Method for producing organic compound

Abstract

The purpose of the present invention is to provide a technique for producing a useful substance by using microorganisms without competing with a food supply and without consuming large amounts of finite resources. A method for producing an organic compound, the method comprising a substance production step for, in a sugar-containing liquid containing a non-natural sugar, causing microorganisms to use the non-natural sugar as a substrate to produce a substance.

Description

Organic compound manufacturing method

[0001] The present invention relates to a technique for producing organic compounds using chemically synthesized sugars as materials. More specifically, the present invention relates to a technique for causing microorganisms to produce organic compounds using a sugar-containing liquid containing unnatural sugars as a substrate.

[0002] Sugars derived from agricultural crops are the most common substrates used for microbial production of useful substances. Such bioproduction processes have been shown to be more environmentally friendly, with lower greenhouse gas emissions than conventional petrochemical processes (Non-Patent Document 1). However, the Earth's biomass production capacity is limited by biophysical boundaries and cannot meet the enormous demand for fuel and chemical production (Non-Patent Document 2). Despite the limited availability of biomass compared to fossil fuels, approximately 13% of the world's agricultural land is currently used for biofuel and fiber production (Non-Patent Document 3). Therefore, expanding the use of industrial biomass could lead to competition with food demand (Non-Patent Document 3). Furthermore, industrial agriculture to meet this enormous demand has negative aspects, such as the massive consumption of vast amounts of land, exhaustible resources such as water, nitrogen, and phosphorus, water pollution due to eutrophication, and loss of biodiversity (Non-Patent Documents 3, 5). In fact, some have suggested that human material demands in agriculture could potentially lead to transgressing planetary boundaries (Non-Patent Document 6).

[0003] On the other hand, the formose reaction, a catalytic chemical synthesis method for sugars from formaldehyde, is known and has attracted attention as an upgrading technology for carbon dioxide and formaldehyde. In recent years, progress has been made in identifying formose reaction products (Non-Patent Document 7), selecting appropriate catalysts and reaction conditions (Non-Patent Document 8), and studying reaction kinetics (Non-Patent Document 9), leading to improved reaction selectivity. Formose reaction products contain branched sugar chains that do not exist in nature (Non-Patent Document 10) and are generally toxic to living organisms. In fact, there have been reported cases of rats dying from diet containing 50% synthetic sugar solution (Non-Patent Document 11).

[0004] WJ Groot, T. Boren, The International Journal of Life Cycle Assessment 2010, 15, 970-984K. H. Erb, C. Lauk, T. Kastner, A. Mayer, MC Theurl, H. Haberl, Nat Commun 2016, 7, 11382J. Poore, T. Nemecek, Science 2018, 360, 987-992.A. Muscat, EM de Olde, IJM de Boer, R. Ripoll-Bosch, Global Food Security 2020, 25.Y. Lu, S. Song, R. Wang, Z. Liu, J. Meng, AJ Sweetman, A. Jenkins, RC Ferrier, H. Li, W. Luo, T. Wang, Environ Int 2015, 77, 5-15J. A. Foley, N. Ramankutty, KA Brauman, ES Cassidy, JS Gerber, M. Johnston, ND Mueller, C. O'Connell, DK Ray, PC West, C. Balzer, EM Bennett, SR Carpenter, J. Hill, C. Monfreda, S. Polasky, J. Rockstrom, J. Sheehan, S. Siebert, D. Tilman, DP Zaks, Nature 2011, 478, 337-342.WE Robinson, E. Daines, P. van Duppen, T. de Jong, WTS Huck, Nat. Chem. 2022, 14, 623-631J. B. Lambert, S.A. Gurusamy-Thangavelu, K. Ma, Science 2010, 327, 984-986.P. van Duppen, E. Daines, W. E. Robinson, W. T. S. Huck, J. Am. Chem.Soc. 2023, 145, 7559-7568.RD Partridge, AH Weiss, D. Todd, Carbohydr. Res. 1972, 24, 29-44.T. Mizuno, AH Weiss, Adv. Carbohydr. Chem. Biochem. 1974, 29, 173-227.

[0005] To achieve sustainable production of useful substances using microorganisms, it is necessary to address the issues related to the supply of biomass-derived sugars as substrates (i.e., competition with food supply and large-scale consumption of finite resources) as mentioned above. Formose reaction products are also sugar mixtures, but whereas biomass-derived sugars consist of natural sugars, formose reaction products contain unnatural sugars and are biotoxic. Therefore, they are recognized as essentially unable to replace biomass-derived sugars.

[0006] Therefore, an object of the present invention is to provide a technology that enables useful substances to be produced by microorganisms without competing with the food supply and without requiring the large-scale consumption of finite resources.

[0007] The present inventors have unexpectedly discovered that formose reaction products, which should not be able to replace sugars derived from biomass, can be used as substrates for substance production using microorganisms, and have found that organic compounds can be produced in microorganisms by using unnatural sugars as substrates, which do not compete with the food supply and do not require the large-scale consumption of finite resources.

[0008] That is, the present invention provides the following aspects of the invention: Item 1. A method for producing an organic compound, comprising a substance producing step of causing a microorganism to produce a substance in a sugar-containing solution containing an unnatural sugar using the unnatural sugar as a substrate. Item 2. The production method according to Item 1, further comprising a step of culturing the microorganism prior to the substance producing step. Item 3. The production method according to Item 1, wherein the concentration of the microorganism in the sugar-containing solution is 0.5 to 25 w / v %. Item 4. The production method according to any one of Items 1 to 3, wherein the unnatural sugar comprises at least one of a triose to a hexose. Item 5. The production method according to any one of Items 1 to 4, wherein the unnatural sugar is selected from the group consisting of stereoisomers of 1,3,4,5-tetrahydroxypentan-2-one, 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one, and 1,2,4,5,6-pentahydroxyhexan-3-one. Item 6. Item 7. The method for producing a sugar-containing liquid according to any one of Items 1 to 5, wherein the sugar-containing liquid contains formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), and at least one of erythrose (C4a) and threose (C4a). Item 8. The method for producing a sugar-containing liquid according to any one of Items 1 to 7, wherein the sugar-containing liquid contains erythrulose (C4k) in an amount greater than 0 mM and not greater than 20 mM. Item 9. The method for producing a sugar-containing liquid according to any one of Items 1 to 7, wherein the sugar-containing liquid contains erythrose and / or threose (C4a) in a total amount greater than 0 mM and not greater than 25 mM. Item 10. The method according to any one of Items 1 to 8, wherein the sugar-containing liquid contains formaldehyde (C1a) in an amount greater than 0 mM and less than 0.6 mM, glycolaldehyde (C2a) in an amount greater than 0 mM and less than 4 mM, glyceraldehyde (C3a) in an amount greater than 0 mM and less than 7 mM, and / or dihydroxyacetone (C3k) in an amount greater than 0 mM and less than 4 mM. Item 10. The method according to any one of Items 1 to 9, wherein the sugar-containing liquid is a formose reaction product. Item 11. The method according to any one of Items 1 to 9, wherein the sugar-containing liquid is a base-treated formose reaction product. Item 12. The method according to any one of Items 1 to 11, wherein the total amount of sugar in the sugar-containing liquid is 0.1 to 60 w / v %. Item 13. The method according to any one of Items 1 to 12, wherein the organic compound comprises lactic acid and / or a salt thereof.Item 14. The production method according to any one of Items 1 to 13, wherein the organic compound comprises acetic acid, succinic acid, fumarate, and / or salts thereof. Item 15. The production method according to any one of Items 1 to 14, wherein the organic compound comprises β-glucan. Item 16. The production method according to any one of Items 1 to 15, wherein the microorganism is selected from the group consisting of bacteria, yeast, and microalgae.

[0009] The present invention makes it possible to produce useful substances using microorganisms without competing with the food supply and without requiring the consumption of large amounts of finite resources.

[0010] The photograph of the synthetic sugar solution, which is the formose reaction product, and the HPLC chromatogram and identification results of the formose reaction product are shown. (a) OD when C. glutamicum was cultured in a mineral medium containing the synthetic sugar solution. 600 The time course of OD was measured. N = 3, error bars indicate 95% confidence intervals. (b) HPLC chromatogram of the supernatant before cultivation (before cultivation) and after 57 hours of cultivation in a medium containing 0.5% (w / v) sugar (after cultivation). The OD was measured when various concentrations of C1a were added to a mineral medium containing 0.1% glucose. 600 The time course of OD2 was measured when various concentrations of C2a were added to a mineral medium containing 0.1% glucose. N = 3, error bars indicate 95% confidence intervals. 600 The time course of OD was measured when various concentrations of C3a were added to a mineral medium containing 0.1% glucose. N = 3, error bars indicate 95% confidence intervals. 600 The time course of OD was measured when various concentrations of C3k were added to a mineral medium containing 0.1% glucose. N = 3, error bars indicate 95% confidence intervals. 600 The time course of OD was measured when various concentrations of C4a were added to a mineral medium containing 0.1% glucose. N = 3, error bars indicate 95% confidence intervals. 600 The time course of OD was measured when various concentrations of C4k were added to a mineral medium containing 0.1% glucose. N = 3, error bars indicate 95% confidence intervals. 600The graph shows the time course of OD when 5% synthetic sugar solution was added to the mineral medium (Syn-sugar 5%) and when base-treated synthetic sugar solution was added (Base treatment). N=3, error bars indicate 95% confidence interval. 600The results are comparisons of the time course of the lactic acid fermentation and the control treatment. The results of the base treatment were normalized to the final growth value of a control experiment (0.1% glucose) performed with the same batch for comparison with the value of 5% syn-sugar. N = 3, error bars indicate 95% confidence intervals. (a) HPLC chromatograms of C5-C6 sugars in the supernatant immediately (0 h) and 24 h after lactic acid fermentation in the presence of C. glutamicum and with the addition of 1% (w / v) synthetic sugar (formose reaction product - Ca). (b) HPLC chromatograms of C5-C6 sugars in the supernatant immediately (0 h) and 24 h after lactic acid fermentation in the absence of C. glutamicum and with the addition of 1% (w / v) synthetic sugar (formose reaction product - Ca). HPLC chromatograms of the supernatant collected 24 hours after the start of lactic acid fermentation in the presence of C. glutamicum (a) with 1% (w / v) synthetic sugar (formose reaction product-Ca) added (w / syn-sugar) and without added sugar (w / o sugar) are shown. (b) Quantification of organic acids after the reaction based on HPLC analysis in (a). N = 3, error bars indicate 95% confidence intervals. Quantification of organic acids obtained by lactic acid fermentation in the presence of C. glutamicum using different concentrations of 1% (w / v) synthetic sugar (formose reaction product-W) is shown in the same manner as in Figure 6(b). HPLC chromatograms of C5-C6 sugars in the supernatant immediately (0 h) and 24 h after the start of the reaction in the presence of E. coli and with 1% (w / v) synthetic sugar (formose reaction product-Ca) added are shown. The figure shows HPLC chromatograms of C5-C6 sugars in the supernatant immediately (0 h) and 24 hours after the start of the reaction (24 h) in the presence of E. coli and with the addition of 1% (w / v) synthetic sugar (formose reaction product-W). The figure also shows HPLC chromatograms of the supernatant collected 24 hours after the start of the reaction in the presence of E. coli, with the addition of 1% (w / v) synthetic sugar (formose reaction product-Ca) (w / syn-sugar) and without the addition of sugar (w / o sugar).This figure shows HPLC chromatograms of the supernatant collected 24 hours after the start of the reaction in the presence of E. coli, with and without the addition of 1% (w / v) synthetic sugar (formose reaction product-W). This figure also shows HPLC chromatograms of C5-C6 sugars in the supernatant collected immediately (0 h) and 24 hours after the start of the reaction in the presence of S. cerevisiae, with and without the addition of 1% (w / v) synthetic sugar (formose reaction product-Ca). This figure also shows HPLC chromatograms of C5-C6 sugars in the supernatant collected immediately (0 h) and 24 hours after the start of the reaction in the presence of S. cerevisiae, with and without the addition of 1% (w / v) synthetic sugar (formose reaction product-W). (a) HPLC chromatograms of the supernatant collected 24 hours after the start of the reaction in the presence of S. cerevisiae, with 1% (w / v) synthetic sugar (formose reaction product-Ca) added (w / syn-sugar) and without added sugar (w / o sugar). (b) HPLC chromatograms of the supernatant collected 24 hours after the start of the reaction in the presence of S. cerevisiae, with 1% (w / v) synthetic sugar (formose reaction product-W) added (w / syn-sugar) and without added sugar (w / o sugar). (c) C1 sugar concentrations of C2-C4 sugars in the supernatant immediately (0 h) and 24 hours after the start of the reaction (24 h) in the presence of E. gracilis, with 1% (w / v) synthetic sugar (formose reaction product-Ca) added. (b) The C1 sugar-equivalent concentration of β-glucan in the supernatant collected 24 hours after the start of the reaction in the presence of E. gracilis, with 1 w / v% synthetic sugar (formose reaction product-Ca) added (w / sugar) and without added sugar (w / o sugar).

[0011] The method for producing an organic compound of the present invention is characterized by including a substance production step in which a microorganism is caused to produce a substance in a sugar-containing solution containing an unnatural sugar using the unnatural sugar as a substrate. The method for producing a glycoside of the present invention is described in detail below. In this specification, sugars with N carbon atoms (N is an integer of 2 or more) are referred to as CN (N-carbon sugar), and aldoses and ketoses are particularly referred to as CNa and CNk, respectively. Furthermore, for substances other than sugars, formaldehyde is also referred to as C1a.

[0012] 1. Substance Production Process 1-1. Sugar-Containing Liquid 1-1-1. Sugars Contained in the Sugar-Containing Liquid The sugars contained in the sugar-containing liquid containing unnatural sugars are not particularly limited and include C2 sugars, C3 sugars, C4 sugars, C5 sugars, C6 sugars, C7 sugars, C8 sugars, etc., and the liquid may contain any one of these sugars or a combination of two or more of them.

[0013] A sugar-containing liquid containing unnatural sugars typically contains sugar products obtained by chemical synthesis. Many known sugar products obtained by chemical synthesis exist, and in the present invention, any known sugar product containing an unnatural sugar can be used without particular limitation. Preferred examples of sugars contained in the sugar product in the present invention include the following C2 sugars (the compounds shown in (C2-1) and (C2-2)); C3 sugars (the compounds shown in (C3-1) to (C3-3)); C4 sugars (the compounds shown in (C4-1) to (C4-5)); C5 sugars (the compounds shown in (C5-1) to (C5-9)); and C6 sugars (the compounds shown in (C6-1) to (C6-18). The following compounds include any stereoisomers (e.g., enantiomers (D-sugars, L-sugars), and racemates thereof).

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] The unnatural sugar preferably includes at least one of C3 sugars, C4 sugars, C5 sugars, and C6 sugars, more preferably C5 sugars and / or C6 sugars, and even more preferably linear C5 sugars such as 1,3,4,5-tetrahydroxypentan-2-one (the compound shown in (C5-7) above), branched C5 sugars such as 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one (the compound shown in (C5-5) above), branched C6 sugars such as 1,2,4,5,6-pentahydroxyhexan-3-one (the compound shown in (C6-3) above), and any stereoisomers thereof. Unnatural sugars also include L-sugars. These unnatural sugars may be used alone or in combination of two or more. In the present invention, at least one of these unnatural sugars can be used as a substrate for substance production.

[0020] The sugar-containing liquid may contain natural sugars in addition to unnatural sugars. Natural sugars may have the same structure as sugars found in nature, and may be derived from natural products or produced by chemical synthesis. Examples of natural sugars include formaldehyde (C1a), glycolaldehyde (C2a, the compound shown in (C2-2) above), glyceraldehyde (C3a, the compound shown in (C3-3) above), dihydroxyacetone (C3k, the compound shown in (C3-1) above), erythrose or threose (C4a, the compound shown in (C4-1) above), erythrulose (C4k, the compound shown in (C4-2) above), and glucose (the compound shown in (C6-4) above). These natural sugars may be used alone or in combination of two or more. In the present invention, at least one of these natural sugars may be used as a substrate for substance production.

[0021] 1-1-2. Other Components Contained in the Sugar-Containing Solution In addition to sugar, the sugar-containing solution may contain at least one of formaldehyde (C1a), sugar alcohols, and sugar carboxylic acids. These sugar alcohols and sugar carboxylic acids may also be known compounds contained in sugar products obtained by chemical synthesis (e.g., those reported in Life 2020, 10(8), 125; https: / / doi.org / 10.3390 / life10080125).

[0022] 1-1-3. Sugar Content When the sugar-containing liquid contains at least one of formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), and erythrose and threose (C4a), the concentration of the sugar in the sugar-containing liquid is not particularly limited, but from the viewpoint of improving the growth environment for microorganisms, the following concentrations are preferred.

[0023] Formaldehyde (C1a): preferably more than 0 mM and not more than 0.6 mM, more preferably more than 0 mM and not more than 0.5 mM, even more preferably more than 0 mM and not more than 0.4 mM, even more preferably more than 0 mM and not more than 0.2 mM, or more than 0 mM and not more than 0.1 mM. Glycolaldehyde (C2a): preferably more than 0 mM and not more than 4 mM, more preferably more than 0 mM and not more than 2 mM, even more preferably more than 0 mM and not more than 1 mM, and even more preferably more than 0 mM and not more than 0.5 mM. Glyceraldehyde (C3a): preferably more than 0 mM and not more than 7 mM, more preferably more than 0 mM and not more than 6 mM, more preferably more than 0 mM and not more than 5 mM, more than 0 mM and not more than 2 mM, more preferably more than 0 mM and not more than 1 mM, or more than 0 mM and not more than 0.5 mM. Dihydroxyacetone (C3k): preferably more than 0 mM and not more than 4 mM, more preferably more than 0 mM and not more than 3 mM, and even more preferably more than 0 mM and not more than 2 mM. Erythrulose (C4k): preferably more than 0 mM and not more than 20 mM, more preferably more than 0 mM and not more than 20 mM, even more preferably more than 0 mM and not more than 10 mM Erythrose and / or threose (C4a): preferably more than 0 mM and not more than 25 mM, more preferably more than 0 mM and not more than 20 mM, even more preferably more than 0 mM and not more than 15 mM, even more preferably more than 0 mM and not more than 10 mM, and even more preferably more than 0 mM and not more than 5 mM in total

[0024] The total amount of sugars contained in the sugar-containing liquid is, for example, 0.1 to 60 w / v%, or 0.5 to 10 w / v%, preferably 0.8 to 10 w / v%, more preferably 1.8 to 10 w / v%, 1.8 to 6 w / v%, or 1.8 to 4 w / v%. The total amount of sugars refers to all sugars contained in the sugar-containing liquid (CHO). n (n is an integer of 2 or more) is a value derived by the method described in the examples based on total organic carbon (TOC).

[0025] 1-1-4. Specific Examples of Sugar-Containing Liquids (Sugar-Containing Liquids Containing Formose Reaction Products) The sugar-containing liquid may be obtained by any synthesis method as long as it contains an unnatural sugar. Preferably, the sugar-containing liquid is a sugar-containing liquid containing a formose reaction product. The formose reaction method for obtaining a formose reaction product is publicly known, and is not particularly limited as long as it is a method for non-enzymatically chemically synthesizing sugars from formaldehyde, and can be appropriately selected by a person skilled in the art.

[0026] Typically, the formose reaction can be carried out using formaldehyde as a substrate under suitable liquid conditions, preferably using a suitable initiator and catalyst.

[0027] The liquid pH (pH) may be appropriately selected by a person skilled in the art depending on the initiator and catalyst selected, and may be, for example, neutral (6.5 or more and 8.5 or less) or basic (more than 8.5).

[0028] Examples of the initiator include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Of these four types of sugars, any one type may be used, or multiple types may be used in combination. Examples of monosaccharides include diose, triose, tetrose, pentose, hexose, and heptose. These may be aldoses or ketoses, and may have either a chain (linear or branched) structure or a cyclic structure. Specific examples of monosaccharides include glycolaldehyde, dihydroxyacetone, glyceraldehyde, erythrose, threose, erythrulose, ribose, arabinose, xylose, lyxose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, and heptulose. Specific examples of disaccharides include lactose, maltose, isomaltose, sucrose, and trehalose. Oligosaccharides are formed by condensing 3 to 10 monosaccharides, and specific examples include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, soybean oligosaccharides, isomaltooligosaccharides, lactoferrin oligosaccharides, and raffinose. Polysaccharides are formed by condensing 11 or more monosaccharides, and specific examples include starch, dextrin, cellulose, glycogen, dextran, mutan, levan, and inulin. Each of these initiators may be used alone or in combination of two or more.

[0029] More specific examples of the initiator include intermediate products of the formose reaction, and more specific examples include glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), erythrose, and threose (C4a). These may be used alone or in combination of two or more.

[0030] The catalyst may be either a homogeneous catalyst or a heterogeneous catalyst. Specific examples of the catalyst include alkali metal hydroxides (such as NaOH and KOH), zinc hydroxide (Zn(OH)), alumina, alkaline earth metal hydroxides (such as Ca(OH), Mg(OH), Ba(OH), and Sr(OH)), alkaline earth metal oxides (such as CaO, SrO, and BaO), zeolites, oxides of Group 5 elements (such as V, Nb, Ta, and Db) or Group 6 elements (such as Cr, Mo, W, and Sg), oxoacids, and their salts (such as alkali metal salts (such as potassium salts and sodium salts), and alkaline earth metal salts (such as calcium and barium salts)), and complexes. Specific examples of oxides, oxoacids, salts thereof, and complexes of Group 5 or Group 6 elements include niobium oxide, molybdenum oxide, tungsten oxide, aluminum oxide, niobic acid and salts thereof, molybdic acid and salts thereof, tungstic acid and salts thereof, and aluminic acid and salts thereof. These catalysts may be used alone or in combination of two or more.

[0031] Examples of the solvent include water and lower alcohols (e.g., monohydric lower alcohols such as methanol, ethanol, isopropyl alcohol, and butanol), and these may be used alone or in combination of two or more.

[0032] The formose reaction product can be used after appropriate post-treatment. The post-treatment can be appropriately selected from methods for removing components other than the sugar product (e.g., catalyst, solvent, raw material formaldehyde, etc.) and / or methods for increasing the concentration of the sugar product. Specific examples of post-treatment include solid-liquid separation, ion exchange resin, freeze-drying, etc. Sterilization can also be used as post-treatment.

[0033] When the formose reaction product contains at least one of the sugars formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), and erythrose and threose (C4a), post-treatment can also include a treatment to reduce the content of the sugar in the culture solution to the preferred content described above in "1-1-3. Sugar content" from the viewpoint of improving the growth environment of the microorganism. Such treatment is not particularly limited, and examples include dilution of the formose reaction product and a treatment that can promote the aldol reaction of C1 to C4 sugars (e.g., base treatment, etc.). Among these treatments, a treatment that can promote the aldol reaction of C1 to C4 sugars (particularly base treatment) is preferred from the viewpoint of reducing the concentration of the sugar without diluting the useful substrate. Examples of basic compounds used in the base treatment include alkali metal hydroxides (NaOH, KOH, etc.), alkaline earth metal hydroxides (Ca(OH)2, Mg(OH)2, Ba(OH)2, Sr(OH)2, etc.), etc. In the base treatment, the pH of the formose reaction product can be increased to, for example, 10 to 14, preferably 11 to 13. After the base treatment, the pH can be returned to neutral (specifically, to a pH that allows microorganisms to grow).

[0034] 1-2. Microorganisms The type of microorganism is not particularly limited as long as it is a microorganism that can grow heterotrophically using sugar as a substrate (i.e., a heterotrophic microorganism with a glycolytic pathway), and examples thereof include bacteria, yeast, microalgae, etc. Examples of bacteria include actinomycetes such as bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum), and bacteria of the genera Ralstonia and Escherichia (e.g., Escherichia coli). Examples of yeast include budding yeast (e.g., Saccharomyces cerevisiae), and examples of microalgae include bacteria of the genus Spirulina, Chlorella, and Euglena (e.g., Euglena gracilis).

[0035] Furthermore, the microorganism may be a wild-type strain, or a mutant strain or a transformant into which a mutant gene has been introduced. When a mutant strain or a transformant into which a mutant gene has been introduced is used as the microorganism, the type of mutation is not particularly limited. Examples of the type of mutation include those containing a gene designed to produce a substance different from a substance produced by the wild-type and / or a gene designed to improve the efficiency of substance production.

[0036] These mutations may be appropriately selected by those skilled in the art based on the type and / or production efficiency of the target compound (organic compound) set in the production method of the present invention. Techniques relating to mutant microorganisms designed to produce substances different from those produced by wild-type microorganisms are known. For example, for mutant Corynebacterium glutamicum, see "Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products," Applied Microbiology and Biotechnology, October 2018, Volume 102, Issue 20, pp. 8685-8705; for mutant Escherichia coli, see "Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol," Nature Chemical Biology, July 2011, Volume 7, Issue 7, pp. 445-452; and for mutant Saccharomyces cerevisiae, see "Production of aromatics in Saccharomyces cerevisiae—A feasibility study," Journal of Biotechnology, Volume 163, Issue 2, January 2018. 2013, Pages 184-193, etc.; for mutated Euglena gracilis, examples include those described in Bioproducts From Euglena gracilis: Synthesis and Applications, Frontiers in Bioengineering and Biotechnology, May 2019, Volume 7, Article 1083, etc.

[0037] The specific concentration of the microorganism in the culture medium is not particularly limited, and examples thereof include 0.5 to 25 w / v%, 5 to 25 w / v%, 10 to 20 w / v%, 12 to 15 w / v%, 0.5 to 20 w / v%, 0.5 to 10 w / v%, 0.5 to 5 w / v%, 0.5 to 3 w / v%, 0.5 to 2 w / v%, and 0.8 to 1.5 w / v%, regardless of the type of microorganism. For example, when the microorganism is a bacterium of the genus Corynebacterium, the concentration may be, for example, 0.5 to 25 w / v%, preferably 5 to 25 w / v%, more preferably 10 to 20 w / v%, and even more preferably 12 to 15 w / v%. When the microorganism is Escherichia coli, yeast, or microalgae, the concentration may be, for example, 0.5 to 25 w / v%, preferably 0.5 to 20 w / v%, 0.5 to 10 w / v%, more preferably 0.5 to 5 w / v%, 0.5 to 3 w / v%, and even more preferably 0.5 to 2 w / v%, or 0.8 to 1.5 w / v%.

[0038] Furthermore, specific concentrations of microorganisms in the culture medium, regardless of the type of microorganism, in terms of optical density OD600, include, for example, 0.5 to 150, 0.5 to 100, 0.5 to 50, 0.5 to 30, 0.5 to 20, 0.5 to 15, 1 to 10, and 2 to 8. For example, when the microorganism is Escherichia coli, yeast, or microalgae, the concentration is, for example, 0.5 to 50, preferably 0.5 to 30, more preferably 0.5 to 20, 0.5 to 15, and even more preferably 1 to 10 or 2 to 8.

[0039] 1-3. Substance Production Conditions Conditions for substance production can be determined appropriately based on the growth conditions of the microorganism. Specifically, substance production can be carried out under anaerobic conditions, oxygen-limited conditions (including trace amounts of oxygen), or aerobic conditions, preferably anaerobic or oxygen-limited conditions. For example, when at least one of branched sugars, such as branched C5 sugars (e.g., 1,3,4,5-tetrahydroxypentan-2-one and 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one) and branched C6 sugars (e.g., stereoisomers of 1,2,4,5,6-pentahydroxyhexan-3-one) is used as the substrate, and a Corynebacterium bacterium is used as the microorganism, substance production can be carried out under anaerobic or oxygen-limited conditions. Substance production can be carried out in any of batch, fed-batch, and continuous systems.

[0040] 1-4. Products (organic compounds) The organic compounds obtainable by the production method of the present invention are not particularly limited as long as they are obtained by converting a substrate into a substance through a biological reaction in a microorganism, and are determined depending on the type of substrate and microorganism, and further depending on the design of the mutation if the microorganism has a mutation.

[0041] Examples of organic compounds include organic acids and / or salts thereof, such as lactic acid and / or salts thereof. Organic compounds can also include acetic acid, succinic acid, fumaric acid, and / or salts thereof. Examples of salts include alkali metal salts (e.g., sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, when bacteria or yeast are used as the microorganism, acetic acid, succinic acid, fumaric acid, and / or salts thereof can be obtained as products in addition to lactic acid. Another example of an organic compound is β-glucan. In particular, when the microorganism is a Euglena species, β-glucan can be obtained as a product.

[0042] 2. Culturing Step The method for producing an organic compound of the present invention preferably includes a culturing step of growing the microorganisms described above in "1-2. Microorganisms" before the substance production step. This allows the microorganisms to be subjected to the substance production step in a state where their concentration is sufficiently increased.

[0043] In the culturing step, a culture solution containing a substrate and a microorganism may be incubated under aerobic conditions.

[0044] The substrate used for culturing the microorganism may be a natural sugar, an unnatural sugar, or both. The types of natural sugar and unnatural sugar can be selected from those described above in "1-1-1. Sugars contained in the sugar-containing liquid."

[0045] When the substrate used for culturing the microorganism contains a natural sugar, a preferred example of the natural sugar is glucose. When the substrate used for culturing the microorganism contains an unnatural sugar, the formose reaction product described in "1-1-4. Specific examples of sugar-containing liquids (sugar-containing liquids containing formose reaction products)" above can be used as the substrate.

[0046] When the culture solution containing the substrate and the microorganism contains at least any one of the sugars formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), and erythrose and threose (C4a) (for example, when it contains a formose reaction product), it is preferable to adopt, as the concentration of the sugar in the culture solution containing the substrate and the microorganism, the concentration shown as the concentration of the sugar in the sugar-containing solution in "1-1-3. Sugar content" above, from the viewpoint of improving the growth rate of the microorganism.

[0047] The total amount of sugars in the culture medium containing the substrate and the microorganism is not particularly limited, but is preferably 0.1 to 60 w / v%, or 0.5 to 10 w / v%, more preferably 1 to 8 w / v%, even more preferably 3 to 7 w / v%, and even more preferably 4 to 6 w / v%.

[0048] Those skilled in the art can appropriately determine the medium components contained in the culture solution containing the substrate and the microorganism depending on the growth characteristics of the microorganism, etc. The culture solution used for culturing can also contain any additives as long as they do not inhibit the culture. Examples of such additives include sugar stabilizers, more specifically boric acid, silicic acid, and derivatives thereof. Examples of derivatization reagents include 2-(hydroxymethyl)phenylboronic acid, 4-formylphenylboronic acid, 4-(methylcarbamoyl)phenylboronic acid, 3-acetamidophenylboronic acid, 2-formylphenylboronic acid, and 2-(dimethylaminomethyl)phenylboronic acid.

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following, concentration values ​​expressed as % mean values ​​expressed in w / v % unless otherwise specified.

[0050] (1) Materials Used. All materials were used as purchased without purification. 1,3-Dihydroxyacetone dimer, D-erythrose, D-(-)-ribose, D-(-)-lyxose, D-psicose, D-(+)-xylose, D-tagatose, D-(+)-talose, L-(-)-sorbose, D-(+)-allose, L-gulose, D-(+)-mannose, D-(+)-galactose, D-biotin, and 3,4-dihydroxybenzoic acid were obtained from Tokyo Chemical Industry Co., Ltd. Glycolaldehyde dimer, DL-glyceraldehyde, L-(+)-erythrulose, D-ribulose, D-altrose, (2,4-dinitrophenyl)hydrazine, and yeast nitrogen base (amino acid-free) were obtained from Sigma-Aldrich Japan. Formaldehyde solution, D-(-)-arabinose, D-(-)-fructose, D-(+)-glucose, sodium hydroxide, calcium hydroxide, methanol, acetonitrile, phosphoric acid, urea, ammonium sulfate ((NH4)2SO4), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), magnesium sulfate heptahydrate (MgSO4.7H2O), iron(II) sulfate heptahydrate (FeSO4.7H2O), thiamine hydrochloride, 3-(N-morpholino)propanesulfonic acid (MOPS), calcium chloride dihydrate (CaCl2-2H2O), zinc sulfate heptahydrate (ZnSO4-7H2O), copper(II) sulfate pentahydrate (CuSO4-5H2O), nickel(II) chloride hexahydrate (NiCl2-6H2O), hydrochloric acid, acetic acid, sulfuric acid, sodium chloride, disodium hydrogen phosphate (Na2HPO4 ), ammonium chloride, Bis-Tris, 4H (EDTA-free acid), sodium lactate, sodium pyruvate, sodium L-(-)-malate, disodium succinate hexahydrate, disodium fumarate, and sodium acetate were purchased from Fujifilm Wako. Phenylhydrazine was obtained from Kanto Chemical. Manganese(II) sulfate monohydrate (MnSO4-H2O) was obtained from Alfa Aesar. Bacto TM Yeast Extract, Bacto TM peptone, Polypeptone TMPeptone was purchased from Thermo Fisher Scientific. Casein acid hydrolysate was obtained from Biokar Diagnostics. Deionized and distilled water was supplied by a Millipore system.

[0051] (2) Instruments used: High-performance liquid chromatography (HPLC) analysis of HCHO and C2-C4 sugars was performed using a Chromaster equipped with a UV / Vis detector (360 nm; 5430 diode array detector). (R) The system was used with an InertSustain C18 column (150 mm, GL Sciences) and a mixed solvent of water and acetonitrile (6:4, v / v) as the eluent, at a flow rate of 1.0 mL min -1 HPLC analysis of C5-C6 sugars was performed using an HPLC Chromaster equipped with a fluorescence detector (5440 FL detector) and an NH2P-50 4E column (250 mm, Shodex) as the stationary phase. (R) The analysis was performed using a Sugar Analysis System (Hitachi). The mobile phases were solvent A (acetonitrile), solvent B (water), and solvent C (10% aqueous phosphoric acid solution), and the flow rate was 1.0 mL min -1 An aqueous solution of acetic acid (44.3%), phosphoric acid (54.2%), and phenylhydrazine (1.5%) was added at a rate of 0.4 mL min -1 The sample was added to the post-column flow path at a flow rate of 0.01 s, and analysis was performed.

[0052] In the example using [I] C. glutamicum as the microorganism, organic acids were quantified using an HPLC system (Shimadzu Corporation) equipped with an electric conductivity detector (CDD-10AVP) and three Shim-pack Fast-OA columns (Shimadzu Corporation), with a 2.75 mM H2SO4 mobile phase and a flow rate of 0.8 mL min -1The column temperature was 50°C. Total organic carbon was quantified using a TOC-L (Shimadzu Corporation). In the example using [II] E. coli and [III] S. cerevisiae as microorganisms, organic acids were quantified using an HPLC system (Shimadzu Corporation) equipped with an electric conductivity detector (CDD-10ASP) and one TSKgel OApak-P (40 mm, Tosoh) and two TSKgel OApak-A (300 mm, Tosoh). The mobile phase was a 0.75 mM MHCl aqueous solution at a flow rate of 0.7 mL min. -1 The column temperature was 50 °C. An aqueous solution of 0.75 mM H2SO4, 20 mM Bis-Tris, and 0.1 mM EDTA-4H was added at a flow rate of 0.7 mL min -1 The total organic carbon was quantified using a TOC-L (Shimadzu Corporation).

[0053] (3) Formose Reaction (3-1) Preparation of Formose Reaction Product-Ca The substrate (2 M formaldehyde (C1a), 20 mM dihydroxyacetone (C3k)) was dissolved in a solvent (10% CH3OH aqueous solution) to prepare a 90 mL solution. This solution was placed in a screw-top vial equipped with a stir bar. Before starting the formose reaction, the solution in the vial was heated to 80 °C while stirring using an organic synthesis stirrer (HHE-19G-US IV, KPI). The reaction was initiated by adding a catalyst (100 mM Ca(OH)2), and the solution was maintained at 80 °C for approximately 1.5 minutes. After the solution turned yellow (an indicator of the progress of the formose reaction), the vial was cooled in ice water to terminate the reaction. Subsequently, Ca was extracted using a cation exchange resin, Amberlite IR120BHAG (Organo). 2+ The unreacted HCHO and CH3OH were also removed by freeze-drying. The resulting solid powder was redissolved in distilled water. The concentrated sugar solution was again passed through Amberlite IR120BHAG (Organo) to remove Ca. 2+The cation concentration was reduced. This resulted in the formation of the formose reaction product -Ca. The total organic carbon content was quantified using a TOC-L (Shimadzu Corporation). The measurement method used was the NPOC (Non-Purgeable Organic Carbon) method. The composition of all monosaccharides produced by the formose reaction was (CHO) n Therefore, if the solution contains only (CHO) n Assuming this, the sugar concentration was calculated using the following formula (1).

[0054]

[0055] (3-2) Preparation of Formose Reaction Product-W A formose reaction was carried out in the same manner as in (3-1), except that sodium tungstate was used as the catalyst instead of 100 mM Ca(OH) to obtain Formose Reaction Product-W.

[0056] (4) Derivatization and preparation for analysis Derivatization for HPLC analysis of HCHO and C2-C4 sugars was carried out as follows. After the reaction, the solution (2.5 μL) obtained was diluted with water (748 μL). Acetonitrile (1 mg mL -1 375 μL of 2,4-dinitrophenylhydrazine (DNPH) in HCl (HCl) and 22.5 μL of aqueous phosphoric acid (20 vol%) were added dropwise to each diluted sample. The mixed solution was stirred at room temperature for 60 min before HPLC analysis. Samples for HPLC analysis of C5-C6 sugars were prepared by adding water (250 μL) and acetonitrile (500 μL) to 250 μL of the reaction mixture.

[0057] (5) Phenol-sulfuric acid method: First, 1.5 g of phenol was dissolved in 48.5 mL of ultrapure water to prepare a 3% phenol solution. Next, 100 μL of sample (10-100 μg / mL) and 200 μL of 3% phenol solution were added to a test tube and stirred. 750 μL of concentrated sulfuric acid was then added, stirred, and allowed to stand for 20 minutes. The absorbance of the solution was measured at 490 nm and 480 nm, and the concentrations of C6 and C5 sugars, respectively, were estimated. Glucose and xylose were used as standards for the calibration curves of C6 and C5 sugars, respectively.

[0058] (6) Cultivation of Microorganisms Using Synthetic Sugars (6-[I]) Cultivation of C. glutamicum ATCC 13032 (Wild-Type Strain) A glycerol stock of C. glutamicum ATCC 13032 (wild-type strain) was inoculated into 5 mL of medium A (containing 2 g urea, 2 g yeast extract, 7 g casamino acids, 7 g (NH4)2SO4, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 6 mg FeSO4·7H2O, 4.2 mg MnSO4·H2O, 0.2 mg biotin, 0.2 mg thiamine, and 4% (w / v) glucose per L) and cultured with shaking at 30°C to prepare a seed culture. This main culture was centrifuged at 3300 x g for 5 minutes at 25°C, and the supernatant was removed.

[0059] For the growth experiments of C. glutamicum, modified minimal medium CGXII (mCGXII) was used, containing (NH4)2SO4 (5 g L -1 ), urea (5 g L -1 ), KH2PO4 (1 g L -1 ), K2HPO4 (1 g L -1 ), MgSO4·7H2O (0.25 g L -1 ), CaCl2 (10 mg L -1 ), FeSO4-7H2O (10 mg L -1 ), MnSO4-H2O (0.1 mg L -1 ), ZnSO4-7H2O (1 mg L -1 ), CuSO4-5H2O (0.2 mg L -1 ), NiCl2-6H2O (0.02 mg L -1 ), biotin (0.2 mg L -1 ), 3,4-dihydroxybenzoic acid, MOPS (21 g L -1 The pH of the medium was adjusted to pH 7 with NaOH.

[0060] The formose reaction product-Ca obtained in (3-1) above was filter-sterilized and added to mCGXII medium at various sugar concentrations (0.5%, 1%, 2%, 3%, 4%, and 5%). Furthermore, moist cells were added to the mCGXII medium containing the formose reaction product and cultured at 30°C with stirring in a 12-well plate. Separately, similar cultures were performed using various concentrations of C1-C4 sugars (specifically, formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrose (C4a), or erythrulose (C4k)) together with 0.1% glucose instead of the formose reaction product-Ca. Microbial growth was confirmed by measuring the absorbance at 600 nm using a plate reader (Infinite F Nano+, TECAN). The absorbance data obtained were expressed as the optical density at 600 nm (OD ). 600 After incubation, the samples were filtered to obtain the supernatants for further analysis.

[0061] (6-[II]) Cultivation of E. coli K-12 (wild-type strain) A glycerol stock of E. coli K-12 (wild-type strain) was inoculated into 5 mL of LB medium (containing 10 g of Polypepton, 5 g of yeast extract, 10 g of sodium chloride, 0.2 mg of thiamine, and 4% (w / v) glucose per liter) and cultured with shaking at 37°C to prepare a seed culture. This main culture was centrifuged at 3300 xg at 25°C for 5 minutes, and the supernatant was removed.

[0062] The formose reaction product-Ca obtained in (3-1) above was filter-sterilized and added to 1.25x M9 medium (12.5 mL of 5x M9 medium containing 12 g of NaHPO, 6 g of KHPO, 1 g of NaCl, and 2 g of NHCl in 200 mL) diluted with 1.25x M9 medium (12.5 mL of 5x M9 medium supplemented with 125 μL of 1 M MgSO, 12.5 μL of 1 M CaCl, 125 μL of 1% thiamine, and 37.2375 mL of ultrapure water). The moist cells were then added to the 1.25x M9 medium containing the formose reaction product and incubated at 37°C with stirring in a 96-well plate. Microbial growth was confirmed by measuring the absorbance at 600 nm using a plate reader (Infinite F Nano+, TECAN). The absorbance data obtained were expressed as the optical density at 600 nm (OD ). 600 After incubation, the samples were filtered to obtain the supernatants for further analysis.

[0063] (6-[III]) Cultivation of wild-type yeast strain BY21391 A glycerol stock of wild-type yeast strain BY21391 was inoculated into 5 mL of YPD medium (containing 10 g of yeast extract, 20 g of peptone, and 20 g of glucose per liter) and cultured with shaking at 30°C to prepare a seed culture. This main culture was centrifuged at 3300 xg at 25°C for 5 minutes, and the supernatant was removed.

[0064] The formose reaction product-Ca obtained in (3-1) above was filter-sterilized and added to YNB medium (containing 6.7 g of YNB per 1 L). Furthermore, moist cells were added to the YNB medium containing the formose reaction product, and the mixture was cultured at 37°C with stirring in a 96-well plate. Microbial growth was confirmed by measuring the absorbance at 600 nm using a plate reader (Infinite F Nano+, TECAN). The obtained absorbance data were expressed as the optical density at 600 nm (OD 600 ) was converted.

[0065] (7) Production of Organic Compounds from Synthetic Sugars Using Microbial Cells (7-1) Cultivation Step (7-1-[I]) A glycerol stock of C. glutamicum ATCC 13032 was inoculated into 25 mL of BA medium containing 4% glucose and cultured with shaking at 33°C to prepare a seed culture. This seed culture was then inoculated into 200 mL of BA medium containing 4% glucose and cultured overnight with shaking at 33°C in a 500 mL flask. This main culture was centrifuged three times in BS buffer at 5,500 xg, 4°C, and 15 minutes, and the supernatant was removed.

[0066] (7-1-[II]) A glycerol stock of E. coli K-12 was inoculated into 40 mL of LB medium containing 4% glucose and 0.002% thiamine and cultured overnight with shaking at 37°C. This culture was centrifuged three times in M9 medium at 3,300 xg, 4°C, and 5 minutes, and the supernatant was removed.

[0067] (7-1-[III]) A glycerol stock of yeast strain BY21391 was inoculated into 5 mL of YPD medium containing 4% glucose and cultured with shaking at 30°C to prepare a seed culture. This seed culture was inoculated into 10 mL of YBD medium containing 4% glucose and cultured with shaking at 30°C overnight. This main culture was centrifuged in YNB medium once at 3,300 x g for 5 minutes at 4°C and twice at 1,800 x g for 5 minutes at 4°C, and the supernatant was removed.

[0068] (7-1-[IV]) An appropriate volume of KH medium containing 1.2% glucose was inoculated with a subculture stock of E. gracilis and cultured with shaking at 25°C. The culture was then centrifuged at 800 x g for 3 minutes in a 50 mL centrifuge tube, and the supernatant containing KH medium components was removed. The cells were then suspended in CM medium and cultured. This main culture was then centrifuged at 800 x g for 3 minutes, and the supernatant was removed.

[0069] (7-2) Substance Production Step (7-2-[I]) The wet bacterial cells prepared in (7-1-[I]) above were suspended in BS buffer to a cell concentration of 12% (w / v). Filter-sterilized solutions of the formose reaction product-Ca obtained in (3-1) above or the formose reaction product-W obtained in (3-2) above were added to the bacterial cell suspension to a final concentration of 1% (w / v) or 2% (w / v). This solution was placed in a screw-top vial equipped with a stir bar. Lactic acid fermentation was carried out by maintaining the vial at 33°C while stirring under oxygen-limited conditions (including trace amounts of oxygen) using an organic synthesis stirrer (HHE-19G-US IV, KPI). After the reaction, the sample was filtered to obtain the supernatant, which was quantitatively analyzed by HPLC equipped with an electrical conductivity detector.

[0070] (7-2-[II]) The wet cells prepared in (7-1-[II]) above were added to M9 medium at a cell concentration of 1% (w / v) and a cell optical density (OD 600 The bacterial cell suspension was suspended to a final concentration of 8% (w / v). The formose reaction product-Ca obtained in (3-1) above or the formose reaction product-W obtained in (3-2) above was filter-sterilized and added to the bacterial cell suspension to a final concentration of 1% (w / v). This solution was placed in a screw-top vial. Organic acid fermentation was carried out by shaking the vial in a constant-temperature incubator at 37°C under oxygen-limited conditions (including trace amounts of oxygen) using Ar gas substitution. After the reaction, the sample was filtered to obtain the supernatant, which was then quantitatively analyzed by HPLC with an electrical conductivity detector.

[0071] (7-2-[III]) The wet cells prepared in (7-1-[III]) above were added to YNB medium at a cell concentration of 1% (w / v) and a cell optical density (OD 600The bacterial cell suspension was suspended to a final concentration of 8% (w / v). The formose reaction product-Ca obtained in (3-1) above or the formose reaction product-W obtained in (3-2) above was filter-sterilized and added to the bacterial cell suspension to a final concentration of 1% (w / v). This solution was placed in a screw-top vial. Organic acid fermentation was carried out by shaking the vial in a constant-temperature incubator at 37°C under oxygen-limited conditions (including trace amounts of oxygen) using Ar gas substitution. After the reaction, the sample was filtered to obtain the supernatant, which was then quantitatively analyzed by HPLC with an electrical conductivity detector.

[0072] (7-2-[IV]) The wet cells prepared in (7-1-[IV]) above were added to nitrogen-deficient CM medium at a cell concentration of 1% (w / v) and a cell optical density (OD 600 ) was suspended to a concentration of 2. The formose reaction product-Ca obtained in (3-1) above was filter-sterilized and added to the bacterial cell suspension to a final concentration of 1% (w / v). 1300 μL of this solution was added to a 5 mL test tube, and incubation was carried out at a shaking speed of 240 rpm and a temperature of 25°C. After the reaction, β-glucan was extracted and quantitatively analyzed by the phenol-sulfuric acid method.

[0073] (8) Results (8-1) Composition of Formose Reaction Product The chromatogram of the formose reaction product-Ca obtained in (3-1) above by high-performance liquid chromatography (HPLC) is shown in Figure 1. As shown in Figure 1, the production of linear sugars (specifically, ribose (3), sorbose (7), fructose (8), etc.) and branched sugars (specifically, 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one (1) and stereoisomers of 1,2,4,5,6-pentahydroxyhexan-3-one (3-hexuloses (4) and (5))) was confirmed.

[0074] Table 1 also shows the concentrations of each sugar, including C5-C6 sugars (measured by the phenol-sulfuric acid method), C2-C4 monosaccharides (specifically, glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrose (C4a), and erythrulose (C4k)), and unreacted formaldehyde (C1a).

[0075]

[0076] (8-2-[I]) Cultivation of C. glutamicum using formose reaction products (8-2-[I]-1) Substrates In the above (6), the OD of C. glutamicum was measured when the formose reaction products were used as substrates at various sugar concentrations (0.5%, 1%, 2%, 3%, 4%, and 5%). 600 The time course of the change in OD is shown in Figure 2(a). The concentrations shown in Figure 2(a) were calculated based on the total organic carbon (TOC) values ​​of the synthesized sugars. 600 increased only when formose reaction products were added, and when no formose reaction products were added, OD 600 No increase in the final OD was observed with increasing amounts of formose reaction product added. 600 These results indicate that the synthetic sugars contained in the formose reaction products served as substrates for C. glutamicum.

[0077] Figure 2(b) shows the HPLC analysis results of the culture supernatant before and 57 hours after the start of cultivation when 0.5% synthetic sugar solution was added. As shown in Figure 2(b), even when the lowest concentration of synthetic sugar solution shown in Figure 2(a) was used, the peak intensities of several sugar components decreased after cultivation. The decreased peaks were due to linear sugars (specifically, 1,3,4,5-tetrahydroxypentan-2-one (2), ribose (3), and fructose (8)), branched sugars (specifically, 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one (1), and the stereoisomers of 1,2,4,5,6-pentahydroxyhexan-3-one (3-hexulose, (4) and (5))). These results demonstrate that wild-type C. glutamicum cells can metabolize several synthetic sugars, including branched sugars (especially unnatural sugars).

[0078] (8-2-[I]-2) Controlling Factors for Controlling Microbial Growth Rate The effect of short-carbon chain monosaccharides (i.e., intermediates of the formose reaction) in the synthetic sugar solution on growth was evaluated. Table 2 shows the concentrations of each C1-C4 sugar when the sugar concentration of the formose reaction solution-Ca obtained in (3-1) above was 5%.

[0079]

[0080] On the other hand, in the above (6), when the above C1-C4 sugars were added at different concentrations together with 0.1% glucose instead of the formose reaction product, the OD 600 The time course of the changes is shown in Figures 3a to 3f. As shown in Figures 3a to 3f, it was revealed that C1 to C4 sugars are factors that control the growth rate of microorganisms, and that reducing the coexisting amounts of these sugars can increase the growth rate of microorganisms. In other words, reducing the coexisting amounts of the C1 to C4 sugars provides a desirable growth environment for microorganisms, and it can be reasonably inferred that this can be applied as a desirable condition not only when microorganisms are grown using a liquid containing unnatural sugars such as formose reaction products, but also when substances are produced using microorganisms in the liquid containing unnatural sugars.

[0081] Treatment for controlling the growth rate of microorganisms NaOH was added to the formose reaction product-Ca obtained in (3-1) above to raise the pH to 12, and the mixture was left at room temperature overnight. The pH was then returned to neutral and quantitatively analyzed by HPLC. The compositions of the synthetic sugar solution before and after the base treatment are shown in Table 3.

[0082]

[0083] As shown in Table 3, the C1 to C4 sugars were each reduced by base treatment of the formose reaction product. This is thought to be due to the fact that the aldol reaction of the C1 to C4 sugars, which are formose intermediate products, proceeded due to the function of the base catalyst.

[0084] In particular, C4k was contained at a relatively high ratio in the formose reaction product-Ca obtained by (3-1) above, but was reduced to about 1 / 5 (4.96 mM) by base treatment. According to the results in Figure 3f, when the C4k concentration is 5 mM or less, the growth rate is significantly increased to the extent that the delay in growth rate compared to when glucose alone is used as a substrate is negligible. Therefore, it is expected that the growth rate will be significantly increased when a base-treated formose reaction product is used. To confirm this, we actually measured the OD of cells given a base-treated formose reaction product instead of the formose reaction product (before base treatment) in (6) above. 600 The time course of the change in is shown in Figure 4. As shown in Figure 4, when a base-treated formose reaction product was used as a substrate, the cell growth rate was significantly increased. In other words, the base treatment of a formose reaction product provides a desirable growth environment for microorganisms, and it can be reasonably inferred that the base treatment can be applied not only as a desirable condition for growing microorganisms using a formose reaction product, but also as a desirable condition for producing substances using microorganisms in a sugar-containing solution containing a formose reaction product.

[0085] Cultivation of E. coli using (8-2-[II]) formose reaction products The findings regarding the substrate and the control of microbial growth rate were similar to those obtained in the above-mentioned "Cultivation of C. glutamicum using (8-2-[I]) formose reaction products."

[0086] Cultivation of Yeast Using (8-2-[III]) Formose Reaction Products The findings regarding the substrate and the control of microbial growth rate were similar to those obtained in the above-mentioned "Cultivation of C. glutamicum Using (8-2-[I]) Formose Reaction Products."

[0087] Cultivation of E. gracilis using (8-2-[IV]) formose reaction products The findings regarding the substrate and the control of microbial growth rate were similar to those obtained in the above-mentioned "Cultivation of C. glutamicum using (8-2-[I]) formose reaction products."

[0088] (8-3) Substance Production Using Formose Reaction Product (8-3-[I]) Substance Production by C. glutamicum Using Formose Reaction Product Figure 5(a) shows the HPLC chromatogram of the supernatant obtained after the reaction (24 hours) using 1 w / v% formose reaction product-Ca in (7) above, along with the HPLC chromatogram of the supernatant immediately after the start of the reaction (0 hours). As shown in Figure 5(a), consumption of C5-C6 sugars was observed, similar to the cell growth results in Figure 2(b). Figure 5(b) also shows the HPLC chromatogram obtained by performing the same procedure as in (7) above, except that no cells were added. As shown in Figure 5(b), no decrease in peak intensity was observed when cells were not added.

[0089] Furthermore, the results of HPLC analysis of the reaction product obtained by the production of substances using 1 w / v% formose reaction product-Ca in (7) above are shown in Figure 6(a). As shown in Figure 6(a), when the formose reaction product was added (w / syn-sugar), lactic acid was produced in sync with the decrease in C5-C6 sugars, but this lactic acid production was not observed when no sugar was added (w / o sugar).

[0090] Based on the results in Figure 6(a), the amounts of lactic acid and other organic acids produced by the production of substances using 1 w / v% formose reaction product-Ca were quantified based on the difference in concentration between immediately after the start of the reaction (0 hours) and after the reaction (24 hours). The results are shown in Figure 6(b). Figure 6(b) shows the results when the formose reaction product was added as a substrate (w / syn-sugar) and when no sugar was added (w / o sugar), as well as when only glucose (1.5%) was added as a substrate (glucose).

[0091] As shown in Figure 6(b), the main product from glucose alone (Glucose) was lactate (62 mM), with small amounts of succinate (4.1 mM) and acetate (2.9 mM). The amount of glucose consumed over 24 h was 45% of the initial amount, and the lactate yields relative to the initial and consumed amounts of glucose were approximately 33% and 83%, respectively. Even without added sugar (w / o sugar), succinate (12 mM) and acetate (6.7 mM) were produced. These organic acids under w / o sugar conditions may be derived from organic matter already accumulated within the cells. On the other hand, with the addition of formose reaction product-Ca (w / syn-sugar), approximately 11 mM lactate was produced, and the lactate yield from 1% (w / v) formose reaction product-Ca was 10.2%. Additionally, by-products such as succinic acid (5.5 mM), acetic acid (6.2 mM), and a small amount of fumaric acid (0.04 mM) were also identified.

[0092] Similar to Fig. 6(b), Fig. 7 shows the quantitative results of the organic acids obtained by producing substances using 2 w / v% formose reaction product-Ca in (7) above, and by producing substances using 1 w / v% or 2 w / v% formose reaction product-W. Fig. 7 also shows the quantitative results of the organic acids obtained by w / syn-sugar (production using 1 w / v% formose reaction product-Ca) shown in Fig. 6(b).

[0093] In addition, for the w / syn-sugar (formose reaction product - substance produced using 1 w / v% Ca) in Figure 6(b), the concentrations of C1 to C4 sugars before and after the lactic acid fermentation reaction are shown in Table 4.

[0094]

[0095] As shown in Table 4, the concentrations of C3k, C4k, and other sugars also decreased after lactic acid fermentation, suggesting that these sugars, as well as unquantified sugar alcohols and sugar carboxylic acids, partially served as carbon sources for lactic acid production.

[0096] (8-3-[II]) Substance Production by E. coli Using Formose Reaction Product Figure 8 shows an HPLC chromatogram of the supernatant obtained after the reaction (24 hours) using 1 w / v% formose reaction product-Ca in (7) above, together with an HPLC chromatogram of the supernatant immediately after the start of the reaction (0 hours). Similarly, Figure 9 shows an HPLC chromatogram of the supernatant obtained after the reaction (24 hours) using 1 w / v% formose reaction product-W in (7) above, together with an HPLC chromatogram of the supernatant immediately after the start of the reaction (0 hours). As shown in Figures 8 and 9, consumption of C5-C6 sugars was observed.

[0097] Furthermore, the HPLC analysis results (w / syn-sugar) of the reaction product obtained by using 1 w / v% formose reaction product-Ca in (7) above is shown in Figure 10 , and the HPLC analysis results (w / syn-sugar) of the reaction product obtained by using 1 w / v% formose reaction product-W in (7) above is shown in Figure 11 . Both figures also show the HPLC analysis results (w / o sugar) of the reaction product obtained by the same procedure except that formose reaction product-Ca was not used. As shown in Figures 10 and 11 , when the formose reaction product was added (w / syn-sugar), lactic acid was produced in sync with the decrease in C5-C6 sugars, whereas this lactic acid production was not observed when no sugar was added (w / o sugar).

[0098] (8-3-[III]) Substance Production by Yeast Using Formose Reaction Product Figure 12 shows an HPLC chromatogram of the supernatant obtained after the reaction (24 hours) using 1 w / v% formose reaction product-Ca in (7) above, together with an HPLC chromatogram of the supernatant immediately after the start of the reaction (0 hours). Similarly, Figure 13 shows an HPLC chromatogram of the supernatant obtained after the reaction (24 hours) using 1 w / v% formose reaction product-W in (7) above, together with an HPLC chromatogram of the supernatant immediately after the start of the reaction (0 hours). As shown in Figures 12 and 13, consumption of C5-C6 sugars was observed.

[0099] Furthermore, Figure 14 shows the HPLC analysis results (w / syn-sugar) of the reaction product obtained by using 1 w / v% formose reaction product-Ca in (7) above, and Figure 15 shows the HPLC analysis results (w / syn-sugar) of the reaction product obtained by using 1 w / v% formose reaction product-W in (7) above. Both figures also show the HPLC analysis results (w / o sugar) of the reaction product obtained by performing the same procedure except without using formose reaction product-Ca. As shown in Figures 14 and 15, when formose reaction product was added (w / syn-sugar), organic acids such as acetic acid were produced in sync with the decrease in C5-C6 sugars.

[0100] (8-3-[IV]) Substance Production by E. gracilis Using Formose Reaction Product Figure 16(a) shows the C2-C4 sugar concentrations (converted to C1 sugars) in the supernatant after the reaction (24 hours) obtained by the substance production using 1 w / v% formose reaction product-Ca in (7) above, along with the C2-C4 sugar concentrations (converted to C1 sugars) in the supernatant immediately after the start of the reaction (0 hours). As shown in Figure 16(a), consumption of C2-C4 sugars was observed.

[0101] Furthermore, Figure 16(b) shows the β-glucan concentration (concentration converted to C1 sugar) (w / o sugar) in the reaction product obtained by producing a substance using 1 w / v% formose reaction product-Ca in (7) above, together with the β-glucan concentration (concentration converted to C1 sugar) (w / o sugar) in the reaction product obtained by the same procedure except that formose reaction product-Ca was not used. As shown in Figure 16(b), the production of β-glucan was observed.

[0102] (9) Summary The coryneform bacteria and Escherichia coli shown in the above test examples are heterotrophic prokaryotes, yeast is a heterotrophic eukaryote, and Euglena is a heterotrophic and autotrophic eukaryote. As shown in the above test examples, it was confirmed that these wide range of biological species are capable of producing organic compounds such as organic acids and β-glucan from unnatural sugars. Because sugar metabolism in living organisms generally occurs via the glycolytic pathway, organic acids are thought to be products of unnatural sugars metabolized via the glycolytic pathway. Furthermore, β-glucan is thought to be a product of the paramylon (β-1,3-glucan) synthesis pathway in the Euglena genus. In other words, it can be reasonably inferred that even microbial species different from those described above are similarly capable of producing substances via in vivo pathways such as glycolysis or the paramylon synthesis pathway.

Claims

1. A method for producing an organic compound, comprising a substance production step of causing a microorganism to produce a substance in a sugar-containing liquid containing an unnatural sugar using the unnatural sugar as a substrate.

2. The method according to claim 1, further comprising a step of culturing the microorganism prior to the substance producing step.

3. The method according to claim 1, wherein the concentration of the microorganism in the sugar-containing liquid is 0.5 to 25 w / v %.

4. The method according to claim 1, wherein the unnatural sugar comprises at least one of a triose to a hexose.

5. The method of claim 1, wherein the unnatural sugar is selected from the group consisting of stereoisomers of 1,3,4,5-tetrahydroxypentan-2-one, 1,3,4-trihydroxy-3-(hydroxymethyl)butan-2-one, and 1,2,4,5,6-pentahydroxyhexan-3-one.

6. The method of claim 1, wherein the sugar-containing liquid contains at least one of formaldehyde (C1a), glycolaldehyde (C2a), glyceraldehyde (C3a), dihydroxyacetone (C3k), erythrulose (C4k), and erythrose (C4a) and threose (C4a).

7. The method of claim 1, wherein the sugar-containing liquid contains erythrulose (C4k) in an amount greater than 0 mM and less than 20 mM.

8. The method according to claim 1, wherein the sugar-containing liquid contains erythrose and / or threose (C4a) in a total amount greater than 0 mM and not more than 25 mM.

9. The manufacturing method of claim 1, wherein the sugar-containing liquid contains formaldehyde (C1a) in an amount greater than 0 mM and less than 0.6 mM, glycolaldehyde (C2a) in an amount greater than 0 mM and less than 4 mM, glyceraldehyde (C3a) in an amount greater than 0 mM and less than 7 mM, and / or dihydroxyacetone (C3k) in an amount greater than 0 mM and less than 4 mM.

10. The process of claim 1, wherein the sugar-containing liquid comprises a formose reaction product.

11. The method of claim 1, wherein the sugar-containing liquid comprises a base-treated formose reaction product.

12. The method according to claim 1, wherein the total amount of sugar in the sugar-containing liquid is 0.1 to 60 w / v %.

13. The method of claim 1, wherein the organic compound comprises lactic acid and / or a salt thereof.

14. The process of claim 1, wherein the organic compounds include acetic acid, succinic acid, fumaric acid and / or salts thereof.

15. The method of claim 1, wherein the organic compound comprises β-glucan.

16. The method of claim 1, wherein the microorganism is selected from the group consisting of bacteria, yeast, and microalgae.

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