WO2020050113A1 - Procédé de fabrication d'une substance utile par l'utilisation d'un procédé de fermentation - Google Patents

Procédé de fabrication d'une substance utile par l'utilisation d'un procédé de fermentation Download PDF

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WO2020050113A1
WO2020050113A1 PCT/JP2019/033690 JP2019033690W WO2020050113A1 WO 2020050113 A1 WO2020050113 A1 WO 2020050113A1 JP 2019033690 W JP2019033690 W JP 2019033690W WO 2020050113 A1 WO2020050113 A1 WO 2020050113A1
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useful substance
producing
escherichia coli
culture
photosynthetic
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清敬 原
陽子 原
吉博 戸谷
史生 松田
健太郎 鎌田
涼 田中
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静岡県公立大学法人
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid

Definitions

  • 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.
  • Patent Document 1 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.
  • Patent Document 3 There is a method for producing a light-driven high-energy Saccharomyces submonum yeast (Patent Document 3).
  • dR Delta-rhodopsin
  • Patent Document 3 targets only Saccharomyces submonum yeast and requires that a nucleic acid (gene) encoding a mitochondrial localization signal besides rhodopsin be incorporated.
  • 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.
  • 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. 3.
  • the method of manufacturing the substance 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.
  • a 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 ⁇ mol / 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.
  • 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.
  • the useful substance is acetic acid and / or glutathione.
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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).
  • 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).
  • Example 3-4 It is a figure which shows the glutathione production result about the transformed Escherichia coli (pR expression strain 1).
  • 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 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • dR delta-rhodopsin
  • bacteriorhodopsin including archrhodopsin and crux rhodopsin
  • sensory rhodopsin I include archrhodopsin and crux rhodops
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • pH 2-9 preferably pH 4-9, more preferably pH 5-8
  • 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.
  • 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.
  • a culture may be collected and observed with a microscope, or may be observed with absorbance.
  • 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.
  • 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.
  • the culture medium for culturing the transformed non-photosynthetic prokaryote may be simply referred to as “culture solution”.
  • 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 ⁇ mol / m 2 / s, more preferably under light irradiation with a light amount of 10 to 200 ⁇ mol / 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.
  • 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.
  • 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.
  • 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.
  • the organic acid include acetic acid, lactic acid, and succinic acid.
  • Examples of the peptide include glutathione, alanylglutamine, and ⁇ -glutamyl valylglycine
  • examples of the polypeptide include polylysine and polyglutamic acid.
  • 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), ⁇ -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.
  • the nucleoside include inosine, guanosine, inosinic acid, guanylic acid, adenylic acid and the like.
  • vitamins include riboflavin, thiamine, and ascorbic acid.
  • sugar examples include xylose and mannose
  • examples of the sugar alcohol include xylitol and mannitol
  • examples of the alcohol include ethanol.
  • 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).
  • acetic acid is particularly preferable for organic acids, glutathione for peptides, mevalonic acid and isoprenol for isoprenoids, and hydroxypropionic acid for lipids.
  • 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.
  • Example 1 Preparation of transformed Escherichia coli (dR expression strain)
  • Example 1 shows a method of preparing deltarhodopsin (dR) transformed Escherichia coli.
  • 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”.
  • deltarhodopsin (dR) expressed in E. coli has the amino acid sequence shown in SEQ ID NO: 1 below.
  • Example 2 Preparation of transformed Escherichia coli (pR expression strain)
  • pR expression strain 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”.
  • 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).
  • SEQ ID NO: 3 see Biochimica et Biophysica Acta 1777: 2008, 504-513.
  • 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.
  • 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 °C Incubation time 15 hours without light irradiation
  • the sample prepared above was irradiated with light having a light intensity of 50, 70, 200 and 1500 ⁇ mol / 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.
  • Example 3-2 Measurement of ATP
  • 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 ⁇ mol / m 2 / s using a halogen lamp projector (JCD100V-300WL). .
  • the dark condition means 0.02 ⁇ mol / 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 ⁇ l 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).
  • Example 3-3 Measurement of metabolites by transformed Escherichia coli
  • production of metabolites by the transformed Escherichia coli (dR expression strain 1) cultured in Example 3 was confirmed.
  • light with a light amount of 50 ⁇ mol / m 2 / s was irradiated as a bright condition, and 0.02 ⁇ mol / 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 ⁇ m 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.
  • an internal standard solution 5 mM isobutyric acid
  • HPLC high performance liquid chromatography
  • Example 3-4 Glutathione production by transformed Escherichia coli 1
  • the production of glutathione (GSH) by the transformed Escherichia coli (dR expression strain 1) cultured in Example 3 was confirmed.
  • light with a light amount of 50 ⁇ mol / m 2 / s was irradiated as a bright condition, and 0.02 ⁇ mol / m 2 / s or less in a dark condition.
  • FIG. 4 shows the E. coli cell concentration (OD 600 ) after 24 hours of culture.
  • OD 600 E. coli cell concentration
  • 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.
  • 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.
  • 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.
  • Example 3-5 Glutathione production 2 by transformed E. coli
  • Glutathione production 2 by transformed E. coli
  • GSH glutathione
  • Example 4 Preparation of transformed Escherichia coli (dR expression strain 2)
  • 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.
  • dR expression strain 2 a method for preparing dR expression strain 2 will be described.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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).
  • HPLC high performance liquid chromatography
  • 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)
  • 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.
  • dR expression strain 3 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.
  • nudF 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.
  • 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.
  • the production of isoprenol, a metabolite of dR-expressing strain 3 was also confirmed.
  • the metabolic pathway of isoprenol is as shown in FIG.
  • 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 ⁇ m 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.
  • 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 ⁇ % 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).
  • 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)
  • 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.
  • dR expression strain 4 a method for preparing dR expression strain 4 will be described.
  • 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.
  • 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.
  • 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.
  • the initial cell concentration OD 660 0.05, temperature 37 ° C., with stirring 150 rpm, the light irradiation in the bright condition (50 ⁇ mol / m 2 / s) to about
  • 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 ⁇ m 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).
  • HPLC high performance liquid chromatography
  • Shimadzu HPLC was operated under the following conditions to measure central metabolites including 3HP.
  • 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.
  • 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.
  • 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.
  • 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).
  • JP-A-2017-55777 an energy-saving microorganism
  • the energy of basal metabolism can be deleted, and the energy of producing useful substances can be improved.
  • 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.
  • microalgae eg, Euglena
  • microalgae utilized for producing useful substances other than microorganisms such as Escherichia coli can also compete for producing useful substances.
  • microalgae can originally utilize light energy, so they are not the destination to provide this technology.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de production d'une substance utile par l'utilisation d'un procédé de fermentation de micro-organismes pouvant produire et fabriquer plus efficacement la substance utile. Ce procédé est basé sur la fourniture d'une capacité de photophosphorylation à des procaryotes non photosynthétiques par bio-ingénierie. En donnant la capacité de photophosphorylation de prokarkyotes non photosynthétiques par bio-ingénierie, des cellules qui ont une action d'énergie élevée entraînée par la lumière, tout en étant des procaryotes non photosynthétiques, sont obtenues. En utilisant ces procaryotes non photosynthétiques transformés, la substance utile peut être produite et fabriquée plus efficacement. Plus spécifiquement, la substance utile peut être fabriquée plus efficacement à l'aide d'un procédé de fermentation de micro-organismes par culture des procaryotes non photosynthétiques transformés, dans lesquels un gène qui code la rhodopsine a été introduit par génie génétique et a engagé l'expression, sous irradiation avec de la lumière.
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EP4163292A1 (fr) 2021-10-08 2023-04-12 Shizuoka Prefectural University Corporation Nouveaux mutants de rhodopsine

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WO2022044979A1 (fr) * 2020-08-24 2022-03-03 静岡県公立大学法人 Opsine exogène et cellules exprimant ladite rhodopsine
EP4163292A1 (fr) 2021-10-08 2023-04-12 Shizuoka Prefectural University Corporation Nouveaux mutants de rhodopsine

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