Microorganisms of the genus Aspergillus, and methods for avoiding glucose-induced growth inhibition of Aspergillus microorganisms.

Abstract

To provide a technology that enables the successful growth of Aspergillus microorganisms even in the presence of glucose. [Solution] This technology provides an Aspergillus microorganism in which glucose-induced growth inhibition is avoided by deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD). This technology also provides a method for avoiding glucose-induced growth inhibition of an Aspergillus microorganism, which includes a culture step of culturing an Aspergillus microorganism in which the function of the gene encoding manganese superoxide dismutase (manganese SOD) is deficient, or a step of deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD) in an Aspergillus microorganism.

Description

[Technical Field]

[0001] The present technology relates to a microorganism of the genus Aspergillus. More specifically, the present technology relates to a microorganism of the genus Aspergillus in which growth inhibition by glucose is avoided, and to a method for avoiding growth inhibition by glucose in a microorganism of the genus Aspergillus. [Background Art]

[0002] Microorganisms of the genus Aspergillus are a type of fungi widely present in natural environments such as soil, air, and foods such as grains, and are also called koji molds. Microorganisms of the genus Aspergillus have long been used in the production of fermented foods such as soy sauce, miso, sake, and bonito flakes. In recent years, they are also used in the production process of useful substances such as enzymes, heterologous proteins, and organic acids. Furthermore, they are very useful microorganisms that can also be used for the decomposition of biomass.

[0003] Techniques for propagating useful microorganisms such as those of the genus Aspergillus have also been developed. For example, Patent Document 1 proposes a method capable of easily promoting the growth of microorganisms without adjusting the concentration of components added to a medium before culture by performing a step of bringing vanillin and / or ionone as gas components into contact with the microorganisms when propagating the microorganisms.

[0004] In addition, Patent Document 2 proposes a technique for regulating the growth of microorganisms by adding a Gynostemma pentaphyllum extract to a medium and culturing the microorganisms when culturing the microorganisms. And in Patent Document 2, the genus Aspergillus is exemplified as an example of microorganisms. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2021-145637 [Patent Document 2] Japanese Patent Application Publication No. 112976 / 1983 [Overview of the initiative] [Problems that the invention aims to solve]

[0006] As mentioned above, many technologies are being developed to propagate useful microorganisms such as Aspergillus microorganisms. However, as will be shown in the examples described later, it has been found that the growth of Aspergillus microorganisms is inhibited by glucose.

[0007] However, since glucose is a relatively inexpensive and energy-efficient carbon source, it would be extremely useful if it could be used as a carbon source for the growth of Aspergillus microorganisms.

[0008] Therefore, the primary objective of this technology is to provide a method that enables the successful growth of Aspergillus microorganisms even in the presence of glucose. [Means for solving the problem]

[0009] The inventors of this invention conducted intensive research on the gene function of Aspergillus microorganisms and discovered that Aspergillus microorganisms lacking the function of the gene encoding manganese superoxide dismutase (manganese SOD) exhibit good growth even in the presence of glucose, leading to the completion of this technology.

[0010] In other words, this technology first provides Aspergillus microorganisms in which glucose-induced growth inhibition is avoided by deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD). The Aspergillus microorganism related to this technology may also be Aspergillus oryzae.

[0011] This technology provides a method for avoiding glucose-induced growth inhibition of Aspergillus microorganisms, which includes either a culture step of culturing Aspergillus microorganisms in which the function of the gene encoding manganese superoxide dismutase (manganese SOD) is deficient, or a step of deficient the function of the gene encoding manganese superoxide dismutase (manganese SOD) in Aspergillus microorganisms. [Brief explanation of the drawing]

[0012] [Figure 1] This is a photograph of the agar medium after culturing in the example. [Figure 2] This graph shows the measurement results of the number of colonies after culturing in the example. [Modes for carrying out the invention]

[0013] The following describes preferred embodiments for implementing this technology. Note that the embodiments described below are merely examples of typical embodiments of this technology, and this should not be interpreted as narrowing the scope of this technology.

[0014] 1. Microorganisms of the genus Aspergillus The Aspergillus microorganisms that can be used in this technology are not particularly limited, as long as they do not impair the action or effect of this technology. Examples of Aspergillus microorganisms that can be used in this technology include Aspergillus oryzae, Aspergillus nidulans, Aspergillus sojae, Aspergillus fumigatus, Aspergillus luchuensis, Aspergillus niger, Aspergillus tamarii, Aspergillus kawachii, and Aspergillus glaucus. Among these, Aspergillus oryzae is preferred in this technology.

[0015] 2. Functional deficiency of the gene encoding manganese SOD The Aspergillus microorganisms related to this technology are characterized by a lack of function in the gene encoding manganese superoxide dismutase (manganese SOD) (hereinafter also referred to as the "manganese SOD gene").

[0016] The gene modification that causes the manganese SOD gene to lose function is not particularly limited as long as it reduces or completely stops the function of manganese SOD, and general gene modification methods can be freely selected and used. Specific examples of gene modification that causes the manganese SOD gene to lose function include, for example, methods of modification such as substitution, addition, insertion, deletion, or inversion of all or part of the base sequence of the manganese SOD gene. More specifically, a strain deficient in manganese SOD gene function can be produced by any combination of common methods, such as disruption of the manganese SOD gene by homologous recombination or induction of manganese SOD gene function deficiency by mutagenesis.

[0017] As a method for disrupting the manganese SOD gene by homologous recombination, for example, there can be mentioned a method comprising inserting a DNA fragment, which is obtained by combining a fragment of the manganese SOD gene or an upstream / downstream region thereof with a marker gene, into a vector, introducing the vector into a microorganism by the protoplast-PEG method, electroporation, or the like, and integrating the DNA fragment into the genome of the microorganism by homologous recombination. Other methods for introducing a DNA fragment into a microorganism include, for example, the particle gun method, the Agrobacterium method, the microinjection method, and the like.

[0018] Examples of methods for inducing manganese SOD gene function deficiency by mutagenesis include physical methods such as irradiation with ultraviolet rays, ion beams, radiation, and the like, and chemical methods using mutagens such as ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, acridine dyes, and the like.

[0019] It can be mentioned that, compared with a non-deficient strain, the manganese SOD activity in a strain deficient in manganese SOD gene function is, for example, 30% or less, preferably 20% or less, more preferably 10% or less, and still more preferably 5% or less. The manganese SOD activity can be measured using a known method.

[0020] 3. Culturing The Aspergillus microorganism according to the present technology, which is deficient in the function of the gene encoding manganese superoxide dismutase (manganese SOD), can be cultured using a common medium. The form of the medium is not particularly limited, and any form of medium such as a solid medium such as an agar medium, a liquid medium, or the like can be used; however, in order to form a large amount of conidia of the Aspergillus microorganism, it is preferable to select a solid medium.

[0021] Any nutrient source that can be included in the medium may be freely selected and used as long as it can support the growth of a microorganism of the genus *Aspergillus*, and one or more nutrient sources commonly used in media may be freely selected and used. Examples thereof include a carbon source, a nitrogen source, a phosphorus source, a sulfur source, an organic component, and an inorganic component.

[0022] Examples of the carbon source include saccharides such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, sucrose, molasses, starch, starch hydrolysates (e.g., dextrin and the like), and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol (glycerin), and crude glycerol; and fatty acids such as lecithin.

[0023] Among carbon sources, it is preferable to use glucose, which is a relatively inexpensive carbon source with high energy production efficiency. However, as shown in the Examples described later, there is a concern that glucose may suppress the growth of a microorganism of the genus *Aspergillus*. However, the inventors of the present application succeeded in avoiding growth suppression by glucose by disabling the function of a gene encoding manganese superoxide dismutase (manganese SOD) in a microorganism of the genus *Aspergillus*. That is, the microorganism of the genus *Aspergillus* according to the present technique can satisfactorily grow even when glucose is used as the carbon source.

[0024] Examples of the nitrogen source include ammonium salts such as sodium nitrate, ferric ammonium citrate, ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein hydrolyzate; ammonia; and urea.

[0025] Examples of the phosphorus source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0026] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.

[0027] Examples of organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, zinc, manganese, magnesium, potassium, sodium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and peptones, casamino acids, yeast extracts, and hydrolyzed soy protein containing these components.

[0028] Furthermore, this technology can also utilize commercially available agar media. Specifically, examples include DHL (Desoxycholate-hydrogen sulfide-lactose) agar, standard agar, desoxycholate agar, X-SAL agar, MLCB (Mannitol lysine crystal violet brilliant green) agar, and SS-SB (Salmonella-Shigella Sucrose Bromcresolpurple) agar.

[0029] The culture conditions can also be freely set within the range of conditions that allow Aspergillus microorganisms to grow, according to the properties of the Aspergillus microorganisms being grown, such as heat resistance.

[0030] The lower limit of the culture temperature is, for example, 15°C or higher, preferably 20°C or higher, and more preferably 25°C or higher. The upper limit of the culture temperature is, for example, 40°C or lower, preferably 35°C or lower.

[0031] The lower limit of the culture pH is, for example, pH 6.0 or higher, preferably pH 6.5 or higher. The upper limit of the culture pH is, for example, pH 8.0 or lower, preferably pH 7.5 or lower.

[0032] The culture process described above and the process of deleting the function of the gene encoding manganese SOD, as mentioned above, may be performed by the same institution or by different institutions. For example, an institution different from the one that performed the process of deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD) in Aspergillus microorganisms may perform the culture of said Aspergillus microorganisms. In other words, both the form in which the process of deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD) in Aspergillus microorganisms is performed in order to avoid glucose-induced growth inhibition of Aspergillus microorganisms, and the form in which the culture process of culturing Aspergillus microorganisms in which the function of the gene encoding manganese superoxide dismutase (manganese SOD) is deficient is included in the method of avoiding growth inhibition related to this technology.

[0033] 4. Uses of Aspergillus microorganisms The Aspergillus microorganisms involved in this technology can be used in a variety of applications, including the production of enzymes and heterologous proteins, the manufacture of food and beverages, the decomposition of biomass, and the production of recycled resources.

[0034] <Protein production> The method for producing proteins using Aspergillus microorganisms according to this technology comprises at least a culture step of culturing Aspergillus microorganisms according to this technology to express proteins, and a recovery step of recovering the expressed proteins.

[0035] Examples of proteins that can be produced using this technology include enzymes. Examples of enzymes that can be produced by microorganisms of the genus Aspergillus include amylase, cellulase, xylanase, arabinase, pectinase, mannanase, protease, and lipase.

[0036] The culture conditions are the same as described above, so we will omit the explanation here.

[0037] In the recovery process, one or more general protein recovery methods used in protein production can be freely combined and used depending on the type of protein being produced.

[0038] The recovered proteins can be further processed, etc., depending on the form of the final product, as long as it does not impair the function or effect of this technology.

[0039] <Manufacturing of food and beverages> The method for producing food and beverages using Aspergillus microorganisms related to this technology involves either a propagation step in which Aspergillus microorganisms related to this technology are propagated in food and beverage raw materials, or a fermentation step in which food and beverage raw materials are fermented using Aspergillus microorganisms related to this technology.

[0040] The various conditions in the breeding and fermentation processes can be freely set according to the type of food or beverage being manufactured, as long as they do not impair the function or effect of this technology.

[0041] Examples of foods and beverages that can be manufactured using this technology include koji, soy sauce, miso, mirin, rice vinegar, amazake, sake, shochu, pickles, and other fermented foods. Furthermore, this technology can also be used to produce starter koji for manufacturing foods and beverages.

[0042] In the manufacture of food and beverages, an inactivation step may be performed after the propagation or fermentation step to inactivate Aspergillus microorganisms. The method used in the inactivation step can be freely selected and used, as long as it does not impair the action or effect of this technology, and may include general methods for inactivating microorganisms such as heating or disinfection.

[0043] The manufactured food and beverages may be further processed, etc., depending on the form of the final product, to the extent that the function and effect of this technology are not impaired.

[0044] <Biomass decomposition, production of recycled resources> The method for decomposing biomass and producing recycled resources using Aspergillus microorganisms related to this technology is a method for performing a decomposition process in which biomass is decomposed using Aspergillus microorganisms related to this technology.

[0045] Examples of biomass that can be decomposed by this technology include agricultural waste, food waste, and wood residues, and it is particularly effective at decomposing biomass containing cellulose, hemicellulose, lignin, starch, chitin, and the like. [Examples]

[0046] The present technology will be described in more detail below based on the following examples. The examples described below are representative examples of the present technology and should not be interpreted as narrowing the scope of the present technology.

[0047] 1.Bacterial strains used In this example, Aspergillus oryzae was used as an example of an Aspergillus microorganism. More specifically, in this example, Aspergillus oryzae strain RIB40Δku70ΔpyrG (SEQ ID NO: 1) was used as the wild-type strain. The Aspergillus oryzae RIB40Δku70ΔpyrG strain is a strain of Aspergillus oryzae RIB40 (M. Machida et al, Genome sequencing and analysis of Aspergillus oryzae, (2005) Nature 438, 1157-1161) in which the ku70 gene related to the non-homologous recombination repair mechanism and the pyrG gene of the carboxylase with orotidine-5'-phosphate have been disrupted (Ishikawa, K. et al, (2018). Comparison of the paralogous transcription factors AraR and XlnR in Aspergillus oryzae. Current genetics, 64(6), 1245-1260).

[0048] 2. Creation of gene knockout strains encoding manganese superoxide dismutase (manganese SOD) The ΔsodB strain (SEQ ID NO: 2) was created by replacing the region (Chr2 4951314-4952495) of the sodB gene (gene number: AO090003001062), which encodes manganese superoxide dismutase (manganese SOD), in the Aspergillus oryzae RIB40Δku70ΔpyrG strain with the region (1061690-1063938) containing the pyrG gene (AN6157) of Aspergillus nidulans. As a comparative example, a ΔsodA strain was also created in which the region (Chr6 483219-484910) of the sodA gene (gene number: AO090020000521), which encodes copper / zinc superoxide dismutase (copper / zinc SOD) in the Aspergillus oryzae RIB40Δku70ΔpyrG strain, was replaced with the region (1061690-1063938) containing the pyrG gene (AN6157) of Aspergillus nidulans.

[0049] 3.Culture The growth of the wild-type strain, the ΔsodB strain prepared as described above, and the ΔsodA strain were investigated in media with different carbon sources. Specifically, the wild-type strain, the ΔsodB strain prepared as described above, and the ΔsodA strain were seeded on agar plates prepared by adding agar (Becton Dickinson Co., Ltd. "bacto agar") to the media shown in Table 1 below to a final concentration of 1.4%, and cultured at 30°C for 4 days. As the carbon source, 10g of starch, 10g of cellulose, or 20g of glucose were used.

[0050] [Table 1]

[0051] 4.Results Figure 1 shows a photograph of the culture medium after 4 days of incubation. Figure 2 shows a graph of the colony count measured after 3 days of incubation. The complete culture medium shown in Figure 2 was an agar medium prepared by adding agar (Becton Dickinson Co., Ltd. "bacto agar") to the culture medium shown in Table 2 below to a final concentration of 1.4%.

[0052] [Table 2]

[0053] 5. Discussion As shown in Figures 1 and 2, the wild-type (WT) strain's growth was inhibited by glucose, whereas the ΔsodB strain, which lacked the function of the gene encoding manganese superoxide dismutase (manganese SOD), avoided glucose-induced growth inhibition and showed good growth. The ΔsodA strain, however, exhibited growth inhibition by glucose, similar to the wild-type strain.

Claims

1. Aspergillus microorganisms in which the function of the gene encoding manganese superoxide dismutase (manganese SOD) is lost, thereby avoiding glucose-induced growth inhibition.

2. The Aspergillus microorganism described above is Aspergillus oryzae, as per claim 1.

3. A method for avoiding glucose-induced growth inhibition of Aspergillus microorganisms, comprising a culture step of culturing Aspergillus microorganisms lacking the function of the gene encoding manganese superoxide dismutase (manganese SOD).

4. A method for avoiding glucose-induced growth inhibition of Aspergillus microorganisms, comprising the step of deleting the function of the gene encoding manganese superoxide dismutase (manganese SOD) in Aspergillus microorganisms.

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