WO2020050113A1

WO2020050113A1 - Method for manufacturing useful substance through use of fermentation process

Info

Publication number: WO2020050113A1
Application number: PCT/JP2019/033690
Authority: WO
Inventors: 清敬原; 陽子原; 吉博戸谷; 史生松田; 健太郎鎌田; 涼田中
Original assignee: 静岡県公立大学法人
Priority date: 2018-09-03
Filing date: 2019-08-28
Publication date: 2020-03-12
Also published as: JPWO2020050113A1

Abstract

Provided is a method for producing a useful substance through the use of a microorganism fermentation process that can more effectively produce and manufacture the useful substance. This method is based on giving photophosphorylation ability to nonphotosynthetic prokaryotes by bioengineering. By giving the nonphotosynthetic prokarkyotes photophosphorylation ability by bioengineering, cells that have a light-driven high-energy action, while being nonphotosynthetic prokaryotes, are obtained. By using these transformed nonphotosynthetic prokaryotes, the useful substance can be more effectively produced and manufactured. More specifically, the useful substance can be more effectively manufactured using a microorganism fermentation process by culturing the transformed nonphotosynthetic prokaryotes, in which a gene that encodes rhodopsin has been introduced by genetic engineering and allowed to express, under irradiation with light.

Description

Method for producing useful substances by using fermentation process

(4) The present invention relates to a method for producing a useful substance by culturing a non-photosynthetic prokaryote having photophosphorylation ability. More specifically, the present invention relates to a method for producing a useful substance, which comprises culturing, under light conditions, a non-photosynthetic prokaryote in which a gene encoding rhodopsin has been introduced and expressed by genetic engineering.

This application claims the priority of Japanese Patent Application No. 2018-164907, which is incorporated herein by reference.

In an era where clean energy that is friendly to living organisms and the environment is desired, use solar cells that use inexhaustible light energy and plants that store light energy as organic matter as energy sources and fermentation materials for chemical products. Research on biorefinery is active. Research has been conducted on microorganisms having photosynthetic ability, such as photosynthetic bacteria (bacteria) and cyanobacterium, to produce useful substances using light energy. However, these microorganisms generally have a low growth rate and the entire metabolic pathway is unclear, which has been a problem in practical use. On the other hand, research is being conducted to improve the fermentation microorganisms such as Escherichia coli and budding yeast, which have a high growth rate, by metabolic engineering to improve the fermentation productivity of various useful substances such as pharmaceuticals, foods, cosmetics, fuels, and polymer raw materials. Have been. However, as the productivity of the target substance increases, these fermenting microorganisms, which have been subjected to a biased load different from that of the wild type, often fall into intracellular energy (ATP: Adenosine triphosphate) deficiency, which leads to a decrease in growth and a decrease in the target substance. Often face productivity peaks.

Plants and algae of eukaryotes and photoautotrophs of prokaryotes such as cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria plants and algae, photosynthetic bacteria It is disclosed that introduction of proteorhodopsin into a microorganism (photoautotoroph) enables conversion to a highly photosynthetic organism (hyper @ photosynthetic @ organism) (Patent Document 1). The photoautotrophic microorganism described in Patent Document 1 refers to an organism that acquires energy through photosynthesis, and is completely different from a non-photosynthetic prokaryote.

(4) A rapid and inexpensive production method has been disclosed in which rhodopsin is expressed in Escherichia coli and mass production of proteorhodopsin (PR) is expected (Patent Document 2 and Non-Patent Document 1). Although mentioning the formation of light-driven proton driving force, Escherichia coli expressing rhodopsin has a good effect on the energy production of ATP required for the production of useful substances, and has a high effect on the production of useful substances. No indication is given.

がある There is a method for producing a light-driven high-energy Saccharomyces submonum yeast (Patent Document 3). Here, it is disclosed that budding yeast expressed delta-rhodopsin (dR: Delta-rhodopsin) and showed a favorable effect on the production of useful substances through the formation of light-driven proton driving force and the production of ATP. . However, Patent Document 3 targets only Saccharomyces submonum yeast and requires that a nucleic acid (gene) encoding a mitochondrial localization signal besides rhodopsin be incorporated.

において In a method utilizing a fermentation process of a microorganism, development of a method for more effectively producing and producing a useful substance is desired.

Japanese Patent Application Laid-Open No. 2014-87360 (Japanese Patent Application No. 2013-269377) Japanese Patent Application No. 2004-521604 (Japanese Patent Application 2001-580311) Re-published publication WO2015 / 170609 (Japanese Patent Application No. 2016-517869)

The present invention relates to a method for producing a useful substance by utilizing a fermentation process of a microorganism, and an object of the present invention is to provide a method for producing and producing a useful substance more effectively.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by imparting photophosphorylation ability to non-photosynthetic prokaryotes biotechnologically, while being a non-photosynthetic prokaryote, a light-driven high energy action And succeeded in producing a useful substance more effectively, thereby completing the present invention. More specifically, by culturing non-photosynthetic prokaryotes expressed by introducing a gene encoding rhodopsin by genetic engineering under light conditions, it is possible to more effectively produce useful substances using the fermentation process of microorganisms The present invention was completed.

That is, the present invention includes the following.
1. A method for producing a useful substance, which comprises culturing a non-photosynthetic prokaryote in which a gene encoding rhodopsin is introduced and expressed by genetic engineering under light irradiation.
2. Non-photosynthetic prokaryotes expressed by introducing the gene encoding rhodopsin by genetic engineering, the production of useful substances when cultured under light irradiation, the production of useful substances when cultured in the dark 2. The method for producing a useful substance according to the above item 1, which is a non-photosynthetic prokaryote showing a higher production amount.
3. 3. The method for producing a useful substance according to the above 1 or 2, wherein the non-photosynthetic prokaryote is a non-photosynthetic bacterium.
4. The

useful item

1 or 2 above, wherein the non-photosynthetic prokaryote is any one selected from Escherichia coli, lactic acid bacteria, acetic acid bacteria, Bacillus subtilis, actinomycetes, coryneform bacteria, Pseudomonas bacteria, methanogens, and archaea. The method of manufacturing the substance.
5. 5. The method for producing a useful substance according to any one of items 1 to 4, wherein the non-photosynthetic prokaryote is Escherichia coli.
6. 6. The method for producing a useful substance according to any one of items 1 to 5, wherein the culture of the non-photosynthetic prokaryote under light irradiation is a culture under light irradiation of a light amount of 1 to 2000 μmol / m ² / s.
7. A method for producing a useful substance derived from a culture product of Escherichia coli, comprising the following steps:
1) a step of introducing a gene encoding rhodopsin into E. coli;
2) a step of culturing the Escherichia coli into which the gene of the above 1) has been incorporated at 4 to 40 ° C. for 4 to 72 hours under light irradiation of 1 to 2000 μmol / m ² / s;
3) a step of recovering the culture product of Escherichia coli cultured in the step 2) from the culture solution.
8. 8. The method for producing a useful substance according to any one of items 1 to 7, wherein the rhodopsin is bacteriorhodopsin or proteorhodopsin.
9. 8. The method for producing a useful substance according to any one of Items 1 to 7, wherein rhodopsin is delta-rhodopsin.
10. 10. The useful substance according to any one of items 1 to 9, wherein the useful substance is one or more selected from organic acids, peptides, amino acids, proteins, nucleic acids, vitamins, sugars, sugar alcohols, alcohols, isoprenoids, and lipids. For producing useful substances.
11. 10. The method for producing a useful substance according to any one of the above items 1 to 9, wherein the useful substance is acetic acid and / or glutathione.
12. 10. The method for producing a useful substance according to any one of the above items 1 to 9, wherein the useful substance is one or more selected from mevalonic acid, isoprenol, and 3-hydroxypropionic acid.

Non-photosynthetic prokaryotes usually do not have the purpose of producing useful substances, so the productivity of useful substances using non-photosynthetic prokaryotes is low. As non-photosynthetic prokaryotes such as Escherichia coli are improved by metabolic engineering to improve the fermentation productivity of various useful substances such as pharmaceuticals, foods, cosmetics, fuels, and polymer raw materials, microorganisms fall short of intracellular energy. It is not uncommon to face a decline in growth or a plateau in productivity of the target substance. This is thought to be due to the fact that the carbon source is consumed in the fermentation process of the microorganism, such as “material of the cell itself (Cell mass)”, “production of the target product”, and “intracellular energy”. According to the light-driven non-photosynthetic prokaryote of the present invention in which a non-photosynthetic prokaryote is provided with a photophosphorylation ability, it can be a host for producing a renewable useful substance. Thus, light can be used as an energy source in addition to the conventional energy sources, so that the productivity of useful substances can be improved.

It is a figure which shows the result of having confirmed proton transfer activity about the transformed Escherichia coli (dR expression strain 1). It is a figure showing the result of having checked fluctuation of pH by light irradiation. (Experimental example 3-1) It is a figure which shows the result of having confirmed ATP production in Escherichia coli cells by the difference of light irradiation about the transformed Escherichia coli (dR expression strain 1). (Experimental example 3-2) It is a figure which shows the result of having confirmed the production of the metabolite by the transformed Escherichia coli (dR expression strain 1). (Experimental example 3-3) It is a figure which shows the result of the growth of the Escherichia coli cell which used glucose or glycerol as a carbon source about the transformed Escherichia coli (dR expression strain 1). rpm indicates the stirring speed (the number of revolutions per minute). (Experimental example 3-4) It is a figure which shows the result of glutathione production which used glucose or glycerol as a carbon source about the transformed Escherichia coli (dR expression strain 1). rpm indicates the stirring speed (the number of revolutions per minute). (Experimental example 3-4) It is a figure which shows the glutathione production result about the transformed Escherichia coli (pR expression strain 1). (Experimental example 3-5) FIG. 4 shows the results of confirming the production of metabolites by transformed Escherichia coli (dR expression strains 2 to 4). (Examples 4 to 6) It is a figure which shows the result of having confirmed the production amount of the metabolite (mevalonic acid) by the transformed Escherichia coli (dR expression strain 2). (Example 4) It is a figure which shows the result of having confirmed the production amount of the metabolite (isoprenol) by the transformed Escherichia coli (dR expression strain 3). (Example 5) It is a figure which shows the result of having confirmed the production amount of the metabolite (3HP: 3 hydroxypropionic acid) by the transformed Escherichia coli (dR expression strain 4). (Example 6)

方法 The method for producing a useful substance according to the present invention is characterized in that it comprises a step of culturing a non-photosynthetic prokaryote, which has been expressed by introducing a gene encoding rhodopsin by genetic engineering, under light conditions (light irradiation).

As used herein, the term “non-photosynthetic prokaryote” refers to a prokaryote that does not normally perform photosynthesis, such as non-photosynthetic bacteria, non-photosynthetic archea, and non-photosynthetic actinomycetes ( non-photosynthetic actinomycetes). Specifically, Escherichia coli , Bacillus subtilis , lactic acid bacteria ( Lactobacillus ), acetic acid bacteria ( Acetobacteraceae ), coryneform bacteria ( Corynebacterium ), Pseudomonas bacteria, methanogens ( Methanobacterium , Methanosarcina), Streptomyces (Streptomyces), include actinomycetes (Actinomyces), etc., it is particularly suitable E. coli. The non-photosynthetic prokaryote that can be used may be a wild type as long as rhodopsin can be expressed by genetic engineering, or may be a mutated or established prokaryote. For example, in the case of Escherichia coli, Escherichia coli MG1655 (DE3) strain (Journal of Bioscience and Bioengineering Vol. 123 No. 2, 177 e182, 2017) can be used, and expression methods such as constitutive expression and propionic acid, When inducible expression with arabinose, acetic anhydride, or the like is applied, a wild strain or an appropriate strain can be selected and used.

As used herein, “rhodopsin” is a complex of a protein called Opsin and retinal (Retinal), and refers to a photoreceptor protein. Examples of the type of rhodopsin include delta-rhodopsin (dR), bacteriorhodopsin (including archrhodopsin and crux rhodopsin), sensory rhodopsin I, sensory rhodopsin II, proteorhodopsin (pR), channelrhodopsin I, channelrhodopsin II, halo Rhodopsin, xanthrodopsin, sodium pump type rhodopsin, heliorhodopsin and the like. For example, delta-rhodopsin and bacteriorhodopsin have a function of pumping protons from the inside of prokaryotes to the outside under light conditions (light irradiation), and halorhodopsin has a function of taking in chloride ions by light. Rhodopsin of the present invention is preferably delta-rhodopsin, bacteriorhodopsin or proteorhodopsin, more preferably delta-rhodopsin. Deltarhodopsin includes those derived from Haloterrigena turkmenica , Haloterrigena sp. Arg-4. There is a report on deltarhodopsin derived from H. turkmenica expressed in E. coli (BiochemBiophys Res Commun 2006, 341: 285-290, GeneBank Accession No. AB00962).

において In the present specification, the term “gene encoding rhodopsin” includes a nucleic acid sequence consisting of a base sequence capable of expressing rhodopsin. The expressed rhodopsin does not necessarily need to be full-length rhodopsin, but may be activated by light energy and be long enough to produce intracellular energy such as a proton concentration gradient or ATP by activating the electron transport system. I just need. Rhodopsin artificially designed may be used. A gene encoding rhodopsin having such a function may be used.

As a method for introducing a gene encoding rhodopsin by genetic engineering, a method known per se or any method to be developed in the future can be applied. In the method for producing a useful substance of the present invention, the non-photosynthetic prokaryote (hereinafter, also referred to as “transformed non-photosynthetic prokaryote”) into which the gene encoding rhodopsin of the present invention is introduced and expressed by the genetic engineering method is used. Those prepared in advance may be used, or the transformed non-photosynthetic prokaryote may be prepared to produce a useful substance.

The method for producing a useful substance of the present invention comprises the steps of preculturing the transformed non-photosynthetic prokaryote and culturing the transformed non-photosynthetic prokaryote under light conditions (light irradiation) to produce a useful substance. included. Further, a step of collecting the produced useful substance can be included. The step of collecting the produced useful substance may be simultaneous with the step of culturing the transformed non-photosynthetic prokaryote, or may be after the culturing.

培養 The transformed non-photosynthetic prokaryote can be recovered by culturing and growing the transformed non-photosynthetic prokaryote in a conventional manner. Culture conditions can be appropriately set under conditions suitable for the growth of the transformed non-photosynthetic prokaryote to be used. The culture medium used for culturing the transformed non-photosynthetic prokaryote includes a carbon source, a nitrogen source, inorganic ions, and an organic substance required by the transformed non-photosynthetic prokaryote in order to produce a useful substance which is a target product of the present invention. There is no particular limitation as long as it is a commonly used medium containing trace elements, nucleic acids, vitamins and the like. In the cultivation of the present invention, pH and temperature conditions may be selected so as to be most suitable for the growth of the transformed non-photosynthetic prokaryote to be used. For example, pH 2-9, preferably pH 4-9, more preferably pH 5-8, and the culture temperature is 4-50 ° C, preferably 15-45 ° C, more preferably 30-40 ° C. be able to. The culturing period is not particularly limited as long as the production of the desired product can be confirmed. For example, culturing can be performed for 4 to 144 hours, preferably 4 to 72 hours, and more preferably 4 to 36 hours. Subculture may be performed if necessary. The method for confirming the growth of the transformed non-photosynthetic prokaryote is not particularly limited. For example, a culture may be collected and observed with a microscope, or may be observed with absorbance. The concentration of dissolved oxygen in the medium during the culture of the transformed non-photosynthetic prokaryotic organism is not particularly limited, but is usually preferably 0.5 to 20 ppm. For this purpose, the amount of ventilation can be adjusted, agitated, or oxygen can be added to the ventilation. The culture method is not particularly limited under the above conditions, but a liquid culture method is preferred, and any of batch culture, fed-batch culture, continuous culture, or perfusion culture may be used. Further, the culture can be carried out by shaking or stirring with aeration. Hereinafter, the culture medium for culturing the transformed non-photosynthetic prokaryote may be simply referred to as “culture solution”.

In order to produce the useful substance of the present invention from the transformed non-photosynthetic prokaryote of the present invention, it is preferable to culture the transformed non-photosynthetic prokaryote under light conditions (light irradiation). By culturing under light irradiation, it is possible to effectively utilize the energy in the transformed non-photosynthetic prokaryote and to produce a useful substance more effectively. Light irradiation for producing a useful substance is preferably performed under light irradiation with a light amount of 1 to 2000 μmol / m ² / s, more preferably under light irradiation with a light amount of 10 to 200 μmol / m ² / s. Yes, and most preferably under light irradiation (bright conditions) of 50 μmol / m ² / s. The light irradiation may be performed from the start of the culture or during the culture. The irradiation time may be constant irradiation during the culture or intermittent irradiation. As a light source of the irradiation light, any light source such as a fluorescent lamp, an LED, and sunlight may be used, but when the wavelength is limited, 300 to 800 nm is preferable, and 450 to 650 nm is more preferable. Yes, 550 nm is most preferred.

The useful substance can be recovered, for example, by continuously extracting a culture solution containing the product from a culture tank containing the transformed non-photosynthetic prokaryote cultured under the above-mentioned light conditions (light irradiation). The culture vessel containing the transformed non-photosynthetic prokaryote may be continuously supplied with the medium. While continuously withdrawing the culture solution from the culture tank, the transformed non-photosynthetic prokaryote can be cultured under conditions that allow stable growth during the useful substance production phase. Continuous culture of the transformed non-photosynthetic prokaryote and production of a useful substance are achieved by supplying a medium as a substrate solution, extracting the culture solution, and culturing under growth conditions. As a result, the ability of the transformed non-photosynthetic prokaryote to proliferate is maintained even during the useful substance production period, and as a result, the production of the useful substance can be maintained even in continuous culture.

において In the present specification, the “useful substance” is a substance that can be produced by the fermentation process of the transformed non-photosynthetic prokaryote of the present invention, and is not particularly limited as long as it is industrially useful. Examples of useful substances include, for example, organic acids, peptides, amino acids, proteins, nucleosides, vitamins, sugars, sugar alcohols, alcohols, isoprenoids, and lipids. More specifically, the following can be mentioned. Examples of the organic acid include acetic acid, lactic acid, and succinic acid. Examples of the peptide include glutathione, alanylglutamine, and γ-glutamyl valylglycine, and examples of the polypeptide include polylysine and polyglutamic acid. As amino acids, L-alanine, glycine, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, L-lysine, L-methionine, L-threonine, L-leucine, L-valine, L-isoleucine , L-proline, L-histidine, L-arginine, L-tyrosine, L-tryptophan, L-phenylalanine, L-serine, L-cysteine, L-3-hydroxyproline, L-4-hydroxyproline, 5-aminolevulin Acids and the like can be given. Proteins include luciferase, inosine kinase, Glutamate-5-kinase (EC 2.7.2.11), Glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41), Pyrroline-5-carboxylatereductase (EC 1.5.1.2), γ-glutamylcysteine synthase (EC 6.3.2.2), glutathione synthase (EC 6.3.2.3), human granulocyte colony stimulating factor, xylose reductase, P450 and the like. Examples of the nucleoside include inosine, guanosine, inosinic acid, guanylic acid, adenylic acid and the like. Examples of vitamins include riboflavin, thiamine, and ascorbic acid. Examples of the sugar include xylose and mannose, examples of the sugar alcohol include xylitol and mannitol, and examples of the alcohol include ethanol. Examples of the isoprenoids include mevalonate (MVA), isoprenol, astaxanthin, isoprene, isopentenol, limonene, pinene, farnesene, and bisabolene. Examples of the lipid include propionic acid, hydroxypropionic acid, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Among useful substances in the present invention, acetic acid is particularly preferable for organic acids, glutathione for peptides, mevalonic acid and isoprenol for isoprenoids, and hydroxypropionic acid for lipids.

方法 A method for isolating a useful substance from a culture solution containing the product is not particularly limited, and a method known per se or any method to be developed in the future can be applied.

In order to deepen the understanding of the present invention, the contents of the present invention will be specifically described with reference to examples and experimental examples, but it is apparent that the present invention is not limited to the description contents of these examples and the like. is there.

(Example 1) Preparation of transformed Escherichia coli (dR expression strain) Example 1 shows a method of preparing deltarhodopsin (dR) transformed Escherichia coli. In this example, transformed Escherichia coli capable of expressing the following delta-rhodopsin (dR) was prepared in Escherichia coli MG1655 (DE3) strain (Journal of Bioscience and Bioengineering Vol. 123 No. 2, 177 e182, 2017). The transformed Escherichia coli prepared in this example is hereinafter referred to as “dR-expressing strain 1”.

In this example, deltarhodopsin (dR) expressed in E. coli has the amino acid sequence shown in SEQ ID NO: 1 below.
MCCAALAPPMAATVGPESIWLWIGTIGMTLGTLYFVGRGRGVRDRKMQEFYIITIFITTIAAAMYFAMATGFGVTEVMVGDEALTIYWARYADWLFTTPLLLLDLSLLAGANRNTIATLIGLDVFMIGTGAIAALSSTPGTRIAWWAISTGALLALLVVLVGTLSENARNRVLVTFLL

The amino acid sequence, using the GeneArt ^(R) GeneOptimizer ^(R) program (ThermoFisher), codon-optimized for E. coli, obtaining the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 2 below (gene).
(SEQ ID NO: 2)

Recognition sites for restriction enzymes NdeI and KpnI were added to both ends of the sequence shown in SEQ ID NO: 2 and inserted into the pCDFDuet ^™ -1 vector (Novagen). Escherichia coli MG1655 (DE3) was transformed and introduced into cells. Expression strain 1 was prepared.

(Example 2) Preparation of transformed Escherichia coli (pR expression strain) In Example 2, as in Example 1, transformed Escherichia coli capable of expressing the following proteorhodopsin (pR) in Escherichia coli MG1655 (DE3) was prepared. did. The transformed Escherichia coli prepared in this example is hereinafter referred to as “pR expression strain 1”.

In this example, proteorhodopsin (pR) expressed in Escherichia coli has the following amino acid sequence shown in SEQ ID NO: 3 (see Biochimica et Biophysica Acta 1777: 2008, 504-513).
MKLLLILGSVIALPTFAAGGGDLDASDYTGVSFWLVTAALLASTVFFFVERDRVSAKWKTSLTVSGLVTGIAFWHYMYMRGVWIETGDSPTVFRYIDWLLTVPLLICEFYLILAAATNVAGSLFKKLLVGSLVMLVFGYMGEAGIMAAWPAFIIGCLAWVYMIYNVLAGSVNGYNAVLAGSVN

The amino acid sequence, using the GeneArt ^(R) GeneOptimizer ^(R) program (ThermoFisher), codon-optimized for E. coli, obtaining the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 4 below (gene).
(SEQ ID NO: 4)

Recognition sites for the restriction enzymes NdeI and KpnI were added to both ends of the sequence shown in SEQ ID NO: 4 and inserted into the pCDFDuet ^™ -1 vector (Novagen). Escherichia coli MG1655 (DE3) was transformed and introduced into cells. Expression strain 1 was prepared.

(Example 3) Culture of transformed Escherichia coli Example 3 shows a method of culturing the transformed Escherichia coli (dR-expressing strain 1, pR-expressing strain 1) prepared in Examples 1 and 2.

Each transformed E. coli was pre-cultured in LB medium (Luria-Bertani medium) at 37 ° C. with stirring at 200 rpm, and then in M9 medium containing glucose (4-8%) or glycerol (8%) as a carbon source. Main culture was performed at 37 ° C. and 200 rpm. The medium contains an appropriate amount of streptomycin, and the same applies to the culture in each of the following Examples and Experimental Examples. Growth of transformed E. coli, the culture medium containing the E. coli cells and the absorbance of OD ₆₀₀ ultraviolet-visible spectrophotometer; by measuring in (UVmini-1240 Shimadzu), it was confirmed from the cell concentration. After culturing until the cell concentration (OD ₆₀₀ ) reaches 0.6, 0.025 to 1 mM isopropyl-β-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside) and 10 μM all-trans-retinal (all-trans- Retinal) was added for induction expression, and the cells were cultured at 37 ° C. at 50 to 200 rpm for 24 hours under light conditions under light irradiation of 0 to 135 μmol / m ² / s.

Preculture medium LB medium volume 5 ml
Addition amount of transformed Escherichia coli to culture medium Inoculate one colony from LB agar plate Temperature 37 ℃
Incubation time 15 hours without light irradiation

Main culture medium M9 medium containing glucose (4-8%) or glycerol (8%) as carbon source 100 ml
Add 2 ml of transformed E. coli to the medium
Temperature 37 ℃
Incubation time 24 hours stirring 50-200 rpm
Light irradiation 0-135 μmol / m ² / s

(Experimental example 3-1) Proton transport activity The proton transport activity of the above-described transformed Escherichia coli (dR expression strain 1) was confirmed.
The culture solution (100 ml) containing dR-expressing strain 1 was centrifuged, the supernatant was removed, and 5 to 10 ml of physiological saline (100 mM NaCl) was added to the obtained E. coli cells and equilibrated for 10 minutes. This was repeated three times to obtain a sample for measuring proton transport activity.

Using a halogen lamp projector (JCD100V-300WL), the sample prepared above was irradiated with light having a light intensity of 50, 70, 200 and 1500 μmol / m ² / s, and the change in pH caused by ion transport of rhodopsin was measured. It was measured using a meter (F-72; HORIBA). The proton transport activity of the transformed Escherichia coli (dR-expressing strain 1) was confirmed by the change in pH, and it was confirmed whether the transformed Escherichia coli (dR-expressing strain 1) was a light-driven bacterium. The fact that the pH change was due to the rhodopsin proton pump activity was confirmed by adding the proton ionophore CCCP (Carbonyl Cyanide3-Chlorophenylhydrazone) to the solution. The results are shown in FIG.

(4) The rate of pH decrease in the sample prepared above increased with the increase in the amount of irradiated light, confirming that delta-rhodopsin transported protons extracellularly in a light-dependent manner.

(Experimental example 3-2) Measurement of ATP In this experimental example, for the transformed Escherichia coli (dR expression strain 1) cultured in Example 3, ATP production in Escherichia coli cells due to differences in light irradiation was confirmed. The sample prepared in the same manner as the sample for measuring the proton transport activity in Experimental Example 3-1 was irradiated with light of 25, 50 and 100 μmol / m ² / s using a halogen lamp projector (JCD100V-300WL). . In the examples and thereafter, the dark condition means 0.02 μmol / m ² / s or less.

The culture solution (100 ml) containing the dR-expressing strain 1 was centrifuged at 3000 g for 5 minutes at room temperature, and the supernatant was removed to give 1 ml of 100 ml of 100 mM Tris-HCl, 1 mM EDTA (pH 7.5). Was added, and the mixture was centrifuged again to remove the supernatant. 500 μl of 100 mM Tris-HCl, 1 mM EDTA (pH 7.5) was added to E. coli cells, and transferred to a cell disruption tube. Zirconia beads having a diameter of 0.6 mm were added, and the cells were crushed at 1500 rpm for 0 minutes using a cell crusher (shake master neo). The Escherichia coli cell solution after cell disruption was centrifuged at 16000 g for 20 minutes at 4 ° C., and the supernatant was used as an ATP measurement sample.

ATP production was measured by a luciferase luminescence method using a commercially available ATP Bioluminescent Assay Kit (FLAA-1KT; Sigma) according to the method described in the manual. It was confirmed that the ATP production increased according to the amount of light irradiation (FIG. 2).

(Experimental example 3-3) Measurement of metabolites by transformed Escherichia coli In this experimental example, production of metabolites by the transformed Escherichia coli (dR expression strain 1) cultured in Example 3 was confirmed. In the present experimental example, light with a light amount of 50 μmol / m ² / s was irradiated as a bright condition, and 0.02 μmol / m ² / s or less in a dark condition.

The culture solution (100 ml) containing the dR expression strain 1 was centrifuged at 15000 g at 4 ° C. for 5 minutes, and the supernatant was collected, filtered through a 0.45 μm filter (Millex HV; Merck KGAA), and stored at -30 ° C. The resulting sample was used as a culture solution sample. Next, an equal amount of an internal standard solution (5 mM isobutyric acid) was mixed with a culture solution sample to prepare a metabolite measurement sample.

The sample prepared above was analyzed by high performance liquid chromatography (HPLC; Shimadzu). HPLC was operated under the following conditions to measure central metabolites including acetic acid.
・ Column: Aminex HPX-87X column (Bio-Rad), Column temperature: 65 ℃
And separating the solvent: 1.5 mM H ₂ SO _4, flow rate: 0.5 ml / min Detector: UV / vis detector (SPD-20A; Shimadzu), RI detector (RID-10A; Shimadzu)
・ Detection wavelength: 210 nm

The analysis results are shown in FIG. As a result, the production rate of most metabolites from glucose as a carbon source was almost the same under light and dark conditions, but the carbon flow to the TCA cycle decreased under light conditions compared to dark conditions. And the carbon flow to acetic acid increased. The growth rate of Escherichia coli cells under light and dark conditions was almost the same, but under light conditions glucose consumption decreased during the metabolic cycle, and as a result, acetic acid production increased by about 1.3 times under light conditions. Was done.

(Experimental example 3-4) Glutathione production by transformed Escherichia coli 1
In this experimental example, the production of glutathione (GSH) by the transformed Escherichia coli (dR expression strain 1) cultured in Example 3 was confirmed. In the present experimental example, light with a light amount of 50 μmol / m ² / s was irradiated as a bright condition, and 0.02 μmol / m ² / s or less in a dark condition.

FIG. 4 shows the E. coli cell concentration (OD ₆₀₀ ) after 24 hours of culture. When cultured at 200 rpm with agitation using glucose as a carbon source under aerobic conditions, the value was 1.37 in the light condition and 1.14 in the dark condition. When glycerol was used as a carbon source and cultured aerobically with stirring at 200 rpm, the value was 2.54 under light conditions and 2.28 under dark conditions. When anaerobically cultured at 50 rpm using glycerol as a carbon source, the value was 1.13 in the light condition and 2.28 in the dark condition.

(4) The culture solution after 24 hours of culture was collected, and Escherichia coli cells (dR expression strain) from which the culture solution supernatant was removed by centrifugation were washed with 100 mM Tris-HCl (pH 7.5) and 2 mM EDTA. Thereafter, appropriate amounts of 100 mM Tris-HCl (pH 7.5) and 2 mM EDTA were added to prepare a cell suspension. Next, zirconia beads (diameter = 0.6 mm) were added to the cell suspension, and the cells were crushed for 10 minutes at 1500 rpm using a cell crusher (shake master Neo; Biomedical Science). A sample from which cell debris was removed by centrifugation was used as a sample for GSH measurement.

The amount of GSH was measured by a coloration method using DTNB (5-5′-dithiobis [2-nitrobenzoic acid]) using a commercially available GSH measurement kit (Dojindo Chemical).

G The GSH concentration per cell after 24 hours of culture is shown in the upper part of FIG. When glucose was used as a carbon source and cultured aerobically with stirring at 200 rpm, the concentration was 0.221 mg / l / OD under light conditions and 0.117 mg / l / OD under dark conditions. When glycerol was used as a carbon source and cultured aerobically with stirring at 200 rpm, the light condition was 1.18 mg / l / OD and the dark condition was 0.60 mg / l / OD. When anaerobic cultivation was performed at 50 ° rpm using glycerol as a carbon source, it was 4.01 mg / l / OD under light conditions and 2.52 mg / l / OD under dark conditions. The GSH concentration per culture solution after 24 hours of culture is shown in the lower part of FIG. When glucose was used as a carbon source and cultured aerobically with stirring at 200 rpm, the concentration was 0.302 mg / l under light conditions and 0.135 mg / l under dark conditions. When glycerol was used as a carbon source and cultured aerobically with stirring at 200 rpm, the concentration was 2.17 mg / l in the light condition and 1.55 mg / l in the dark condition. When anaerobically cultured at 50 ° rpm using glycerol as a carbon source, the concentration was 4.53 mg / l in the light condition and 2.07 mg / l in the dark condition.

(Example 3-5) Glutathione production 2 by transformed E. coli
In this experimental example, production of glutathione (GSH) by the transformed Escherichia coli (pR expression strain 1) cultured in Example 3 was confirmed.

In the present experimental example, light with a light amount of 50 μmol / m ² / s was irradiated as a bright condition, and 0.02 μmol / m ² / s or less in a dark condition. The cells were cultured aerobically at 150 rpm with glucose as a carbon source. In the same manner as in Experimental Example 3-4, a sample for GSH measurement was prepared for Escherichia coli cells (pR expression strain 1), and the amount of GSH was determined using a commercially available GSH measurement kit (Dojindo) using DTNB (5-5'-dithiobis). [2-nitrobenzoic acid]). The GSH concentration per cell after 24 hours of culture is shown in FIG. The light condition was 0.91 mg / l / OD, and the dark condition was 0.54 mg / l / OD.

(Example 4) Preparation of transformed Escherichia coli (dR expression strain 2) In Example 4, a nucleic acid consisting of the nucleotide sequence specified by SEQ ID NO: 2 was introduced into Escherichia coli MG1655 (DE3) in the same manner as in Example 1, Transformed E. coli (dR expression strain 2) was prepared. PCOLADuet-1-mvaES and pBbS7a-dR were introduced into Escherichia coli MG1655 (DE3) to prepare an MVADR strain, which was designated as dR-expressing strain 2. Hereinafter, a method for preparing dR expression strain 2 will be described.

According to the method described in Journal of Bioscience and Bioengineering 124: 2017, 177-182, pCOLADuet-1-mvaES attaches recognition sites for the restriction enzymes NdeI and KpnI to the NdeI-XhoI site of MCS2 of pCOLADuet-1 (Merck KGaA). It was prepared by introducing the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, which were codon-optimized. mvaE and mvaS are both mevalonate synthases.

Nucleotide sequence of codon-optimized gene encoding mvaE
(SEQ ID NO: 5)

Nucleotide sequence of codon-optimized gene encoding mvaS
(SEQ ID NO: 6)

pBbS7a-dR was prepared by treating pBbS7a-RFP (Plasmid # 35313; Addgene) with restriction enzymes NdeI and KpnI, and replacing the RFP gene of pBbS7a-RFP with the Rd gene consisting of the nucleotide sequence specified by SEQ ID NO: 2. . The purified plasmid was treated with AvrII and SacI, and replaced with the replication origin SC101 of BbS47a-RFP (Plasmid ## 35301; #Addgene). Escherichia coli MG1655 (DE3) was transformed with the above plasmid to prepare dR expression strain 2.

(Experimental example 4-1) Culture method of transformed Escherichia coli (dR expression strain 2) and production of mevalonic acid The dR expression strain 2 prepared above was subjected to preculture and main culture under the following culture conditions. In this experimental example, production of mevalonic acid, which is a metabolite of dR expression strain 2, was also confirmed. The metabolic pathway of mevalonic acid is as shown in FIG.

In the preculture, 20 ml of M9 medium containing 4 g / L glucose was used as a carbon source, and the cells were cultured for 20 hours at an initial cell concentration of OD ₆₆₀ = 0.05, a temperature of 37 ° C., and a stirring of 165 rpm. At this time, no light irradiation was performed.

In the main culture, 40 ml of M9 medium containing 4 g / L glucose was used as a carbon source, the initial cell concentration was OD ₆₆₀ = 0.05, the temperature was 37 ° C., the stirring was 165 rpm, and light irradiation (50 μmol / m ² / s) was performed under light conditions. After culturing for nearly 4 hours, after the cell concentration reached OD ₆₆₀ = 0.5, 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and 10 μM retinal were added. Further, at 6 and 7 hours after the start of the culture, 5 μM retinal was added.

The culture solution (100 ml) containing the dR expression strain 2 was centrifuged at 15000 g at 4 ° C. for 5 minutes, and the supernatant was collected, filtered through a 0.45 μm filter (Millex HV; MerckAKGaA), and stored at -30 ° C. This was used as a mevalonic acid measurement sample. The mevalonic acid concentration was measured by high performance liquid chromatography (HPLC).

The sample prepared above was analyzed by high performance liquid chromatography (HPLC; Shimadzu). HPLC was operated under the following conditions to measure central metabolites including mevalonic acid.
・ Column: Aminex HPX-87X column (Bio-Rad), Column temperature: 65 ℃
And separating the solvent: 1.5 mM H ₂ SO _4, flow rate: 0.5 ml / min Detector: UV / vis detector (SPD-20A; Shimadzu), RI detector (RID-10A; Shimadzu)
・ Detection wavelength: 210 nm

FIG. 8 shows the measurement results of the cell concentration and the mevalonic acid concentration. The production rate of mevalonic acid was 0.64 ± 0.07 mmol / g / h when cultured under light conditions, and 0.49 ± 0.020.0mmol / g / h when cultured under dark conditions. The production rate of mevalonate is determined by the cell concentration during the phase in which the cell concentration and the mevalonate concentration increase linearly (culture time 6 to 7.5 hours) and the amount of mevalonate increased per time (1.5 hours) Was obtained. Mevalonic acid is a very important compound as an intermediate hub compound of isoprenoids, which are natural pigments having various structures and physiological functions, and pharmaceutical intermediates.

(Example 5) Preparation of transformed Escherichia coli (dR expression strain 3) In Example 5, a nucleic acid having the nucleotide sequence specified by SEQ ID NO: 2 was introduced into Escherichia coli MG1655 (DE3) as in Example 1, Transformed E. coli (dR expression strain 3) was prepared. E. coli MG1655 (DE3) was introduced with pCOLADuet-1-mvaES-nudF into which an ADP-ribose pyrophosphatase expression gene was inserted, pMevB and pBbS7a-dR, and an IPDR strain was prepared to be dR expression strain 3.

Hereinafter, a method for preparing dR expression strain 3 will be described. As an ADP-ribose pyrophosphatase expression gene, a nudF sequence (SEQ ID NO: 7) obtained from the genome of Bacillus subtilis was used. pCOLADuet-1-mvaES-nudF was prepared by introducing a nudF sequence into the Nco1-BamH1 site of MCS1 of pCOLADuet-1-mvaES prepared in Example 4.

Nucleotide sequence of codon-optimized gene encoding nudF
(SEQ ID NO: 7)

PMevB was obtained from Plasmid # 17819 (Addgene). pBbS7a-dR was produced in the same manner as in Example 4. Escherichia coli MG1655 (DE3) was transformed using the above plasmid to prepare dR expression strain 3.

(Experimental example 5-1) Culture method of transformed Escherichia coli (dR expression strain 3) and production of isoprenol The dR expression strain 3 prepared above was subjected to preculture and main culture under the following culture conditions. In this experimental example, the production of isoprenol, a metabolite of dR-expressing strain 3, was also confirmed. The metabolic pathway of isoprenol is as shown in FIG.

In the pre-culture, 100 ml of LB medium was used, and the cells were cultured for about 18 hours at an initial cell concentration of OD ₆₆₀ = 0.1, a temperature of 37 ° C., and agitation at 165 rpm. At this time, no light irradiation was performed. Escherichia coli cells were collected from the culture solution by centrifugation (7000 g, 25 ° C., 4 minutes).

In the main culture, 50 ml of M9 medium containing 4 g / L glucose was used as a carbon source, and the cell concentration was OD ₆₆₀ = 5, temperature 37 ° C, stirring 165 rpm, and light irradiation (50 μmol / m ² / s) under bright conditions did. 0.1 mM IPTG and 10 μM retinal were added at the start of the main culture.

The culture solution (100 ml) containing dR-expressing strain 3 was centrifuged at 15000 g at 4 ° C. for 5 minutes, and the supernatant was collected, filtered through a 0.45 μm filter (Millex HV; Merck KKGA), and stored at -30 ° C. The resulting sample was used as a culture solution sample. Next, an equal amount of an internal standard solution (0.1% 3-methyl-1-butanol) was mixed with a culture solution sample to prepare a sample for isoprenol measurement.

Isoprenol was measured by GC (Gas Chromatography) -FID (Flame Ionization Detector, flame ionization type detector). The GC-FID was operated under the following conditions, and isoprenol was measured.
・ Column: Stabiliwax (length 60 m, inner diameter 0.32 mm, film thickness 1 μm) (Restek)
・ Carrier gas: Helium ・ Rising temperature condition: 70 ℃ (3 min) ～ 10 ℃ / min ～ 200 ℃
・ Injector temperature and detection temperature: 250 ℃

FIG. 9 shows the measurement results of the cell concentration, glucose concentration, and isoprenol concentration. The isoprenol yield was calculated from the changes in the isoprenol concentration and the glucose concentration at the 0th and 10th hours of the culture. As a result, after 10 hours of culture, the concentration was 1.78 ± 0.14 ° C.-mol で% under the bright condition, and 1.17 ± 0.10 ° C-mol% under the dark condition. Isoprenol has an energy density (35 MJ / L) about 1.8 times that of ethanol (19.6 MJ / L), and is expected as an alternative fuel to gasoline (32 MJ / L). Also, isoprenol can be converted into general chemical raw materials such as isoprene by a chemical process. Isoprene is a raw material for synthetic rubber.Since synthetic rubber produces 15 million tons worldwide and 1,68,000 tons in Japan, it is expected to form a large market as a synthetic petroleum alternative biomaterial. You.

(Example 6) Preparation of transformed Escherichia coli (dR expression strain 4) In Example 6, a nucleic acid consisting of the nucleotide sequence specified by SEQ ID NO: 2 was introduced into Escherichia coli MG1655 (DE3) as in Example 1, Transformed E. coli (dR expression strain 4) was prepared. PETM6-mcrNC and pBbS7a-dR were introduced into Escherichia coli MG1655 (DE3) to prepare an MVADR strain, which was designated as dR-expressing strain 4. Hereinafter, a method for preparing dR expression strain 4 will be described.

According to the method described in Metabolic Engineering 52: 2019, 215-223, pETM6-mcrNC is an N-terminal side (mcrN, amino acids 1 to 549) and a C-terminal side (mcrC, amino acid number 550) of Malonyl-CoA reductase derived from Chloroflexus aurantiacus. -1219), which was prepared by introducing a codon-optimized sequence for E. coli. pBbS7a-dR was produced in the same manner as in Example 4. Escherichia coli MG1655 (DE3) was transformed with the above plasmid to prepare dR expression strain 4.

(Experimental example 6-1) Culture method of transformed Escherichia coli (dR expression strain 4) and production of 3HP The dR expression strain 4 prepared above was subjected to preculture and main culture under the following culture conditions. In this experimental example, the production of 3HP (3-hydroxypropionic acid), which is a metabolite of dR-expressing strain 4, was also confirmed. The metabolic pathway of 3HP is as shown in FIG.

In the pre-culture, 50 ml of M9 medium containing 10 g / L glucose was used as a carbon source, and the cells were cultured at an initial cell concentration of OD ₆₆₀ = 0.05, a temperature of 37 ° C., and a stirring of 150 rpm. At this time, no light irradiation was performed.

Using M9 medium 50 ml of containing 4g / L glucose as carbon source in the culture, the initial cell concentration OD ₆₆₀ = 0.05, temperature 37 ° C., with stirring 150 rpm, the light irradiation in the bright condition (50μmol / m ² / s) to about The cells were cultured for 4.5 hours. After reaching a cell concentration of OD ₆₆₀ = 0.5, 0.1 mM IPTG and 10 μM retinal were added. Further, at 6 and 8 hours after the start of the culture, 10 μM retinal was added.

The culture solution (100 ml) containing the dR expression strain 4 was centrifuged at 15000 g at 4 ° C. for 5 minutes, and the supernatant was collected, filtered through a 0.45 μm filter (Millex HV; Merck KGaA), and stored at -30 ° C. This was used as a sample for measuring mevalonic acid. 3HP was measured by high performance liquid chromatography (HPLC).

The sample prepared above was analyzed by high performance liquid chromatography (HPLC; Shimadzu). HPLC was operated under the following conditions to measure central metabolites including 3HP.
・ Column: Aminex HPX-87X column (Bio-Rad), Column temperature: 65 ℃
And separating the solvent: 1.5 mM H ₂ SO _4, flow rate: 0.5 ml / min Detector: UV / vis detector (SPD-20A; Shimadzu), RI detector (RID-10A; Shimadzu)
・ Detection wavelength: 210 nm

The measurement results of the cell concentration and the 3HP concentration are shown in FIG. The production rate of 3HP was 1.2 ± 0.05 mmol / g / h when cultured under light conditions, whereas it was 1.0 ± 0.09 mmol / g / h when cultured under dark conditions. The 3HP production rate is calculated by converting the cell concentration between the cell concentration and the phase in which the 3HP concentration increases linearly (culture time 6 to 10 hours) and the amount of 3HP increased per time (4 hours). I got it. 3HP and its esters are useful compounds as raw materials for aliphatic polyesters, and polyesters synthesized therefrom have attracted attention as biodegradable polyesters. For this reason, it also leads to the solution of the microplastic problem, which is concerned about recent marine pollution and adverse effects on the human body.

As described in detail above, non-photosynthetic prokaryotes in which a gene encoding rhodopsin has been introduced and expressed by genetic engineering become a new light-driven energy-regenerating useful substance production host that can use light energy. It is advantageous in gaining.

(4) In the production of useful substances using ordinary non-photosynthetic prokaryotes, the productivity is usually low because non-photosynthetic prokaryotes do not have the purpose of producing useful substances. Research is being conducted to improve metabolic engineering of non-photosynthetic prokaryotes such as Escherichia coli to improve fermentation productivity of various useful substances such as pharmaceuticals, foods, cosmetics, fuels, and polymer raw materials. However, as the productivity of the target substance increases, the microorganisms often fall into a deficiency in intracellular energy, and often face a decrease in growth and a peak in productivity of the target substance. This is the process of distributing the carbon source, which is the input of the fermentation itself, into three outputs: “cell material”, “production of target product”, and “intracellular energy”. It is thought that the root cause exists in the off relationship. Thus, the technology of the present invention can separate "intracellular energy" from the fermentation process in the three-phase state, and imparts photophosphorylation ability to non-photosynthetic prokaryotes that originally cannot use light energy, This is an advantage in that it can be a new light-driven energy-renewable useful material production host that makes energy available.

As described above, the technology of the present invention can improve the productivity of useful substances because light can be used as an energy source in addition to the conventional energy sources. As a competing technology, there is an energy-saving microorganism (see JP-A-2017-55777). In this technique, by deleting genes that perform basal metabolism, the energy of basal metabolism can be deleted, and the energy of producing useful substances can be improved. However, there is a limit to the saving of basal metabolic energy, and it is assumed that the growth speed is slowed by reducing the energy of basal metabolism, and the improvement in productivity seems to be limited.

微細 In addition, microalgae (eg, Euglena) utilized for producing useful substances other than microorganisms such as Escherichia coli can also compete for producing useful substances. In addition, microalgae can originally utilize light energy, so they are not the destination to provide this technology. However, microalgae have a slow growth rate, and it is considered that practical use as a host for producing substances is difficult when compared with non-photosynthetic prokaryotes such as Escherichia coli. Therefore, according to the technology of the present invention, which imparts photophosphorylation ability to non-photosynthetic prokaryotes to produce microorganisms capable of utilizing light energy, light can be used as an energy source in addition to conventional energy sources, and therefore, a useful substance. Can be improved in productivity. Useful substances include substances that can be produced by utilizing the fermentation process of microorganisms, such as proteins, peptides, amino acids, nucleic acids, vitamins, sugars, sugar alcohols, alcohols, organic acids, isoprenoids, bioactive low molecular weight compounds and And lipids. Particularly useful substances in the present invention are glutathione as a peptide and acetic acid as an organic acid.

On the other hand, useful substances produced by the method of the present invention can be effectively used as raw materials for pharmaceuticals, foods, cosmetics, fuels, polymers, and the like, and have extremely excellent industrial applicability.

Claims

A method for producing a useful substance, which comprises culturing a non-photosynthetic prokaryote in which a gene encoding rhodopsin is introduced and expressed by genetic engineering under light irradiation.
Non-photosynthetic prokaryotes expressed by introducing the gene encoding rhodopsin by genetic engineering, the production of useful substances when cultured under light irradiation, the production of useful substances when cultured in the dark The method for producing a useful substance according to claim 1, which is a non-photosynthetic prokaryote exhibiting a higher production amount.
The method for producing a useful substance according to claim 1 or 2, wherein the non-photosynthetic prokaryote is a non-photosynthetic bacterium.
The non-photosynthetic prokaryote is any one selected from Escherichia coli, lactic acid bacteria, acetic acid bacteria, Bacillus subtilis, actinomycetes, coryneform bacteria, Pseudomonas bacteria, methanogens, and archaea. Method for producing useful substances.
The method for producing a useful substance according to any one of claims 1 to 4, wherein the non-photosynthetic prokaryote is Escherichia coli.
The method for producing a useful substance according to any one of claims 1 to 5, wherein the culture of the non-photosynthetic prokaryote under light irradiation is a culture under light irradiation of a light amount of 1 to 2000 μmol / m 2 / s.
A method for producing a useful substance derived from a culture product of Escherichia coli, comprising the following steps:
1) a step of introducing a gene encoding rhodopsin into E. coli;
2) a step of culturing the Escherichia coli into which the gene of the above 1) has been incorporated at 4 to 40 ° C. for 4 to 72 hours under light irradiation of 1 to 2000 μmol / m 2 / s;
3) a step of recovering the culture product of Escherichia coli cultured in the step 2) from the culture solution.
The method for producing a useful substance according to any one of claims 1 to 7, wherein the rhodopsin is bacteriorhodopsin or proteorhodopsin.
The method for producing a useful substance according to any one of claims 1 to 7, wherein the rhodopsin is delta-rhodopsin.
10. The method according to claim 1, wherein the useful substance is at least one selected from organic acids, peptides, amino acids, proteins, nucleic acids, vitamins, sugars, sugar alcohols, alcohols, isoprenoids, and lipids. A method for producing the useful substance described above.
The method for producing a useful substance according to any one of claims 1 to 9, wherein the useful substance is acetic acid and / or glutathione.
The method for producing a useful substance according to any one of claims 1 to 9, wherein the useful substance is one or more kinds selected from mevalonic acid, isoprenol, and 3-hydroxypropionic acid.