Screening method for filamentous fungi and screening method for culture conditions of filamentous fungi

Abstract

To provide a technique for efficient screening filamentous fungi or culture conditions for filamentous fungi.SOLUTION: The present invention provides a screening method for filamentous fungi or culture conditions for filamentous fungi, the method comprising: a culture step of culturing filamentous fungi; an observation image acquisition step of acquiring an observation image by observing the cultured filamentous fungi under a microscope at a predetermined time point or for a predetermined period in the culture step; an observation value acquisition step of extracting morphological features of the filamentous fungi in the observation image and acquiring an observation value based on the morphological features; an observation value evaluation step of evaluating whether or not the observation value meets a predetermined threshold; and an identification step of identifying the filamentous fungi or the culture conditions thereof that meet the predetermined threshold in the evaluation step.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for screening filamentous fungi and a method for screening culture conditions for filamentous fungi. [Background technology]

[0002] Conventionally, filamentous fungi are known as microorganisms that are used in fermented foods such as miso, soy sauce, and sake, and in the industrial production of useful enzymes, antibiotics, organic acids, and alcohol. In recent years, with the development of biotechnology, attempts have been made to utilize strains isolated through breeding and strains constructed by gene recombination and editing techniques. For example, according to Patent Document 1, for M. oryzae and M. sojae, which are known as soy sauce koji starters, the hydrolysis activity is improved in modified strains of the rseA gene, and the use of this is said to contribute to the efficient production of fermented foods. In addition, according to Patent Document 2, in Trichoderma filamentous fungi that can be used as a microorganism for producing cellulase-based biomass decomposition enzymes, the Zn(II) of ACE3, which is known as a positive transcription factor involved in cellulase induction expression, has been reported. 2 Cys 6 It is believed that by constitutively expressing a mutant in which the DNA-binding domain (DBD) is completely deleted, high production of cellulase and the like can be achieved without using expensive inducers. In addition, according to Patent Document 3, it is believed that highly stable production of exogenous proteins can be achieved by reducing the expression or activity of KEX2 and / or ALP7 protease in a genetically modified Ascomycota filamentous fungus. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2016-136843 A [Patent Document 2] JP 2022-161526 A [Patent Document 3] Special Publication No. 2023-525833 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the method of differentiation using enzyme activity or substance production as an indicator has a problem in that it takes time and effort to measure the activity or production amount.

[0005] Therefore, an object of the present invention is to provide a technique for more efficiently screening filamentous fungi and their culture conditions. [Means for solving the problem]

[0006] As a result of intensive research into achieving the above object, the present inventors came up with the idea of ​​conducting screening that makes use of the external characteristics of filamentous fungi, and thus completed the present invention.

[0007] That is, in a first aspect, the present invention provides a pre-screening step of preparing a filamentous fungus to be screened; A culturing step of culturing the filamentous fungus; an image acquisition step of observing the cultured filamentous fungus under a microscope at a predetermined time or for a predetermined period in the culture step to obtain an image of the cultured filamentous fungus; an observation value acquisition step of extracting morphological characteristics of the filamentous fungus in the observation image and obtaining an observation value based on the morphological characteristics; evaluating the observed value to determine whether the observed value satisfies a predetermined threshold; and an identification step of identifying the filamentous fungus that satisfies a predetermined threshold value in the evaluation step from the screening target prepared in the pre-screening step. A method for screening filamentous fungi is provided.

[0008] According to the method of the first aspect of the present invention, the morphological characteristics of a filamentous fungus are observed under a microscope as it grows, and the filamentous fungus of interest can be easily selected because the filamentous fungus is cultured and differentiated based on the observed image. In addition, because the intended screening target is differentiated under certain culture conditions, the filamentous fungus that meet the requirements for industrial production can be accurately selected.

[0009] In the screening method according to the first aspect of the present invention, the morphological characteristic may be hyphal width or hyphal volume, which allows more accurate acquisition of an observation value based on the morphological characteristics in an observation image obtained by observation under a microscope.

[0010] In the screening method according to the first aspect of the present invention, the morphological characteristic may be the number of cell nuclei present in a predetermined area of ​​the hyphae, which allows more accurate acquisition of observation values ​​based on the morphological characteristics in an observation image obtained by observation under a microscope.

[0011] In the screening method according to the first aspect of the present invention, the threshold value may be a threshold value that correlates with the production capacity of a predetermined substance, which allows more accurate selection of filamentous fungi that meet the requirements for industrial production, etc.

[0012] On the other hand, in a second aspect, the present invention provides A pre-screening step of preparing culture conditions for the filamentous fungus to be screened; A culturing step of culturing a filamentous fungus under the culture conditions for the filamentous fungus; an image acquisition step of observing the cultured filamentous fungus under a microscope at a predetermined time or for a predetermined period in the culture step to obtain an image of the cultured filamentous fungus; an observation value acquisition step of extracting morphological characteristics of the filamentous fungus in the observation image and obtaining an observation value based on the morphological characteristics; evaluating the observed value to determine whether the observed value satisfies a predetermined threshold; A process for identifying a condition that satisfies a predetermined threshold value in the evaluation process from the screening target prepared in the pre-screening process. A method for screening culture conditions for filamentous fungi is provided.

[0013] According to the method of the second aspect of the present invention, the morphological characteristics of a filamentous fungus as it grows are observed under a microscope and differentiation is performed based on the observed images, so that the culture conditions for the desired filamentous fungus can be easily selected. Moreover, because differentiation is performed under fixed culture conditions for the intended screening target, it is possible to accurately select culture conditions for a filamentous fungus that meet the requirements for industrial production, etc.

[0014] In the screening method according to the second aspect of the present invention, the morphological characteristic may be hyphal width or hyphal volume, which allows more accurate acquisition of observation values ​​based on morphological characteristics in images obtained by observation under a microscope.

[0015] In the screening method according to the second aspect of the present invention, the morphological characteristic may be the number of cell nuclei present per predetermined area of ​​the hyphae, which allows more accurate acquisition of observation values ​​based on the morphological characteristics in images obtained by observation under a microscope.

[0016] In the screening method according to the second aspect of the present invention, the threshold value may be a threshold value that is correlated with the production amount of a predetermined substance, thereby enabling more accurate selection of culture conditions that meet the requirements for industrial production, etc. Effect of the Invention

[0017] According to the present invention, a filamentous fungus is cultured, and its morphological characteristics are observed under a microscope as it grows, and differentiation is performed based on the observed images, so that the target filamentous fungus or its culture conditions can be easily selected. In addition, because differentiation is performed under certain culture conditions for the intended screening target, it is possible to accurately select a filamentous fungus or its culture conditions that meet the requirements for industrial production, etc. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a process diagram showing one embodiment of the method for screening filamentous fungi according to the present invention. [Diagram 2] FIG. 1 is a process diagram showing one embodiment of a method for screening culture conditions for filamentous fungi according to the present invention. [Diagram 3] FIG. 3 is an explanatory diagram illustrating a method for measuring the hypha width and number of cell nuclei of a filamentous fungus from images obtained by microscopic observation. FIG. 3(a) shows an example of a fluorescent field image of a cell nucleus obtained by microscopic observation (a strain in which green fluorescent protein GFP has been added to histone), FIG. 3(b) shows a bright field image of the image shown in FIG. 3(a), FIG. 3(c) shows another example of a fluorescent field image of a nucleus obtained by microscopic observation, FIG. 3(d) shows a bright field image of the image shown in FIG. 3(c), and FIG. 3(e) is an explanatory diagram showing an example of an increase in cell nuclei within a hypha. [Figure 4] 1 is a chart showing an example of an image observed under a fluorescence microscope when each filamentous fungus was cultured on a plate medium of a minimal medium in Test Example 1. [Diagram 5] FIG. 1 is a diagram showing an example of an image observed by a fluorescence microscope when Aspergillus oryzae RIB915 was cultured in a minimal medium or a minimal medium supplemented with 1% yeast extract in Test Example 2. [Figure 6]FIG. 6 is a graph showing the results of measuring the number of cell nuclei within a hyphal length range of 100 μm from the hyphal tip in Test Example 3 based on the observation images obtained in Test Examples 1 and 2, and the results of measuring α-amylase activity for each filamentous fungus used in Test Examples 1 and 2. FIG. 6(a) is a graph in which the type of each filamentous fungus is plotted on the X-axis and the number of cell nuclei and α-amylase activity are plotted on both Y-axes, and FIG. 6(b) is a graph in which α-amylase activity is plotted on the X-axis and the number of cell nuclei is plotted on the Y-axis. [Figure 7] In Test Example 4, a graph is shown which summarizes the results of measuring the hyphal width at a point 100 μm from the hyphal tip based on the observation images obtained in Test Examples 1 and 2, and the results of measuring the α-amylase activity of each filamentous fungus used in Test Examples 1 and 2, with the α-amylase activity plotted on the X-axis and the hyphal width plotted on the Y-axis. [Figure 8] 1 is a chart showing an example of an image observed by a fluorescence microscope when Aspergillus oryzae RIB915 was cultured in a minimal medium containing various substances in Test Example 5. [Figure 9] FIG. 13 is a diagram showing an example of an image observed by a fluorescence microscope when Aspergillus oryzae RIB915 was cultured in a minimal medium containing various substances in Test Example 6. [Figure 10]FIG. 10 is a table showing examples of images observed by a fluorescence microscope for Trichoderma reesei and Penicillium chrysogenum when cultured in minimal medium or minimal medium supplemented with 1% yeast extract in Test Example 7, where FIG. 10(a) is an example of an image observed for Trichoderma reesei, FIG. 10(b) is an example of an image observed for Penicillium chrysogenum, and FIG. 10(c) is a graph summarizing the results of measuring the number of cell nuclei within a hyphal length range of 100 μm from the tip of the hyphal for each filamentous fungus based on the images observed by a fluorescence microscope. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The filamentous fungi that are the subject of the method according to the present invention are not particularly limited in genus and species, and include, for example, filamentous fungi belonging to the genera Aspergillus, Trichoderma, Penicillium, Neurospora, Rasamsonia, Fusarium, Chrysosporium, Humicola, Hypocrea, Acremonium, Chrysosporium, Corynascus, Myceliophthora, Piromyces, Talaromyces, Thermoascus, and Thielavia. Among these, the filamentous fungi may be, but are not limited to, filamentous fungi selected from the group consisting of the genus Aspergillus, the genus Trichoderma, and the genus Penicillium. Examples of the filamentous fungi belonging to the genus Aspergillus include Aspergillus oryzae, Aspergillus nidualns, Aspergillus flavus, Aspergillus sojae, Aspergillus kawachii, and Aspergillus luchuensis. Examples of filamentous fungi belonging to the genus Trichoderma include Trichoderma reesei and Trichoderma vuride.Examples of filamentous fungi belonging to the genus Penicillium include Penicillium chrysogenum, Penicillium gaxaucum, and Penicillium griseofluvum.

[0020] Many of the above-mentioned filamentous fungi are known to be useful for the industrial production of useful enzymes such as amylase, glucoamylase, cellulase, endoglucanase, xylanase, pectinase, acid protease, neutral protease, alkaline protease, lipase, phytase, catalase, lactase, etc., antibiotics such as penicillin, cephalosporin, griseofulvin, etc., organic acids, alcohols, etc. The screening method according to the present invention can be suitably used as a method for screening filamentous fungi or culture conditions for filamentous fungi suitable for the production of an arbitrarily selected predetermined substance.

[0021] Generally, filamentous fungi grow while extending their hyphae. In other words, in terms of their life cycle, when spores (conidia) of filamentous fungi are placed under appropriate conditions, hyphae germinate, and the hyphae continue to grow by extending. During the process of hyphae growth, depending on the timing, a site where the hyphae branch out from the tip side facing the extension direction or a site where the hyphae branch out from the side side of the formed hyphae is generated, and new hyphae are generated from the branched sites, and the hyphae extend from each of them. In addition, as the hyphae extend, the cell cycle progresses and nuclear division is repeated, and the inside of the hyphae becomes multinucleated, and at a certain timing, partitions are formed to separate the inside of the hyphae. Therefore, in one aspect, filamentous fungi can be said to be multicellular organisms that grow in a multicellular form in which tubular, elongated cells are connected and separated by partitions.

[0022] The present invention relates to a method for screening filamentous fungi or culture conditions for filamentous fungi by observing the filamentous fungi in the process of growth under a microscope and making an evaluation based on the observed images.

[0023] [1. Screening method for filamentous fungi] One embodiment of the screening method for filamentous fungi according to the present invention is shown in Figure 1. Specifically, this embodiment of the screening method can be composed of a pre-screening step (S1), a culturing step (S2), an observation image acquisition step (S3), an observation value acquisition step (S4), an observation value evaluation step (S5), and an identification step (S6), which are described below.

[0024] (Pre-screening process) This step is a step of preparing the filamentous fungus to be screened. This allows the filamentous fungus to be screened in the following steps to be set to be from the target range. Then, filamentous fungus that satisfy a predetermined threshold can be selected from the target filamentous fungus. Here, the filamentous fungus to be screened may be a random cell population of filamentous fungi or an individual strain. It may also be a cell population or an individual strain of filamentous fungi after treatment with a mutagen, or it may be a modified form in which a foreign gene has been introduced into a random cell population or a specific strain of filamentous fungus. Furthermore, a strain having typical properties of filamentous fungi belonging to the genus or species, such as a typical strain, may be obtained and used as the screening target as it is or after modification such as treatment with the above-mentioned mutagen. In addition, filamentous fungi belonging to the same genus or species may be used as the screening target, or filamentous fungi belonging to different genuses and species may be used as the screening target.

[0025] (Culture process) This step is a step of culturing filamentous fungi. This allows the filamentous fungi to be in a state where they can grow, and in particular allows the state of mycelial growth to be observed. The culture conditions for this can be appropriately selected according to the type of filamentous fungi to be screened and the conditions in accordance with the desired production requirements. Examples include minimal medium and PDA medium with the compositions shown below. Commercially available products for PDA medium include "Potato Dextrose Broth 254920" (BD Difco).

[0026] Minimal medium: Glucose 10g NaNO 3 6g 0.52g KCl KH 2 PO 4 1.52g MgSO 4 7H 2 O 0.52g Trace element 2mL The above can be made up to 1 L with deionized water. In this case, the pH may be adjusted with a dilute hydrochloric acid solution, typically to pH 6.5.

[0027] (Trace element composition) ZnSO 4 H 2 O 2.2g H 3 BO 3 1.1g MnCl 2 4H 2 O 0.5g FeSO 4 7H 2 O 0.5g CoCl 2 6H 2 O 0.16g (NH 4 )Mo 7 O 2 4H 2 O 0.11g The above can be made up to 100mL with deionized water.

[0028] PDA medium: Potato dextrose broth 15g 15g agar The above can be made up to 1 L with deionized water. In this case, the pH may be adjusted with a dilute hydrochloric acid solution, typically to pH 6.5.

[0029] The culture may be performed by inoculating the culture medium into a liquid medium and culturing it under conditions of, for example, 25°C to 37°C, by stirring or static culture, or by preparing a plate medium by adding agar and spreading the culture medium on the plate and culturing it under similar temperature conditions.

[0030] (Observation image acquisition process) This step is a step of observing the cultured filamentous fungus under a microscope at a predetermined time or for a predetermined period in the culture step, and obtaining an observation image. The obtained observation image can then be subjected to a subsequent step of obtaining an observation value. In particular, but not limited to, for example, the obtained observation image can be subjected to image processing and analysis, thereby extracting morphological characteristics of the filamentous fungus during its growth process and obtaining an observation value based on the extracted morphological characteristics. The time and period of observation can be set, for example, after inoculating a spore suspension onto an agar medium with a sterilized toothpick and initiating culture in an incubator (for example, maintaining a constant temperature at any temperature condition in the range of 25°C to 37°C), at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Alternatively, a period of any of these hours or days may be set. Examples of microscopes for observation include optical microscopes, phase contrast microscopes, stereo microscopes, fluorescent microscopes, and confocal fluorescent microscopes. However, the microscope is not limited to these, and any microscope can be used as long as it can extract the morphological characteristics of filamentous fungi and obtain observation values ​​based on the extracted characteristics. For fluorescent observation, a part of the agar medium used for the culture may be cut out and immersed in a fluorescent reagent solution for staining, or a cover glass covering the sample may be wetted with the fluorescent reagent solution before microscopic observation. Examples of staining reagents for cell nuclei include SYBR Green, DAPI, Hoechst 33258, Hoechst 33342, and Hoechst 34580.

[0031] On the other hand, the culture and observation of filamentous fungi can also be performed by liquid culture using, for example, a 96-well plate container. In this case, the above-mentioned observation image can be obtained by filling each well with an appropriate liquid medium, inoculating a spore suspension of the filamentous fungus, and observing under a microscope after a predetermined period of time has passed or for a predetermined period of time. In addition, when observing by fluorescence, a fluorescent reagent solution can be added to an appropriate concentration before observation. According to such an embodiment, it is possible to perform high-throughput processing of the culture and observation of multiple different samples at once.

[0032] (Observation value acquisition process) This step is a step of extracting the morphological characteristics of the filamentous fungus in the obtained observation image, and obtaining an observation value based on the morphological characteristics. The obtained observation value can be used in the subsequent step of evaluating the observation value. The obtained observation value may be any value, and may in particular be, for example, a characteristic of the hyphal width or hyphal volume. It may also be the number of cell nuclei present in a given area of ​​the hyphal. As shown in the test example described below, the observation value based on these morphological characteristics is correlated with the production ability of useful enzymes by the filamentous fungus. That is, for example, the longer the hyphal width, the higher the production ability. Also, the more the number of cell nuclei, the higher the production ability. The observation value may be any numerical value, data, etc. that can be evaluated in the subsequent step by comparing it with a predetermined threshold value, and may be, for example, statistically processed data such as an average or distribution over multiple samples of the same sample or over a given period of time, or a data set that can be statistically processed.

[0033] In any non-limiting embodiment of the present invention, the observation value based on the morphological characteristics used as an index as described above is preferably the hyphal width or hyphal volume at a point from the tip side of the growing hyphae that has grown sufficiently from the tip side, for example, arbitrarily determined within a range of 80 to 150 μm, more typically, for example, at a point 100 μm from the tip side. Alternatively, it is preferably the hyphal width or hyphal volume at the point of the partition that is located first from the tip side of the growing hyphae. Also, it is preferably the number of cell nuclei located in the intrahyphal region surrounded by the hyphal length from the tip side that is sufficiently grown from the tip side of the growing hyphae, for example, arbitrarily determined within a range of 80 to 150 μm, more typically, for example, the number of cell nuclei located in the intrahyphal region surrounded by the hyphal length 100 μm from the tip side. Alternatively, it is preferably the number of cell nuclei located in the intrahyphal region surrounded by the partition that is located first from the tip side of the growing hyphae.

[0034] The above-mentioned observed values ​​of hyphal width or hyphal volume, number of cell nuclei, etc. are not limited, but can be obtained, for example, by measuring an image observed under a microscope after image processing using image processing software well known to those skilled in the art. In this case, the hyphal volume can be obtained from distance data of the focal position in the depth direction by image analysis when a confocal microscope or the like is used, or the cylindrical volume can be calculated by assuming that the shape of the hyphae is a cylinder with the hypha width as the diameter and the hypha length from the tip as the height.

[0035] (Evaluation process of observed values) This step is a step of evaluating whether the observed value satisfies a predetermined threshold value. For example, when the hypha width is used as an index, the threshold value may be, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, etc., without being limited thereto. That is, when the observed value exceeds the threshold value or is equal to or greater than the threshold value, the threshold value may be evaluated as being satisfied. Furthermore, for example, when the number of cell nuclei present per given region of hyphae is used as an index, the threshold value may be, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, etc. In other words, when the number exceeds the threshold value or is equal to or greater than the threshold value, the threshold value may be evaluated as being satisfied.

[0036] More specifically, for example, in Aspergillus oryzae, the hyphal width at the site of the first septum located from the tip side of the growing hyphae in typical strains is about 2 to 5 μm, so the threshold value is preferably 7 μm, more preferably 8 μm, even more preferably 9 μm, and particularly preferably 10 μm. In this case, the threshold value is preferably a value obtained by adding a certain width to the standard hyphal width in typical strains, for example, a value obtained by adding 2 μm, preferably adding 3 μm, preferably adding 4 μm, and most preferably adding 5 μm. In Aspergillus oryzae, in typical strains, the number of cell nuclei present in the intrahyphal region surrounded by a septum located first from the tip of the growing hyphae is about 10 to 20, so the threshold is preferably set to 50, more preferably to 100, even more preferably to 150, and particularly preferably to 200. In this case, the threshold is preferably set to a value obtained by adding a predetermined number to the standard number of cell nuclei in typical strains, for example, a value obtained by adding 30, preferably adding 80, preferably adding 130, and most preferably adding 180.

[0037] For example, in Trichoderma reesei, the hyphal width at the site of the first septum from the tip of the growing hyphae in typical strains is about 3 to 5 μm, so the threshold is preferably 7 μm, more preferably 8 μm, even more preferably 9 μm, and particularly preferably 10 μm. In this case, the threshold is preferably a value obtained by adding a certain width to the standard hyphal width in typical strains, for example, a value obtained by adding 2 μm, preferably adding 3 μm, preferably adding 4 μm, and most preferably adding 5 μm. In Trichoderma reesei, in typical strains, the number of cell nuclei present in the intrahyphal region surrounded by a septum located first from the tip of the growing hyphae is about 40, so the threshold is preferably set to 100, more preferably to 150, even more preferably to 200, and particularly preferably to 300. In this case, the threshold is preferably set to a value obtained by adding a predetermined number to the standard number of cell nuclei in typical strains, for example, by adding 60, preferably adding 110, preferably adding 160, and most preferably adding 260.

[0038] For example, in Penicillium chrysogenum, the hyphal width at the site of the first septum located from the tip of the growing hyphae in typical strains is about 2 to 4 μm, so the threshold value is preferably 5 μm, more preferably 6 μm, even more preferably 7 μm, and particularly more preferably 8 μm. In this case, the threshold value is preferably a value obtained by adding a certain width to the standard hyphal width in typical strains, for example, a value obtained by adding 1 μm, preferably adding 2 μm, preferably adding 3 μm, and most preferably adding 4 μm. In Penicillium chrysogenum, the number of cell nuclei present in the intrahyphal region surrounded by a septum located first from the tip of the growing hyphae in typical strains is about 20, so the threshold is preferably set to 50, more preferably to 100, even more preferably to 150, and particularly preferably to 200. In this case, the threshold is preferably set to a value obtained by adding a predetermined number to the standard number of cell nuclei in typical strains, for example, a value obtained by adding 30, preferably adding 80, preferably adding 130, and most preferably adding 180.

[0039] The preferred value of the mycelium volume may be a value obtained by assuming the shape of the growing mycelium from the tip to a point 100 μm away as a cylinder and calculating the value of the preferred range of mycelium width as the diameter. When evaluating whether or not a predetermined threshold is satisfied as described above, the predetermined threshold value may be any value or data that can be evaluated, and may be, for example, statistically processed data such as averages or distributions over multiple samples of the same sample or over a predetermined period of time, or a data set that can be statistically processed.

[0040] (Specific process) This step is a step of identifying filamentous fungi that satisfy a predetermined threshold value in the evaluation step, thereby enabling accurate selection of filamentous fungi that meet the requirements for industrial production, etc., from among the intended screening targets.

[0041] [2. Screening method for culture conditions for filamentous fungi] One embodiment of the screening method for culture conditions for filamentous fungi according to the present invention is shown in Figure 2. Specifically, this embodiment of the screening method can be composed of a pre-screening step (S11), a culturing step (S12), an observation image acquisition step (S13), an observation value acquisition step (S14), an observation value evaluation step (S15), and an identification step (S16), which are described below.

[0042] (Pre-screening process) This method can be carried out in the same manner as described above in [1. Screening method for filamentous fungi]. However, instead of filamentous fungi as the subject of screening, the culture conditions are set. That is, for example, various components of the culture medium can be added to the medium, or not added, or the concentrations of the components can be changed, or conditions such as the culture temperature, culture cycle, and culture atmosphere can be changed, and a certain filamentous fungus can be subjected to the subsequent steps and screened in the same manner.

[0043] (Culture process) The method can be carried out in the same manner as described above in [1. Screening method for filamentous fungi]. That is, apart from the fact that the subject of screening is culture conditions set arbitrarily, appropriate preferred aspects such as the setting of the culture period can be applied.

[0044] (Observation image acquisition process) The method can be carried out in the same manner as described above in [1. Screening method for filamentous fungi]. In other words, apart from the fact that the subject of screening is culture conditions set arbitrarily, appropriate preferred modes, such as the form of obtaining observation images under a microscope, can be applied.

[0045] (Observation value acquisition process) The method can be carried out in the same manner as described above in [1. Screening method for filamentous fungi]. In other words, apart from the fact that the subject of screening is culture conditions set arbitrarily, suitable preferred aspects such as extraction of morphological characteristics of filamentous fungi can be applied.

[0046] (Evaluation process of observed values) The method can be carried out in the same manner as described above in [1. Screening method for filamentous fungi]. That is, apart from the fact that the subject of screening is a culture condition set arbitrarily, appropriate preferred aspects such as the setting of evaluation criteria can be applied.

[0047] (Specific process) This method can be carried out in the same manner as described in [1. Screening method for filamentous fungi] above. However, instead of a filamentous fungus being the subject of screening, its culture conditions are arbitrarily set, and a certain filamentous fungus is subjected to the culture conditions, and the growth process is observed under a microscope, and the above evaluation is carried out based on the observed image. This can be suitably used for the purpose of identifying a medium suitable for the production of a specific substance such as a useful enzyme, but is not limited thereto.

[0048] The method according to the present invention can also be constituted by any combination of the various aspects described above. EXAMPLES

[0049] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0050] 1. Materials and Methods <1> Filamentous fungi used in the test Aspergillus oryzae RIB40 Aspergillus oryzae RIB128 Aspergillus oryzae RIB915 Aspergillus flavus Trichoderma reesei Penicillium chrysogenum

[0051] <2> Morphological observation <2.1> Preparation of agar medium (composition) Glucose 10g NaNO 3 6g 0.52g KCl KH 2 PO 4 1.52g MgSO 4 7H 2 O 0.52g Trace element 2mL Yeast extract (*add as needed) 10g The above was dissolved in deionized water, adjusted to pH 6.5, and then diluted to 1 L. Agar was added to this to a final concentration of 1.5% by mass, autoclaved, and dispensed into culture dishes to prepare agar medium. When yeast extract was added, 10 g was added before adjusting the pH.

[0052] (Trace element composition) ZnSO 4 H 2 O 2.2g H 3 BO 3 1.1g MnCl 2 4H 2 O 0.5g FeSO 4 7H 2 O 0.5g CoCl 2 6H 2 O 0.16g (NH 4 )Mo 7 O 2 4H 2O 0.11g The above was dissolved in deionized water and made up to 100 mL to prepare the trace element.

[0053] <2.2> Culture The spore suspension was inoculated onto the agar medium using a sterilized toothpick and cultured at 30°C. Spore suspension: After growth on PDA medium until spores were formed, the spores were suspended using a suspension solution (10% glycerol, 0.9% NaCl) and stored at -80°C.

[0054] <2.3> Microscopic observation The periphery of the colony formed on the agar medium was cut out together with the agar using a microspatula, and this was placed on a cover glass to which 1 μL of a fluorescent reagent solution (a 100-fold dilution of Molecular Probes' "SYBR Green I × 10,000") had been added, and observed using an inverted fluorescent microscope (Axio Observer.Z1, Carl Zeiss). The objective lens (oil immersion) was used at 10x, 40x, or 63x magnification as required. The cell nuclei were observed by exciting the fluorescence of SYBR Green at 488 nm.

[0055] <2.4> Image analysis Images acquired by the fluorescence microscope were processed using image processing software (ZEN Software Version 3.5, Carl Zeiss) and ImageJ (freeware), and noise was removed using filters such as the Despeckle node. After that, measurements were made of the hypha width and the number of cell nuclei.

[0056] <2.5> Obtaining observations FIG. 3 explains a method for measuring the hypha width and the number of cell nuclei of a filamentous fungus from an acquired image.

[0057] FIG. 3(a) shows an example of a fluorescent field image of a cell nucleus obtained by microscopic observation. In this image range, 26 fluorescent dots are observed, which indicates that there are 26 cell nuclei. Meanwhile, FIG. 3(b) is a bright field image of the image shown in FIG. 3(a). In this bright field image, a septum is observed at the location indicated by the arrow in the figure, and the location of the first septum from the tip of the hyphae can be identified. Therefore, it is understood that the number of cell nuclei located in the intrahyphal region surrounded by the first septum from the tip of the hyphae is 22. It is also understood that the hypha width at the first septum from the tip of the hyphae can be measured from the acquired image.

[0058] FIG. 3(c) shows another example of a fluorescent field image of a cell nucleus obtained by microscopic observation. More than 200 dots of fluorescent color are observed in this image range, which indicates that more than 200 cell nuclei are present. Meanwhile, FIG. 3(d) is a bright-field image of the image shown in FIG. 3(c). In this bright-field image, a septum is observed at the location indicated by the arrow in the figure, and the location of the first septum from the tip of the hyphae can be identified. Therefore, it can be seen that the number of cell nuclei present in the intrahyphal region surrounded by the first septum from the tip of the hyphae is at least 200 or more. It can also be seen that the hypha width at the first septum from the tip of the hyphae can be measured from the acquired image.

[0059] Figure 3(e) shows a schematic diagram of an increase in cell nuclei within a hypha. As shown in this schematic diagram, when filamentous fungi are generally cultured, cell nuclei increase in number and accumulate within the cells as the hyphae grow. When the increase in cell nuclei is significant, the cell nuclei may be observed overlapping in two-dimensional observations, but it is possible to accurately measure the number of cell nuclei by three-dimensional analysis such as Z-stack observation with a confocal microscope.

[0060] <3> Enzyme activity measurement <3.1> Preparation of liquid medium (composition) Glucose 10g NaNO 3 6g 0.52g KCl KH 2 PO 4 1.52g MgSO 4 7H 2 O 0.52g Trace element 2mL Yeast extract (**add as needed) 10g The above was dissolved in deionized water, adjusted to pH 6.5, and then made up to 1 L and autoclaved. When yeast extract was added, 10 g was added before adjusting the pH.

[0061] (Trace element composition) ZnSO 4 H 2 O 2.2g H 3 BO 3 1.1g MnCl 2 4H 2 O 0.5g FeSO 4 7H 2 O 0.5g CoCl 2 6H 2 O 0.16g (NH 4 )Mo 7 O 2 4H 2 O 0.11g The above was dissolved in deionized water and made up to 100 mL to prepare the trace element.

[0062] <3.2> Culture The spore suspension was inoculated into 200 mL of medium (500 mL baffled flask) containing a carbon source suitable for each filamentous fungus, and cultured at 30°C and 120 to 150 rpm for 3 to 5 days. Spore suspension: After growth on PDA medium until spores were formed, the spores were suspended using a suspension solution (10% glycerol, 0.9% NaCl) and stored at -80°C.

[0063] <3.3> Measurement The culture medium was centrifuged to recover the supernatant, and each measurement was carried out.

[0064] A) Protein concentration measurement Protein quantification was performed using Bradford reagent (BIO-RAD Protein Assay Kit, BIO-RAD). Protein concentration was calculated using a calibration curve prepared using bovine serum albumin (BSA) as a standard.

[0065] B) SDS-PAGE Electrophoresis was performed on a 12.5% ​​polyacrylamide gel and stained with Coomassie brilliant blue R250.

[0066] C) α-amylase The measurement method of the α-amylase measurement kit (Kikkoman Biochemifa Corporation) was followed. The amount of enzyme that liberates 1 μmol of CNP from the synthetic substrate N3-G5-β-CNP per minute of reaction was defined as 1 U.

[0067] 2. Tests and Results [Test Example 1] Figure 4 shows examples of images (fluorescent field images of cell nuclei) observed by a fluorescence microscope for Aspergillus oryzae RIB40, Aspergillus oryzae RIB128, Aspergillus oryzae RIB915, and Aspergillus flavus when each filamentous fungus was cultured on a plate of minimal medium for three days. The figure also shows the outline of the mycelium observed in the bright field image of the same observation. In addition, the right column of the fluorescence microscope image for each filamentous fungus shows an example of a section cut out of the agar medium as a sample for observation by the fluorescence microscope.

[0068] As shown in Figure 4, when each filamentous fungus was cultured under the same conditions, the number of cell nuclei located in a given area of ​​the hyphae differed between the filamentous fungi. In this way, the tendency of the number of cell nuclei located in a given area of ​​the hyphae was similar among the same filamentous fungi, that is, it was recognized that the tendency of the number of cell nuclei is an inherent property of the type or strain of filamentous fungus.

[0069] [Test Example 2] Figure 5 shows an example of an image observed by a fluorescence microscope for Aspergillus oryzae RIB915 when it was cultured for three days on a plate medium of minimal medium or minimal medium supplemented with 1% yeast extract.

[0070] As shown in Figure 5, when Aspergillus oryzae RIB915 was cultured in minimal medium supplemented with 1% yeast extract, the number of cell nuclei present per given area of ​​the mycelium was increased compared to when it was cultured in minimal medium.

[0071] [Test Example 3] FIG. 6 shows the results of counting the number of cell nuclei within a range of 100 μm of hyphal length from the tip of the hyphal based on the observation images obtained in Test Example 1 and Test Example 2, and the results of the above-mentioned " <3> The graphs shown in Fig. 6 summarize the results of measuring α-amylase activity using the "Enzyme Activity Measurement" method. Fig. 6(a) is a graph in which the type of filamentous fungus is plotted on the X-axis and the number of cell nuclei and α-amylase activity are plotted on both Y-axes, and Fig. 6(b) is a graph in which α-amylase activity is plotted on the X-axis and the number of cell nuclei is plotted on the Y-axis.

[0072] As shown in FIG. 6, the number of cell nuclei present in a given area of ​​a hypha, which is a morphological characteristic of a filamentous fungus, correlated well with the activity of α-amylase secreted by the filamentous fungus.

[0073] [Test Example 4] FIG. 7 shows the results of the hyphal width measured at a point 100 μm from the hyphal tip based on the observation images obtained in Test Example 1 and Test Example 2, and the results of the above-mentioned " <3> 1 is a graph showing the results of measuring α-amylase activity using the "Enzyme Activity Measurement" method, in which α-amylase activity is plotted on the X-axis and hyphal width is plotted on the Y-axis.

[0074] As shown in FIG. 7, the hyphal width, which is a morphological characteristic of filamentous fungi, correlated well with the activity of α-amylase secreted by the filamentous fungi.

[0075] [Test Example 5] Figure 8 shows an example of an image observed by a fluorescence microscope for Aspergillus oryzae RIB915 when it was cultured for three days on a plate medium of minimal medium supplemented with various substances.

[0076] As shown in Figure 8, when Aspergillus oryzae RIB915 was cultured in a minimal medium supplemented with 10 mM nucleic acid (deoxyribonucleic acid MERCK D8515) or 100 mM vitamin (containing biotin, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, para-aminobenzoic acid, and nicotinic acid), cell nuclei were present within the mycelium at almost the same level as when cultured in a minimal medium (results of Test Example 2).

[0077] [Test Example 6] Figure 9 shows an example of an image observed by a fluorescence microscope for Aspergillus oryzae RIB915 when it was cultured for three days on a plate medium of minimal medium supplemented with various substances.

[0078] As shown in Figure 9, when Aspergillus oryzae RIB915 was cultured in a minimal medium supplemented with 1% peptone, 1% casamino acids, 0.1% isoleucine, 0.1% leucine, or 0.1% valine, the number of cell nuclei present per given area of ​​the hyphae was increased compared to when it was cultured in a minimal medium (results of Test Example 2).

[0079] [Test Example 7] Figure 10 shows examples of bright-field images of Trichoderma reesei and Penicillium chrysogenum observed by a fluorescent microscope after culturing each filamentous fungus for three days on a plate medium of minimal medium or minimal medium supplemented with 1% yeast extract. Figure 10(a) shows an example of an image of Trichoderma reesei, and Figure 10(b) shows an example of an image of Penicillium chrysogenum. Figure 10(c) shows the results of counting the number of cell nuclei within a 100 μm hyphal length range from the hyphal tip side for each filamentous fungus based on the images observed by the fluorescent microscope.

[0080] As shown in Figure 10, when Trichoderma reesei and Penicillium chrysogenum were cultured on minimal medium supplemented with 1% yeast extract, the number of cell nuclei present per given area of ​​the mycelium tended to increase compared to when cultured on minimal medium.

Claims

1. a pre-screening step of preparing a filamentous fungus to be screened; A culturing step of culturing the filamentous fungus; an observation image acquisition step of observing the cultured filamentous fungus under a microscope at a predetermined time or for a predetermined period in the culture step to obtain an observation image; an observation value acquisition step of extracting morphological characteristics of the filamentous fungus in the observation image and obtaining an observation value based on the morphological characteristics; evaluating the observed value to determine whether the observed value satisfies a predetermined threshold; and an identification step of identifying the filamentous fungus that satisfies a predetermined threshold value in the evaluation step from the screening target prepared in the pre-screening step. Screening methods for filamentous fungi.

2. The method of claim 1 , wherein the morphological characteristic is hyphal width or hyphal volume.

3. The method of claim 1 , wherein the morphological characteristic is the number of cell nuclei present per given area of ​​hyphae.

4. The method of claim 1 , wherein the threshold value correlates with a productivity of a given material.

5. A pre-screening step of preparing culture conditions for the filamentous fungus to be screened; A culturing step of culturing a filamentous fungus under the culture conditions for the filamentous fungus; an observation image acquisition step of observing the cultured filamentous fungus under a microscope at a predetermined time or for a predetermined period in the culture step to obtain an observation image; an observation value acquisition step of extracting morphological characteristics of the filamentous fungus in the observation image and obtaining an observation value based on the morphological characteristics; evaluating the observed value to determine whether the observed value satisfies a predetermined threshold; A process for identifying a condition that satisfies a predetermined threshold value in the evaluation process from the screening target prepared in the pre-screening process. A method for screening culture conditions for filamentous fungi.

6. The method of claim 5 , wherein the morphological characteristic is hyphal width or hyphal volume.

7. The method of claim 5 , wherein the morphological characteristic is the number of cell nuclei present per given area of ​​hyphae.

8. The method according to claim 5 , wherein the threshold value is correlated with a production amount of a predetermined substance.

Images