JP5582628B2 - Neisseria gonorrhoeae that can be used for the production of seasonings lacking the function of the gene encoding creC and use thereof - Google Patents

Description

The present invention relates to a gonococcus deficient in the function of a gene encoding a protein creC that controls and positively controls gonococcal carbon catabolite repression, and uses thereof.

In the production of seasonings such as shochu, sake, mirin, miso, soy sauce, Aspergillus kawachii, Aspergillus awamori, A. niger, Aspergillus oryzae ory z ) And Aspergillus sojae (A. sojae) are used. Aspergillus is suitable for the production of various enzymes, and depending on its use, a method of making a koji suitable for the expression of the various enzyme systems is employed. For example, in the production of soy sauce, koji molds are produced by spraying koji mold spores on a solid raw material such as cooked cereal and allowing the koji mold to grow on the surface. Various types of koji making, such as temperature, humidity, incubation time, maintenance conditions, ventilation / ventilation conditions, etc., depending on the type of soybean, wheat, and rice used for koji, as well as their pregelatinization and moisture content In general, the conditions are adjusted so that various enzymes are efficiently produced.

In particular, the activity of various enzymes such as protease, amylase, and xylanase correlates very well with the raw material utilization rate. Therefore, conventionally, in order to produce seasonings with high yield, breeding for obtaining koji molds having high various enzyme activities has been repeated by ultraviolet irradiation or mutagenesis with drugs.

On the other hand, in Aspergillus nidulans (A. nidulans), which is closely related to Aspergillus oryzae that can be used for the production of seasonings, the mutant strain lacking the function of the gene encoding the protein creC that positively controls carbon catabolite repression It is known that protease production is improved (Non-patent Document 1).

However, it has not been reported whether the expression of genes other than protease is altered in creC gene-deficient strains except for β-galactosidase and quinate dehydrogenase. In addition, since Aspergillus nidulans is not a koji mold used for brewing or the like, it is not known at all whether or not the same gene exists in a highly safe koji mold that can be used for seasoning production. Therefore, in koji molds used for seasoning production, whether or not a strain having a high enzymatic activity such as a protease can be obtained by a similar gene deficiency in a situation where the presence of the gene is unknown by those skilled in the art. Even if it was, it was not something that could be expected, and it was a completely unknown matter.

Mol. gen. Genet. 150, 193-204, 1977

Therefore, the object of the present invention is to first examine whether or not a similar gene exists in koji molds that can be used for seasoning production, and if present, the function of the same gene is lost by deleting the function of the same gene. The purpose is to investigate whether the activity of various enzymes of the protease changes and to determine whether such changes are useful in the production of seasonings.

In order to achieve the above object, the present inventor attempted to clone a similar gene in koji mold that can be used for seasoning production. First, a homology search was performed in the Aspergillus nidulans creC gene sequence using the Aspergillus nidulans gonococcal genome database DOGAN (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao) In the genome of Aspergillus oryzae, a gene having ID: AO090003000149 was found as a highly homologous gene. However, since the function of the gene was unknown, a primer was designed based on the sequence on the database, and the full length of the gene was cloned by PCR.

Since the exact region encoding the gene was unknown on the genome database, the gene region was determined by the 5′-RACE method. As a result, the gene consists of 2371 bp (start codon ATG: stop codon TAA) and 595 amino acid residues. It was found that it encodes a protein of about 65.4 kDa, and the same gene as the creC gene (hereinafter referred to as “creC gene”) was also found in Aspergillus oryzae (see the attached sheet). The base sequence of the creC gene is shown as “sequence 1”, and the amino acid sequence is shown as “sequence 2”). Therefore, a creC gene-deficient strain was prepared by homologous recombination method using Aspergillus oryzae, which is a koji mold used in seasoning production, and the activities of various enzymes involved in seasoning production were measured.

As a result, surprisingly, the present inventor improves not only protease activity but also amylase activity and xylanase activity, which play an important role in the production of seasonings, by deleting the function of the creC gene of Aspergillus oryzae. And the present invention was completed by finding that a koji strain suitable for seasoning production can be produced.

Therefore, the present invention is as follows.
(1) By deleting only the function of the gene encoding creC consisting of the base sequence shown in SEQ ID NO: 1, each enzyme activity of protease, amylase and xylanase is less than that of the parent strain in which the function of the gene is not deleted. Improved Aspergillus oryzae.
(2) Crec are deficient in the gene encoding function, deletion of one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 1, resulting from substitution, or addition, the (1), wherein the Aspergillus oryzae (Aspergilluls oryzae).
(3) A method for producing koji, characterized in that the koji is made using the Aspergillus oryzae described in (1) above.
(4) A method for producing a seasoning, comprising preparing a koji using the Aspergillus oryzae as described in (1) above, charging the koji using a koji method in a conventional manner, fermenting and aging.

Since the koji mold of the present invention has high protease activity, high amylase activity and high xylanase activity, and the koji mold usually used for seasoning production is used as a parent strain, it is directly applicable to the efficient production of seasonings. Therefore, the industrial applicability is very high. Furthermore, since the creC gene deficiency is thought to be related to the derepression of carbon catabolite repression, it is susceptible to carbon catabolite repression and is used for the production of liquid koji and hydrolysis used in the saccharification stage of bioethanol production. It can be applied to enzyme production.

In the present invention, the koji mold that can be used for the seasoning production is particularly a koji mold belonging to the genus Aspergillus that can be used for the production of seasonings such as shochu, sake, mirin, soy sauce, miso, etc. and has established safety. There is no limitation. Specifically, Aspergillus kawachi, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus soya and the like are preferable. Of these, Aspergillus oryzae and Aspergillus soya are particularly preferred.

In the present invention, the creC gene refers to a gene whose ID is AO090003000149 in the genome database DOGAN (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao).

A function-deficient strain of the creC gene can be obtained by gene disruption by homologous recombination or induction of function deficiency by mutagenesis.

As a method for disrupting a gene by homologous recombination, a known method can be used. For example, a creC gene fragment or a DNA fragment that combines the upstream / downstream region and a marker gene is incorporated into a vector, the vector is incorporated into a filamentous fungus by the protoplast-PEG method or electroporation method, and homologous recombination is performed. Examples thereof include a method for introducing the DNA fragment into the genome of a filamentous fungus. Other methods for incorporating the DNA fragment into the gonococcal cell include the particle gun method, the Agrobacterium method, and the microinjection method.

As a method for confirming that a desired gene has been introduced into koji mold by homologous recombination, a known method can be used. For example, when a gene is introduced, an auxotrophic mutant is used as a parent strain, and a gene having a function that compensates for the auxotrophy is used as a marker gene. For example, a method of selecting a grown strain can be mentioned. However, such auxotrophy alone cannot confirm whether the target gene locus has been replaced with the introduced marker gene. Therefore, it is necessary to confirm that the target gene locus has been replaced with a marker by using a PCR method, Southern hybridization method or the like as appropriate in accordance with auxotrophy.

In addition, as a method for introducing mutation, a known treatment method can be used, such as a physical method of irradiating with ultraviolet rays, ion beams, radiation, etc., ethylmethanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, There are chemical methods that use mutagens such as nitrous acid and acridine dyes. Particularly preferred is a method of irradiating with an ion beam or ultraviolet rays.

As a method of screening for a strain in which the function of creC is lost and the protease activity is improved by the gene disruption method or the mutation introduction method as described above, a halo assay is performed on a plate containing milk casein, so that Examples include a method in which a strain having an increased halo is used as a candidate strain, genomic DNA is extracted from the selected candidate strain, and confirmed by sequence analysis. As another method, a colony hybridization method or the like can be used.

Moreover, the strain in which various enzyme activities have increased in the present invention refers to a strain in which enzyme activity has increased 1.1 times or more compared to the parent strain in a soy sauce raw material medium or the like.

In addition, as a method for measuring various enzyme activities, a commonly used method can be used. For example, a method for measuring protease activity is described in “Shoyu Test Method” (published by Japan Soy Sauce Research Institute). This method can be used.

As a method for producing a koji using the koji mold of the present invention and a method for producing a seasoning using the koji, known methods can be used. For example, in the production of soy sauce, a koji mold prepared by inoculating a koji mold lacking the function of the above creC gene into a mixture of a koji raw material, for example, a soy material that has been boiled and steamed, and a wheat koji material that has been cracked with fried rice. Then, the obtained koji is charged into a normal charging tank with an appropriate concentration of saline solution, and fermented and aged for about 3 to 6 months with proper stirring to obtain soy sauce moromi. To make product soy sauce (raw soy sauce or fire soy sauce).

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples. In addition,% in an Example means all w / v%.

Example 1 Preparation of creC gene-deficient strain by homologous recombination method (1) Preparation of creC gene-deficient strain by homologous recombination Utilizing sulfuric acid (sulfur source) as a parent strain for preparing a creC gene-deficient strain Aspergillus aspergillus oryzae ΔligD strain, which is an auxotrophic strain that cannot be used, was used. The ΔligD strain is a strain prepared based on the Aspergillus oryzae RIB40 strain (deposit number: ATCC 42149) according to known literature (Fungal Genet Biol. 45 (2008), 878-889). As a marker gene, Aspergillus nidulans sC gene (Mol Gen Genet. 1995 May 20; 247 (4): 423-429.) Having a function to assimilate sulfuric acid (sulfur source) was used.

For this reason, the strain into which the intended marker gene has been successfully introduced is restored to the auxotrophy of the ΔligD strain by introduction of the sC gene, and therefore grows in a medium containing only sulfate as a sulfur source. It becomes possible. From this, a creC gene-deficient strain can be easily isolated.

That is, using the genomic DNA of the Aspergillus oryzae ΔligD strain as a template, primers 1 and 2 from an upstream region of about 1.5 kbp (upstream region of the creC ORF) to a downstream region of about 1.5 kbp (downstream region of the creC ORF) of the creC gene coding region. Was amplified by the PCR method. Amplification by the PCR method is performed by a conventional method, the obtained amplification product is introduced into the vector pENTR D / TOPO (Invitrogen), and the completed vector is PCR again using the primers 3 and 4 to which the restriction enzyme AatII site and XbaI site are added. Amplified by the method. The obtained amplification product was treated with restriction enzymes AatII and XbaI to obtain a linear sequence (A) lacking the ORF portion of creC.

Next, sC gene region was amplified by PCR method using Aspergillus nidulans genomic DNA as a template and primers 5 and 6 to which AatII site and XbaI site were added, and then introduced into pCRBlunt vector (Invitrogen). The obtained vector was treated with AatII and XbaI to obtain the sequence (B) of the sC region.

  The sequence A and B thus obtained were fused using DNA ligase to create a creC gene disruption construct (FIG. 1). This vector was treated with NotI, and the resulting linear vector was introduced into Neisseria gonorrhoeae ΔligD using the protoplast-PEG method. Selection medium containing sulfuric acid (glucose 1%, sodium nitrite 0.2%, dipotassium phosphate 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, iron sulfate 0.001%, trace element 0.1%, 4.68% salt) was transferred to the same medium three times to obtain a creC gene-deficient candidate strain.

  Genomic DNA was extracted from these candidate strains according to a conventional method, and amplification of the creC gene was confirmed by PCR using this as a template. When the creC gene was disrupted, gene amplification was not observed. Therefore, a strain that did not recognize amplification was designated as a creC gene-deficient strain.

  The primers used are shown in Table 1.

(2) Growth confirmation A broth culture medium was prepared in a plastic petri dish having a diameter of 8 cm, and a creC gene-deficient strain and a parent strain ΔligD as a control strain were seeded at the center of the plate. Colony diameters were measured every 30 hours at 30 ° C. As a result, it became clear that there was no difference in growth between the parent strain ΔligD strain and the creC gene-deficient strain (FIG. 2).

(3) Halo assay A halo assay medium (milk plate, starch plate) is prepared in a plastic petri dish having a diameter of 8 cm, and a creC gene-deficient strain and a parent strain ΔligD as a control strain are seeded at the center of the plate to form a halo. The size of was measured. As a result, the creC gene-deficient strain formed a larger halo than the parent strain, suggesting that protease activity and amylase activity were improved (FIG. 3).

In addition, the composition of the used milk plate is as follows.
2% glucose, 0.3% sodium nitrite, 0.1% dipotassium phosphate, 0.5% magnesium sulfate, 0.5% potassium chloride, 0.001% iron sulfate, 0.1% milk casein, trace Element 0.1%, Agar 1.5%

Moreover, the composition of the used starch plate is as follows.
2% glucose, 0.3% sodium nitrite, 0.1% dipotassium phosphate, 0.5% magnesium sulfate, 0.5% potassium chloride, 0.001% iron sulfate, 0.1% starch, trace element 0.1%, agar 1.5%

(3) Measurement of protease activity To 5 g of defatted soybeans, 8 ml of sterilized distilled water was sprinkled, 5 g of ground wheat was added, and the mixture was stirred well and subjected to an autoclave treatment for 40 minutes to obtain a soy sauce raw material medium. The creC gene-deficient strain and the spore of the parent strain ΔligD as a control strain were sprayed so that the number of spores was 10 ⁶ per gram of the soy sauce raw material medium, stirred well, and cultured at 28 ° C. for about 48 hours. As a result of the culture, no difference in growth was observed between the creC gene-deficient strain and the parent strain.

To the cultured koji, 140 ml of cold water was added and stirred well, then allowed to stand for 4 hours, and filtered with a filter paper to prepare an enzyme solution. Protease activity was measured using the prepared enzyme solution. The measurement was performed according to the following procedure.

1 ml of 1.5% milk casein solution and 1 ml of distilled water were placed in a test tube and preheated in a thermostatic bath at 30 ° C. for 5 minutes. After 5 minutes, 1 ml of enzyme solution was added and reacted for 10 minutes, and then 3 ml of 0.4 M trichloroacetic acid solution was added to stop the reaction. Further, the mixture was allowed to stand at 30 ° C. for 30 minutes, and the precipitate was removed with a filter paper. Then, 2 ml of the filtrate was taken, 0.55M sodium carbonate solution 5 ml and 1 ml of Folin reagent were added and reacted at 30 ° C. for 30 minutes, and 660 nm with a spectrophotometer. The absorbance was measured. In the same manner, a calibration curve was prepared using a tyrosine standard solution, and the enzyme activity was calculated with 1 unit being the amount of enzyme that liberates 1 μg of tyrosine per minute. As a result, the creC gene-deficient strain had significantly higher protease activity than the parent strain ΔligD (FIG. 4).

(4) Measurement of amylase activity The amylase activity was measured according to the following procedure.
1 ml of 50 mM MacL Lvine buffer (pH 5.0) was added to 1 ml of enzyme solution, 2 ml of 1% starch aqueous solution was added, and the reaction was performed at 30 ° C. for 30 minutes. Thereafter, 10 ml of 0.5 N acetic acid and 10 ml of 0.0003 normal iodine solution were added, and the absorbance at 700 nm was measured with a spectrophotometer. The enzyme activity was calculated by setting the amount of enzyme that reduces iodine blue coloration by 10% as 1 unit. As a result, the creC gene-deficient strain had significantly higher amylase activity than the parent strain ΔligD (FIG. 5).

(5) Measurement of xylanase activity The xylanase activity was measured according to the following procedure.
Add 40 μl of enzyme solution to 160 μl of 0.5% xylan aqueous solution, react for about 60 minutes at 40 ° C., add 400 μl of DNS reagent, boil for 5 minutes, then add 2.4 ml of pure water. After mixing, the absorbance at 500 nm was measured with a spectrophotometer. A calibration curve was prepared using a xylose standard solution in the same manner, and the enzyme activity was calculated with 1 unit being the amount of enzyme that liberates 1 μg of xylose per minute. As a result, the creC gene-deficient strain had significantly higher protease activity than the parent strain ΔligD (FIG. 6).

The DNS reagent used here was prepared by the following procedure.
To 300 ml of 4.5% aqueous sodium hydroxide solution, 880 ml of 1% 3,5-dinitrosalicylic acid aqueous solution and 255 g of Rochelle salt were added. To this solution, 10 g of phenol was mixed with 22 ml of 10% aqueous sodium hydroxide solution, 6.9 g of sodium bicarbonate was dissolved in 69 ml of a solution that had been filled up to 100 ml, and stored in a dark place at room temperature for 2 days or more. used.

The results of comparing the numerical values of various enzyme activities of the parent strain ΔligD and the creC gene-deficient strain and the ratio of the enzyme activity of the creC gene-deficient strain to the control strain, ie ΔligD, in the above (3) to (5) are summarized in Table 2 below. It became like this. In the table, ΔcreC indicates a creC gene-deficient strain.

From the above results, it was possible to obtain koji mold suitable for seasoning production in which protease activity, amylase activity, and xylanase activity were higher than those of the parent strain.

Example 2 Production of soy sauce using creC gene-deficient strain 7 L of water was added to 5 kg of defatted soybean and mixed for 1 hour, and then steamed in a high pressure steaming can at a steaming pressure of 4 kg / cm ² for 10 minutes. To this steamed defatted soybean, 5.2 kg of wheat that had been cracked after heat denaturation was added and mixed with the seed meal to make a koji. Note that the creC gene-deficient strain obtained in Example 1 was used as the seed pod, and the parent strain was used as a control.

The obtained koji was charged with cold salt water, immediately after charging, the temperature was raised to 10-15 ° C., and one month later, from 25 ° C. to 30 ° C., and brewed for a total of 5 months to obtain a standard thick soy sauce. In addition, this
All these studies were conducted in a P1 level closed system. As a result of analyzing the obtained moromi, etc., it was found that when the creC gene-deficient strain was used, the nitrogen utilization rate was higher and the soy sauce cake was less than the parent strain.

FIG. 1 schematically shows a gene disruption vector production process performed in the examples. The arrow indicates the position and direction of primer preparation, the number appended to the arrow indicates the primer number shown in the Examples, and AnsC indicates the sC gene derived from Aspergillus nidulans. FIG. 2 shows the results of comparison of colony diameters between the control strain ΔligD and the creC gene-deficient strain. ◇ indicates a measured value in a parent strain, and □ indicates a measured value in a creC gene-deficient strain (ΔcreC). FIG. 3 shows the results of the halo assay. The upper row shows the results for the milk plate, and the lower row shows the results for the starch plate. FIG. 4 shows the results of comparing the protease activity of the parent strain ΔligD and the creC gene-deficient strain (ΔcreC) in the soy sauce raw material medium. FIG. 5 shows the results of comparing the amylase activities of the parent strain ΔligD and the creC gene-deficient strain (ΔcreC) in the soy sauce material medium. FIG. 6 shows the results of comparing the xylanase activity of the parent strain ΔligD and the creC gene-deficient strain (ΔcreC) in the soy sauce raw material medium.

Claims (4)

By deleting only the function of the gene encoding creC consisting of the base sequence shown in SEQ ID NO: 1, the enzyme activities of protease, amylase and xylanase are improved compared to the parent strain not having the function of the gene. -Aspergillus oryzae. loss of gene function encoding creC is, deletion of one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 is provided by substitution or addition, Aspergillus of claim 1, wherein oryzae (Aspergilluls oryzae). A method for producing a cocoon, wherein the cocoon is made using the Aspergillus oryzae according to claim 1. A method for producing a seasoning, comprising preparing a koji using the Aspergillus oryzae according to claim 1, charging the koji using a koji method by a conventional method, fermentation and aging.

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