METHOD FOR PRODUCING USEFUL SUBSTANCE

Abstract

The present disclosure concerns a method for producing peptides such as glutathione and a microorganism that can be used for such method. One or more embodiments of the first aspect of the present disclosure concern a method for producing peptides such as glutathione comprising culturing a prokaryotic microbial strain in which the expression levels of one or more genes selected from among the gshA gene, the gshB gene, and the gshF gene are enhanced, compared with the expression levels thereof in the wild-type strain thereof in a medium in which the total concentration of cysteine and cystine is 0.5 g/l or lower. The second aspect of the present disclosure concerns a microorganism comprising disruptions of the γ-glutamyltransferase gene and the glutathione reductase gene and exhibiting the enhanced expression levels of the gshA gene and the gshB or gshF gene.

Claims

1. A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and/or oxidized glutathione, comprising culturing a gram-negative bacterium in a medium, wherein the total concentration of cysteine and cystine in the medium is 0.5 g/l or lower before inoculation of the gram-negative bacterium, thereby increasing the expression levels of one or more genes selected from genes encoding glutamate-cysteine ligase, glutathione synthetase, and bifunctional glutathione synthetase, when compared with expression levels in a wild-type strain thereof, wherein the gram negative bacterium is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and/or oxidized glutathione by induced expression of the one or more genes when cultured in medium.

2. The method according to claim 1, wherein the gram-negative bacterium carries one or more genes selected from genes encoding glutamate-cysteine ligase, glutathione synthetase, and bifunctional glutathione synthetase, operably linked to an inducible promoter, wherein, when the one or more genes is the gene encoding glutamate-cysteine ligase, the inducible promoter increases the expression level of the gene encoding glutamate-cysteine ligase in the gram-negative bacterium by at least 20 times greater than that of the wild-type strain thereof.

3. The method according to claim 2, wherein the inducible promoter is IPTG inducible promoter, photoinducible promoter, araBAD promoter, rhaBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter comprising, as an operator sequence, tetO or lacO operator.

4. The method according to claim 3, wherein the inducible promoter is T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, araBAD promoter, rhaBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter comprising, as an operator sequence, tetO or lacO operator.

5. The method according to claim 4, wherein the inducible promoter is T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, or tac promoter.

6. The method according to claim 5, wherein the inducible promoter is T5 promoter.

7. The method according to claim 1, wherein the gram-negative bacterium is a transformed enteric bacterium.

8. The method according to claim 1, wherein the gram-negative bacterium is a transformed Escherichia coli strain.

9. A microorganism comprising disruptions of the gene [1] and the gene [2] below and exhibiting enhanced expression levels of the genes [3] or the gene [4] below: [1] a gene encoding γ-glutamyltransferase (EC:2.3.2.2); [2] a gene encoding glutathione reductase (EC:1.8.1.7); [3] a gene encoding glutamate-cysteine ligase (EC:6.3.2.2) and a gene encoding glutathione synthetase (EC:6.3.2.3); and [4] a gene encoding bifunctional glutathione synthetase.

10. The microorganism according to claim 9, comprising a disruption of the gene [5] below: [5] a gene encoding tripeptide peptidase (EC:3.4.11.4).

11. The microorganism according to claim 9, wherein the microorganism is a transformed bacterium.

12. The microorganism according to claim 9, wherein the microorganism is a transformed enteric bacterium.

13. The microorganism according to claim 9, wherein the microorganism is a transformed Gram-negative bacterium.

14. The microorganism according to claim 9, wherein the microorganism is a transformed E. coli strain.

15. A method for producing glutathione comprising culturing the microorganism according to claim 9 in a medium.

Description

EXAMPLES

[0377] Hereafter, the first aspect and the second aspect of the present disclosure are described in greater detail with reference to the examples, although the first aspect and the second aspect of the present disclosure are not limited to these examples.

[0378] Genetic engineering described below can be performed with reference to Molecular Cloning (Cold Spring Harbor Laboratory Press, 1989). Enzymes, cloning hosts, and materials used for genetic engineering may be purchased from commercial providers and used in accordance with the instructions. The enzymes are not particularly limited, provided that they can be used for genetic engineering.

<The First Aspect of the Present Disclosure>

[0379] The results of experimentation demonstrating the first aspect of the present disclosure are provided below.

[Example 1-1] Construction of Glutathione Synthetic Gene Expression Vector (1)

[0380] The T5 promoter, the E. coli-derived gshA gene (SEQ ID NO: 12), and the E. coli-derived gshB gene (SEQ ID NO: 14) were inserted into a space between the SmaI site and the HindIII site of the plasmid vector pQEK1-term as shown in SEQ ID NO: 4. Primers were designed in accordance with the instructions of the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), and the vector was constructed in accordance with the designated procedure.

[Example 1-2] Construction of Glutathione Synthetic Gene Expression Vector (2)

[0381] The T5 promoter, the E. coli-derived gshA gene (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding the mutant enzyme (WO 2018/084165) of glutathione synthetase derived from sulfur bacteria Thiobacillus denitrificans were inserted into a space between the SmaI site and the HindIII site of the plasmid vector pQEK1-term as shown in SEQ ID NO: 4. Primers were designed in accordance with the instructions of the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), and the vector was constructed in accordance with the designated procedure. The plasmid vector constructed is designated to be “pQEK1-PT5-ABTd(V260A)-term.”

[Example 1-3] Construction of Glutathione Synthetic Gene Expression Vector (3)

[0382] The T5 promoter and the SA gshF gene (SEQ ID NO: 19) encoding the bifunctional glutathione synthetase gene derived from Streptococcus agalactiae were inserted into a space between the SmaI site and the HindIII site of the plasmid vector pQEK1-term as shown in SEQ ID NO: 4. Primers were designed in accordance with the instructions of the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), and the vector was constructed in accordance with the designated procedure. The plasmid vector constructed is designated to be “pQEK1-PT5-FSa-term.”

[Example 1-4] Construction of Glutathione Synthetic Gene Expression Vector (4)

[0383] The lac promoter, the E. coli-derived gshA gene (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding the mutant enzyme (WO 2018/084165) of glutathione synthetase derived from sulfur bacteria Thiobacillus denitrificans were inserted into a space between the SmaI site and the HindIII site of the plasmid vector pQEK1-term as shown in SEQ ID NO: 4. Primers were designed in accordance with the instructions of the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), and the vector was constructed in accordance with the designated procedure. The plasmid vector constructed is designated to be “pQEK1-Plac-ABTd(V260A)-term.”

[Example 1-5] Construction of Glutathione Synthetic Gene Expression Vector (5)

[0384] The lacUV5 promoter, the E. coli-derived gshA gene (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding the mutant enzyme (WO 2018/084165) of glutathione synthetase derived from sulfur bacteria Thiobacillus denitrificans were inserted into a space between the SmaI site and the HindIII site of the plasmid vector pQEK1-term as shown in SEQ ID NO: 4. Primers were designed in accordance with the instructions of the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), and the vector was constructed in accordance with the designated procedure. The plasmid vector constructed is designated to be “pQEK1-PlacUV5-ABTd(V260A)-term.”

[Example 1-6] Preparation of Host Strains

[0385] The E. coli strain BW25113 obtained from the National Institute of Genetics (Japan) was subjected to a treatment using the plasmid pTH18cs1 obtained from the National Institute of Genetics in accordance with the method of preparing a cytosine deaminase-deficient strain disclosed in JP 2004-344029 A to prepare strains comprising disruptions of the γ-glutamyltransferase gene (SEQ ID NO: 21) and the tripeptide peptidase gene (SEQ ID NO: 23).

[Example 1-7] Preparation of Glutathione Synthetic Gene Expression-Enhanced Strains

[0386] Competent cells of the host cells prepared in Example 1-6 were prepared in accordance with a conventional technique and transformed with the plasmid vectors prepared in Example 1-1 to Example 1-5. Thus, transformants were obtained.

[Example 1-8] Evaluation of Production of Glutathione by Fermentation

[0387] The host strains prepared in Example 1-6 (without plasmid) or the glutathione synthetic gene expression-enhanced strains prepared in Example 1-7 were inoculated into 5 ml of LB medium containing 20 μg/ml tetracycline and shake-cultured at 300 rpm and 30° C. for 8 hours. The culture solution (1 ml) was inoculated into 100 ml of M9 medium (6 g/l disodium hydrogen-phosphate, 3 g/l potassium dihydrogen-phosphate, 0.5 g/l sodium chloride, 1 g/l ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose) supplemented with 20 sg/ml tetracycline. After inoculation, the culture solution was cultured using a culture apparatus (Bio Jr.8, Able Corporation) at 34° C. and pH 6.5 with shaking at 1,000 rpm and aeration of 100 ml/min for 18 hours. The culture solution 18 hours after the initiation of culture (20 ml) was inoculated into 2 liters of M9 medium supplemented with 20 μg/ml tetracycline. After the second inoculation, the culture solution was cultured using a culture apparatus (Bioneer-Neo, Marubishi Bioengineering Co., Ltd.) at 34° C. and pH 6.7 with shaking at 600 rpm and aeration of 4 l/min. During culture, a 50 w/v % glucose solution was added, according to need, so as to maintain the glucose concentration to 15 g/l or higher in the system, 0.1 mM isopropyl-β-thiogalactopyranoside was added 6 hours after the initiation of culture, and, at the same time, 780 mM glycine and 780 mM sodium sulfate were added. An adequate amount of the culture solution was sampled 24 hours after the initiation of culture, and cells were separated from the supernatant via centrifugation. The supernatant was adequately diluted with distilled water, GSH and GSSG in the culture supernatant were quantified by the method described in WO 2016/002884, and the total concentration was determined. The results of quantification of the total concentration of GSH and GSSG in the culture supernatant are shown in Table 1. The culture solution used in the above experiment does not substantially contain cysteine or cystine and the total concentration thereof is less than 0.5 g/l therein.

TABLE-US-00001 TABLE 1 Total amount of GSH + GSH accumulated in supernatant (g/l) Host strain (without plasmid) 0.2 pQEK1-PT5-ABTd(V260A)-term 5.8 pQEK1-Plac-ABTd(V260A)-term 0.9 pQEK1-PlacUV5-ABTd(V260A)-term 1.3 pQEK1-PT5-ABEc-term 5.9 pQEK1-PT5-FSa-term 4.7

[Example 1-9] Transcription Analysis of the Glutamate-Cysteine Ligase Gene and the Glutathione Synthetase Gene

[0388] The expression levels of the overexpressed genes were analyzed using plasmids via real-time PCR. The culture solution after the second inoculation in the culture described in Example 1-8 was cultured for 6 hours, 0.1 mM isopropyl-β-thiogalactopyranoside was added thereto, and an adequate amount of the sample was obtained 1 hour later. RNA was extracted using NucleoSpin RNA purchased from Takara Bio Inc. in accordance with the instructions. RNA samples were diluted with water to adjust the concentration to 50 ng/μl. Reverse transcription was performed using the PrimeScript RT reagent Kit (Perfect Real Time) purchased from Takara Bio Inc. in accordance with the instructions and cDNA was synthesized from RNA. With the use of TB Green Premix Ex Taq II (Tli RNaseH Plus) purchased from Takara Bio Inc. and the QuantStudio 3 real-time PCR system (Thermo Fisher Scientific), gshA, TDgshB (V260A), gshB, and SAgshF in the samples were quantified. As the internal standard, hcaT (SEQ ID NO: 27) known as a housekeeping gene was used. hcaT, gsh4, and gshB were subjected to real-time PCR simultaneously with the samples using genomic DNA of E. coli host strains, and calibration curves were prepared. On the basis of the calibration curves, the amounts of genes contained in the cDNAs were quantified. The calibration curve of TDgshB (V260A) was prepared using the pTDGSH2m15 plasmid described in WO 2018/084165, and the calibration curve of SAgshF was prepared using the pNGSHF plasmid described in WO 2016/017631. The quantified value of each gene in the same sample was divided by the quantified value of the internal standard hcaT, and the standardized value was designated to be the expression level of each gene. The forward primer shown in SEQ ID NO: 28 and the reverse primer shown in SEQ ID NO: 29 were used for hcaT amplification. The forward primer shown in SEQ ID NO: 30 and the reverse primer shown in SEQ ID NO: 31 were used for gshA amplification. The forward primer shown in SEQ ID NO: 32 and the reverse primer shown in SEQ ID NO: 33 were used for TDgshB (V260A) amplification. The forward primer shown in SEQ ID NO: 34 and the reverse primer shown in SEQ ID NO: 35 were used for gshB amplification. The forward primer shown in SEQ ID NO: 36 and the reverse primer shown in SEQ ID NO: 37 were used for SAgshF amplification.

[0389] The expression level of the E. coli-derived gshA gene in a transformant into which a plasmid vector comprising the E. coli-derived gsh4 gene; i.e., pQEK1-PT5-ABTd(V260A)-term, pQEK1-Plac-ABTd(V260A)-term, pQEK1-PlacUV5-ABTd(V260A)-term, or pQEK1-PT5-ABEc-term, had been introduced (i.e., the expression level standardized by dividing the quantified gshA gene expression level in a transformant by the quantified hcaT gene expression level in the same transformant) was determined as a relative value base on the E. coli-derived gshA gene expression level in a untransformed host strain prepared in Example 1-6 (i.e., the expression level standardized by dividing the quantified gshA gene expression level in a host strain by the quantified hcaT gene expression level in the same host strain) designated to be 1. The relative value of 5 to less than 10 was evaluated “+,” that of 10 to less than 20 was evaluated “++,” and that of 20 or more was evaluated “+++.” Also, the expression level of the E. coli-derived gshB gene in a transformant into which a plasmid vector comprising the E. coli-derived gshB gene; i.e., pQEK1-PT5-ABEc-term, had been introduced (i.e., the expression level standardized by dividing the quantified gshB gene expression level in a transformant by the quantified hcaT gene expression level in the same transformant) was determined as a relative value base on the E. coli-derived gshB gene expression level in a untransformed host strain (i.e., the expression level standardized by dividing the quantified gshB gene expression level in a host strain by the quantified hcaT gene expression level in the same host strain) designated to be 1. The relative value of 5 to less than 10 was evaluated “+,” that of 10 to less than 20 was evaluated “++,” and that of 20 or more was evaluated “+++.” The results are shown in Table 2.

TABLE-US-00002 TABLE 2 E. coli gshA E. coli gshB pQEK1-PT5-ABTd(V260A)-term +++ − pQEK1-Plac-ABTd(V260A)-term + − pQEK1-PlacUV5-ABTd(V260A)-term ++ − PQEK1-PT5-ABEc-term +++ +++ Relative to the gene expression level in the untransformed host strain, 1 +: 5 to less than 10 of the host strain ++: 10 to less than 20 of the host strain +++: 20 or more of the host strain

[0390] Concerning the TDgshB(V260A) or SAgshF gene expression level in a transformant into which a plasmid vector comprising the TDgshB (V260A) or SAgshF gene that is not inherent to the E. coli host strain; i.e., pQEK1-PT5-ABTd(V260A)-term, pQEK1-Plac-ABTd(V260A)-term, pQEK1-PlacUV5-ABTd(V260A)-term, or pQEK1-PT5-FSa-term, had been introduced, the quantified gene expression level was divided by the quantified hcaT gene expression level in the same sample to obtain a standardized value. As a negative control, the gene expression level in the untransformed host strain (without plasmid) was determined. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 TDgshB (V260A) SAgshF Host strain (without plasmid) 0 0 pQEK1-PT5-ABTd(V260A)-term 1.39 pQEK1-Plac-ABTd(V260A)-term 0.08 pQEK1-PlacUV5-ABTd(V260A)-term 0.13 pQEK1-PT5-FSa-term 12.44 Relative to the hcaT gene expression level in the same sample, 1

<The Second Aspect of the Present Disclosure>

[0391] Subsequently, the results of experimentation demonstrating the second aspect of the present disclosure are provided below.

(Analysis of Glutathione Concentration in Culture Solution)

[0392] The glutathione concentration in the culture solution was determined by high-performance liquid chromatography (HPLC, Shimadzu Corporation).

[0393] HPLC conditions are as described below.

Column: Develosil ODS-HG-3 4.6 mm×250 mm (Nomura Chemical Co., Ltd.)
Mobile phase: A solution of 30.5 g of potassium dihydrogen-phosphate and 18 g of sodium heptane sulfonate in 4.5 liters of distilled water was prepared, a pH of the solution was adjusted to 3 with phosphoric acid, 250 ml of methanol was added thereto, and a pH of the solution was readjusted to 3 with phosphoric acid.
Flow rate: 1 ml/min
Detection: UV detector, λ=210 nm
Column temperature: 40° C.
Amount of injection: 10 μl

[0394] When analyzing the glutathione concentration in the culture solution, cells were removed via centrifugation, and the supernatant was allowed to pass through a syringe filter (φ=0.2 μm, Advantech Co., Ltd.) to obtain a culture supernatant. The culture supernatant was diluted to 10-fold with distilled water and the resultant was then subjected to HPLC.

(Production Example 2-1) Preparation of BW25113Δggt Strain

[0395] At the outset, a plasmid vector for disrupting the ggt (γ-glutamyltransferase) gene (SEQ ID NO: 21) was prepared. A DNA fragment comprising the upstream sequence and the downstream sequence of the ggt gene (SEQ ID NO: 1) was obtained by PCR using synthetic oligo DNA. The resulting fragment was digested with XbaI and HindIII, the temperature-sensitive plasmid pTH18cs1 (GenBank Accession Number: AB019610, Hashimoto-Gotoh, T., Gene, 241, 185-191, 2000) was digested with XbaI and HindIII, and the digested fragments were ligated to each other with the aid of Ligation high Ver. 2 (Toyobo Co., Ltd.) to obtain the plasmid vector, pTH18cs1-ggt-UD.

[0396] Subsequently, the BW25113Δggt strain was prepared using pTH18cs1-ggt-UD. pTH18cs1-ggt-UD was introduced into the E. coli BW25113 strain via electroporation, applied to an LB agar plate containing chloramphenicol at 10 μg/ml, and cultured at 30° C. to obtain transformants. The resulting transformants were shake-cultured in an LB liquid medium containing chloramphenicol at 10 μg/ml at 30° C. overnight, the culture solution was applied to an LB agar plate containing chloramphenicol at 10 μg/ml, and culture was performed at 42° C. to obtain transformants. The resulting transformants were cultured in an LB liquid medium at 42° C. overnight and applied to an LB agar plate to obtain colonies. The resulting colonies were replica-plated to an LB agar plate and an LB agar plate containing chloramphenicol at 10 μg/ml, and chloramphenicol-sensitive transformants were selected. The selected transformants were analyzed by PCR and using a DNA sequencer to isolate a strain having deletion of a region from the start codon to the stop codon of the ggt gene on the chromosome. This gene-disrupted strain was designated to be the BW25113Δggt strain.

[0397] The BW25113Δggt strain is derived from the E. coli BW25113 host strain, and it has deletion of a region from the start codon to the stop codon of the ggt gene on the chromosome.

(Production Example 2-2) Preparation of BW25113ΔggtΔpepT Strain

[0398] At the outset, a plasmid vector for disrupting the pepT (tripeptide peptidase) gene (SEQ ID NO: 23) was prepared. A DNA fragment comprising the upstream sequence and the downstream sequence of the pepT gene (SEQ ID NO: 2) was obtained by PCR using synthetic oligo DNA. The resulting fragment was digested with XbaI and HindIII, pTH18cs1 was digested with XbaI and HindIII, and the digested fragments were ligated to each other with the aid of Ligation high Ver. 2 to obtain the plasmid vector, pTH18cs1-pepT-UD.

[0399] Subsequently, a strain derived from a parent strain, i.e., the BW25113Δggt strain prepared in Production Example 2-1, and having deletion of a region from the start codon to the stop codon of the pepT gene on the chromosome was isolated using pTH18cs1-pepT-UD in the same manner as in Production Example 2-1. This gene-disrupted strain was designated to be the BW25113ΔggtΔpepT strain.

[0400] The BW25113ΔggtΔpepT strain is derived from the E. coli BW25113 host strain, and it has deletion of a region from the start codon to the stop codon of the ggt gene and that of the pepT gene on the chromosome.

(Production Example 2-3) Preparation of pQEK1-PT5-ABTd(V260A)-Term

[0401] At the outset, the pQEK1 vector as shown in SEQ ID NO: 3 was constructed from pQE-80L (QIAGEN) by replacing the drug-resistant marker with a tetracycline-resistant gene, so as to construct a vector for introducing a gene into E. coli. In addition, a lambda phage-derived terminator sequence was inserted into the HindIII locus of pQEK1 to construct the pQEK1-term vector as shown in SEQ ID NO: 4.

[0402] Subsequently, a DNA fragment comprising the T5 promoter, the E. coli-derived gsh4 gene, and the Thiobacillus denitrificans-derived gshB gene (with V260A mutation) (SEQ ID NO: 5) was obtained by PCR using synthetic oligo DNA. The resulting fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) to obtain pQEK1-PT5-ABTd(V260A)-term shown in SEQ ID NO: 6.

(Production Example 2-4) Preparation of BW25113ΔGgtΔpepT/pQEK1-PT5-ABTd(V260A)-Term Strain

[0403] pQEK1-PT5-ABTd(V260A)-term prepared in Production Example 2-3 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-ABTd(V260A)-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepT/pQEK1-PT5-ABTd(V260A)-term strain.

(Production Example 2-5) Preparation of BW25113ΔggtΔpepTΔgor Strain

[0404] At the outset, a plasmid vector for disrupting the gor (glutathione reductase) gene was prepared. A DNA fragment comprising the upstream sequence and the downstream sequence of the gor gene (SEQ ID NO: 7) was obtained by PCR using synthetic oligo DNA. The resulting fragment was digested with XbaI and HindIII, pTH18cs1 was digested with XbaI and HindIII, and the digested fragments were ligated to each other with the aid of Ligation high Ver. 2 to obtain the plasmid vector, pTH18cs1-gor-UD.

[0405] Subsequently, a strain derived from a parent strain; i.e., the BW25113ΔggtΔpepT strain prepared in Production Example 2-2, and having deletion of a region from the start codon to the stop codon of the gor gene on the chromosome was isolated using pTH18cs1-gor-UD in the same manner as in Production Example 2-1. This gene-disrupted strain was designated to be the BW25113ΔggtΔpepTΔgor strain.

(Production Example 2-6) Preparation of BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABTd(V260A)-Term Strain

[0406] pQEK1-PT5-ABTd(V260A)-term prepared in Production Example 2-3 was introduced into the BW25113 ΔggtΔpepTΔgor strain prepared in Production Example 2-5 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-ABTd(V260A)-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABTd(V260A)-term strain.

(Production Example 2-7) Preparation of pQEK1-PT5-ABEc-Term

[0407] A DNA fragment comprising the T5 promoter, the E. coli-derived gshA gene, and the E. coli-derived gshB gene (SEQ ID NO: 8) was obtained by PCR using synthetic oligo DNA. The resulting fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix to obtain pQEK1-PT5-ABEc-term shown in SEQ ID NO: 9.

(Production Example 2-8) Preparation of BW25113ΔggtΔpepT/pQEK1-PT5-ABEc-Term Strain

[0408] pQEK1-PT5-ABEc-term prepared in Production Example 2-7 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-ABEc-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepT/pQEK1-PT5-ABEc-term strain.

(Production Example 2-9) Preparation of BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABEc-Term Strain

[0409] pQEK1-PT5-ABEc-term prepared in Production Example 2-3 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Production Example 2-5 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-ABEc-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABEc-term strain.

[0410] (Production Example 2-10) Preparation of pQEK1-PT5-FSa-term A DNA fragment comprising the T5 promoter and the Streptococcus agalactiae-derived gshF gene (SEQ ID NO: 10) was obtained by PCR using synthetic oligo DNA. The resulting fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix to obtain pQEK1-PT5-FSa-term shown in SEQ ID NO: 11.

(Production Example 2-11) Preparation of BW25113ΔggtΔpepT/pQEK1-PT5-FSa-Term Strain

[0411] pQEK1-PT5-FSa-term prepared in Production Example 2-10 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-FSa-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepT/pQEK1-PT5-FSa-term strain.

(Production Example 2-12) Preparation of BW25113ΔggtΔpepTΔgor/pQEK1-PT5-FSa-Term Strain

[0412] pQEK1-PT5-FSa-term prepared in Production Example 2-10 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Production Example 2-5 via electroporation, and the resultant was applied to an LB agar plate containing tetracycline at 20 μg/ml to select transformants. The selected transformants were subjected to PCR analysis to isolate a strain comprising pQEK1-PT5-FSa-term introduced thereinto. This strain was designated to be the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-FSa-term strain.

(Example 2-1) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABTd(V260A)-Term Strain

[0413] The BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABTd(V260A)-term strain obtained in Production Example 2-6 was cultured under the conditions described below to produce GSH and GSSG. The resultant was inoculated into 5 ml of LB medium containing 20 μg/ml tetracycline and shake-cultured therein at 300 rpm and 30° C. for 8 hours. The culture solution (1 ml) was inoculated into 100 ml of M9 medium (6 g/l disodium hydrogen-phosphate, 3 g/l potassium dihydrogen-phosphate, 0.5 g/l sodium chloride, 1 g/l ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose) supplemented with 20 μg/ml tetracycline. After inoculation, the culture solution was cultured using a culture apparatus (Bio Jr.8, Able Corporation) at 34° C. and pH 6.5 with shaking at 1,000 rpm and aeration of 100 ml/min for 18 hours. The culture solution 18 hours after the initiation of culture (20 ml) was inoculated into 2 liters of M9 medium supplemented with 20 μg/ml tetracycline and then cultured using a culture apparatus (Bioneer-Neo, Marubishi Bioengineering Co., Ltd.) at 34° C. and pH 6.7 with shaking at 600 rpm and aeration of 4 l/min. During culture, a 50 w/v % glucose solution was added, according to need, so as to maintain the glucose concentration to 15 g/l or higher in the system. 0.1 mM isopropyl-β-thiogalactopyranoside was added 6 hours after the initiation of culture, and, at the same time, glycine and sodium sulfate were added to adjust the final concentration to 100 mM. An adequate amount of the culture solution was sampled 30 hours after the initiation of culture, and cells were separated from the supernatant via centrifugation. The supernatant was adequately diluted with distilled water, and GSH and GSSG were quantified by HPLC analysis. The results of quantification are shown in Table 4.

(Comparative Example 2-1) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepT/pQEK1-PT5-ABTd(V260A)-Term Strain

[0414] The BW25113ΔggtΔpepT/pQEK1-PT5-ABTd(V260A)-term strain obtained in Production Example 2-4 was cultured under the same conditions as those in Example 2-1 to produce GSH and GSSG. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 GSH + Strains GSSG (g/l) Ex. 2-1 BW25113 ΔggtΔpepTΔgor/ 8.1 pQEK1-PT5-ABTd*-term Comp. BW25113 ΔggtΔpepT/ 6.8 Ex. 2-1 pQEK1-PT5-ABTd*-term

<Examination>

[0415] The results of Example 2-1 and the results of Comparative Example 2-1 shown in Table 4 demonstrate that glutathione productivity (GSH+GSSG) is increased to a significant extent by disruption of the gor gene. This indicates that disruption of the gor gene is effective for glutathione production by fermentation.

(Example 2-2) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABEc-Term Strain

[0416] The BW25113ΔggtΔpepTΔgor/pQEK1-PT5-ABEc-term strain obtained in Production Example 2-9 was cultured under the same conditions as those in Example 2-1 to produce GSH and GSSG. The results are shown in Table 5.

(Comparative Example 2-2) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepT/pQEK1-PT5-ABEc-Term Strain

[0417] The BW25113ΔggtΔpepT/pQEK1-PT5-ABEc-term strain obtained in Production Example 2-8 was cultured under the same conditions as those in Example 2-1 to produce GSH and GSSG. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 GSH + Strains GSSG (g/l) Ex. 2-2 BW25113 ΔggtΔpepTΔgor/ 8.5 pQEK1-PT5-ABEc-term Comp. BW25113 ΔggtΔpepT/ 7.0 Ex. 2-2 pQEK1-PT5-ABEc-term

<Examination>

[0418] The results of Example 2-2 and the results of Comparative Example 2-2 shown in Table 5 demonstrate that glutathione productivity (GSH+GSSG) is increased to a significant extent by disruption of the gor gene. This indicates that disruption of the gor gene is effective for glutathione production by fermentation.

(Example 2-3) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepTΔgor/pQEK1-PT5-FSa-Term Strain

[0419] The BW25113ΔggtΔpepTΔgor/pQEK1-PT5-FSa-term strain obtained in Production Example 2-12 was cultured under the same conditions as those in Example 2-1 to produce GSH and GSSG. The results are shown in Table 6.

(Comparative Example 2-3) Production of Glutathione by Fermentation Using the BW25113ΔggtΔpepT/pQEK1-PT5-FSa-Term Strain

[0420] The BW25113ΔggtΔpepT/pQEK1-PT5-FSa-term strain obtained in Production Example 2-11 was cultured under the same conditions as those in Example 2-1 to produce GSH and GSSG. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 GSH + Strains GSSG (g/l) Ex. 2-3 BW25113 ΔggtΔpepTΔgor/ 6.6 pQEK1-PT5-FSa-term Comp. BW25113 ΔggtΔpepT/ 5.0 Ex. 2-3 pQEK1-PT5-FSa-term

<Examination>

[0421] The results of Example 2-3 and the results of Comparative Example 2-3 shown in Table 6 demonstrate that glutathione productivity (GSH+GSSG) is increased to a significant extent by disruption of the gor gene. This indicates that disruption of the gor gene is effective for glutathione production by fermentation.

[0422] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.