second aspect, the application provides the following steps:
(1) culturing a microorganism having an arginine-dependent acid resistance mechanism under acidic conditions in the presence of arginine;
(2) obtaining a culture supernatant from the culture of step (1);
(3) A microorganism having an AgDI pathway is added to the culture supernatant of step (2) and cultured; and (4) putrescine is separated from the culture solution of step (3);
A method for producing putrescine is provided.

In the second aspect, the microorganisms having an arginine acid resistance mechanism, the microorganisms having an AgDI pathway, the culture conditions such as the amount of arginine and acidic conditions, and the range intended as a method for separating putrescine, As described in the embodiment.

In the step (1) of the second aspect, the culture time is not particularly limited, and may be, for example, 6 hours, 12 hours, 24 hours, 48 hours.

The time for culturing in the step (3) of the second aspect is not particularly limited. A person skilled in the art can appropriately set by sampling the mixed culture solution and measuring the amount of putrescine in the culture solution. For example, it may be 6 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours.

In the step (2) of the second aspect, the method for obtaining the culture supernatant may be performed by any method including a known method. For example, it can be performed by centrifugation, filter sterilization, a technique using a cell immobilization technique, or the like.

<II. Method for producing putrescine using ornithine-producing microorganism and iODC-containing microorganism>
In another embodiment, the present invention relates to a method for producing putrescine using a microorganism producing ornithine and a microorganism having iODC which is ornithine decarboxylase. Specifically, the method includes a step of culturing a microorganism that produces ornithine and a microorganism having iODC.

The ornithine-producing microorganism is not particularly limited as long as it has the characteristic that ornithine is produced, but it may be a wild-type microorganism that produces ornithine, or a microorganism that has been modified to improve the production of ornithine (for example, And microorganisms described in JP-A-2013-544532). Wild-type microorganisms that produce ornithine include microorganisms having the ADI pathway described later, microorganisms belonging to the genus Corynebacterium, Saccharomyces, Candida, or Pichia. included.

In another aspect, the present invention relates to a method for producing putrescine using a microorganism having an ADI pathway (arginine deiminase metabolic pathway) and a microorganism having iODC which is an ornithine decarboxylase. That is, as exemplified in FIG. 2, ornithine is produced from arginine by a microorganism having an ADI pathway, and a microorganism having iODC produces putrescine using the produced ornithine.

The ADI pathway is a pathway possessed by some microorganisms, and is a metabolic pathway in which arginine is absorbed, then arginine is decomposed using arginine deiminase, and ornithine is produced via citrulline (Simon, J. P. Et al., Journal of Bacteriology, (1982), 150 (3): 1085-1090).

IODC is one of the ornithine decarboxylases and is encoded by the SpeF gene. iODC works inductively with ornithine and putrescine antiporter when ornithine is present outside the cells, and biosynthesizes putrescine from the incorporated ornithine. The biosynthesized putrescine is released outside the cells (Kashiwagi, K., et al., The Journal of Biological Chemistry, (1991), 266 (31): 20922-20927).

The present application provides a method for producing putrescine, comprising a step of culturing a microorganism having an ADI pathway and a microorganism having iODC in the presence of arginine.

Microorganisms having the ADI pathway include microorganisms belonging to the family Enterococcus, Streptococcus, Lactobacillus, Pseudomonas, Vibrionaceae, Streptomycetaceae, or Mycobacteriaceae, It is not limited to this. In a preferred embodiment, the microorganism having the ADI pathway is a microorganism belonging to the family Enterococcus, more preferably Enterococcus faecalis.

The microorganism having iODC is not particularly limited as long as it is a microorganism expressing iODC. In a preferred embodiment, the microorganism having iODC is E. coli having iODC, more preferably E. 好ましく coli JCM5491 (ATCC25922) or E. coli JCM1649 (ATCC11775).

In the mixed culture process, the microorganism is cultured in the presence of arginine. The concentration of arginine in the culture solution is not particularly limited, but may be 0.1 μmM or higher, 0.5 μmM or higher, 1.0 μmM or higher, 50 μmM or higher, 100 μmM or higher, 200 μmM or higher. The upper limit of the arginine concentration is not particularly limited, but may be 1.0 M or less, 800 mM or less, 600 mM or less, or 400 mM or less. The upper and lower limits of the arginine concentration can be appropriately selected from the above ranges. In a preferred embodiment, the concentration of arginine in the culture solution is 0.1 to 500 μm, 200 to 400 μm.

Culture in the presence of arginine may be performed by culturing in a medium to which arginine has been added, and / or by adding a microorganism that produces arginine and performing mixed culture. When culturing in a medium to which arginine is added, arginine may be added to the medium in advance and / or arginine may be appropriately added during the culture. The mixed culture by adding microorganisms that produce arginine means that mixed culture of at least three kinds of microorganisms, that is, microorganisms having an ADI pathway, microorganisms having iODC, and microorganisms that produce arginine is performed. The microorganisms that produce arginine are not particularly limited as long as they produce arginine. For example, the microorganisms include genus Corynebacterium, Brevibacterium, Bacillus, Serratia, Escherichia or Saccharomyces. The microorganism to which it belongs is mentioned.

In the mixed culture process, the microorganism is cultured under acidic conditions or under weakly acidic to neutral conditions. The acidic condition is not particularly limited as long as it is in a range of pH 4.0 or more and less than pH 7.0. The mildly acidic to neutral conditions are not particularly limited as long as they are in the range of pH 5.0 or more and pH 7.5 or less. In a preferred embodiment, the microorganism is cultured under mildly acidic to neutral conditions, more preferably at a pH in the range of 6.0 to 7.5.

In the mixed culture step, the microorganism may be cultured under either aerobic conditions or anaerobic conditions. Preferably, the mixed culture is performed under aerobic conditions.

The type of medium, the amount of medium components, the culture method, etc. in the mixed culture process are described in I. And can be appropriately selected by those skilled in the art in consideration of various situations.

The culture time for mixed culture is the same as that described in I. above. As described above.

In the mixed culture step, primary magnesium phosphate (Mg (H ₂ PO ₄ ) ₂ ), secondary magnesium phosphate (magnesium hydrogen phosphate (MgHPO ₄ )), and tertiary magnesium phosphate (Mg ₃ (PO ₄₎ ₂ ), and mixed culture may be performed using a medium to which an ammonia removing agent selected from the group consisting of magnesium pyrophosphate (Mg ₂ P ₂ O ₇ ) is added. An example of a preferred ammonia scavenger is magnesium hydrogen phosphate. The ammonia removing agent is used to remove ammonia generated when ornithine is produced from arginine by a microorganism having an ADI pathway. By suppressing the inhibition of iODC activity by ammonia, putrescine production from ornithine by microorganisms having iODC is improved. The concentration of the ammonia removing agent added to the medium is 400 mM or more, preferably 600 mM or more, 700 mM or more, or 800 mM or more. The upper limit of the ammonia removing agent concentration is not particularly limited, but is, for example, 2.0 M or less and 1.5 M or less. The lower limit and the upper limit of the concentration of the ammonia removing agent can be appropriately selected from the above ranges. In particularly preferred embodiments, the concentration of the ammonia removal agent added to the medium is 400 mM to 1.5 M, 600 mM to 1.5 M, or 800 mM to 1.5 M.

The above-described method for producing putrescine may further include a step of separating putrescine from the culture solution of the mixed culture. Regarding the step of separating putrescine, the above-mentioned I.I. As described in.

Specific examples of the present invention are shown below. These examples are intended to provide an illustration for understanding the invention and are not intended to limit the scope of the invention.

<Example 1: Search for combinations of microorganisms that produce putrescine from arginine>
A search was made for a combination of two bacterial species having the largest amount of putrescine production among the 14 intestinal bacterial species shown in Table 1.

Among the above 14 bacterial species, combinations of 2 bacterial species, that is, 91 combinations in total, were mixed and cultured in a culture solution (GAM bouillon) to which arginine (final concentration: 1 mM) was added for 24 hours. The putrescine concentration in the culture broth 7 and 24 hours after the start of the culture was measured by ultra high performance liquid chromatography (UPLC).

As a result, in 8 combinations (Table 2) including Enterococcus faecalis, it was confirmed that the putrescine concentration in the culture solution was 5 mM or more. In addition, when the following combination of bacterial species was cultured, the concentration of putrescine in the culture increased by an average of about 11.1 times compared to the case where only one bacterial species was cultured. This indicates that putrescine is synthesized by the cooperative action of multiple bacterial species.

<Example 2: Influence of pH of culture solution>
Of the eight combinations of bacterial species found in Example 1, the combination of E. coli and Enterococcus faecalis that produced the largest amount of putrescine was used as a model case in the culture solution. Changes in the amount of putrescine production due to the difference in pH were examined.

E. coli and En. Faecalis were treated in the same manner as in Example 1 in a culture solution supplemented with arginine at different pH (pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5). The mixed culture was performed, and the putrescine concentration in the culture medium was measured. As a result, the amount of putrescine produced by the mixed culture increased in the weakly acidic range (pH 6.0, pH 5.5, pH 5.0) rather than the neutral range (pH 7.0 and 7.5).

<Example 3: Two-stage culture>
The following experiment was conducted for the purpose of confirming whether metabolites were transported between E. coli and Enterococcus faecalis.

About one of the two types of bacterial species, single bacterial culture was performed in an arginine-added culture solution (pH 6.0). The culture supernatant was sterilized by filter, and the other bacterial species was inoculated and cultured. Thereafter, the putrescine concentration in the culture solution was measured in the same manner as in Example 1.

As a result, an increase in the amount of putrescine production was observed only when En. Faecalis was cultured using the culture supernatant of E. coli. This indicates that En. Faecalis biosynthesizes and releases putrescine using the metabolite produced by E. coli.

<Example 4: Putrescine production by mixed culture of two bacterial species>
The following experiment was conducted using Escherichia coli MG1655 strain (ATCC700926) and Enterococcus faecalis V583 strain (ATCC700802) (both purchased from ATCC (purchased from American Type Culture Collection)).
(1) Medium The following medium was used. The culture conditions are anaerobic culture at 37 ° C. unless otherwise specified.

(2) Experiment E. coli MG1655 strain was precultured overnight in A medium. Thereafter, using B medium containing arginine, the initial OD ₆₀₀ = 0.01 equivalent amount of E. coli MG1655 strain was added, and main culture was started. Then, pre-culture of En. Faecalis V583 strain was performed in A medium. Sampling was carried out 24 hours after the start of the main culture, and at the same time, cells of En. Faecalis V583 strain were added in an amount equivalent to the initial OD ₆₀₀ = 0.01, and the culture was continued. Sampling was performed 48 hours after the start of the main culture, and the concentration of putrescine in the culture medium was confirmed.
(3) Results As a result, it was confirmed that putrescine was accumulated in the culture solution (Table 4).

Similarly, putrescine production experiments were carried out using Escherichia coli JCM5491 strain (ATCC25922) and Enterococcus faecalis JCM5803 strain (ATCC19433) (both purchased from RIKEN). The results are shown in Table 5.

From the above, it has been clarified that putrescine can be produced in a high yield by using a combination of E. coli and E.cofaecalis regardless of the strain type.

<Example 5: Increase in yield by examining culture conditions>
Example 4 is the same as Example 4 except that GAM bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) adjusted to pH (pH 4.0 to pH 7.0) by adding 50 mM arginine instead of A medium and B medium was used. A similar experiment was conducted. The composition of GAM bouillon is as shown in Table 6.

The results are shown in Table 7.

By changing the culture conditions, the amount of putrescine produced was improved in both combinations compared to Experimental Example 4. This result shows that an improvement in the amount of putrescine production can be expected by further changing the culture conditions in addition to pH.

<Example 6: Identification of E. coli retaining iODC activity>
The E. coli strains (purchased from ATCC or RIKEN) shown in Table 8 were examined to retain iODC activity.

(1) Medium composition As the basal medium shown in Table 9 and the ornithine-added basal medium, media each containing 400 mM ornithine hydrochloride were prepared.

(2) Culture The strain was pre-cultured in a basal medium for 24 hours, and the main culture was ornithine-added basal medium, 1% of the pre-culture solution was added, and cultured at 37 ° C. under aerobic conditions for 24 hours. . After the culture, the ornithine and putrescine concentrations in the culture were measured.
(3) Results E. coli JCM5491 and E. coli JCM1649 retain iODC activity because the ornithine concentration in the culture solution decreases and the putrescine concentration increases in the E. coli JCM5491 and E. coli JCM1649 culture solutions. This was confirmed (FIG. 3). However, in E. coli JCM1246 and E. coli MG1655 culture media, the ornithine concentration in the culture solution does not decrease and the putrescine concentration does not increase, so E. coli JCM1246 and E. coli MG1655 do not retain iODC activity. It was confirmed.

<Example 7: Putrescine production from arginine by mixed culture of E. coli and En. Faecalis>
Using the E. coli and En. Faecalis strains shown in Table 10, production of putrescine from arginine by mixed culture of E. coli and En. Faecalis was examined.

(1) Culture The basal medium was prepared as described in Example 6. Further, as a medium for main culture, a liquid medium in which 400 mM arginine was added to a basal medium was prepared.
Each strain was precultured in basal medium for 24 hours. In the main culture, En. Faecalis is added to the main culture solution in a 1% amount of the pre-culture solution, E. coli is added to the main culture solution in an amount of 0.001%, and the aerobic condition is 37 ° C. It was performed by culturing under 30 hours. And the putrescine density | concentration in a culture solution was measured. During the culture, the pH was raised by ammonia produced by En. Faecalis, so that the pH was maintained between 6.0 and 7.5 by adding phosphoric acid.
(2) Results When E. coli JCM5491 and E. coli JCM1649 retaining iODC activity were used, 40 mM or more putrescine was produced in the culture solution in the mixed culture with all En. Faecalis (FIG. 4). . However, when using E. coli JCM1246 and E. coli MG1655 that do not retain iODC activity, putrescine was not detected in all mixed cultures with En. Faecalis. The above results indicate that iODC activity retained by some E. coli is important for the production of putrescine from arginine by mixed culture of E. coli and En. Faecalis (weakly acidic to neutral and aerobic conditions). It is shown that. The combination with the highest amount of putrescine production was about 70 mM for the combination of E. coli JCM1649 and En. Faecalis JCM8726.

<Example 8: Inhibition of iODC activity of E. coli by ammonia>
The production of putrescine from arginine by the route of FIG. 2 has been problematic in that the production of putrescine from ornithine in E. coli is suppressed with the progress of ornithine production from arginine by En. Faecalis. En. Faecalis produces 2 moles of ammonia simultaneously when producing 1 mole of ornithine from arginine. It was thought that this ammonia inhibited putrescine production from ornithine. Therefore, the inhibition of iODC activity of E. coli by ammonia was examined using E. coli JCM5491.

(1) Culture The basal medium was prepared as described in Example 6. As a medium for main culture, a liquid medium was prepared by adding 400 mM ornithine hydrochloride and 0 to 200 mM ammonium chloride to the basal medium.
The strain is precultured in a basal medium for 24 hours. In the main culture, 1% of the preculture is added to the main culture, and cultured at 37 ° C. under aerobic conditions. Ornithine concentration and putrescine concentration were measured.
(2) Results It was found that when the ammonia concentration in the culture solution increased, the amount of decrease in ornithine concentration in the culture solution decreased and the putrescine concentration did not increase (FIG. 5). From this, it was confirmed that the ammonia concentration in the culture solution inhibited the iODC activity of E. coli.

<Example 9: Effect of addition of magnesium hydrogen phosphate on putrescine production from arginine by mixed culture of E. coli and En. Faecalis>
In order to improve the inhibition of iODC activity by ammonia shown in Example 8, a method using struvite was sought. The struvite is a crystal of magnesium ammonium hydrogen phosphate, and has the property that it is extremely difficult to dissolve in neutral to alkaline water. So far, it has been reported that ammonia is recovered as struvite in wastewater treatment (Japanese Patent Laid-Open No. 2006-289168). In this study, we tried to improve the inhibition of E. coli iODC activity by adding magnesium hydrogen phosphate to the medium and reacting with the ammonia produced by En. Faecalis to precipitate it as struvite.

Specifically, the combination of E. coli JCM1649 and En. Faecalis JCM8726, and E. coli JCM5491 and En. Faecalis JCM5803 are mixed and cultured in the presence of magnesium hydrogen phosphate to reduce the amount of putrescine production. We examined improvement.

(1) Culture A basal medium was prepared in the same manner as in Example 6. In addition, as a medium for main culture, a medium in which 400 mM arginine and 0, 200, 400, 600, or 800 mM magnesium hydrogen phosphate were added to a basal medium was prepared.

Experiments were performed using a combination of E. coli JCM1649 and En. Faecalis JCM8726, and a combination of E. coli JCM5491 and En. Faecalis JCM5803. The strains are cultured in a basal medium for 24 hours. En. Faecalis JCM8726 and En. Faecalis JCM5803 add 1% of the preculture to the main culture, and E. coli JCM1649 and E. coli JCM5491 A preculture solution was added in an amount of 0.001%, and cultured at 37 ° C. under aerobic conditions for 24 hours. During the culture, the pH was raised by ammonia produced by En. Faecalis, so the pH was maintained between 6.0 and 7.5 using phosphoric acid. After the culture, the ornithine and putrescine concentrations in the culture were measured.

Also, in the combination of E. coli JCM5491 and En. Faecalis JCM5803, 1.2 M of magnesium hydrogenphosphate was added to the basal medium and cultured under the above conditions, and arginine, ornithine and putrescine concentrations were measured over time.

(2) Results Ornithine in the culture solution decreased and putrescine increased depending on the concentration of magnesium hydrogen phosphate (FIG. 6). In particular, when 400 mM or more of magnesium hydrogen phosphate was added, the amount of putrescine produced by E. coli and En. Faecalis increased greatly. Theoretically, En. Faecalis produces 800 mM ammonia from 400 mM arginine added to the culture solution, and at least the ammonia concentration in the culture solution is 400 mM or less in order for E. coli iODC to work. It is suggested that it is desirable. From the above, it was shown that it is important to remove ammonia produced by En. Faecalis as a by-product of ornithine for putrescine production from arginine by mixed culture of E. coli and En. Faecalis. As one of them, a method for precipitating and removing ammonia as struvite has been demonstrated.

As shown in a series of experimental results, ornithine is produced with a decrease in arginine, and then putrescine is produced with a decrease in ornithine in the culture solution supplemented with 1.2 M magnesium hydrogen phosphate, which gave the maximum putrescine yield. It was recognized that The putrescine yield after 24 hours of main culture was 3.43%, and the yield of putrescine after 40 hours of culture was 4.17% (FIG. 7).

Claims

A method for producing putrescine, comprising the step of mixing and culturing a microorganism producing ornithine and a microorganism having iODC which is ornithine decarboxylase.
The method according to claim 1, wherein the microorganism producing ornithine is a microorganism having an ADI pathway (arginine deiminase metabolic pathway), and the mixed culture is performed in the presence of arginine.
The method according to claim 1 or 2, wherein the microorganism having iODC is Escherichia coli having iODC.
The method according to any one of claims 1 to 3, wherein the Escherichia coli having iODC is Escherichia coli JCM5491 strain (ATCC25922) or Escherichia coli JCM1649 strain (ATCC11775).
The method according to any one of claims 2 to 4, wherein the microorganism having the ADI pathway is Enterococcus faecalis.
The conditions for the mixed culture are the following (a)-(c):
(A) pH 4.5 to pH 7.5;
(B) arginine is present in the range of 0.1 mM to 500 mM; and (c) aerobic conditions;
The method according to any one of claims 2 to 5, which has at least one of the following conditions.
Mixed culture in the presence of arginine
Incubate in a medium supplemented with arginine; and / or add a microorganism that produces arginine and cultivate mixedly;
The method according to any one of claims 2 to 6, which is carried out by
In the mixed culture step, primary magnesium phosphate (Mg (H 2 PO 4 ) 2 ), secondary magnesium phosphate (magnesium hydrogen phosphate (MgHPO 4 )), and tertiary magnesium phosphate (Mg 3 (PO 4 )) The method according to any one of claims 2 to 7, wherein a medium containing an ammonia removing agent selected from the group consisting of 2 ) and magnesium pyrophosphate (Mg 2 P 2 O 7 ) is used. .
A method for producing putrescine, comprising a step of mixing and culturing a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) in the presence of arginine under acidic conditions. Said method.
A method for producing putrescine comprising the following steps:
(1) culturing a microorganism having an arginine-dependent acid resistance mechanism under acidic conditions in the presence of arginine;
(2) obtaining a culture supernatant from the culture of step (1);
(3) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture supernatant of step (2) and cultured; and (4) putrescine is separated from the culture solution of step (3);
Said method.