Bacteria having high carbon yield of carbon dioxide and metabolic enzyme gene-disrupted strain thereof

Abstract

Provided are a bacterium having a high carbon yield of carbon dioxide and a metabolic enzyme gene-disrupted strain of the bacterium. Also provided is a method for isolating carbon dioxide from bacteria having a high carbon yield. Also provided are a method for producing, culturing, and using a metabolic enzyme gene-destroyed strain of the bacterium. According to the present invention, by disrupting a gene encoding an enzyme capable of generating a metabolic reaction of carbon dioxide in a bacterium having a high carbon yield of carbon dioxide, for example, the environmental burden caused by the culture of the bacterium can be reduced and / or the bacterium can be changed to a state more suitable for the production of a substance.

Description

TECHNICAL FIELD

[0001] The present application relates to a bacterium having a high carbon yield of carbon dioxide and a metabolic enzyme gene disruption strain of the bacterium. The present application also relates to a method for isolating a bacterium having a high carbon yield of carbon dioxide. The present application also relates to a method for producing, a method for culturing, and a method for using a metabolic enzyme gene disruption strain of the bacterium. BACKGROUND

[0002] Techniques for reducing the activity of isocitrate dehydrogenase (ICD) of Corynebacterium in the production of lysine have been disclosed (Patent Documents 1 to 5, Non-Patent Document 4). It has been disclosed that the proliferation ability and sugar consumption amount are reduced by making the isocitrate dehydrogenase gene (icd) of Escherichia coli deficient (Non-Patent Documents 1 and 2). It has been disclosed that glutamate auxotrophy is caused by disrupting icd of Corynebacterium (Non-Patent Document 3). Various studies on metabolic enzyme disruption are disclosed in Non-Patent Documents 4 to 9.

[0003] PRIOR ART DOCUMENTS

[0004] PATENT DOCUMENTS

[0005] Patent Document 1: JP 2015-154768 A

[0006] Patent Document 2: JP 2013-533740 A

[0007] Patent Document 3: JP 2011-518571 A

[0008] Patent Document 4: JP 5719434 B

[0009] Patent Document 5: JP 5395893 B

[0010] NON-PATENT DOCUMENTS

[0011] Non-Patent Document 1: Appl. Microbiol. Biotechnol., 2004, 65: 84-96

[0012] Non-Patent Document 2: FEMS Microbiol. Lett., 2019, 366

[0013] Non-Patent Document 3: J. Bacteriol., 1995, 177(3): 774-782

[0014] Non-Patent Document 4: Appl. Environ. Microbiol., 2009. 75(24): 7866-7869

[0015] Non-patent literature 5: Microbial. Biotechnol., 2019. 12(5): 932-945

[0016] Non-patent literature 6: J. bacteriol., (2014). 3045-3057

[0017] Non-patent literature 7: BMC Biotechnol., (2016) 16:52

[0018] Non-patent literature 8: Metabolomics, (2022) 18:56

[0019] Non-patent literature 9: Appl. Environ. Microbiol., 2012. 78(19): 6975-6986 SUMMARY

[0020] The present application provides a bacterium having a high carbon yield of carbon dioxide and a genetically modified strain (particularly, a metabolic enzyme gene disruption strain) of the bacterium. The present application also provides a method for isolating a bacterium having a high carbon yield of carbon dioxide. The present application also provides a method for producing, a method for culturing, and a method for using the bacterium (particularly, a metabolic enzyme gene disruption strain).

[0021] According to the present application, for example, the following application can be provided.

[0022] (1) A metabolic enzyme gene disruption strain, which is a metabolic enzyme gene disruption strain of an actinobacteria (preferably, a streptomyces actinobacteria) having a carbon yield of carbon dioxide (CO2) of more than 50% in at least a logarithmic growth phase or a stationary phase in aerobic culture in a culture medium containing a sufficient amount of a carbon source and other nutrients, wherein at least one of genes encoding enzymes responsible for metabolic reactions that produce CO2 is disrupted, and the carbon yield of CO2 is decreased compared to a control actinobacteria of the same species in which none of the above genes is disrupted.

[0023] (2) The metabolic enzyme gene disruption strain according to the above (1), wherein the enzyme responsible for the metabolic reactions that produce CO2 is an enzyme of any one or more metabolic pathways selected from the group consisting of glycolysis, the pentose phosphate cycle, and the citric acid cycle.

[0024] (3) The metabolic enzyme gene disruption strain according to the above (1) or (2), wherein one or more selected from the group consisting of phosphogluconate dehydrogenase, pyruvate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, isocitrate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, and a-ketoglutarate dehydrogenase is disrupted.

[0025] (4) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (3), wherein the proliferation ability thereof is equivalent to that of a control actinomycete or shows a decrease of 50% or more in the presence of a sufficient amount of a carbon source in aerobic culture.

[0026] (5) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (4), wherein at least one or more genes of isocitrate dehydrogenase, phosphogluconate dehydrogenase, pyruvate dehydrogenase, pyruvate carboxykinase, and malate dehydrogenase are disrupted.

[0027] (5A) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (4), wherein at least isocitrate dehydrogenase is disrupted.

[0028] (5B) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (4), wherein at least phosphogluconate dehydrogenase is disrupted.

[0029] (5C) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (4), wherein at least malate dehydrogenase is disrupted.

[0030] (5D) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (4), wherein at least pyruvate carboxykinase is disrupted.

[0031] (6) The metabolic enzyme gene disrupted strain according to any one of the above (1) to (5), wherein the actinomycete is Streptomyces.

[0032] (7) A composition for producing a metabolic product, which contains the metabolic enzyme gene disrupted strain according to any one of the above (1) to (6).

[0033] (8) A composition for producing a metabolic product derived from the citric acid cycle, which contains the metabolic enzyme gene disrupted strain according to any one of the above (1) to (6).

[0034] (9) A method for culturing a metabolic enzyme gene disrupted strain of an actinomycete, wherein the metabolic enzyme gene disrupted strain contains the metabolic enzyme gene disrupted strain according to any one of the above (1) to (6), and the method includes a step of culturing the metabolic enzyme gene disrupted strain in a medium containing a sufficient amount of a carbon source and other nutrients under aerobic conditions.

[0035] (10) The method according to the above (9), wherein the culturing includes a step of culturing the metabolic enzyme gene disrupted strain in the logarithmic growth phase and a step of culturing the metabolic enzyme gene disrupted strain in the stationary phase.

[0036] (11) A method for producing a metabolite by a metabolic enzyme gene-disrupted strain of an actinomycete, comprising the step of culturing a metabolic enzyme gene-disrupted strain, the metabolic enzyme gene-disrupted strain comprising the metabolic enzyme gene-disrupted strain according to any one of (1) to (6) above, the method comprising the steps of culturing the metabolic enzyme gene-disrupted strain under aerobic conditions in a culture medium containing a sufficient amount of a carbon source and other nutrients, so that the metabolic enzyme gene-disrupted strain produces the metabolite, and recovering the metabolite.

[0037] (12) The method according to (11) above, wherein the metabolite is derived from the citric acid cycle.

[0038] (13) A method for selecting an actinomycete, comprising the steps of providing an isolated actinomycete, measuring a carbon dioxide-to-carbon yield of the actinomycete in a logarithmic growth phase or a stationary phase in aerobic culture in a culture medium containing a sufficient amount of a carbon source and other nutrients, and selecting or obtaining an actinomycete having a carbon dioxide-to-carbon yield of more than 50%.

[0039] (14) A method for genetically modifying an actinomycete to obtain a genetically modified actinomycete, comprising the steps of providing an isolated actinomycete, measuring a carbon dioxide-to-carbon yield of the actinomycete in a logarithmic growth phase or a stationary phase in aerobic culture in a culture medium containing a sufficient amount of a carbon source and other nutrients, selecting or obtaining an actinomycete having a carbon dioxide-to-carbon yield of more than 50%, disrupting at least one of genes of the obtained actinomycete encoding enzymes responsible for metabolic reactions that produce carbon dioxide by genetic modification, and obtaining a genetically modified actinomycete. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 A metabolic pathway including glycolysis, the pentose phosphate cycle, and the TCA cycle is shown.

[0041] Figure 2 The optical density (OD600), dry cell weight, and glucose consumption after the start of formal culture of the wild-type (WT) and Δicd strains of S. peucetius are shown.

[0042] Figure 3 The production amount of undecaprenyl pyrophosphate (Udp) of the wild-type (WT) and Δicd strains of S. peucetius is shown.

[0043] Figure 4 A synthetic pathway of Udp is shown.

[0044] Figure 5The formal culture start after the optical density (OD600), dry cell weight and glucose consumption amount of a deletion strain of the gene (pckA) encoding an enzyme that produces phosphoenolpyruvate from oxaloacetate of Streptomyces lividans (ΔpckA), a deletion strain of the gene (maeA) encoding an enzyme that produces pyruvate from malate (ΔmaeA), a deletion strain of the gene (gnd) encoding an enzyme that produces ribulose-5-phosphate from 6-phosphogluconate (Δgnd) are shown.

[0045] Figure 6 The production amount of undecaprenyl pyrophosphate (Udp) of the ΔpckA strain is shown. DETAILED DESCRIPTION

[0046] <Definitions of Terms>

[0047] In the present specification, "about" means a range of ±10%, ±5% or ±1% of the numerical value following it.

[0048] In the present specification, "aerobic bacteria" means microorganisms having a mechanism to consume oxygen to produce energy. Aerobic bacteria utilize oxygen in order to obtain energy from sugars and lipids and the like. As aerobic bacteria, facultative aerobic bacteria and obligate aerobic bacteria can be exemplified. Facultative aerobic bacteria can utilize oxygen, but can also produce energy in an anaerobic manner. In the present specification, aerobic bacteria can include microorganisms classified as anaerobic bacteria such as facultative anaerobic bacteria, as long as the above definition is satisfied. As examples of facultative aerobic bacteria, yeasts, bacteria, molds can be exemplified. Aerobic bacteria in which metabolic enzymes are disrupted have been established. Therefore, in the present specification, aerobic bacteria having metabolic enzymes after disruption are sometimes referred to simply as metabolic enzyme gene disruption strains.

[0049] In the present specification, "actinomycete" means a bacterium belonging to the phylum Actinobacteria. As the actinomycete, there is no particular limitation, and examples thereof include actinomycetes of the genus Streptomyces, the genus Actinomyces, the genus Mycobacterium, and the genus Corynebacterium. The actinomycete is a facultative aerobic bacterium, and is capable of producing energy under anaerobic conditions, but is cultured under aerobic conditions when used for production of a substance. As the actinomycete of the genus Streptomyces, there can be mentioned, for example, Streptomyces lividans, Streptomyces violaceoruber, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces rapamycinicus, Streptomyces kanamyceticus, Streptomyces peucetius, Streptomyces galilaeus, Streptomyces purvulus, Streptomyces albus, Streptomyces albulus, and the like as representative examples. As the actinomycete of the genus Streptomyces, there can also be mentioned S. albaduncus, S. albiaxialis, S. albidochromogenes, S. albidoflavus, S. albiflaviniger, S. albireticuli, S. albofaciens, S. alboflavus, S. albogriseolus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subsp. labilomyceticus, S. albolongus, S. alboniger, S. albospinus, S. albosporeus, S. albo.subsp. albosporeus, S. albo.subspalboverticillatus), S. albovinaceus, S. alboviridis, S. albulus, S. albus, S. albu. subsp. albus, S. albu. subsp. pathocidicus, S. aldersoniae, S. almquistii, S. alni, S. althioticus, S. amakusaensis, S. ambofaciens, S. aminophilus, S. amritsarensis, S. anandii, S. angustmyceticus, S. anthocyanicus, S. antibioticus, S. antimycoticus, S. anulatus, S. aomiensis, S. arabicus, S. araujoniae, S. ardus, S. arenae, S. argenteolus, S. armeniacus, S. artemisiae, S. ascomycinicus, S. asiaticus, S. asterosporus, S. atacamensis, S. atratus, S. atriruber, S. atroaurantiacus, S. atroolivaceus, S. atrovirens, S. aurantiacus, S. auran tiogriseus, S. auratus, S. aureocirculatus, S. aureofaciens, S. aureus, S. auroopictus, S. aurosepiens, S. auroviridis, S. aurozonatus, S. aurozonus, S. aurum, S. aurus, S. australiensis, S. australinus, S. australis, S. austrinus, S. austroafricanus, S. austrosinensis, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus, S. austrosinicus,Streptomyces aureorectus, Streptomyces aureoversilis, Streptomyces aureoverticillatus, Streptomyces aureus, Streptomyces avellaneus, Streptomyces avermectinius, Streptomyces avermitilis, Streptomyces avicenniae, Streptomyces avidinii, Streptomyces axinellae, Streptomyces azaticus, Streptomyces azureus, Streptomyces baarnensis, Streptomyces bacillaris, Streptomyces badius, Streptomyces baldaccii, Streptomyces baliensis, Streptomyces bambergiensis, Streptomyces benjaminus. Streptomyces barkulensis, Streptomyces beijiangensis, Streptomyces bellus, Streptomyces bikiniensis, Streptomyces biverticillatus, Streptomyces blastmyceticus, Streptomyces bluensis, Streptomyces bobili, Streptomyces bottropensis, Streptomyces brasiliensis, Streptomyces brevispora, Streptomyces bullii, Streptomyces bungoensis, Streptomyces burgazadensis, Streptomyces cacaoi, Streptomyces cac. subsp. asoensis, Streptomyces cac. subsp. Streptomyces cacaoi, Streptomyces caelestis, Streptomyces caeruleatus, Streptomyces caeruleus, Streptomyces calidiresistens, Streptomyces californicus, Streptomyces calvus, Streptomyces canarius, Streptomyces candidus, Streptomyces canescens, Streptomyces yogyakarta (S.Streptomyces cangkringensis, Streptomyces caniferus, Streptomyces canus, Streptomyces capillispiralis, Streptomyces capoamus, Streptomyces carpaticus, Streptomyces carpinensis, Streptomyces castelarensis, Streptomyces catbensis, Streptomyces catenulae, Streptomyces caviscabies, Streptomyces cavourensis, Streptomyces cav. subsp. cavourensis, Streptomyces cav. subsp. washingtonensis, Streptomyces cellostaticus, Streptomyces floccosum (S. Streptomyces celluloflavus, Streptomyces cellulolyticus, Streptomyces cellulosae, Streptomyces champavatii, Streptomyces chartreusis, Streptomyces chattanoogensis, Streptomyces cheonanensis, Streptomyces chiangmaiensis, Streptomyces chibaensis, Streptomyces chilikensis, Streptomyces chlorus, Streptomyces chrestomyceticus, Streptomyces chromofuscus, Streptomyces chryseus, Streptomyces chrysomallus, Streptomyces chrysomallus subsp. chrysomallus, Streptomyces chrysomallus subsp. chrysomallus. *Streptomyces fumigatus*, *Streptomyces chumphonensis*, *Streptomyces cinereorectus*, *Streptomyces cinereoruber*, *Streptomyces ci. subsp. cinereoruber*, *Streptomyces ci. subsp. fructofermentans*, *Streptomyces cinereospinus*, *Streptomyces cinereus*, and *Streptomyces griseus*.*Streptomyces cinerochromogenes*, *Streptomyces cinnabarinus*, *Streptomyces cinnamonensis*, *Streptomyces cinnamoneus*, *Streptomyces cirratus*, *Streptomyces ciscaucasicus*, *Streptomyces citreofluorescens*, *Streptomyces clavifer*, *Streptomyces clavuligerus*, *Streptomyces coacervatus*, *Streptomyces cochleatus*, *Streptomyces cocklensis*, *Streptomyces coelescens*, *Streptomyces coelicoflavus*, *Streptomyces coelicolor*, *Streptomyces coeruleoflavus*, *Streptomyces coeruleoflavus*. Streptomyces coeruleofuscus, Streptomyces coeruleoprunus, Streptomyces coeruleorubidus, Streptomyces coerulescens, Streptomyces collinus, Streptomyces colombiensis, Streptomyces corchorusii, Streptomyces costaricanus, Streptomyces cremeus, Streptomyces crystallinus, Streptomyces curacoi, Streptomyces cuspidosporus, Streptomyces cyaneofuscatus, Streptomyces cyaneus, Streptomyces cyanoalbus, Streptomyces cystargineus, Streptomyces daghestanicus, Streptomyces daliensis, Streptomyces decanensis. *Streptomyces deccanensis*, *Streptomyces decoyicus*, *Streptomyces demainii*, *Streptomyces deserti*, *Streptomyces diastaticus*, *Streptomyces d. subsp. ardesiacus*, *Streptomyces d. subsp. diastaticus*, *Streptomyces diastatochromogenes*, *Streptomyces ecdystomum*.Streptomyces distallicus, Streptomyces djakartensis, Streptomyces drozdowiczii, Streptomyces durhamensis, Streptomyces durmitorensis, Streptomyces echinatus, Streptomyces echinoruber, Streptomyces ederensis, Streptomyces ehimensis, Streptomyces emeiensis, Streptomyces endophyticus, Streptomyces endus, Streptomyces enissocaesilis, Streptomyces erringtonii, Streptomyces erumpens, Streptomyces erythraeus, Streptomyces erythrogriseus, Streptomyces euromyces (S. erythrogriseus). Streptomyces eurocidicus, Streptomyces europaeiscabiei, Streptomyces eurythermus, Streptomyces exfoliatus, Streptomyces felleus, Streptomyces fenghuangensis, Streptomyces ferralitis, Streptomyces fervens, Streptomyces filamentosus, Streptomyces fildesensis, Streptomyces filipinensis, Streptomyces fimbriatus, Streptomyces fimicarius, Streptomyces finlayi, Streptomyces flaveolus, Streptomyces flaveus, Streptomyces flavidofuscus, Streptomyces flavidovirens, Streptomyces microsclerotium. *Streptomyces flaviscleroticus*, *Streptomyces flavofungini*, *Streptomyces flavofuscus*, *Streptomyces flavogriseus*, *Streptomyces flavopersicus*, *Streptomyces flavotricini*, *Streptomyces flavovariabilis*, *Streptomyces flavovirens*, *Streptomyces flavoviridis*, *Streptomyces pubescens*.Streptomyces flocculus, Streptomyces floridae, Streptomyces fluorescens, Streptomyces fradiae, Streptomyces fragilis, Streptomyces fukangensis, Streptomyces fulvissimus, Streptomyces fulvorobeus, Streptomyces fumanus, Streptomyces fumigatiscleroticus, Streptomyces galbus, Streptomyces galilaeus, Streptomyces gancidicus, Streptomyces gardneri, Streptomyces gelaticus, Streptomyces geldanamycininus, Streptomyces geysersis. *Streptomyces geysiriensis*, *Streptomyces ghanaensis*, *Streptomyces gibsonii*, *Streptomyces glaucescens*, *Streptomyces glauciniger*, *Streptomyces glaucosporus*, *Streptomyces glaucus*, *Streptomyces globisporus*, *Streptomyces gl. subsp. caucasicus*, *Streptomyces gl. subsp. flavofuscus*, *Streptomyces gl. subsp. globisporus*, *Streptomyces globosus*, *Streptomyces glomeratus*, *Streptomyces glomeroaurantiacus*, *Streptomyces glomeratiacus*. glycovorans), Gobi streptococcus, Goshikiensis, Gougerotii, graminearus, gramineus, graminifolii, graminilatus, graminisoli, graminofaciens, griseiniger, griseinus, and griseinus.Streptomyces griseoaurantiacus, Streptomyces griseobrunneus, Streptomyces griseeocarneus, Streptomyces griseochromogenes, Streptomyces griseoflavus, Streptomyces griseofuscus, Streptomyces griseoincarnatus, Streptomyces griseoloalbus, Streptomyces griseolosporeus, Streptomyces griseolus, Streptomyces griseoluteus, Streptomyces griseomycini, Streptomyces griseoplanus, Streptomyces griseorubens, Streptomyces griseoruber, Streptomyces griseorubiginosus, Streptomyces griseorubiginosus. Streptomyces griseosporeus, Streptomyces griseostramineus, Streptomyces griseoverticillatus, Streptomyces griseoviridis, Streptomyces griseus, Streptomyces griseus alpha, Streptomyces griseus cretosus, Streptomyces griseus solvifaciens, Streptomyces guanduensis, Streptomyces gulbargensis, Streptomyces hachijoensis, Streptomyces hainanensis, Streptomyces haliclonae, Streptomyces halophytocola, Streptomyces holsteredii. Streptomyces hawaiiensis, Streptomyces hebeiensis, Streptomyces heilongjiangensis, Streptomyces heliomycini, Streptomyces helvaticus, Streptomyces herbaceus, Streptomyces herbaricolor, Streptomyces nigermostatin (S.Streptomyces himastatinicus, Streptomyces hiroshimensis, Streptomyces hirsutus, Streptomyces hokutonensis, Streptomyces hoynatensis, Streptomyces humidus, Streptomyces humiferus, Streptomyces hyderabadensis, Streptomyces hydrogenans, Streptomyces hygroscopicus, Streptomyces h. subsp. angustmyceticus, Streptomyces h. subsp. decoyicus, Streptomyces h. subsp. glebosus, Streptomyces h. subsp. hygroscopicus, Streptomyces h. subsp. *Streptomyces ossamyceticus*, *Streptomyces hypolithicus*, *Streptomyces iakyrus*, *Streptomyces iconiensis*, *Streptomyces incanus*, *Streptomyces indiaensis*, *Streptomyces indicus*, *Streptomyces indigoferus*, *Streptomyces indonesiensis*, *Streptomyces intermedius*, *Streptomyces inusitatus*, *Streptomyces ipomoeae*, *Streptomyces iranensis*, *Streptomyces janthinus*, *Streptomyces javensis*, *Streptomyces jietaisiensis*, *Streptomyces jiujiangensis*, *Streptomyces composurei*. *Streptomyces kaempferi*, *Streptomyces kanamyceticus*, *Streptomyces karpasiensis*, *Streptomyces kashmirensis*, *Streptomyces kasugaensis*, *Streptomyces katrae*, *Streptomyces kentuckensis*, *Streptomyces kifunensis*, *Streptomyces kishiwadensis*, *Streptomyces koyangensis*, *Streptomyces kunmingensis*, *Streptomyces kuryi*.*Streptomyces kurssanovii*, *Streptomyces labedae*, *Streptomyces laceyi*, *Streptomyces lacticiproducens*, *Streptomyces laculatispora*, *Streptomyces ladakanum*, *Streptomyces lanatus*, *Streptomyces lannensis*, *Streptomyces lateritius*, *Streptomyces laurentii*, *Streptomyces lavendofoliae*, *Streptomyces lavendulae*, *Streptomyces la. subsp. grasserius*, *Streptomyces la. subsp. lavendulae*, *Streptomyces lavenduligriseus*, *Streptomyces lavandulae*. *Streptomyces lavendulocolor*, *Streptomyces leeuwenhoekii*, *Streptomyces levis*, *Streptomyces libani*, *Streptomyces li. subsp. libani*, *Streptomyces li. subsp. rufus*, *Streptomyces lienomycini*, *Streptomyces lilacinus*, *Streptomyces limosus*, *Streptomyces lincolnensis*, *Streptomyces lipmanii*, *Streptomyces litmocidini*, *Streptomyces lividans*, *Streptomyces lomondensis*, *Streptomyces longisporoflavus*, *Streptomyces longisporoflavus*. Streptomyces longispororuber, Streptomyces longisporus, Streptomyces longwoodensis, Streptomyces lopnurensis, Streptomyces lucensis, Streptomyces lunalinharesii, Streptomyces luridiscabiei, Streptomyces luridus, Streptomyces lusitania, Streptomyces luteireticuli, Streptomyces luteogriseus, Streptomyces luteosporeus, Streptomyces luteosporeus.*Streptomyces luteoverticillatus*, *Streptomyces lydicus*, *Streptomyces macrosporus*, *Streptomyces malachitofuscus*, *Streptomyces malachitospinus*, *Streptomyces malaysiensis*, *Streptomyces marinus*, *Streptomyces marokkonensis*, *Streptomyces mashuensis*, *Streptomyces massasporeus*, *Streptomyces matensis*, *Streptomyces mauvecolor*, *Streptomyces mayteni*, *Streptomyces mediocidicus*, *Streptomyces mediolani*, *Streptomyces megasporus*, *Streptomyces megasporus*, *Streptomyces meiflora*. Streptomyces melanogenes, Streptomyces melanosporofaciens, Streptomyces mexicanus, Streptomyces michiganensis, Streptomyces microflavus, Streptomyces milbemycinicus, Streptomyces minutiscleroticus, Streptomyces mirabilis, Streptomyces misakiensis, Streptomyces misionensis, Streptomyces mobaraensis, Streptomyces monomycini, Streptomyces mordarskii, Streptomyces morookaense, Streptomyces muensis, Streptomyces murinus, Streptomyces mutabilis, Streptomyces mumutamycinus (S. Streptomyces mutomycini, Streptomyces naganishii, Streptomyces nanhaiensis, Streptomyces nanshensis, Streptomyces narbonensis, Streptomyces nashvillensis, Streptomyces netropsis, Streptomyces neyagawaensis, Streptomyces niger, Streptomyces nigrescens, Streptomyces nigrifaciens, Streptomyces nigrifaciens.nitrosporeus), Streptomyces niveiscabiei, Streptomyces niveoruber, Streptomyces niveus, Streptomyces noboritoensis, Streptomyces nodosus, Streptomyces nogalater, Streptomyces nojiriensis, Streptomyces nooursei, Streptomyces novaecaesareae, Streptomyces ochraceiscleroticus, Streptomyces odorifer, Streptomyces olivaceiscleroticus, Streptomyces olivaceoviridis, Streptomyces olivaceus, Streptomyces olivaceus, Streptomyces olivaceiscleroticus. *Streptomyces olivochromogenes*, *Streptomyces olivomycini*, *Streptomyces olivoreticuli*, *Streptomyces olivoverticillatus*, *Streptomyces olivoviridis*, *Streptomyces omiyaensis*, *Streptomyces orinoci*, *Streptomyces osmaniensis*, *Streptomyces pactum*, *Streptomyces panacagri*, *Streptomyces panaciradicis*, *Streptomyces paracochleatus*, *Streptomyces paradoxus*, *Streptomyces parvisporogenes*, *Streptomyces parvulus*, *Streptomyces parvus*, *Streptomyces pathocidini*, *Streptomyces oligosporus*. Streptomyces paucisporeus, Streptomyces peucetius, Streptomyces phaeochromogenes, Streptomyces phaeofaciens, Streptomyces phaeogriseichromatogenes, Streptomyces phaeoluteichromatogenes, Streptomyces phaeoluteigriseus, Streptomyces phaeopurpureus, Streptomyces phaeoviridis, Streptomyces pharetrae, Streptomyces pharetrae (p. paucisporeus), Streptomyces peucetius, Streptomyces phaeoluteigriseus, Streptomyces phaeopurpureus, Streptomyces phaeoviridis, Streptomyces pharetrae, Streptomyces pharetrae (p. paucisporeus), Streptomyces peucetius, Streptomyces phaeoluteigriseus, Streptomyces phaeopurpureus, Streptomyces phaeoviridis, Streptomyces pharetrae (p. paucisporeus), Streptomyces phaeoluteigriseus ...luteigriseus, Streptomyces phaeoStreptomyces pharmamarensis, Streptomyces phosalacineus, Streptomyces phytohabitans, Streptomyces pilosus, Streptomyces platensis, Streptomyces plicatus, Streptomyces plumbiresistens, Streptomyces pluricolorescens, Streptomyces pluripotens, Streptomyces polyantibioticus, Streptomyces polychromogenes, Streptomyces poonensis, Streptomyces praecox, Streptomyces prasinopilosus, Streptomyces prasinosporus, Streptomyces prasinus, Streptomyces pratens, Streptomyces prasiatum. *Streptomyces pratensis*, *Streptomyces prunicolor*, *Streptomyces psammoticus*, *Streptomyces pseudoechinosporeus*, *Streptomyces pseudodogriseolus*, *Streptomyces pseudovenezuelae*, *Streptomyces pulveraceus*, *Streptomyces puniceus*, *Streptomyces puniciscabiei*, *Streptomyces purpeofuscus*, *Streptomyces purpurascens*, *Streptomyces purpureus*, *Streptomyces purpurogeneiscleroticus*, *Streptomyces qinglanensis*, *Streptomyces racemochromogenes*, *Streptomyces radiopugnans*, *Streptomyces cladoides*. *Streptomyces rameus*, *Streptomyces ramulosus*, *Streptomyces rangoonensis*, *Streptomyces rapamycinicus*, *Streptomyces recifensis*, *Streptomyces rectiverticillatus*, *Streptomyces rectiviolaceus*, *Streptomyces regensis*, *Streptomyces resistomycificus*, *Streptomyces reticulatus*.*Streptomyces reticuliscabiei*, *Streptomyces rhizosphaericus*, *Streptomyces rhizophilus*, *Streptomyces rimosus*, *Streptomyces ri. subsp. paromomycinus*, *Streptomyces ri. subsp. rimosus*, *Streptomyces rishiriensis*, *Streptomyces rochei*, *Streptomyces rosealbus*, *Streptomyces roseiscleroticus*, *Streptomyces roseodiastaticus*, *Streptomyces roseoflavus*, *Streptomyces roseofulvus*, *Streptomyces roseolilacinus*, *Streptomyces roseolus*, *Streptomyces roseosporeus*. *Streptomyces roseosporus*, *Streptomyces roseoverticillatus*, *Streptomyces roseoviolaceus*, *Streptomyces roseoviridis*, *Streptomyces ruanii*, *Streptomyces ruber*, *Streptomyces rubidus*, *Streptomyces rubiginosohelvolus*, *Streptomyces rubiginosus*, *Streptomyces rubrogriseus*, *Streptomyces rubrus*, *Streptomyces rutgersensis*, *Streptomyces ru. subsp. castelarensis*, *Streptomyces ru. subsp. rutgersensis*, *Streptomyces salmonis*, *Streptomyces sampsonii*, *Streptomyces samsonii*. Streptomyces samsunensis, Streptomyces sanglieri, Streptomyces sannanensis, Streptomyces sanyensis, Streptomyces sapporonensis, Streptomyces scabiei, Streptomyces scabrisporus, Streptomyces sclerotialus, Streptomyces scopiformis, Streptomyces scopuliridis, Streptomyces sedi, Streptomyces serrulata.Streptomyces seoulensis, Streptomyces septatus, Streptomyces seranimatus, Streptomyces setae, Streptomyces setonii, Streptomyces shaanxiensis, Streptomyces shenzhenensis, Streptomyces showdoensis, Streptomyces silaceus, Streptomyces sindenensis, Streptomyces sioyaensis, Streptomyces smyrnaeus, Streptomyces sodiiphilus, Streptomyces somaliensis, Streptomyces sparsogenes, Streptomyces sparsus, Streptomyces specialis, Streptomyces spectabilis, Streptomyces fasciatus. *Streptomyces speibonae*, *Streptomyces speleomycini*, *Streptomyces spheroides*, *Streptomyces spinoverrucosus*, *Streptomyces spiralis*, *Streptomyces spiroverticillatus*, *Streptomyces spitsbergensis*, *Streptomyces spongiae*, *Streptomyces sporocinereus*, *Streptomyces sporoclivatus*, *Streptomyces spororaveus*, *Streptomyces sporoverrucosus*, *Streptomyces staurosporininus*, *Streptomyces stelliscabiei*, *Streptomyces stramineus*, *Streptomyces subrutilus*, *Streptomyces sulfonicum*. *Streptomyces sulfonofaciens*, *Streptomyces sulphureus*, *Streptomyces sundarbansensis*, *Streptomyces synnematoformans*, *Streptomyces syringium*, *Streptomyces tacrolimicus*, *Streptomyces tanashiensis*, *Streptomyces tateyamensis*, *Streptomyces tauricus*, *Streptomyces tendae*, *Streptomyces termites*.The following are listed: *Streptomyces thermoalitolerans*, *Streptomyces thermoautotrophicus*, *Streptomyces thermocarboxydovorans*, *Streptomyces thermocarboxydus*, *Streptomyces thermocoprophilus*, *Streptomyces thermodiastaticus*, *Streptomyces thermogriseus*, *Streptomyces thermolineatus*, *Streptomyces thermomonitrificans*, *Streptomyces thermospinosisporus*, *Streptomyces thermomoviolaceus*, *Streptomyces thermoviolaceus* subsp. *apingens*, *Streptomyces thermoviolaceus* subsp. *thermoviolaceus*, *Streptomyces thermovulgaris*, and *Streptomyces thermovulgaris*. Streptomyces thioluteus, Streptomyces torulosus, Streptomyces toxytricini, Streptomyces tricolor, Streptomyces tritolerans, Streptomyces tubercidicus, Streptomyces tuirus, Streptomyces tunisiensis, Streptomyces turgidiscabies, Streptomyces umbrinus, Streptomyces variabilis, Streptomyces variegatus, Streptomyces varsoviensis, Streptomyces vastus, Streptomyces venezuelae, Streptomyces vietnamensis, Streptomyces vinaceus, Streptomyces rubrumensis. vinaceusdrappus), purple-colored Streptomyces (S. violaceochromogenes), purple-broad Streptomyces (S. violaceolatus), purple straight-filament Streptomyces (S. violaceorectus), purple-red Streptomyces (S. violaceoruber), deep red-purple Streptomyces (S. violaceorubidus), purple Streptomyces (S.Streptomyces violaceus, Streptomyces violaceusniger, Streptomyces violarus, Streptomyces violascens, Streptomyces violatus, Streptomyces violens, Streptomyces virens, Streptomyces virginiae, Streptomyces viridiflavus, Streptomyces viridiviolaceus, Streptomyces viridis, Streptomyces viridobrunneus, Streptomyces viridochromogenes, Streptomyces viridodiastaticus, Streptomyces viridosporus, Streptomyces vitaminophilus, Streptomyces wedmorensis, Streptomyces wellingtoniae, Streptomyces velutipes. Streptomyces werraensis, Streptomyces willmorei, Streptomyces wuyuanensis, Streptomyces xanthochromogenes, Streptomyces xanthocidicus, Streptomyces xantholiticus, Streptomyces xanthophaeus, Streptomyces xiamenensis, Streptomyces xinghaiensis, Streptomyces xishensis, Streptomyces yaanensis, Streptomyces yanglinensis, Streptomyces yanii, Streptomyces yatensis, Streptomyces yeochonensis, Streptomyces yerevanensis, Streptomyces yogyakartensis, Streptomyces yosuka. *Streptomyces yokosukanensis*, *Streptomyces youssoufiensis*, *Streptomyces yunnanensis*, *Streptomyces zaomyceticus*, *Streptomyces zhaozhouensis*, *Streptomyces zinciresistens*, and *Streptomyces ziwulingensis*.(Streptomyces ziwulingensis). Actinomycetes of the genus Streptomyces can be antibiotic-producing organisms. Examples of antibiotics include streptomycin, kanamycin, rapamycin, daunorubicin, aclarubicin, tetracycline, erythromycin, rifampin, bleomycin, and vancomycin.

[0050] In this manual, "aerobic conditions" refers to the conditions under which aerobic bacteria perform aerobic respiration. Aerobic respiration is handled by a metabolic system including glycolysis, the citric acid cycle, and the electron transport chain. Typically, carbohydrates consume oxygen to produce carbon dioxide, water, and ATP as energy. Glycolysis, for example... Figure 1 The diagram illustrates the pathway from glucose to pyruvate via glucose-6-phosphate. Pyruvate produced in glycolysis is converted to acetyl-CoA by the pyruvate dehydrogenase complex and enters the citric acid cycle (for convenience, this specification categorizes the pathway up to the production of acetyl-CoA as glycolysis). Acetyl-CoA then enters the citric acid cycle for metabolism. The citric acid cycle is also known as the TCA cycle. The citric acid cycle produces NADH, FADH2, and GTP, among others. The electron transport chain oxidizes NADH and FADH2 to produce ATP. By culturing aerobic bacteria under aerobic conditions, ATP can be produced from carbohydrates in a metabolic system that includes glycolysis, the citric acid cycle, and the electron transport chain. In contrast, "anaerobic conditions" refer to conditions under which anaerobic organisms produce energy and multiply without utilizing oxygen. Those skilled in the art can appropriately culture aerobic bacteria under aerobic conditions.

[0051] Bacterial culture consists of a lag phase, a logarithmic growth phase, and a stationary phase. For a certain period after the start of cell culture, bacteria do not divide, and the cell number remains constant. This period is called the lag phase. It is believed that bacteria repair their cells, adjust their metabolism, or accumulate carbonic acid during the lag phase. Afterward, bacteria begin to divide, slowly proliferate, gradually increasing the rate of proliferation until the generation time becomes constant, and the bacterial cells multiply logarithmically. This period is called the logarithmic growth phase. As culture continues and cell density increases, the period of logarithmic proliferation (logarithmic growth phase) ends. Typically, the cell division rate gradually decreases, and the viable cell number becomes constant; however, depending on the bacterial species, there are also cases where the bacterial cells continue to proliferate slowly. This period is usually called the stationary phase. Material production using bacteria mainly takes place during the logarithmic growth phase to the stationary phase. Typically, the boundary between the logarithmic growth phase and the stationary phase is not clearly defined. Therefore, in this specification, the logarithmic growth phase will be defined as the period after the start of culture when the cell proliferation becomes active and the doubling time becomes constant, and the stationary phase will be defined as the period when the cell doubling time is significantly prolonged and the viable cell count becomes constant. Alternatively, in the case of a continuously slowly proliferating strain, the stationary phase will be defined as the period when the doubling time is significantly prolonged compared to the logarithmic growth phase (e.g., more than 2, 3, 4, 5, or 10 times longer than the logarithmic growth phase, depending on the strain) and the cell proliferation is stable.

[0052] In this instruction manual, "carbon yield" refers to the amount of carbon contained in a nutrient's carbon source (such as glucose) that is absorbed in the form of carbon from a specific substance.

[0053] In this specification, "carbon source" refers to organic matter that provides energy and carbon components for heterotrophic cells such as aerobic bacteria. In one embodiment, the carbon source is not particularly limited; for example, it may be a sugar, such as a monosaccharide, and glucose is particularly preferred.

[0054] <The bacteria and the method for selecting bacteria of the present invention>

[0055] According to the embodiments described later, the carbon yield of CO2 varies considerably among bacteria. In particular, there are bacteria whose carbon yield of CO2 exceeds approximately 50% at least during the logarithmic growth phase or stationary phase (preferably the logarithmic growth phase). The present invention provides such bacteria, as well as methods for selecting, obtaining, or isolating such bacteria.

[0056] According to the present invention, bacteria with a CO2 carbon recovery rate of glucose exceeding, for example, about 50% at least during the logarithmic growth phase or stationary phase can be provided. These bacteria are capable of metabolizing glucose to produce energy. In one embodiment, bacteria with a CO2 carbon recovery rate of glucose exceeding, for example, about 60%, about 70%, about 80%, about 90%, or about 95% at least during the logarithmic growth phase or stationary phase can be provided. In a preferred embodiment, the bacteria can be actinomycetes of the genus *Streptomyces*. In a particular embodiment, the actinomycete can be *Streptomyces pulvinatus*. According to the present invention, strains of these bacteria (e.g., actinomycetes) with disrupted metabolic enzyme genes can be provided. Bacteria with high CO2 carbon recovery rates of glucose are advantageous as targets for modification according to the present invention. In one embodiment, the CO2 carbon recovery rate of glucose is higher than that of *Escherichia coli* (e.g., more than 1.1 times, more than 1.2 times, more than 1.3 times, more than 1.4 times, or more than 1.5 times) at least during the logarithmic growth phase or stationary phase. Alternatively, the carbon yield of CO2 from glucose is higher than that of Corynebacterium glutamicum, which is a Corynebacterium, at least during the logarithmic growth phase or the stationary phase (e.g., more than 1.1 times, more than 1.2 times, more than 1.3 times, more than 1.4 times, or more than 1.5 times). In a preferred embodiment, the carbon yield is the carbon yield during the logarithmic growth phase.

[0057] According to the present invention, a method for selecting, obtaining, or isolating the aforementioned bacteria can be provided. The method of the present invention includes the step of providing isolated bacteria. Isolation is preferably cloning. Furthermore, the method of the present invention may also include the step of determining the carbon yield relative to carbon dioxide of the aforementioned bacteria during the logarithmic growth phase or stationary phase in an aerobic culture medium containing sufficient amounts of carbon source and other nutrients (e.g., TSB medium containing 1.8 g / L relative to actinomycetes, 50 g / L glucose, pH 7.0). The carbon yield relative to carbon dioxide can be calculated from the amount of glucose consumed and the amount of metabolites generated. The amount of glucose and the amount of metabolites can be determined according to conventional methods. Additionally, the amount of carbon dioxide emitted can be determined according to conventional methods using an exhaust gas analyzer (e.g., manufactured by OFF-GAS Jr. Bio-Ot Co.) and a mass spectrometer. The amount of glucose and the amount of metabolites are not particularly limited, and can be determined, for example, by component separation using high-performance liquid chromatography and subsequent component analysis. Compositional analysis can be performed using various methods such as mass spectrometry, differential refractive index detectors, and UV detectors, or by combining two or more of these methods.

[0058] Based on the determination of carbon yield from carbon dioxide, bacteria with a carbon yield exceeding, for example, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% can be selected, obtained, or isolated. This allows for the acquisition of bacteria with high carbon yields from carbon dioxide. In this method, the bacteria can be isolated from nature (soil, organisms, biological samples, water, etc.). The bacteria can be pre-established strains. Preferably, the bacteria are actinomycetes of the genus *Streptomyces*. The obtained bacteria can be cloned. Cloning can be achieved, for example, by inoculating onto a solid culture medium and picking a single colony. Regarding actinomycetes, for example, they can be cloned by inoculating spores or protoplastized cells onto a solid culture medium and picking a single colony. Therefore, those skilled in the art can appropriately clone bacteria using suitable methods.

[0059] <The Bacterial Metabolic Enzyme Gene Destruction Strain and its Manufacturing Method of the Invention>

[0060] According to the present invention, a method for producing bacterial strains with disrupted metabolic enzyme genes can be provided.

[0061] The bacteria can be aerobic. In one embodiment, the bacteria can be actinomycetes belonging to the genus Streptomyces.

[0062] In a preferred embodiment, the bacteria can be those obtained by the above method. That is, the bacteria can be those whose carbon yield to CO2 from glucose exceeds, for example, about 50% during at least the logarithmic growth phase or the stationary phase, more preferably those exceeding about 60%, about 70%, about 80%, about 90%, or about 95% during the logarithmic growth phase or the stationary phase. The bacteria can be aerobic, preferably actinomycetes of the genus *Streptomyces*.

[0063] A method for producing a bacterial strain with disrupted metabolic enzyme genes includes the step of providing the aforementioned bacteria.

[0064] In the method for creating bacterial metabolic enzyme gene-destroying strains, the modified bacteria are isolated and preferably cloned.

[0065] Decreasing the carbon yield of CO2 from glucose in bacteria is beneficial. The metabolic reactions that produce CO2 in metabolic pathways are known. To reduce the carbon yield of CO2, one or more enzymes catalyzing these metabolic reactions can be disrupted through genetic engineering.

[0066] A method for producing a bacterial metabolic enzyme gene-disrupted strain includes the step of disrupting enzymes in the metabolic pathways of the aforementioned bacteria that catalyze the metabolic reaction of CO2 production. Disruption is achieved through one or more modifications selected from the group consisting of expression inhibition and annulment, function inhibition and annulment, and gene deletion. There are no particular limitations on the disruption, as long as the yield of carbon dioxide to sugar decreases; for example, the metabolic activity of the enzyme is reduced to less than half, less than 30%, less than 20%, less than 10%, or less than 5%, or below the detection limit. In cases where a metabolic reaction is handled by two or more enzymes, disruption is performed on at least the gene encoding one of these enzymes (e.g., the gene encoding all enzymes). According to the present invention, it is possible to provide metabolic enzyme gene-disrupted strains in the bacterial metabolic pathways that catalyze the metabolic reaction of CO2 production by disrupting one or more of these enzymes.

[0067] The following examples illustrate enzymes responsible for the metabolic reactions that produce CO2, but are not limited to these. Furthermore, enzymes that are direct homologs of the enzymes described below and have equivalent functions are also preferred to be destroyed as enzymes responsible for the metabolic reactions that produce CO2.

[0068] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to any particular enzyme, but examples include isocitrate dehydrogenase (EC1.1.1.41). Isocitrate dehydrogenase (EC1.1.1.41) catalyzes the reaction from isocitrate to CO2 and α-ketoglutarate. Genes encoding such enzymes include icd and SLI_7202.

[0069] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to any particular enzyme; examples include pyruvate decarboxylase (EC4.1.1.1). Pyruvate decarboxylase (EC4.1.1.1) is an enzyme that catalyzes the reaction from pyruvate to CO2 and acetaldehyde.

[0070] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to a few examples; phosphoglucose dehydrogenases (EC 1.1.1.44, 1.1.1.343, or 1.1.1.351) are mentioned as examples. Phosphoglucose dehydrogenases (EC 1.1.1.44, 1.1.1.343, or 1.1.1.351) catalyze the reaction of 6-phosphoglucose and NADP in the pentose phosphate cycle. + Ribulose-5-phosphate and NADPH2 are produced. + Enzymes that react with CO2. Genes encoding such enzymes include gnd, SLI_4134, SLI_7003, SLI_1209, and combinations thereof.

[0071] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to a few examples, such as pyruvate dehydrogenase (EC1.2.4.1), dihydrolipoic acid transacetylase (EC2.3.1.12), and dihydrolipoic acid dehydrogenase (EC1.8.1.4). Pyruvate dehydrogenase (EC1.2.4.1), dihydrolipoic acid transacetylase (EC2.3.1.12), and dihydrolipoic acid dehydrogenase (EC1.8.1.4) can form the pyruvate dehydrogenase complex, which is composed of pyruvate, CoA-SH, and NAD+. + Enzymes that produce acetyl-CoA, NADH, and CO2. Genes encoding such enzymes include aceE, SLI_2705, SLI_2510, SLI_7339, and combinations thereof.

[0072] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to a few examples, but can include α-ketoglutarate dehydrogenase or oxoglutarate dehydrogenase (EC1.2.4.2, EC1.2.7.3, or EC1.2.7.11), dihydrolipoamide S-succinylate transferase (EC2.3.1.61), and dihydrolipoate dehydrogenase (EC1.8.1.4). Oxoglutarate dehydrogenase (EC1.2.4.2, EC1.2.7.3, or EC1.2.7.11), dihydrolipoamide S-succinylate transferase (EC2.3.1.61), and dihydrolipoate dehydrogenase (EC1.8.1.4) form the oxoglutarate dehydrogenase complex, which is then converted from α-ketoglutarate and NAD+. + It reacts with CoA-SH to produce succinyl-CoA, NADH, and H. + And CO2. Genes encoding such enzymes include kgd, SLI_2508, SLI_5576, and combinations thereof.

[0073] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to a single enzyme, but examples include malate dehydrogenases (EC1.1.1.38, EC1.1.1.39, or EC1.1.1.40). Malate dehydrogenases (EC1.1.1.38, EC1.1.1.39, or EC1.1.1.40) are composed of malate and NAD+. + It produces pyruvate, CO2, and NADH. Genes encoding such enzymes include maeA, SLI_5552, SLI_3297, and combinations thereof.

[0074] Enzymes responsible for CO2 production in metabolic pathways are not specifically limited to a few examples, but such enzymes include phosphoenolpyruvate carboxykinase (EC4.1.1.32, EC4.1.1.38, and EC4.1.1.49). All three enzymes (EC4.1.1.32, EC4.1.1.38, and EC4.1.1.49) produce phosphoenolpyruvate and CO2 from oxaloacetate. Examples of such enzyme genes include pckA and SLI_5252.

[0075] Enzymes responsible for CO2 production in metabolic pathways are not particularly limited, but examples include glutamate decarboxylase, hydroxypyruvate decarboxylase, 3-oxoacyl synthase, and aspartate decarboxylase. The details of cellular metabolic reactions are well understood, and those skilled in the art can appropriately identify the enzymes responsible for CO2 production and appropriately disrupt their genes.

[0076] In the method of this invention, one, two, three, four, or five or more enzymes responsible for the metabolic reaction that produces CO2 can be destroyed. Therefore, it is expected that the carbon yield of carbon dioxide from glucose in the destroyed strain will decrease.

[0077] According to the present invention, bacteria (metabolic enzyme gene-damaged strains) with one, two, three, four, or five or more enzymes responsible for the metabolic reaction of CO2 production destroyed can be provided. In one embodiment, the bacteria are isolated. In another embodiment, the bacteria are present in a culture. In another embodiment, the bacteria are cloned. In one embodiment, the bacteria to be modified can be aerobic bacteria. In another embodiment, the bacteria can be actinomycetes of the genus *Streptomyces*. In a preferred embodiment, the bacteria to be modified can be bacteria with a carbon recovery rate of at least about 50% for CO2 from glucose during the logarithmic growth phase or stationary phase, and more preferably bacteria with a carbon recovery rate of at least about 60%, about 70%, about 80%, about 90%, or about 95% during the logarithmic growth phase or stationary phase (preferably actinomycetes, such as actinomycetes of the genus *Streptomyces*). In a preferred embodiment, the bacteria can be *Streptomyces pulvinatus* (e.g., strain 1326).

[0078] The carbon recovery of CO2 from glucose by the metabolic enzyme gene-damaged strain, at least during the logarithmic growth phase or stationary phase, can be at least about 90%, preferably about 80%, more preferably about 70%, further preferably about 60%, and even more preferably about 50%. The carbon recovery of CO2 from glucose by the metabolic enzyme gene-damaged strain, at least during the logarithmic growth phase or stationary phase, can be, for example, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, or about 50% or more. The carbon yield of CO2 from glucose in strains with disrupted metabolic enzyme genes can be approximately 5% to approximately 90%, 10% to approximately 90%, 20% to approximately 90%, 30% to approximately 90%, 35% to approximately 80%, 40% to approximately 70%, 45% to approximately 60%, or approximately 50%, at least during the logarithmic growth phase or stationary phase, compared to strains before disruption of the metabolic enzyme gene.

[0079] In a preferred embodiment, the strain with the metabolic enzyme gene disrupted maintains a proliferation capacity of approximately 50%, 60%, 70%, 80%, or 90% or more compared to the strain before the disruption of the metabolic enzyme gene. The proliferation capacity can be determined, for example, by the weight of dried cells in 1 mL of culture medium after culturing (e.g., 40 hours after the start of formal culturing).

[0080] <Bacterial Culture Method of the Invention>

[0081] According to the present invention, a method for culturing the genetically modified bacteria of the present invention can be provided. The method includes the steps of preparing the genetically modified bacteria and culturing the genetically modified bacteria in a suitable culturing environment. In a preferred embodiment, the suitable culturing environment can be conditions suitable for culturing the corresponding wild-type bacteria. In another embodiment, the suitable culturing environment can be an environment suitable for culturing genetically modified bacteria. In one embodiment, the bacteria are cultured under anaerobic conditions. In a preferred embodiment, the bacteria are cultured under aerobic conditions, for example, simultaneously with shaking or aeration.

[0082] Solid media can be used as culture media, but liquid media are preferred. The culture medium can be one containing components necessary for bacterial survival and / or material production (metabolic product production), such as carbon sources and other nutrient sources (e.g., nitrogen sources and inorganic salts). Those skilled in the art can appropriately prepare such media. The culture medium can be synthetic or semi-synthetic. The culture medium may contain: basal media such as TSB, LB, YT (or 2×YT), NZY, M9, SOC, and YPD; buffers; salts; pH adjusters; alkali metal ions such as potassium and sodium; alkaline earth metal ions such as calcium; and metal ions such as magnesium, iron, tin, cobalt, manganese, and zinc; phosphorus and sulfur. The culture medium may contain nitrogen sources selected from the group consisting of ammonium salts, nitrates, amino acids, peptone, casein, casein amino acids, urea, yeast extract, protein hydrolysates, yeast autodigested products, casein pancreatic digests, soybean papain digests, tryptone, plant peptone, meat extract, meat peptone, and tryptone. Carbon sources may include, for example, glucose, dextran, starch, beef extract, pyruvate, and acetic acid. Inorganic salts may include: sodium salts such as sodium phosphate, sodium chloride, and sodium molybdate; potassium salts such as potassium phosphate, potassium iodide, and potassium chloride; calcium salts such as calcium chloride and calcium phosphate; magnesium salts such as magnesium chloride and magnesium sulfate; transition metal salts such as copper sulfate, manganese sulfate, zinc sulfate, and ferric chloride; and borates. The pH of the culture medium is not particularly limited and may be, for example, approximately 7, or approximately 6.5 to approximately 7.5. The culture temperature varies depending on the bacteria and may be, for example, 25°C to 37°C, or 25°C to 30°C. There is no particular limitation on the culture time; it can be, for example, 1 day to 2 months, 1 day to 1 month, 1 day to 2 weeks, or 1 day to 1 week. During the culture process, carbon sources or other nutrient sources can be added to the culture medium. Those skilled in the art can select appropriate culture conditions based on the bacteria.

[0083] In the method of this invention, the culture includes bacteria in a logarithmic growth phase and a stationary phase, or a culture of a metabolic enzyme gene-disrupted strain. During the stationary phase, the bacteria or metabolic enzyme gene-disrupted strain can produce various metabolites. In particular, because the enzymes that synthesize carbon dioxide are disrupted in metabolic enzyme gene-disrupted strains, the carbon yield of the metabolites is increased, allowing for efficient use of the carbon source to produce these various metabolites. Metabolites are not particularly limited, but examples include undecylnidole rubigin (Udp). Examples of metabolites include metabolites from upstream or downstream metabolic pathways involving CO2 expulsion, or metabolites generated using such metabolites. To obtain metabolites from downstream metabolic pathways involving CO2 expulsion, or metabolites generated using such metabolites, it is possible to introduce an intermediate from the downstream metabolic pathway into the cells. The introduction of the intermediate into the cells can be carried out by adding the intermediate to the culture medium. This intermediate can be used intracellularly as a raw material for the aforementioned metabolites.

[0084] According to one method, modification can reduce the carbon yield of CO2 compared to unmodified cells, which can help reduce the amount of CO2 produced during culture. According to a preferred method, modification can redirect carbon that would otherwise be consumed in CO2 calculations to material production; in this case, modification can promote material production compared to unmodified cells. According to a preferred method, modification does not inhibit cell proliferation by more than 50%, 40%, 33%, 30%, 25%, 20%, 15%, or 10%. According to one method, modification does not reduce sugar consumption by more than 50%, 40%, 33%, 30%, 25%, 20%, 15%, or 10% compared to unmodified cells, or preferably, sugar consumption does not decrease or increases. According to a preferred embodiment, the modification reduces the carbon yield of CO2 compared to unmodified cells. On the other hand, the modification ensures that cell proliferation does not differ by more than 20% (preferably not more than 10% or remains unchanged), and / or, sugar consumption does not decrease or increase.

[0085] Example

[0086] Example 1: Carbon yield of bacteria

[0087] Prepare *Escherichia coli*, *Corynebacterium glutamicum*, and *Streptomyces pulmonale*, strain 1326. A synthetic medium was used as the culture medium. The synthetic medium contained 1.8 g / L TSB medium (pH 7.0) as the basal medium. The pH of the synthetic medium was adjusted using NaOH. The synthetic medium also contained an initial glucose concentration of 50 g / L. Each bacterium was cultured in 50 mL of synthetic medium at 28°C and 160 rpm for 48 hours with shaking. Afterward, fermentation was carried out. The bacteria obtained through pre-culture were added to 1 L of synthetic medium to achieve an optical density of 0.2. After 40 hours of culture, glucose was further added at a concentration of 50 g / L. Other antifoaming agents were added to the medium as needed. During culture, the CO2 content in the exhaust gas was measured using an exhaust gas analyzer (manufactured by OFF-GAS Jr. Bio-Ot Co., Ltd.).

[0088] Escherichia coli was recovered after 10 hours (logarithmic growth phase), Corynebacterium was recovered after 10 hours (logarithmic growth phase), and Actinomycetes were recovered after 40 hours (logarithmic growth phase) and 88 hours (stationary phase).

[0089] The recovered culture medium containing bacteria was analyzed by HPLC to determine the carbon recovery rate. The HPLC analysis conditions are as follows.

[0090] Column: Aminex HPX-87H

[0091] Eluent: 5mM H2SO4

[0092] Flow rate: 0.6 mL / min

[0093] Temperature: 50℃

[0094] Detectors: UV detector and Differential Refractive Index Detector (RID)

[0095] The results are shown in Table 1.

[0096] [Table 1]

[0097] As shown in Table 1, the investigated actinomycete strains exhibited high CO2 carbon recovery during the logarithmic growth phase, indicating that more than half of the glucose was converted into CO2. Furthermore, although data are not shown in Table 1, the CO2 carbon recovery of the actinomycetes during the stationary phase shows high values ​​above the logarithmic growth phase. This suggests that species and strains with high CO2 carbon recovery exist among bacteria (especially actinomycetes).

[0098] The carbon yield of CO2 is an indicator of how much carbon from glucose is used to produce CO2. When bacteria are effectively used in material production, the carbon yield of CO2 may negatively impact the production process. This is because the consumption of carbon from glucose in CO2 production may inhibit its efficient utilization in material production.

[0099] Example 2: Preparation of enzyme-destroying strains and CO2 carbon recovery

[0100] Example 2-1: Δicd on carbon yield and glucose consumption

[0101] Enzymatic reactions that produce CO2 occur in metabolic pathways such as glycolysis, the TCA cycle, and the pentose phosphate cycle (see reference). Figure 1 A strain (Δicd) lacking the enzyme (icd) that produces α-ketoglutarate and CO2 from isocitrate was prepared. Cell count, glucose consumption, and CO2-to-carbon recovery during culture were measured. Cells were cultured and recovered under the same conditions as the wild-type strain (WT), except that the initial and added glucose concentrations were both set to 100 g / L. The ICD activity of the Δicd strain was determined by measuring the increase in NADPH generated during the conversion of isocitrate to 2-ketoglutarate by ICD at 340 nm. The results showed that the ICD activity of the Δicd strain was significantly reduced.

[0102] [Table 2]

[0103] As shown in Table 2, during the stationary period, the CO2 carbon yield of the Δicd strain decreased to about half that of the wild-type strain (WT) mentioned above.

[0104] Analysis of carbon yield also revealed that during the logarithmic growth phase of the Δicd strain, the carbon yields of pyruvate, isocitrate or citric acid, and ribulose increased. Furthermore, during the stationary phase of the Δicd strain, the carbon yields of ribulose and pyruvate increased.

[0105] Further analysis of carbon yield revealed a sharp decrease in CO2 carbon yield during the stationary phase of the Δicd strain. Furthermore, the combined carbon yield of various unidentified metabolites accounted for 40.7%, indicating a significant improvement in carbon utilization efficiency within the bacterial cell. Therefore, the disruption of the enzyme responsible for CO2 production in the Δicd strain caused a dramatic change in CO2 carbon yield.

[0106] This means that in bacteria whose carbon source utilization is strictly limited to carbon dioxide emission, it is possible to induce a state that removes this limitation and allows for more efficient use of carbon sources in metabolic reactions. Furthermore, this embodiment demonstrates that disrupting the genes of enzymes responsible for the metabolic reactions that produce carbon dioxide is effective.

[0107] In addition, such as Figure 2 As shown, the Δicd strain was observed to have a 2.6-fold increase in glucose consumption compared to the wild-type strain (WT).

[0108] Example 2-2: Carbon yield and glucose consumption of ΔpckA, ΔmaeA, and Δgnd

[0109] As enzyme genes for CO2 production other than the ICD-disrupted strain, defective strains were created for the enzymes that produce phosphoenolpyruvate from oxaloacetate (pckA) (ΔpckA), the enzyme that produces pyruvate from malate (maeA) (ΔmaeA), and the enzyme that produces ribulose-5-phosphate from 6-phosphogluconic acid (gnd) (Δgnd). Since maeA has two homologs, a double-deficient strain was created. Gnd has three homologs, but a strain deficient in only one of them was created (see Table 9). Both the disrupted strains and the wild-type strains were cultured aerobically in fermenters, and the cell mass, glucose consumption, and CO2-to-carbon recovery were measured. Culture was conducted under the same conditions as the wild-type strains.

[0110] [Table 3]

[0111] As shown in Table 3, after cultivation, the carbon yield of CO2 for each gene-disrupted strain decreased to 0.86 times for strain ΔpckA, 0.88 times for strain ΔmaeA, and 0.86 times for strain Δgnd compared to the wild-type strains (parental strains). Furthermore, during the logarithmic growth phase, the carbon yield of CO2 for each disrupted strain decreased to 0.82 times for strain ΔpckA, 0.86 times for strain ΔmaeA, and 0.92 times for strain Δgnd compared to the wild-type strains. During the stationary phase, the carbon yield of CO2 decreased to 0.97 times for strain ΔpckA, 0.90 times for strain ΔmaeA, and 0.81 times for strain Δgnd.

[0112] [Table 4]

[0113] As shown in Table 4, compared with the above wild-type strains (parental strains), the glucose consumption per unit cell of each gene-damaged strain increased by 1.3 times for strain ΔpckA, 1.9 times for strain ΔmaeA, and 1.2 times for strain Δgnd.

[0114] In addition, such as Figure 5As shown, the carbon yield of CO2 from each gene-damaged strain was reduced compared to the wild-type strain, but cell proliferation was not increased compared to the wild-type strain, and the glucose consumption per cell was increased. Therefore, it can be expected that the reduced CO2 carbon was used for metabolites.

[0115] Example 3: Production of secondary metabolites using enzyme gene-disrupted strains

[0116] Example 3-1: Production of Undecylpyrrolizin (Udp) using Δicd strain

[0117] The production of undecylpyrrolizin (Udp) was confirmed by culturing the wild-type strain and the Δicd strain separately. Each strain was cultured on solid TSB medium (30°C for 48 hours), followed by pre-culture in 10 mL of R2YE medium (refer to Tables 5 and 6) with shaking (28°C, 160 rpm, 48 hours). The entire culture was then added to 50 mL of R2YE medium for formal culture (28°C, 160 rpm, 72 hours). 10 mL of culture was recovered at 48 hours and 72 hours after the start of formal culture, centrifuged, and the bacterial cells were recovered. 10 mL of methanol was added to treat the bacterial cells, and Udp was extracted. The absorbance (530 nm) of the extracted supernatant containing Udp was measured, and the Udp concentration was calculated.

[0118] [Table 5]

[0119] [Table 6]

[0120] The results are as follows Figure 3 As shown, the Δicd strain produced more than 5 times the Udp compared to the wild-type strain (WT). This indicates that the decrease in CO2 carbon yield caused by icd destruction is effective for material production.

[0121] 3-2: Udp production of ΔpckA strain

[0122] The above-mentioned wild-type strain and ΔpckA strain were cultured separately to confirm the production of Udp. Each strain was cultured on solid TSB medium (30℃ for 48 hours), and simultaneously pre-cultured by immersing it in 10 mL of R2YE medium (refer to Tables 5 and 6) (28℃, 160 rpm, 48 hours). All culture was then added to 50 mL of R2YE medium for formal culture (28℃, 160 rpm, 72 hours). 48 hours and 72 hours after the start of formal culture, 10 mL of culture was recovered each time, centrifuged, and the bacterial cells were recovered. 5 mL of methanol was added to treat the bacterial cells, and Udp was extracted. The absorbance (530 nm) of the extracted supernatant containing Udp was measured, and the Udp concentration was calculated.

[0123] The results are as follows Figure 6 As shown, compared with the wild-type strain (WT), the ΔpckA strain produced approximately 2.1 times more Udp at 48 hours and approximately 1.3 times more Udp at 72 hours. This indicates that the decrease in CO2 carbon yield resulting from the destruction of pckA is effective for material production.

[0124] Example 4: Production of primary metabolites using enzyme gene-disrupted strains

[0125] 4-1: Δicd strain and ΔmaeA strain produce ribulose and α-ketoglutarate.

[0126] The above-mentioned wild-type strain, Δicd strain, and ΔmaeA strain were cultured aerobically in a fermenter. After 88 hours of culture, the culture medium was recovered and centrifuged at 15,000 rpm to obtain the supernatant. The obtained culture medium was filtered through a 0.2 μm filter and then analyzed by HPLC. The HPLC analysis conditions for ribulose and α-ketoglutarate are shown in Tables 7 and 8, respectively.

[0127] [Table 7]

[0128] [Table 8]

[0129] Regarding ribulose production, the wild-type strain yielded 0.83 g / L, while the Δicd strain yielded 1.90 g / L and the ΔmaeA strain yielded 1.24 g / L, representing increases of approximately 2 times and 1.5 times, respectively. Regarding α-ketoglutaric acid production, the wild-type strain yielded 0.63 g / L, while the Δicd strain yielded 32.0 g / L and the ΔmaeA strain yielded 0.98 g / L, representing increases of 50.7 times and 1.6 times, respectively. This indicates that the decrease in CO2 carbon yield resulting from the destruction of icd and maeA is effective for material production.

[0130] Udp is synthesized from malonyl-CoA and proline. Malonyl-CoA is produced from acetyl-CoA upstream of the icdase, while proline is produced from α-ketoglutarate downstream of the icdase via glutamate. In this culture, proline was added to the culture medium to compensate for the anticipated reduction in proline due to icdase degradation.

[0131] The following lists the gene-disrupted strains used in this embodiment. All experiments were conducted under aerobic conditions.

[0132] [Table 9]

[0133] As described above, according to this embodiment, a method for reducing carbon dioxide emissions associated with the cultivation of actinomycetes has been discovered. Therefore, it is expected that the environmental burden (especially CO2 emissions) resulting from the cultivation of actinomycetes can be reduced. Furthermore, according to this embodiment, by disrupting the enzymes responsible for the metabolic reactions that produce CO2, the carbon utilization efficiency within the bacteria is significantly improved, successfully transforming the bacteria into a state more suitable for material production. As shown in Example 3, by disrupting the genes of the enzymes responsible for the metabolic reactions that produce CO2, it is possible to substantially increase the production of useful metabolites. It is believed that this method can be effective in all microorganisms (especially bacteria), particularly in microorganisms with high carbon recovery rates from carbon dioxide (especially bacteria), and it is believed that genetically modified strains that reduce environmental burden and / or improve the production of metabolites, their cultivation methods, and pathways for using these produced metabolites can be developed in such microorganisms (especially bacteria).

Claims

1. A metabolic enzyme gene-disrupted strain, which is a metabolic enzyme gene-disrupted strain of actinomycetes that exhibits a carbon recovery rate of more than 50% from carbon dioxide (CO2) during at least the logarithmic growth phase or stationary phase in aerobic culture in a medium containing sufficient carbon sources and other nutrients, wherein, At least one of the genes encoding the enzyme responsible for the metabolic reaction that produces CO2 is disrupted, and the CO2 yield to carbon is reduced compared to the same species of actinomycete control in which none of the genes are disrupted.

2. The metabolic enzyme gene-disrupting strain according to claim 1, wherein, The enzymes responsible for the metabolic reactions that produce CO2 are selected from one or more metabolic pathways in the group consisting of glycolysis, pentose phosphate cycle and citric acid cycle.

3. The metabolic enzyme gene-disrupted strain according to claim 1 or 2, wherein, One or more of the following groups are selected: phosphoglucose dehydrogenase, pyruvate dehydrogenase, phosphoenolpyruvate carboxylkinase, malate dehydrogenase, isocitrate dehydrogenase, dihydrolipoic acid transacetylase, dihydrolipoic acid dehydrogenase, and α-ketoglutarate dehydrogenase.

4. The metabolic enzyme gene-damaged strain according to any one of claims 1 to 3, wherein, In aerobic culture with sufficient carbon source, its proliferation capacity was the same as or did not show a decrease of more than 50% compared with the control actinomycetes.

5. The metabolic enzyme gene-damaged strain according to any one of claims 1 to 4, wherein, At least one of the genes containing isocitrate dehydrogenase, phosphoglucose dehydrogenase, phosphoenolpyruvate carboxykinase, and malate dehydrogenase is disrupted.

6. The metabolic enzyme gene-damaged strain according to any one of claims 1 to 5, wherein, Actinomycetes belong to the genus Streptomyces.

7. A composition comprising a metabolic enzyme gene-destroying strain according to any one of claims 1 to 6 for producing metabolites.

8. A composition comprising a metabolic enzyme gene-destroying strain according to any one of claims 1 to 6 for producing metabolites.

9. A method for culturing a metabolic enzyme gene-destroying strain of actinomycetes, wherein, The metabolic enzyme gene-disrupting strain comprises any one of the metabolic enzyme gene-disrupting strains according to claims 1 to 6. The method includes the step of culturing the metabolic enzyme gene-disrupted strain under aerobic conditions in a culture medium containing sufficient amounts of carbon source and other nutrients.

10. The method according to claim 9, wherein, The culturing process includes steps for culturing metabolic enzyme gene-disrupted strains in the logarithmic growth phase and steps for culturing metabolic enzyme gene-disrupted strains in the stationary phase.

11. A method for producing metabolites from a gene-destroying strain of actinomycetes containing a gene-metabolizing enzyme, wherein, This includes the steps of culturing strains with disrupted metabolic enzyme genes. The metabolic enzyme gene-disrupting strain comprises any one of the metabolic enzyme gene-disrupting strains according to claims 1 to 6. The method includes the following steps: The metabolic enzyme gene-disrupting strain was cultured under aerobic conditions in a medium containing sufficient carbon source and other nutrients, so that the metabolic enzyme gene-disrupting strain produced the metabolic products. as well as The metabolites are recovered.

12. The method according to claim 11, wherein, The metabolites are derived from the citric acid cycle.

13. A method for selecting actinomycetes, comprising the following steps: Provides isolated actinomycetes; The carbon dioxide yield of the actinomycetes during the logarithmic growth phase or stationary phase of aerobic culture in a medium containing sufficient carbon sources and other nutrients was determined; and Select or obtain actinomycetes that have a carbon yield exceeding 50% during either the logarithmic growth phase or the stationary phase.

14. A method for genetically modifying actinomycetes to obtain genetically modified actinomycetes, comprising the following steps: Provides isolated actinomycetes; The carbon yield of the actinomycetes during the logarithmic growth phase or stationary phase in aerobic culture in a medium containing sufficient carbon sources and other nutrients was determined. Select or obtain actinomycetes that have a carbon yield of more than 50% during either the logarithmic growth phase or the stationary phase. The genetic modification disrupts at least one of the genes encoding an enzyme responsible for the metabolic reaction that produces carbon dioxide in the obtained actinomycetes. as well as Genetically modified actinomycetes were obtained.

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