JP2016158523A - Fatty acid release strain of cyanobacteria and production method of fatty acid-resistant strain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple method for obtaining cyanobacteria having free fatty acid high-productivity without requiring complex operations such as genetic recombination.SOLUTION: There is provided a production method of fatty acid release strains of cyanobacteria, including: a step to prepare a plurality of test sections where specimen cyanobacteria are cultured using a medium-chain or long-chain fatty acid-containing culture medium, and among which a type of the medium-chain or long-chain fatty acid contained in the culture medium differs; a step to add and culture specimen cyanobacteria to the culture medium in the plurality of the test sections; a step to select a test section where a colony is not recognized in the early stage of culture but appears when the culture is continued; and a step to collect the cyanobacteria from the colony in the selected test section.SELECTED DRAWING: None

Description

  The present invention relates to an improved technique for cyanobacteria. More specifically, it relates to a method for producing a fatty acid releasing strain / fatty acid resistant strain of cyanobacteria and its use.

  Currently, there is a need to produce energy materials that can replace petroleum. Among them, cyanobacteria that perform oxygen-generating photosynthesis that does not compete with food are attracting attention as model organisms for biofuel (biodiesel) production. Since cyanobacteria are easy to manipulate, new metabolic pathways have been constructed through the introduction of foreign genes, enabling the production of chemicals that cannot be produced by the original cyanobacteria themselves. Until now, the production of cyanobacteria having an increased ability to produce free fatty acids (FFA) has been attempted by genetic recombination (see Patent Document 1 and Non-Patent Documents 1 to 3). For example, in Non-Patent Document 1, a free fatty acid-producing strain is prepared by destroying the acyl-ACP synthase gene (aas gene) of the cyanobacterium Synechocystis sp. Analyzing. In Non-Patent Document 2, a free fatty acid-producing strain is produced from the cyanobacterium Synechococcus sp. PCC 7002 by the same method to increase production efficiency. Also in Non-Patent Document 3, a free fatty acid-producing strain is produced from the cyanobacterium Synechococcus elongatas PCC 7942 strain by the same method to increase production efficiency.

  As described above, attempts have been made to increase the ability of cyanobacteria to produce free fatty acids, but conventional methods require genetic recombination. In genetic recombination, identification of the target gene is a prerequisite, and unless there are special circumstances (for example, when the genomic information has already been decoded and the target gene is known), the decoding of the genetic information and the function of the gene Analysis etc. need to be done. There is also no guarantee that the target gene can be determined. Furthermore, the conventional method requires multi-stage gene recombination, which complicates the operation and causes a problem of restriction of usable marker genes.

Special table 2011-505838 gazette

Gao Q et al., Biotechnol for Biofuels. 2012 Mar 21; 5 (1): 17 Anne M. Ruffing., Front Bioeng Biotechnol. 2014 May 26; 2: 17. Anne M. Ruffing and Howland D.T.Jones Biotechnol, Bioeng. 2012 Sep; 109 (9): 2190-9.

  An object of the present invention is to provide a cyanobacteria that produces a high amount of industrially useful free fatty acids (FFA), such as a precursor for biofuels, without requiring complicated operations such as genetic recombination. Is to get it in a different way.

  Free fatty acids accumulate as lipase-mediated membrane lipid degradation products. The acyl-ACP synthase encoded by the aas gene reincorporates free fatty acids into the fatty acid synthesis pathway and contributes to the reuse of fatty acid residues. When this gene is inactivated, the fatty acid residue recycling pathway is inactivated and free fatty acids accumulate in the cells. Furthermore, a part of them is discharged out of the cell and the product accumulates in the medium (FIG. 1). Research on the construction of fatty acid production strains is advancing using this experimental system.

  The research group of the present inventors has advanced research aiming at high production of fatty acids, which are high-energy substances, in order to achieve practical application of biofuel production technology based on cyanobacteria. Among them, strains were constructed in which mutations were introduced into the aas gene for three species of cyanobacteria (Synecococcus sp. PCC 7942, Synecocystis sp. PCC 6803, Synecococcus sp. PCC 7002). It was confirmed that 100 to 200 μM of free fatty acid was accumulated (according to extracellular FFA) from the culture solution of the second week of each mutant strain (FIG. 2). However, these cells are susceptible to growth inhibition. It was thought that the cause was that free fatty acids accumulated in cells at a high concentration might cause damage to cell physiology (accumulation of free fatty acid accumulation may have resulted in decreased product yield). Is also possible). Therefore, as a result of examining the effects of fatty acids added from the outside on the growth of cyanobacteria, it was confirmed that the three strains of cyanobacteria showed high sensitivity to medium-chain saturated fatty acids and unsaturated fatty acids. The results were reported (The 77th Annual Meeting of the Botanical Society of Japan, September 13, 2013. Fig. 3)

  The conditions were examined to further increase the fatty acid yield, but multiple gene disruptions and gene transfer (enhancement of endogenous gene expression levels) were required, limiting the number of markers required for gene recombination. I faced the challenge of being.

  In the conventional markerless gene modification method, a drug marker and a suicide gene (sacB gene that produces a lethal effect in the presence of sucrose) are once incorporated into the target gene, and then transformed with a construct that disrupts the structure of the gene. Under the lethal conditions (add sucrose to remove sacB) (FIG. 4). Attempts were being made to modify markerless genes via suicide genes in cyanobacteria, but it was impossible to adapt to several genes including the aas gene.

  I decided to explore another approach. In other words, an attempt was made to screen for mutant strains that have acquired fatty acid resistance under the belief that stable free fatty acid production can be achieved if resistance to unsaturated fatty acids can be increased. Specifically, a medium supplemented with a plurality of medium chain or long chain fatty acids each having a different chain length and saturation was prepared, and a cyanobacterial wild strain was cultured. As a result, when cultured in a medium to which a specific fatty acid was added, a surprising phenomenon was observed in which colonies were not formed at the initial stage of culture, and a small number of colonies appeared by continuing the culture ( The remaining fatty acids were sensitive or resistant from the beginning of the culture). This result is a method of preparing a plurality of fatty acid-containing media with different types of fatty acids and culturing cyanobacteria and identifying colonies that do not recognize colonies at the beginning of culture but appear when colonies continue Is effective as a means for obtaining a fatty acid-releasing strain. In other words, according to this method, a highly useful cyanobacterial fatty acid-releasing strain can be produced without complicated operations such as genetic recombination.

  On the other hand, as a result of detailed examination, the resistance of the fatty acid resistant strain that emerged is irreversibly retained, that the fatty acid resistant strain has a high percentage of mutations in the aas gene, and fatty acids that do not depend on the mutation in the aas gene Important and interesting findings were obtained, such as the acquisition of resistance.

The following invention is mainly based on the above results and considerations.
[1] A method for producing a cyanobacterial fatty acid-releasing strain, comprising the following steps (1) to (4):
(1) A plurality of test sections for culturing the test cyanobacteria using medium-chain or long-chain fatty acid-containing medium, wherein the types of medium-chain or long-chain fatty acids contained in the medium are different in the test sections. Preparing a test zone;
(2) adding a test cyanobacteria to the medium and culturing in the plurality of test sections;
(3) a step of selecting a test plot in which colonies are not observed at the initial stage of culture and colonies appear by continuing the culture;
(4) A step of recovering cyanobacteria from the colonies of the selected test section.
[2] The production method according to [1], wherein the medium chain or long chain fatty acid has 10 to 18 carbon atoms.
[3] The production method according to [1] or [2], wherein the medium-chain or long-chain fatty acid contained in the medium of one test section is one kind.
[4] The production method according to any one of [1] to [3], wherein a fatty acid concentration in the medium-chain or long-chain fatty acid-containing medium is 50 μM to 200 μM.
[5] The production method according to any one of [1] to [4], wherein the culture temperature is 25 ° C to 35 ° C.
[6] The production method according to any one of [1] to [5], wherein the initial end point of the culture in step (3) is between the first day after the start of the culture and the tenth day after the start of the culture. .
[7] The production method according to any one of [1] to [6], wherein the time when the colonies appear in the step (3) is from the 5th day after the start of the culture to the 30th day after the start of the culture.
[8] The production method according to any one of [1] to [7], wherein each of the plurality of test sections includes a dilution series in which the concentration of the test cyanobacteria decreases stepwise.
[9] The production method according to any one of [1] to [8], wherein the medium is a solid medium.
[10] The production method according to any one of [1] to [9], wherein the sample cyanobacterium is Synechocystis cyanobacterium, Synecocus genus cyanobacterium, Microcystis genus cyanobacterium, or Arthrospira genus cyanobacterium. .
[11] The production method according to [10], wherein the Synechocystis cyanobacterium is Synechocystis sp. PCC 6803 strain, and the Synechococcus cyanobacterium is Synechococcus sp. PCC 7002 strain.
[12] A method for producing a cyanobacterial fatty acid-releasing strain, comprising the following steps (i) and (ii):
(I) The test cyanobacterium used in the production method according to any one of [1] to [11] is prepared, and the medium used in the test section selected in step (4) of [1] contains Culturing in a medium supplemented with chain or long chain fatty acids;
(Ii) A step of collecting cyanobacteria from the emerged colonies.
[13] A method for producing a cyanobacterial fatty acid-releasing strain comprising the following steps (I) and (II):
(I) Synecocystis sp. PCC 6803 strain or Synecococcus sp. PCC 7002 strain is prepared. In the case of Synecocystis sp. PCC 6803 strain, a medium containing long chain fatty acid C18: 2 or C18: 3 or both of them is used. And in the case of Synechococcus sp. PCC 7002 strain, culturing using a medium containing a long chain fatty acid C12: 0;
(II) A step of collecting cyanobacteria from the appearing colonies.
[14] The production method according to any one of [1] to [13], further including the following step (a) and / or step (b):
(A) confirming that the recovered cyanobacteria release free fatty acids;
(B) identifying the type and / or amount of free fatty acids released by the recovered cyanobacteria.

  The present invention skillfully utilizes the susceptibility of cyanobacteria to free fatty acids, and by culturing in the presence of specific free fatty acids, a fatty acid resistant strain is obtained, which releases free fatty acids. Based on what you have discovered. That is, it is a method in which a fatty acid releasing strain is obtained by screening a fatty acid resistant strain, and does not require a genetic recombination operation. Accordingly, it is not necessary to identify a target gene (typically an acyl-ACP synthase gene) that needs to be destroyed for fatty acid release, and introduction of a foreign gene serving as a marker can be avoided. In this respect, this method is different from the conventional method for producing a high fatty acid-producing strain by genetic recombination. By utilizing the present invention, it is possible to efficiently produce free fatty acids while avoiding the use of genetically modified organisms.

Strategies for constructing fatty acid production strains using genetic recombination technology. When the aas gene is disrupted, free fatty acids accumulate in the cells, and some are excreted outside the cells and accumulate in the medium. Production of free fatty acids in three types of cyanobacterial aas gene mutants. From left to right, free fatty acid production of Synechococcus sp. PCC 7942, free fatty acid production of Synecocystis sp. PCC 6803, and free fatty acid production of Synecococcus sp. PCC 7002. Survival rate of Synechocystis sp. PCC 6803. strain. Synechocystis sp. PCC 6803. strain was cultured in various fatty acid-containing media. High sensitivity to medium chain saturated and unsaturated fatty acids. Conventional fatty acid production strain construction method. Overview of screening for fatty acid resistant strains. Confirmation of C18: 3 resistance of each strain of fatty acid resistant strains. Comparison of free fatty acid releasing ability of fatty acid resistant strains. In the graph on the right, the amount of extracellular free fatty acid of the fatty acid resistant strain Res183 # 1 was compared with the wild type (WT) and the aas gene mutant strain (aas :: sk). Aas gene mutation sites found in fatty acid resistant strains. Percentage of mutations found in the aas gene in fatty acid resistant strains. The presence or absence of mutation in the aas gene was confirmed for those whose genome sequence could be confirmed. The results for Synecocystis sp. PCC 6803. (upper) and those for Synecococcus sp. PCC 7002 (below) are shown. Genes with mutations found in fatty acid resistant strains.

  The present invention provides a method for producing a cyanobacterial fatty acid-releasing strain. In other words, according to the present invention, a cyanobacterial fatty acid releasing strain is obtained. The “cyanobacterium fatty acid releasing strain” is a cyanobacteria that produces free fatty acids and exhibits the ability to secrete at least part of the produced free fatty acids out of the cells (that is, free fatty acid releasing ability). The “cyanobacterial fatty acid-releasing strain” obtained by the present invention has a higher ability to release free fatty acids compared to the original cyanobacterium (also referred to as “tested cyanobacterium” in the present invention). Therefore, the present invention can be regarded as a method for producing cyanobacteria having a high free fatty acid releasing ability. As shown in Examples described later, the cyanobacterial fatty acid-producing release strain produced by applying the method of the present invention has acquired resistance to the specific free fatty acid used in the production. In view of this fact, it can be said that according to the present invention, a cyanobacteria (fatty acid resistant strain) that has acquired resistance to a specific free fatty acid can be obtained.

  As a result of detailed studies by the present inventors, many of the obtained cyanobacterial fatty acid-releasing strains have mutations in the aas gene that has been targeted for increasing the production efficiency of free fatty acids. Therefore, the present invention is a method for adding a mutation to a gene involved in the production efficiency of free fatty acids without involving complicated operations such as gene targeting (which means that identification of the target gene is also unnecessary). It can be said that. On the other hand, some of the obtained cyanobacterial fatty acid-releasing strains had mutations in genes with unknown functions. Considering this fact as well, the method of the present invention is useful as a technique for identifying genes involved in acquiring resistance to fatty acids or releasing free fatty acids.

In the production method of the present invention, the following steps (1) to (4) are performed in this order.
(1) A plurality of test sections for culturing the test cyanobacteria using medium-chain or long-chain fatty acid-containing medium, wherein the types of medium-chain or long-chain fatty acids contained in the medium are different in the test sections. Step of preparing test plots (2) Step of adding test cyanobacteria to the medium and culturing in the plurality of test plots (3) No colonies were observed at the initial stage of culture, and colonies were found by continuing the culture. Step of selecting test plots that appear (4) Step of collecting cyanobacteria from the colonies of the selected test plots

  In step (1), a plurality of test sections (test groups) are prepared. In each test group, the test cyanobacteria are cultured using a medium-chain or long-chain fatty acid-containing medium. In the present invention, the types of medium-chain or long-chain fatty acids contained in the medium are made different in the test section. In this regard, for example, when the number of test sections is set to 3, for example, a specific fatty acid (for example, C12: 0 fatty acid) is used in the first test section, and in the second test section, the first test section is used. Fatty acids with a different carbon number (chain length) (for example, C16: 0 fatty acids) are used than those used, and in the third test zone, carbon used for both the first test zone and the second test zone is carbon. Use different numbers of fatty acids (eg C18: 1 fatty acids).

  The number of test sections is not particularly limited. For example, 2 to 30, preferably 5 to 15 test sections are provided. The small number of test sections affects the efficiency, and the operation becomes complicated if there are too many test sections. In addition, in order to improve the reliability of the experimental results, it is preferable to provide a test medium (control group) that uses a medium containing no fatty acid in addition to the test group.

  The medium chain or long chain fatty acid-containing medium used in each test section contains one or more kinds of medium chain or long chain fatty acids. Preferably, one medium-chain or long-chain fatty acid contained in the medium of one test section is used. Employing such conditions simplifies the configuration of the test section and increases efficiency. In addition, it becomes easy to identify fatty acids useful for producing a fatty acid releasing strain.

  In general, fatty acids are classified into short chain fatty acids (2 to 4 carbon atoms), medium chain fatty acids (5 to 11 carbon atoms), and long chain fatty acids (12 or more carbon atoms) depending on the difference in carbon number. In the present invention, medium chain fatty acids and long chain fatty acids are used. Both saturated fatty acids and unsaturated fatty acids can be used. Moreover, you may decide to use together the fatty acid from which saturation differs. In a preferred embodiment, efficiency is improved by employing a fatty acid having 10 to 18 carbon atoms. Examples of medium chain or long chain fatty acids that can be used in the present invention include C10: 0 (capric acid), C12: 0 (lauric acid), C14: 0 (myristic acid), C15: 0 (pentadecylic acid), C16: 0 (palmitic acid), C16: 1 (palmitoleic acid), C17: 0 (margaric acid), C18: 0 (stearic acid), C18: 1 (9) (oleic acid), C18: 1 (11) (vaccenic acid) ), C18: 2 (9,12) (linoleic acid), C18: 3 (9,12,15) ((9,12,15) -linolenic acid), C18: 3 (6,9,12) (( 6,9,12) -linolenic acid), C18: 3 (9,11,13) (eleostearic acid).

  The fatty acid concentration in the medium-chain or long-chain fatty acid-containing medium is, for example, 50 μM to 200 μM, preferably 70 μM to 150 μM, more preferably 90 μM to 110 μM (specific example is 100 μM). If the fatty acid concentration in the medium is too low, a test plot that appears to be resistant appears or increases, which affects the efficiency and reliability of selection. On the other hand, if the fatty acid concentration in the medium is too high, the growth of test cyanobacteria may be affected more than necessary, and appropriate evaluation may not be possible.

  Either a solid medium or a liquid medium can be used, but a solid medium is preferably used. According to the solid medium, colonies can be easily detected in step (3), and cyanobacteria can be easily recovered from the single colonies in subsequent step (4).

  Various cyanobacteria can be used as the test cyanobacteria. Cyanobacteria (Cyanobacteria) can be categorized into the order of Cleococcus, Nemoptera, Euremo, Pleurocapsa, Stigonema. In the order of the genus Aophanocapsa, Afanoteke, Camaesifon, Chroococcus, Crocosfera, Cyanobacteria, Cyanobium, Cyanoteque, Dactilococcosis, Groebobacter, Gloeocapsa, Gloeotecke, Euhalotheke Genus, halotheke genus, johannes baptistia genus, melismopedia genus, microcystis genus, labdoderma genus, cinecococcus genus, cinecocystis genus, thermosychococcus genus and the like. In the order of the genus Lepidoptera, the genus Coreodesmium, Flemiera, Microcatete, Lexia, Spirirestis, Triposrix, Anabena, Anabenopsis, Aphanizomenon, Aurosila, Cyanospira, Cylindrospermopsis, Cylindros Examples include the genus Sperum, Nodularia, Nenjumo, Liqueria, Kalos Rix, Gloeotricia, Schitonema and the like. In the order of Artemisia, Arthrospira, Gaitrelinema, Halopromine, Halosspirulina, Catagnimene, Leptolymbia, Limnosrix, Lymbia, Microcoleus, Yulemo, Formidium, Planktorikoides, Plantus Examples include the genus Lix, the genus Plectnema, the genus Limnos, the genus Pseudoanabena, the genus Schizothris, the genus Spirulina, the genus Simproca, the genus Trichodesmium, and the genus Chiconema. The genus Pleurocappus includes the genus Chloococdiodiopsis, Delmocarpa, Delmocarpera, Myxosarkina, Pleurocapsa, Staniaria, and Xenococcus. The genus Stagonema includes Capsosilla, Chlorogloeopsis, Fischerella, Haparosifon, Mastigocladopsis, Mastigocladus, Nostochopsis, Stigonema, Symphionema, Symphionemopsis, Umezakia, Westie Includes the genus Eropsis.

  Preferably, cyanobacteria belonging to the genus Synecocystis, Synecococcus, Microcystis or Arthrospira are employed as the test algae. These cyanobacteria are frequently used for research purposes or industrial purposes, and are highly versatile and industrially useful. It also has the advantage of being readily available. Examples of Synechocystis cyanobacterium are Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6714. Similarly, examples of Synechococcus sp. PCC 7002 and Synechococcus elongatus PCC 7942 are examples of Synechococcus sp. PCC 7942, and examples of Microcystis cyanobacterium Microcystis aeruginosa) (for example, NIES-843 strain), and examples of Arthrospira platensis are Arthrospira platensis (for example, NIES-39 strain) and Arthrospira Maxima (Arthrospira Maxima). Arthrospira platensis and Arthrospira maxima are suitable for large-scale culture, and their utility value is particularly high.

In step (2), the test cyanobacteria are added to the medium in each test group and cultured. Cultivating conditions (medium, temperature conditions, illuminance conditions, etc.) of cyanobacteria are based on past reports (for example, refer to Non-Patent Documents 1 to 3) and books (for example, photosynthesis) in consideration of the type of test algae. Research method Low temperature science Vol67. 2008. ISSN1880-7593, Algae handbook (NTS, supervised by Shin Watanabe, 2012) can be used as a reference. For example, the culture temperature is 25 ° C. to 35 ° C. (preferably 27 ° C. to 32 ° C.), the light condition is continuous light or the light / dark cycle of 12 hours light period / 12 hours dark period, and the light intensity is 30 μE / m ² / sec to 300 μE. / m ² / sec.

A dilution series in which the concentration of the test cyanobacteria (number of cells per unit amount) gradually decreases may be provided in each test section. According to this aspect, the evaluation at the time of selection in step (3) is facilitated. The dilution series here can be set, for example, within a concentration range of 1 × 10 ¹⁰ cells / ml to 1 × 10 ² cells / ml.

  When the culture is started, growth or growth inhibition (no growth is observed) of the test cyanobacteria occurs depending on the tolerance or sensitivity to fatty acids in the medium. Specifically, growth of the test cyanobacterium over time is observed in the test plot where the test cyanobacterium is inherently resistant to fatty acids in the medium. On the other hand, no growth of the test cyanobacterium is observed in the test plot where the test cyanobacterium is highly sensitive to fatty acids in the medium. Therefore, typically, a test group in which the growth of the test cyanobacteria is observed and a test group in which the growth of the test cyanobacteria is not observed are observed. However, as shown in the examples described later, according to experiments conducted by the present inventors, colonies were not observed in the initial stage of culture in some test sections (that is, apparently higher than fatty acids in the medium). A surprising phenomenon was observed in which colonies appeared with continued culturing. The present invention is based on knowledge that could not be predicted. That is, in the present invention, a test plot where colonies are not observed at the initial stage of culture and colonies appear is selected by continuing the culture (step (3)). In the test group, genetic mutation occurs when exposed to a fatty acid that is originally sensitive for a long time, and thereby cyanobacteria (fatty acid resistant strain) that have acquired resistance form colonies. In the present invention, the cyanobacteria (fatty acid resistant strain) that formed colonies in this way are recovered as useful, ie, fatty acid releasing strains (step (4)). In other words, the test plots where colonies were formed from the beginning of the culture and the test plots where colonies did not appear even when the culture was continued are excluded.

  When a plurality of colonies appear in the test area to be selected, cyanobacteria are collected from one or more colonies. The recovered cyanobacteria are usually subjected to further culture. Depending on the purpose, a suitable culture scale (for example, a small scale suitable for laboratory culture, a medium scale suitable for practical application testing, and a large scale suitable for industrial production) is adopted. The culture scale may be expanded step by step.

  As described above, no colonies are observed at the initial stage of culture in the selected test plot. “Initiation of culture” is from the start of culture to a specific time point. When the whole culture period is from the start of culture to the time when colonies appear in the test plot to be selected, the initial stage of culture is, for example, 1/3 or less of the total culture period (specific examples are 1/3, 1/4, 1 / 5), preferably 1/5 or less (specific examples are 1/5, 1/7, 1/10), more preferably 1/10 or less (specific examples are 1/10, 1/20, 1/30) It is. As a specific example of the initial stage of culture, from the start of culture to the first day after the start of culture (this is an example of the shortest initial stage of culture, the end point of the initial stage of culture is the first day after the start of culture), Up to the day (this is an example of the longest initial culture period, and the end point of the initial culture period is 10 days after the start of culture). The total culture period is, for example, 5 to 40 days, preferably 10 to 20 days. “Initial culture” may vary depending on the culture conditions, the type of cyanobacteria to be tested, etc., but those skilled in the art can appropriately or optimally carry out preliminary experiments with reference to the description of the present specification. “Initial culture” can be set.

  The time at which colonies appear by continuing the culture may vary depending on the culture conditions, the type of test cyanobacteria, etc., for example, from the 5th day to the 30th day after the start of the culture, preferably the 10th day after the start of the culture. The 30th day.

  The fatty acid released by the cyanobacterial fatty acid-releasing strain produced by the method of the present invention is not particularly limited. Therefore, for example, a cyanobacterial fatty acid-releasing strain releases free fatty acids contained in a selected test medium or free fatty acids contained in a non-selected test medium. Alternatively, the cyanobacterial fatty acid-releasing strain may release free fatty acids that have not been used in any of the test sections. Moreover, the cyanobacterium fatty acid releasing strain may release two or more kinds of fatty acids.

  When steps (1) to (4) are carried out, fatty acids effective for producing a cyanobacterium fatty acid releasing strain can be specified together with the production of a cyanobacterium fatty acid releasing strain. That is, useful information can be obtained in producing a cyanobacterial fatty acid-releasing strain. In another embodiment of the present invention, a cyanobacterium fatty acid-releasing strain is produced using the information. In this embodiment, the test cyanobacteria used in the above step (1) is prepared and cultured in a medium to which medium chain or long chain fatty acid contained in the medium used in the test group selected in the above step (4) is added. (Step (i)). And cyanobacteria are collect | recovered from the colony which appeared by continuing culture | cultivation (step (ii)). According to this method, a cyanobacterial fatty acid-releasing strain can be efficiently obtained. This method is also suitable for mass culture of cyanobacterial fatty acid-releasing strains.

  In yet another aspect of the present invention, a cyanobacterial fatty acid-releasing strain is obtained using the results of an experiment using a specific strain of cyanobacteria (examples described later). Specifically, Synecocystis sp. PCC 6803 strain or Synecococcus sp. PCC 7002 strain is prepared. In the case of Synecocystis sp. In the case of Synechococcus sp. PCC 7002, strain is cultured using a medium containing a long chain fatty acid C12: 0 (step (I)). And cyanobacteria are collect | recovered from the colony which appeared by continuing culture | cultivation (step (II)). According to this aspect, a fatty acid releasing strain that is resistant to long-chain fatty acid C18: 2 or C18: 3 (a mutant strain of Synecocystis sp. PCC 6803), a fatty acid-releasing strain that is resistant to long-chain fatty acid C12: 0 (Synecococcus) sp. PCC 7002 strain). That is, a fatty acid-releasing strain based on cyanobacteria that is highly useful not only academically but industrially will be provided.

In the present invention, the following steps (a) and (b) or both can be performed as additional or optional steps.
(A) The step of confirming that the recovered cyanobacteria release free fatty acids (b) The step of specifying the type and / or amount of free fatty acids released by the recovered cyanobacteria

  If step (a) is additionally performed, the target cyanobacterial fatty acid-releasing strain can be obtained more reliably. Step (a) is a qualitative or quantitative method for measuring free fatty acids in the culture solution of the cyanobacterium (the culture medium in the test section after the culture or the culture liquid after being cultured in another medium after the recovery). What is necessary is just to detect. For this detection, for example, gas chromatography, HPLC, non-ester bond type fatty acid kit (for example, NEFA C-Test Wako (trade name)) and the like can be used. On the other hand, if step (b) is additionally performed, the details of the ability to release fat will be clarified, and useful information for using and applying the recovered cyanobacteria can be obtained. When multiple cyanobacteria are collected, in order to obtain more useful cyanobacteria, the fatty acid releasing ability and / or the type of free fatty acid to be released will be compared among the collected cyanobacteria. Also good. Thin layer chromatography, HPLC, mass spectrometry (MS), etc. can be used to specify the type of free fatty acid (identification of free fatty acid).

  Based on the assumption that stable free fatty acid production would be possible if resistance to unsaturated fatty acids could be increased, we attempted to screen for mutant strains that acquired fatty acid resistance using cyanobacteria as a model. In addition, we applied a screening method using fatty acids to isolate a fatty acid-releasing strain in which a mutation was introduced into the aas gene without a marker.

1. Screening for fatty acid resistant strains Cells (Synecocystis sp. PCC 6803 strain) were suspended in 50 ml of liquid medium BG11, and grown at 30 ° C. with light irradiation of 50 μE / m ² / sec and aeration of 2% CO ₂ . This condition was defined as a normal growth condition. When 100 μL of cyanobacterial culture solution grown to a cell concentration of 10 ⁸ cells / ml in a normal growth environment was dropped on a BG11 agar medium and grown for 1 week, green colonies were observed (FIG. 5). ). As a result of testing the sensitivity to fatty acids, the sensitivity was different depending on the fatty acid species. Therefore, lethal concentration was tested beforehand. Based on the test results, medium-chain fatty acid-containing medium (100 μM lauric acid) or long-chain fatty acid-containing medium (20 μM linolenic acid) was prepared, and the culture solution was dropped to start static culture. No colonies were observed after one week of culture. In this way, the lethal effect of fatty acids was confirmed. However, when the culture was continued for another week, a phenomenon was observed in which several (about 5) cells acquired resistance and formed colonies. It was thought that the natural mutation acquired resistance to external fatty acids and formed colonies.

2. Confirmation of C18: 3 resistance of each line A colony that appeared in a fatty acid-containing medium was picked up and transplanted to a BG11 agar medium without addition of fatty acid. Subculture was continued on a fatty acid-free medium for each strain of the isolated fatty acid resistant strain. Thereafter, when transplanted to a medium supplemented with 100 μM C18: 3, growth similar to that of the medium without additive was shown (FIG. 6). Thus, it was confirmed that the fatty acid resistance was irreversibly retained.

3. Relationship between fatty acid resistance and aas gene mutation in each strain As a result of one week of liquid culture of each strain of resistant strain, a white precipitate was observed in the upper layer of the culture solution, similar to the aas mutant strain (accumulating free fatty acids) . As a result of sampling, a strain in which free fatty acids were accumulated in a culture solution at a concentration of 50 to 100 μM was found in a part of the resistant strain (FIG. 7).

  Considering the previously reported experimental result (the 77th Annual Meeting of the Botanical Society of Japan) that "aas gene mutants have acquired fatty acid tolerance", the presence of mutations at the aas locus is suggested in the genome sequence of strains that have accumulated fatty acids. Is done. Therefore, the genome sequence of the resistant strain was analyzed using a next-generation sequencer. As a result, mutations were found at the aas locus (FIGS. 8 and 9), and it was confirmed that mutations in the aas gene occurred at a high rate by screening in the presence of fatty acids. The above experimental results indicate that it is possible to isolate a strain having an aas mutation using fatty acid resistance and fatty acid release as indicators.

4). Acquisition of fatty acid resistance independent of aas mutation As a result of confirming the mutation sequence of the fatty acid resistant strain, gene mutation was found in the cilia synthesis gene (pilB gene, pilJ gene) in addition to the aas gene mutation (FIG. 10). The pilB gene mutation and the pilJ gene mutation did not affect free fatty acid accumulation in the culture medium, and further did not contribute to the acquisition of free fatty acid resistance. However, when the aas gene mutation and the pilB gene mutation were combined, resistance at a higher concentration (1 mM C18: 3) was shown. Double mutants of aas gene and pilB gene have the same genotype as fatty acid resistant strain line 1. It was suggested that fatty acid tolerance and fatty acid release are caused by a combination of two genetic mutations.

5. Isolation of fatty acid resistant strains in other cyanobacterial species and identification of the causative gene A similar experiment was carried out in the marine cyanobacterial Synechococcus sp. As a result of examining the screening conditions of Synechococcus sp. PCC 7002, it was confirmed that it was appropriate to perform in the presence of 100 μM lauric acid. As a result of screening for a fatty acid resistant strain based on these conditions, colonies were detected 2 weeks after the treatment. Each isolated line was resistant to fatty acids and was characterized by the accumulation of fatty acids in the culture medium. As a result of analysis of the mutant gene, it mainly contained the aas gene (41.2%), and in part, mutation introduction into plsX was confirmed (FIG. 9).

  From the results obtained from experiments using Synecocystis sp. PCC 6803 and experiments using Synecococcus sp. It was also suggested that the phenotype that accumulates free fatty acids is derived from aas mutations. The results obtained by applying the method for producing a fatty acid-releasing strain to Synechococcus sp. PCC 7002 strongly suggest that this method can be applied to various cyanobacteria.

<Summary>
By selecting a resistant strain on a fatty acid-added medium, a strain having a mutation in the aas gene and discharging free fatty acids was found among the resistant strains. In addition to the aas gene, additional genes contributing to “acquisition of resistance to fatty acids” and “release of fatty acids” were confirmed. This method is a screening using natural compounds and does not require genetic manipulation. Therefore, it becomes possible to construct a markerless mutant strain, which leads to reduction of genetic manipulation. In addition, application to organisms (bacteria) in which genetic manipulation is impossible is expected.

  According to the present invention, a cyanobacteria that releases free fatty acids (a cyanobacterial fatty acid-releasing strain) can be produced easily and efficiently. Free fatty acids, for example, serve as precursors for biofuels and are highly industrially useful. The present invention can be an important basic technology related to biofuel (biodiesel), which is expected to expand in future demand. Free fatty acids are also used as raw materials for ester compounds. Therefore, the present invention includes wax (candles, surface brighteners, coating agents), additives (for food, paints, pharmaceuticals, cosmetics, etc.), lubricants (processing processes for metals, rubbers, plastics, fibers, etc.), Use or application to plasticizers, softeners, emulsifiers, etc. is also possible.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (14)

A method for producing a cyanobacterial fatty acid-releasing strain comprising the following steps (1) to (4):
(1) A plurality of test sections for culturing the test cyanobacteria using medium-chain or long-chain fatty acid-containing medium, wherein the types of medium-chain or long-chain fatty acids contained in the medium are different in the test sections. Preparing a test zone;
(2) adding a test cyanobacteria to the medium and culturing in the plurality of test sections;
(3) a step of selecting a test plot in which colonies are not observed at the initial stage of culture and colonies appear by continuing the culture;
(4) A step of recovering cyanobacteria from the colonies of the selected test section.   The production method according to claim 1, wherein the medium-chain or long-chain fatty acid has 10 to 18 carbon atoms.   The production method according to claim 1 or 2, wherein the medium-chain or long-chain fatty acid contained in the medium of one test section is one kind.   The production method according to any one of claims 1 to 3, wherein a fatty acid concentration in the medium-chain or long-chain fatty acid-containing medium is 50 µM to 200 µM.   The production method according to any one of claims 1 to 4, wherein the culture temperature is 25 ° C to 35 ° C.   The production method according to any one of claims 1 to 5, wherein the end point of the initial stage of culture in the step (3) is between the first day after the start of the culture and the tenth day after the start of the culture.   The production method according to any one of claims 1 to 6, wherein a time when a colony appears in the step (3) is from the 5th day after the start of the culture to the 30th day after the start of the culture.   The production method according to any one of claims 1 to 7, wherein each of the plurality of test sections includes a dilution series in which the concentration of the test cyanobacteria decreases stepwise.   The production method according to any one of claims 1 to 8, wherein the medium is a solid medium.   The production method according to any one of claims 1 to 9, wherein the test cyanobacterium is Synechocystis cyanobacterium, Synecococcus genus cyanobacterium, Microcystis genus cyanobacterium, or Arthrospira genus cyanobacterium.   The production method according to claim 10, wherein the Synechocystis cyanobacterium is Synechocystis sp. PCC 6803 strain, and the Synechococcus cyanobacterium is Synechococcus sp. PCC 7002 strain. A method for producing a cyanobacterial fatty acid-releasing strain comprising the following steps (i) and (ii):
(I) The test cyanobacterium used in the production method according to any one of claims 1 to 11 is prepared, and the medium chain contained in the medium used in the test section selected in step (4) of claim 1 or Culturing in a medium supplemented with long chain fatty acids;
(Ii) A step of collecting cyanobacteria from the emerged colonies. A method for producing a cyanobacterial fatty acid-releasing strain comprising the following steps (I) and (II):
(I) Synecocystis sp. PCC 6803 strain or Synecococcus sp. PCC 7002 strain is prepared. In the case of Synecocystis sp. PCC 6803 strain, a medium containing long chain fatty acid C18: 2 or C18: 3 or both of them is used. And in the case of Synechococcus sp. PCC 7002 strain, culturing using a medium containing a long chain fatty acid C12: 0;
(II) A step of collecting cyanobacteria from the appearing colonies. The production method according to any one of claims 1 to 13, further comprising the following step (a) and / or step (b):
(A) confirming that the recovered cyanobacteria release free fatty acids;
(B) identifying the type and / or amount of free fatty acids released by the recovered cyanobacteria.

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