Bacterium having high carbon dioxide yield relative to carbon and metabolic enzyme gene-disrupted strain of same

Abstract

The present disclosure provides a bacterium having high carbon dioxide yield relative to carbon, and a metabolic enzyme gene-disrupted strain of the bacterium. The present disclosure also provides a method for isolating a bacterium having high carbon dioxide yield relative to carbon. The present disclosure also provides a method for producing, a method for culturing and a method for use of a metabolic enzyme gene-disrupted strain of the bacterium. According to the present disclosure, by disrupting a gene encoding an enzyme of a metabolic reaction that produces carbon dioxide in a bacterium having high carbon dioxide yield relative to carbon, an environmental load due to culturing the bacterium, for example, can be reduced and / or the bacterial cells can be changed to a state more suitable for material production.

Description

Bacteria with high carbon dioxide yield and metabolic enzyme gene disruptants of said bacteria

[0001] The present disclosure relates to a bacterium with a high carbon dioxide yield per carbon and a metabolic enzyme gene disruptant of the bacterium. The present disclosure also relates to a method for isolating the bacterium with a high carbon dioxide yield per carbon. The present disclosure also relates to methods for producing, culturing, and using the metabolic enzyme gene disruptant of the bacterium.

[0002] It has been disclosed that in lysine production, the activity of isocitrate dehydrogenase (ICD) in coryneform bacteria is reduced (Patent Documents 1 to 5, Non-Patent Document 4). It has been disclosed that deletion of the isocitrate dehydrogenase gene (icd) in Escherichia coli reduces growth ability and sugar consumption (Non-Patent Documents 1 and 2), and that disruption of the icd gene in coryneform bacteria makes them glutamic acid auxotrophic (Non-Patent Document 3). Non-Patent Documents 4 to 9 disclose various studies on disrupting metabolic enzymes.

[0003] JP2015-154768AJP2013-533740AJP2011-518571AJP5719434BJP5395893B

[0004] Appl. Microbiol. Biotechnol., 2004, 65: 84-96FEMS Microbiol. Lett., 2019, 366J. Bacteriol., 1995, 177(3): 774-782Appl. Environ. Microbiol., 2009. 75(24): 7866-7869Microbial. Biotechnol., 2019. 12(5): 932-945J.bacteriol., (2014). 3045-3057BMC Biotechnol., (2016) 16:52Metabolomics, (2022) 18:56Appl. Environ. Microbiol., 2012. 78(19): 6975-6986

[0005] The present disclosure provides bacteria with a high carbon dioxide yield per carbon and genetically modified strains of the bacteria (particularly metabolic enzyme gene disrupted strains). The present disclosure also provides methods for isolating bacteria with a high carbon dioxide yield per carbon. The present disclosure also provides methods for producing, culturing, and using the bacteria (particularly metabolic enzyme gene disrupted strains).

[0006] According to the present disclosure, for example, the following inventions are provided: (1) A method for producing carbon dioxide (CO ) during at least the logarithmic growth phase or stationary phase in aerobic culture in a medium containing a sufficient amount of a carbon source and other nutrients. ２ A metabolic enzyme gene disruptant of actinomycetes (preferably actinomycetes of the genus Streptomyces) having a carbon yield of more than 50% when CO ２ At least one gene encoding an enzyme responsible for the metabolic reaction that produces CO ２ (2) A metabolic enzyme gene-disrupted strain in which the carbon yield of CO is reduced compared to that of a control actinomycete of the same species in which none of the genes is disrupted. ２(1) The metabolic enzyme gene-disrupted strain according to (1) above, wherein the enzyme responsible for the metabolic reaction to produce actinomycete is an enzyme in any one or more metabolic pathways selected from the group consisting of glycolysis, the pentose phosphate cycle, and the citric acid cycle. (3) The metabolic enzyme gene-disrupted strain according to (1) or (2) above, wherein one or more enzymes selected from the group consisting of phosphogluconate dehydrogenase, pyruvate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, isocitrate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, and α-ketoglutarate dehydrogenase have been disrupted. (4) The metabolic enzyme gene-disrupted strain according to any one of (1) to (3) above, which, when aerobically cultured in the presence of a sufficient amount of carbon source, has the same growth ability as a control actinomycete, or does not show a decrease in growth ability of 50% or more. (5) The metabolic enzyme gene-disrupted strain according to any one of (1) to (4) above, in which one or more genes comprising at least isocitrate dehydrogenase, phosphogluconate dehydrogenase, pyruvate dehydrogenase, pyruvate carboxykinase, and malate dehydrogenase have been disrupted. (5A) The metabolic enzyme gene-disrupted strain according to any one of (1) to (4) above, in which at least isocitrate dehydrogenase has been disrupted. (5B) The metabolic enzyme gene-disrupted strain according to any one of (1) to (4) above, in which at least phosphogluconate dehydrogenase has been disrupted. (5C) The metabolic enzyme gene-disrupted strain according to any one of (1) to (4) above, in which at least malate dehydrogenase has been disrupted. (5D) The metabolic enzyme gene-disrupted strain according to any one of (1) to (4) above, in which at least pyruvate carboxykinase has been disrupted. (6) The metabolic enzyme gene-disrupted strain according to any one of (1) to (5) above, in which the actinomycete belongs to the genus Streptomyces. (7) A composition for use in producing a metabolic product, comprising the metabolic enzyme gene-disrupted strain according to any one of (1) to (6) above. (8) A composition for use in producing a metabolic product derived from the citric acid cycle, comprising the metabolic enzyme gene-disrupted strain according to any one of (1) to (6) above.(9) A method for culturing a metabolic enzyme gene-disrupted strain of actinomycete, wherein the metabolic enzyme gene-disrupted strain comprises the metabolic enzyme gene-disrupted strain of any one of (1) to (6) above, the method comprising culturing the metabolic enzyme gene-disrupted strain under aerobic conditions in a medium containing a sufficient amount of a carbon source and other nutrients. (10) The method according to (9) above, wherein the culturing comprises culturing the metabolic enzyme gene-disrupted strain in logarithmic growth phase and culturing the metabolic enzyme gene-disrupted strain in stationary phase. (11) A method for causing a metabolic enzyme gene-disrupted strain of actinomycete to produce a metabolic product, the method comprising culturing a metabolic enzyme gene-disrupted strain, wherein the metabolic enzyme gene-disrupted strain comprises the metabolic enzyme gene-disrupted strain of any one of (1) to (6) above, the method comprising culturing the metabolic enzyme gene-disrupted strain under aerobic conditions in a medium containing a sufficient amount of a carbon source and other nutrients, to cause the metabolic enzyme gene-disrupted strain to produce the metabolic product, and recovering the metabolite. (12) The method according to (11), wherein the metabolic product is derived from the citric acid cycle. (13) A method for selecting actinomycetes, comprising: providing an isolated actinomycete; measuring the carbon dioxide yield of the actinomycete during logarithmic growth phase or stationary phase in aerobic culture in a medium containing a sufficient amount of a carbon source and other nutrients; and selecting or obtaining an actinomycete with a carbon yield of more than 50%. (14) A method for obtaining a genetically modified actinomycete by genetically modifying an actinomycete, comprising: providing an isolated actinomycete; measuring the carbon dioxide yield of the actinomycete during logarithmic growth phase or stationary phase in aerobic culture in a medium containing a sufficient amount of a carbon source and other nutrients; selecting or obtaining an actinomycete with a carbon yield of more than 50%; genetically disrupting at least one gene encoding an enzyme responsible for a metabolic reaction that produces carbon dioxide in the obtained actinomycete; and obtaining the genetically modified actinomycete.

[0007] Figure 1 shows metabolic pathways including glycolysis, the pentose phosphate pathway, and the TCA cycle. Figure 2 shows the optical density (OD), dry cell weight, and glucose consumption of the wild-type (WT) and Δicd strains of Streptomyces lividans after the start of main culture. Figure 3 shows the production of undecylprodigiosin (Udp) by the wild-type (WT) and Δicd strains of Streptomyces lividans. Figure 4 shows the Udp synthesis pathway. 5 shows the optical density (OD), dry cell weight, and glucose consumption after the start of main culture for a Streptomyces lividans strain (ΔpckA) lacking the gene (pckA) encoding an enzyme that produces phosphoenolpyruvate from oxaloacetate, a strain (ΔmaeA) lacking the gene (maeA) encoding an enzyme that produces pyruvate from malate, and a strain (Δgnd) lacking the gene (gnd) encoding an enzyme that produces ribulose-5-phosphate from 6-phosphogluconate. Figure 6 shows the amount of undecylprodigiosin (Udp) produced by the ΔpckA strain.

[0008] <Definition of Terms> In this specification, the term "about" means within a range of ±10%, ±5%, or ±1% of the following numerical value.

[0009] As used herein, "aerobic bacteria" refers to microorganisms equipped with a mechanism for consuming oxygen to produce energy. Aerobic bacteria use oxygen to obtain energy from sugars, lipids, and the like. Aerobic bacteria include facultative aerobes and obligate aerobes. Facultative aerobes can utilize oxygen but can also produce energy anaerobically. As used herein, aerobic bacteria may also include microorganisms classified as anaerobes, such as facultative anaerobes, as long as they meet the above definition. Examples of facultative aerobes include yeast, bacteria, and mold. Aerobic bacteria with disrupted metabolic enzymes have been established as strains. Therefore, aerobic bacteria with disrupted metabolic enzymes may be simply referred to herein as metabolic enzyme gene-disrupted strains.

[0010] As used herein, "actinomycetes" refers to bacteria belonging to the phylum Actinobacteria. Examples of actinomycetes include, but are not limited to, actinomycetes of the genera Streptomyces, Actinomyces, Mycobacterium, and Corynebacterium. Actinomycetes are facultative aerobic bacteria that can produce energy under anaerobic conditions, but when used for substance production, they are cultured under aerobic conditions. Examples of actinomycetes of the genus Streptomyces include Streptomyces lividans, Streptomyces violaceoruber, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces rapamycinicus, and Streptomyces kanamyceticus. Representative examples include Streptomyces kanamyceticus, Streptomyces peucetius, Streptomyces galilaeus, Streptomyces purvulus, Streptomyces albus, and Streptomyces albulus. Actinomycetes in the genus Streptomyces also include 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. alboverticillatus, 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. annulatus, 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. aurantiogriseus, S. auratus, S. aureocirculatus, S. aureofaciens, S. aureorectus, S. aureoversilis, S. aureoverticillatus, S. aureus, S. avellaneus, S. avermectinius, S. avermitilis, S. avicenniae, S. avidinii, S. axinellae, S. azaticus, S. azureus, S. baarnensis, S. bacillaris, S. badius, S. baldaccii, S. baliensis, S. bambergiensis, S. bangladeshensis, S.barkulensis, S. beijiangensis, S. bellus, S. bikiniensis, S. biverticillatus, S. blastmyceticus, S. bluensis, S. bobili, S. bottropensis, S. brasiliensis, S. brevispora, S. bullii, S. bungoensis, S. burgazadensis, S. cacaoi, S. cac. subsp. asoensis, S. cac. subsp. cacaoi, S. caelestis, S. caeruleatus, S. caeruleus, S. calidiresistens, S. californicus, S. calvus, S. canarius, S. candidus, S. canescens, S. cangkringensis, S. caniferus, S. canus, S. capillispiralis, S. capoamus, S. carpathicus, S. carpinensis, S. castelarensis, S. catbensis, S. catenulae, S. caviscabies, S. cavourensis, S. cav. subsp. cavourensis, S. cav. subsp. washingtonensis, S. cellostatic, S. celluloflavus、S. cellulolyticus、S. Cellulose、S. champavatii、S. Chartreuse, St. Chattanooga、S. Cheonanensis、S. chiangmaiensis、S. chibaensis、S. chilikensis、S. chlorine、S. chrestomyceticus、S. chromofuscus、S. chryseus, St. chrysomallus、S. Ch. subsp. chrysomallus、S. Ch. subsp. smoked、S. chumphonensis、S. cinereorectus、S. ash-red、S. ci. subsp. ash-red、S. ci.subsp. fructofermentans, S. cinereospinus, S. cinereus, S. cinerochromogenes, S. cinnabarinus, S. cinnamonensis, S. cinnamoneus, S. cirratus, S. ciscaucasicus, S. citreofluorescens, S. clavifer, S. clavuligerus, S. coacervatus, S. cochleatus, S. cocklensis, S. coelescens, S. coelicoflavus, S. coelicolor, S. coeruleoflavus, S. coeruleofuscus, S. coeruleoprunus, S. coeruleorubidus, S. coerulescens, S. collinus, S. colombiensis, S. corchorusii, S. costaricanus, S. cremeus, S. crystallinus, S. curacoi, S. cuspidosporus, S. cyaneofuscatus, S. cyaneus, S. cyanoalbus, S. cystargineus, S. daghestanicus, S. daliensis, S. deccanensis, S. decoyicus, S. demainii, S. deserti, S. diastaticus, S. d. subsp. ardesiacus, S. d. subsp. diastaticus, S. diastatochromogenes, S. distallicus, S. djakartensis, S. drozdowiczii, S. durhamensis, S. durmitorensis, S. echinatus, S. echinoruber, S. ederensis, S. ehimensis, S. emeiensis, S. endophyticus, S. endus, S. enissocaesilis, S. erringtonii, S. erumpens, S. erythraeus, S. erythrogriseus, S. eurocidicus, S.europaeiscabiei, S. eurythermus, S. exfoliatus, S. felleus, S. fenghuangensis, S. ferralitis, S. fervens, S. filamentosus, S. fildesensis, S. filipinensis, S. fimbriatus, S. fimicarius, S. finlayi, S. flaveolus, S. flaveus, S. flavidofuscus, S. flavidovirens, S. flaviscleroticus, S. flavofungini, S. flavofuscus, S. flavogriseus, S. flavopersicus, S. flavotricini, S. flavovariabilis, S. flavovirens, S. flavoviridis, S. flocculus, S. floridae, S. fluorescens, S. fradiae, S. fragilis, S. fukangensis, S. fulvissimus, S. fulvorobeus, S. fumanus, S. fumigatiscleroticus, S. galbus, S. galilaeus, S. gancidicus, S. gardneri, S. gelaticus, S. geldanamycininus, S. geysiriensis, S. ghanaensis, S. gibsonii, S. glaucescens, S. glauciniger, S. glaucosporus, S. glaucus, S. globisporus, S. gl. subsp. caucasicus, S. gl. subsp. flavofuscus, S. gl. subsp. globisporus, S. globosus, S. glomeratus, S. glomeroaurantiacus, S. glycovorans, S. gobitricini, S. goshikiensis, S. gougerotii, S. graminearus, S. gramineus, S. graminifolii, S. graminilatus, S. graminisoli, S.graminofaciens、S. griseiniger、S. griseinus、S. gray orange、S. griseobrunneus、S. griseocarneus、S. griseochromogenes、S. greyish yellow、S. greisofuscus、S. griseoincarnatus、S. griseoloalbus、S. griseolosporeus、S. griseolus、S. griseouteus、S. griseomycini、S. griseoplanus、S. red gray、S. grey-red、S. griseorubiginosus、S. griseosporeus、S. griseostramineus、S. griseoverticillatus、S. griseoviridis、S. gray、S. Gr. subsp. alpha、S. Gr. subsp. cretosus、S. Gr. subsp. gray、S. Gr. subsp. Solving、S. guanduensis、S. gulbargensis、S. hachijoensis、S. hainanensis, S. haliclonae, S. halophytocola, S. halstedii, S. hawaiiensis, S. hebeiensis, S. heilongjiangensis, S. heliomycini, S. helvaticus, S. herbaceus, S. herbaricolor, S. himastatinicus, S. hiroshimensis, S. hirsutus, S. hokutonensis, S. hoynatensis, S. humidus, S. humiferus, S. hyderabadensis, S. hydrogenans, S. hygroscopicus, S. h. subsp. angustmyceticus, S. h. subsp. decoyicus, S. h. subsp. glebosus, S. h. subsp. hygroscopicus, S. h. subsp. ossamyceticus, S.hypolithicus, S. iakyrus, S. iconiensis, S. incanus, S. indiaensis, S. indicus, S. indigoferus, S. indonesiensis, S. intermedius, S. inusitatus, S. ipomoeae, S. iranensis, S. janthinus, S. javensis, S. jietaisiensis, S. jiujiangensis, S. kaempferi, S. kanamyceticus, S. karpasiensis, S. kashimirensis, S. kasugaensis, S. katrae, S. kentuckensis, S. kifunensis, S. kishiwadensis, S. koyangensis, S. kunmingensis, S. kurssanovii, S. labedae, S. laceyi, S. lacticiproducens, S. laculatispora, S. ladakanum, S. lanatus, S. lannensis, S. lateritius, S. laurentii, S. lavendofoliae, S. lavendulae, S. la. subsp. grasserius, S. la. subsp. lavendulae, S. lavenduligriseus, S. lavendulocolor, S. leeuwenhoekii, S. levis, S. libani, S. li. subsp. libani, S. li. subsp. rufus, S. lienomycini, S. lilacinus, S. limosus, S. lincolnensis, S. lipmanii, S. litmocidini, S. lividans, S. lomondensis, S. longisporoflavus, S. longispororuber, S. longisporus, S. longwoodensis, S. lopnurensis, S. lucensis, S. lunalinharesii, S. luridiscabiei, S. luridus, S. lusitanus, S.luteireticuli, S. luteogriseus, S. luteosporeus, S. luteoverticillatus, S. lydicus, S. macrosporus, S. malachitofuscus, S. malachitospinus, S. malaysiensis, S. marinus, S. marokkonensis, S. mashuensis, S. massasporeus, S. matensis, S. mauvecolor, S. mayteni, S. mediocidicus, S. mediolani, S. megasporus, S. melanogenes, S. melanosporofaciens, S. mexicanus, S. michiganensis, S. microflavus, S. milbemycinicus, S. minutiscleroticus, S. mirabilis, S. misakiensis, S. misionensis, S. mobaraensis, S. monomycini, S. mordarskii, S. morookaense, S. muensis, S. murinus, S. mutabilis, S. mutomycini, S. naganishii, S. nanhaiensis, S. nanshensis, S. narbonensis, S. nashvillensis, S. netropsis, S. neyagawaensis, S. niger, S. nigrescens, S. nigrifaciens, S. nitrosporeus, S. niveiscabiei, S. niveoruber, S. niveus, S. noboritoensis, S. nodosus, S. nogalater, S. nojiriensis, S. noursei, S. novaecaesareae, S. ochraceiscleroticus, S. odorifer, S. olivaceiscleroticus, S. olivaceoviridis, S. olivaceus, S. olivochromogenes, S. olivomycini, S. olivorreticuli, S.olivoverticillatus, S. olivoviridis, S. omiyaensis, S. orinoci, S. osmaniensis, S. pactum, S. panacagri, S. panaciradicis, S. paracochleatus, S. paradoxus, S. parvisporogenes, S. parvulus, S. parvus, S. pathocidini, S. paucisporeus, S. peucetius, S. phaeochromogenes, S. phaeofaciens, S. phaeogriseichromatogenes, S. phaeoluteichromatogenes, S. phaeoluteigriseus, S. phaeopurpureus, S. phaeoviridis, S. pharetrae, S. pharmamarensis, S. phosalacineus, S. phytohabitans, S. pilosus, S. platensis, S. plicatus, S. plumbiresistens, S. pluricolorescens, S. pluripotens, S. polyantibioticus, S. polychromogenes, S. poonensis, S. praecox, S. prasinopilosus, S. prasinosporus, S. prasinus, S. pratens, S. pratensis, S. prunicolor, S. psammoticus, S. pseudoechinosporeus, S. pseudogriseolus, S. pseudovenezuelae, S. pulveraceus, S. puniceus, S. puniciscabiei, S. purpeofuscus, S. purpurascens, S. purpureus, S. purpurogeneiscleroticus, S. qinglanensis, S. racemochromogenes, S. radiopugnans, S. rameus, S. ramulosus, S. rangoonensis, S. rapamycinicus, S. recifensis, S.rectiverticillatus, S. rectiviolaceus, S. regensis, S. resistomycificus, S. reticuliscabiei, S. rhizosphaericus, S. rhizophilus, S. rimosus, S. ri. subsp. paromomycinus, S. ri. subsp. rimosus, S. rishiriensis, S. rochei, S. rosealbus, S. roseiscleroticus, S. roseodiastaticus, S. roseoflavus, S. roseofulvus, S. roseolilacinus, S. roseolus, S. roseosporus, S. roseoverticillatus, S. roseoviolaceus, S. roseoviridis, S. ruanii, S. ruber, S. rubidus, S. rubiginosohelvolus, S. rubiginosus, S. rubrogriseus, S. rubrus, S. rutgersensis, S. ru. subsp. castelarensis, S. ru. subsp. rutgersensis, S. salmonis, S. sampsonii, S. samsunensis, S. sanglieri, S. sannanensis, S. sanyensis, S. sapporonensis, S. scabiei, S. scabrisporus, S. sclerotialus, S. scopiformis, S. scopuliridis, S. sedi, S. seoulensis, S. septatus, S. seranimatus, S. setae, S. setonii, S. shaanxiensis, S. shenzhenensis, S. showdoensis, S. silaceus, S. sindenensis, S. sioyaensis, S. smyrnaeus, S. sodiiphilus, S. somaliensis, S. sparsogenes, S. sparsus, S. specialis, S. spectabilis, S. speibonae, S.speleomycini, S. spheroides, S. spinoverrucosus, S. spiralis, S. spiroverticillatus, S. spitsbergensis, S. spongiae, S. sporocinereus, S. sporoclivatus, S. spororaveus, S. sporoverrucosus, S. staurosporininus, S. stelliscabiei, S. stramineus, S. subrutilus, S. sulfonofaciens, S. sulphureus, S. sundarbansensis, S. synnematoformans, S. syringium, S. tacrolimicus, S. tanashiensis, S. tateyamensis, S. tauricus, S. tendae, S. termitum, S. thermoalcalitolerans, S. thermoautotrophicus, S. thermocarboxydovorans, S. thermocarboxydus, S. thermocoprophilus, S. thermodiastaticus, S. thermogriseus, S. thermolineatus, S. thermonitrificans, S. thermospinosisporus, S. thermoviolaceus, S. t. subsp. apingens, S. t. subsp. thermoviolaceus, S. thermovulgaris, S. thinghirensis, S. thioluteus, S. torulosus, S. toxytricini, S. tricolor, S. tritolerans, S. tubercidicus, S. tuirus, S. tunisiensis, S. turgidiscabies, S. umbrinus, S. variabilis, S. variegatus, S. varsoviensis, S. vastus, S. venezuelae, S. vietnamensis, S. vinaceus, S. vinaceusdrappus, S.violaceochromogenes, S. violaceolatus, S. violaceorectus, S. violaceoruber, S. violaceorubidus, S. violaceus, S. violaceusniger, S. violarus, S. violascens, S. violatus, S. violens, S. virens, S. virginiae, S. viridiflavus, S. viridiviolaceus, S. viridis, S. viridobrunneus, S. viridochromogenes, S. viridodiastaticus, S. viridosporus, S. vitaminophilus, S. wedmorensis, S. wellingtoniae, S. werraensis, S. willmorei, S. wuyuanensis, S. xanthochromogenes, S. xanthocidicus, S. xantholiticus, S. xanthophaeus, S. Examples of suitable actinomycetes include S. xiamenensis, S. xinghaiensis, S. xishensis, S. yaanensis, S. yanglinensis, S. yanii, S. yatensis, S. yeochonensis, S. yerevanensis, S. yogyakartensis, S. yokosukanensis, S. youssoufiensis, S. yunnanensis, S. zaomyceticus, S. zhaozhouensis, S. zinciresistens, and S. ziwulingensis. Streptomyces can be, for example, antibiotic-producing bacteria. Examples of antibiotics include streptomycin, kanamycin, rapamycin, daunomycin, aclacinomycin, tetracycline, erythromycin, rifampicin, bleomycin, and vancomycin.

[0011] As used herein, "aerobic conditions" refers to conditions under which aerobic bacteria perform aerobic respiration. Aerobic respiration is carried out by a metabolic system including the glycolytic pathway, the citric acid cycle, and the electron transport chain, which normally results in sugars consuming oxygen to produce carbon dioxide, water, and ATP, which serves as energy. As shown in Figure 1, the glycolytic pathway is a pathway that produces pyruvate from glucose via glucose-6 phosphate. Pyruvate produced in the glycolytic pathway is converted to acetyl-CoA by the pyruvate dehydrogenase complex and introduced into the citric acid cycle (for convenience, in this specification, the pathway up to the point of producing acetyl-CoA is classified as the glycolytic pathway). Acetyl-CoA is then metabolized into the citric acid cycle. The citric acid cycle is also known as the TCA cycle. The citric acid cycle is a pathway that converts NADH, FADH, ２ The electron transport chain produces NADH and FADH. ２ By culturing aerobic bacteria under aerobic conditions, ATP is produced from sugars in a metabolic system including the glycolytic pathway, the citric acid cycle, and the electron transport system. In contrast, "anaerobic conditions" refer to conditions under which anaerobic organisms produce energy and grow without using oxygen. Those skilled in the art can appropriately culture aerobic bacteria under aerobic conditions.

[0012] Bacterial cultivation consists of a lag phase, logarithmic growth phase, and stationary phase. For a certain period after the start of cell cultivation, bacteria do not divide and the cell number remains constant. This period is called the lag phase. It is believed that during this period, bacteria repair their cells, regulate their metabolism, and accumulate carbon dioxide. After this, bacteria begin to divide and grow slowly, gradually increasing their growth rate until the generation time becomes constant and the bacterial cells begin to grow logarithmically. This period is called the logarithmic growth phase. As cultivation continues and the cell density increases, the bacterial cells complete the logarithmic growth period. Typically, the rate of cell division gradually decreases and the viable cell count becomes constant, although some bacterial species may continue to grow slowly. This period is generally called the stationary phase. Substance production using bacteria mainly occurs between the logarithmic growth phase and the stationary phase. Generally, the boundary between the logarithmic growth phase and the stationary phase is not clear. Therefore, in this specification, the logarithmic growth phase is defined as the period after the start of culture when bacterial cell growth becomes active and the doubling time can be considered to have become constant, and the stationary phase is defined as the period when the doubling time of bacterial cells is clearly extended and the viable cell count can be considered to have become constant, or, in the case of bacterial species that continue to grow slowly, the doubling time is significantly extended compared to the logarithmic growth phase (for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 10 times or more that of the logarithmic growth phase, depending on the bacterial species), and bacterial cell growth has stabilized.

[0013] As used herein, the term "yield relative to carbon" refers to the amount of carbon contained in a carbon source (e.g., glucose) contained in nutrients that is incorporated as carbon into a specific substance.

[0014] As used herein, the term "carbon source" refers to an organic substance that provides an energy source and carbon components to heterotrophic cells such as aerobic bacteria. In one embodiment, the carbon source is not particularly limited, but may be, for example, a sugar, such as a monosaccharide, particularly glucose.

[0015] <Bacteria and method for selecting bacteria of the present disclosure> According to the examples described below, CO ２ There are large differences among bacteria in the carbon yield of CO ２In some embodiments, bacteria have a carbon yield of greater than about 50% at least during logarithmic or stationary phase (preferably during logarithmic phase). The present disclosure provides such bacteria and methods for selecting, obtaining, or isolating such bacteria.

[0016] According to the present disclosure, CO ２ The bacterium has a carbon yield of, for example, greater than about 50% at least during logarithmic growth or stationary phase. The bacterium can metabolize glucose to produce energy. In one aspect, the CO 2 to glucose ratio is ２ The present disclosure provides bacteria having a carbon yield of more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% at least during the logarithmic growth phase or stationary phase. In a preferred embodiment, the bacterium may be an actinomycete of the genus Streptomyces. In a specific embodiment, the actinomycete may be Streptomyces lividans. According to the present disclosure, metabolic enzyme gene disruptants of these bacteria (e.g., actinomycetes) are provided. ２ Bacteria with high carbon yields are useful targets for modification according to the present disclosure. ２ the carbon yield of the enzyme is higher than that of E. coli at least during the logarithmic growth phase or stationary phase (e.g., about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, or about 1.5 times or more). ２ has a carbon yield higher than that of the coryneform bacterium Corynebacterium glutamicum at least during the logarithmic growth phase or stationary phase (e.g., about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, or about 1.5 times or more). In a preferred embodiment, the carbon yield is during the logarithmic growth phase.

[0017] The present disclosure provides a method for selecting, obtaining, or isolating the bacterium. The method of the present disclosure includes providing an isolated bacterium. Isolation can preferably be cloning. The method of the present disclosure can also further include measuring the carbon dioxide yield of the bacterium during logarithmic or stationary phase aerobic culture in a medium containing a sufficient amount of carbon source and other nutrients (e.g., for actinomycetes, 1.8 g / L TSB medium containing 50 g / L glucose, pH 7.0). The carbon yield can be calculated from the amount of glucose consumed and the amount of metabolites produced. The amounts of glucose and metabolites can be measured by conventional methods. Furthermore, carbon dioxide emissions can be measured by conventional methods using an exhaust gas analyzer (e.g., OFF-GAS Jr., manufactured by Biot) and a mass spectrometer. The amounts of glucose and metabolites can be measured, for example, by separating components using high-performance liquid chromatography followed by component analysis. The component analysis can be carried out by using various techniques such as mass spectrometry, a differential refractive index detector, and a UV detector, either singly or in combination.

[0018] Based on the carbon dioxide yield measurement results, bacteria having a carbon yield of, for example, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% can be selected, obtained, or isolated. In this manner, bacteria with a high carbon dioxide yield can be obtained. In this method, the bacteria can be bacterial cells isolated from nature (soil, a living organism, a biological sample, water, etc.). In this method, the bacteria can be an established strain of bacteria. In this method, the bacteria can preferably be actinomycetes of the genus Streptomyces. The obtained bacteria can be cloned. Cloning can be achieved, for example, by plating on a solid medium and picking up a single colony. In the case of actinomycetes, actinomycetes can be cloned by, for example, plating spores or protoplasted cells on a solid medium and picking up a single colony. In this manner, those skilled in the art can clone bacteria using an appropriate method.

[0019] <Bacterial metabolic enzyme gene disruptant strain and production method thereof according to the present disclosure> The present disclosure provides a method for producing a bacterial metabolic enzyme gene disruptant strain. The bacterium may be an aerobic bacterium. In one aspect, the bacterium may be an actinomycete of the genus Streptomyces.

[0020] In a preferred embodiment, the bacterium can be a bacterium obtained by the above method. That is, the bacterium is a bacterium that can convert CO to glucose. ２ The bacterium may be, for example, a bacterium having a carbon yield of more than about 50% at least during the logarithmic growth phase or stationary phase, and more preferably, a bacterium having a carbon yield of more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% at least during the logarithmic growth phase or stationary phase. The bacterium may be an aerobic bacterium, preferably an actinomycete of the genus Streptomyces.

[0021] A method for producing a metabolic enzyme gene disruptant of a bacterium includes providing the bacterium described above.

[0022] In the method for producing a bacterial metabolic enzyme gene disruptant, the bacterium to be modified is isolated, and preferably cloned.

[0023] Bacterial CO2 response to glucose ２ It is beneficial to reduce the carbon yield of CO in metabolic pathways. ２ There is a known metabolic reaction that produces CO. ２ In order to reduce the carbon yield of a microorganism, one or more enzymes that catalyze such metabolic reactions can be genetically engineered to be disrupted.

[0024] The method for producing a metabolic enzyme gene disruptant of a bacterium includes: ２The present disclosure includes disrupting an enzyme that catalyzes a metabolic reaction that generates CO₂. Disruption is achieved by one or more modifications selected from the group consisting of inhibition and elimination of expression, inhibition and elimination of function, and gene deletion. The disruption is not particularly limited as long as it reduces the metabolic activity of the enzyme, but for example, it reduces the yield of carbon dioxide relative to sugar to about half or less, about 30% or less, about 20% or less, about 10% or less, or about 5% or less, or to below the detection limit. When a metabolic reaction is carried out by multiple enzymes, the disruption is carried out on the gene encoding at least one of the enzymes (e.g., the genes encoding all of the enzymes). According to the present disclosure, CO₂ is generated in the metabolic pathway of the bacterium. ２ A metabolic enzyme gene disruptant is provided in which one or more enzymes that catalyze metabolic reactions that generate the metabolic enzyme are disrupted.

[0025] Below, CO ２ Examples of enzymes that are involved in metabolic reactions that produce CO include, but are not limited to, orthologs of the following enzymes that have equivalent functions. ２ It can be preferably destroyed as an enzyme responsible for the metabolic reaction that produces

[0026] CO in metabolic pathways ２ The enzyme responsible for the metabolic reaction to produce CO is not particularly limited, but is, for example, isocitrate dehydrogenase (EC 1.1.1.41). Isocitrate dehydrogenase (EC 1.1.1.41) converts isocitrate to CO ２ This enzyme catalyzes the reaction of α-ketoglutaric acid with β-ketoglutaric acid. Genes encoding this enzyme include icd and SLI_7202.

[0027] CO in metabolic pathways ２ The enzyme responsible for the metabolic reaction to produce CO2 is, but is not particularly limited to, pyruvate decarboxylase (EC 4.1.1.1). Pyruvate decarboxylase (EC 4.1.1.1) converts pyruvate to CO2. ２ It is an enzyme that catalyzes the reaction that produces acetaldehyde.

[0028] CO in metabolic pathways ２The enzyme responsible for the metabolic reaction to produce 6-phosphogluconate is, but is not limited to, phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, or 1.1.1.351). Phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, or 1.1.1.351) converts 6-phosphogluconate and NADP in the pentose phosphate pathway. ＋ Ribulose-5-phosphate and NADPH ２ ＋ and CO ２ Genes encoding such enzymes include gnd, SLI_4134, SLI_7003, SLI_1209, and the like, and combinations thereof.

[0029] CO in metabolic pathways ２ Enzymes responsible for the metabolic reaction that produces pyruvate include, but are not limited to, pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoyl transacetylase (EC 2.3.1.12), and dihydrolipoyl dehydrogenase (EC 1.8.1.4). Pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoyl transacetylase (EC 2.3.1.12), and dihydrolipoyl dehydrogenase (EC 1.8.1.4) form a pyruvate dehydrogenase complex, which produces pyruvate, CoA-SH, and NAD. ＋ Acetyl CoA, NADH and CO ２ Examples of genes encoding such enzymes include aceE, SLI_2705, SLI_2510, SLI_7339, and the like, and combinations thereof.

[0030] CO in metabolic pathways ２Examples of the enzyme responsible for the metabolic reaction that produces α-ketoglutarate dehydrogenase or oxoglutarate dehydrogenase (EC 1.2.4.2, EC 1.2.7.3, or EC 1.2.7.11), dihydrolipoamide S-succinyltransferase (EC 2.3.1.61), and dihydrolipoyl dehydrogenase (EC 1.8.1.4) include, but are not limited to, α-ketoglutarate dehydrogenase or oxoglutarate dehydrogenase (EC 1.2.4.2, EC 1.2.7.3, or EC 1.2.7.11), dihydrolipoamide S-succinyltransferase (EC 2.3.1.61), and dihydrolipoyl dehydrogenase (EC 1.8.1.4) form an oxoglutarate dehydrogenase complex, and produce α-ketoglutarate, NAD ＋ , and CoA-SH to succinyl-CoA, NADH, H ＋ , and CO ２ Genes encoding such enzymes include kgd, SLI_2508, SLI_5576, and the like, and combinations thereof.

[0031] CO in metabolic pathways ２ The enzyme responsible for the metabolic reaction to produce malate is, but is not limited to, malate dehydrogenase (EC 1.1.1.38, EC 1.1.1.39, or EC 1.1.1.40). Malate dehydrogenase (EC 1.1.1.38, EC 1.1.1.39, or EC 1.1.1.40) converts malate and NAD ＋ From pyruvate, CO ２ , and NADH. Genes encoding such enzymes include maeA, SLI_5552, SLI_3297, etc., and combinations thereof.

[0032] CO in metabolic pathways ２Examples of enzymes responsible for the metabolic reaction to produce CO include, but are not limited to, phosphoenolpyruvate carboxykinase (EC 4.1.1.32, EC 4.1.1.38, EC 4.1.1.49). Phosphoenolpyruvate carboxykinase (EC 4.1.1.32, EC 4.1.1.38, EC 4.1.1.49) converts oxaloacetate to phosphoenolpyruvate and CO ２ Examples of such enzyme genes include pckA and SLI_5252.

[0033] CO in metabolic pathways ２ Enzymes responsible for the metabolic reactions that produce CO include, but are not limited to, glutamate decarboxylase, hydroxypyruvate decarboxylase, 3-oxoacyl synthase, and aspartate decarboxylase. The metabolic reactions of cells have been elucidated in detail, and those skilled in the art will be able to easily understand the mechanisms of CO production. ２ The enzymes responsible for the metabolic reactions that produce the enzymes can be appropriately identified, and their genes can be appropriately disrupted.

[0034] In the method of the present disclosure, CO ２ It is possible to disrupt one or more, two or more, three or more, four or more, or five or more of the enzymes responsible for the metabolic reaction that produces carbon dioxide. This is expected to reduce the carbon yield of carbon dioxide from glucose in the disruptant strain.

[0035] According to the present disclosure, CO ２ Bacteria are provided in which one or more, two or more, three or more, four or more, or five or more enzymes responsible for metabolic reactions to produce CO2 (metabolic enzyme gene disruptant strains). In some embodiments, the bacterium is isolated. In some embodiments, the bacterium is present in culture. In some embodiments, the bacterium is cloned. In some embodiments, the bacterium to be modified may be an aerobic bacterium. In some embodiments, the bacterium may be an actinomycete of the genus Streptomyces. In some preferred embodiments, the bacterium to be modified may be a bacterium that is capable of converting CO2 to glucose. ２The bacterium may be a bacterium having a carbon yield of more than about 50% at least during the logarithmic growth phase or stationary phase, and more preferably a bacterium (preferably an actinomycete, for example, an actinomycete of the genus Streptomyces) having a carbon yield of more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% at least during the logarithmic growth phase or stationary phase. In a preferred embodiment, the bacterium may be Streptomyces lividans (e.g., strain 1326).

[0036] CO2 response to glucose in metabolic enzyme gene disruptants ２ The carbon yield of the metabolic enzyme gene disruptant can be at most about 90% or less, preferably about 80% or less, more preferably about 70% or less, even more preferably about 60% or less, and even more preferably about 50% or less, compared to the strain before disruption of the metabolic enzyme gene, at least during the logarithmic growth phase or stationary phase. ２ The carbon yield of the metabolic enzyme gene disruptant may 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, at least during the logarithmic growth phase or stationary phase, compared to the strain before metabolic modification. ２ The carbon yield of the strain of the present invention may be, for example, about 5% to about 90%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 35% to about 80%, about 40% to about 70%, about 45% to about 60%, or about 50%, at least during the logarithmic growth phase or stationary phase, compared to that of the strain before disruption of the metabolic enzyme gene.

[0037] In a preferred embodiment, the metabolic enzyme gene-disrupted strain maintains about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of the growth ability of the strain before disruption of the metabolic enzyme gene. The growth ability can be determined, for example, by measuring the dry cell weight in 1 mL of culture medium after culture (e.g., 40 hours after the start of main culture).

[0038] <Method for culturing bacteria according to the present disclosure> The present disclosure provides a method for culturing the genetically modified bacteria of the present disclosure. The method of the present disclosure includes preparing a genetically modified bacterium and culturing the genetically modified bacterium in an environment suitable for culturing. In a preferred embodiment, the environment suitable for culturing may be conditions suitable for culturing the corresponding wild-type bacterium. In one embodiment, the environment suitable for culturing may be an environment suitable for culturing the genetically modified bacterium. In one embodiment, the bacterium is cultured under anaerobic conditions. In a preferred embodiment, the bacterium is cultured under aerobic conditions, for example, with shaking or with aeration and agitation.

[0039] Although solid media may be used as the culture medium, liquid media are preferred. The culture medium may contain components necessary for bacterial survival and / or substance production (metabolite production), such as a carbon source and other nutrient sources (e.g., nitrogen source, inorganic salts, etc.). Those skilled in the art can prepare such media appropriately. The culture medium may be a synthetic or semi-synthetic medium. The culture medium may contain a basal medium such as TSB medium, LB medium, YT medium (or 2xYT medium), NZY medium, M9 medium, SOC medium, or YPD medium; a buffer; salts; a pH adjuster; alkali metal ions such as potassium ions and sodium ions; alkaline earth metal ions such as calcium ions; and metal ions such as magnesium ions, iron ions, tin ions, cobalt ions, manganese ions, and zinc ions; phosphorus; and sulfur. The medium may contain a nitrogen source selected from the group consisting of ammonium salts, nitrates, amino acids, peptone, casein, casamino acids, urea, yeast extract, protein hydrolysate, yeast autolysate, casein pancreatic digest, soybean papain digest, tryptone, phytone peptone, meat extract, meat peptone, and tryptose. Carbon sources include, for example, glucose, dextran, starch, beef extract, pyruvic acid, and acetic acid. Inorganic salts include sodium salts such as sodium phosphate, sodium chloride, and sodium molybdenum; 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 iron chloride; and borates. The pH of the medium is not particularly limited, but may be, for example, about 7, e.g., about 6.5 to 7.5. The culture temperature varies depending on the bacterium, but may be, for example, 25°C to 37°C, e.g., 25°C to 30°C. The culture period is not particularly limited, but may be, for example, 1 day to 2 months, e.g., 1 day to 1 month, e.g., 1 day to 2 weeks, e.g., 1 day to 1 week. A carbon source or other nutrient source may be added to the medium during the culture. Those skilled in the art can appropriately select culture conditions according to the bacterium.

[0040] In the method of the present disclosure, culturing includes culturing bacteria or metabolic enzyme gene-disrupted strains in the logarithmic growth phase and stationary phase. In the stationary phase, bacteria or metabolic enzyme gene-disrupted strains can produce various metabolic products. In particular, metabolic enzyme gene-disrupted strains have an improved yield of metabolites per carbon because the enzyme that synthesizes carbon dioxide is disrupted, allowing the carbon source to be utilized for the production of the various metabolic products. Examples of metabolic products include, but are not limited to, undecylprodigiosin (Udp). Examples of metabolic products include, for example, CO ２ Examples of metabolic products include those from metabolic pathways upstream or downstream of enzymatic reactions involving the excretion of CO. ２ In order to obtain a metabolic product of a downstream metabolic pathway of an enzymatic reaction accompanied by CO excretion or a metabolic product produced by utilizing the metabolic product, ２ It is conceivable to introduce into cells an intermediate of a metabolic pathway downstream of an enzymatic reaction involving excretion of a metabolic product. Introduction of the intermediate into cells can be achieved by adding the intermediate to the medium. The intermediate can be used in the cells as a raw material for the metabolic product.

[0041] In some aspects, the modification results in increased CO2 production compared to unmodified cells. ２ The carbon yield of the culture can be reduced, and the CO ２ According to a preferred embodiment, the modification can contribute to a reduction in CO production. ２ According to the calculation of carbon dioxide (CO₂), the carbon that would have been consumed can be used for substance production, and in this case, the modification can promote substance production compared to unmodified cells. According to a preferred embodiment, the modification does not inhibit cell growth by 50% or more, 40% or more, 33% or more, 30% or more, 25% or more, 20% or more, 15% or more, or 10% or more. According to a preferred embodiment, the modification does not reduce sugar consumption by 50% or more, 40% or more, 33% or more, 30% or more, 25% or more, 20% or more, 15% or more, or 10% or more compared to unmodified cells, or preferably does not reduce or increases sugar consumption. According to a preferred embodiment, the modification reduces CO₂ production compared to unmodified cells. ２While the modification reduces the carbon yield of the cells, the modification does not impair cell growth by 20% or more (preferably 10% or more or not at all) and / or does not reduce or increase sugar consumption. ２ While reducing the carbon yield of the yeast, the modification does not impair cell growth by 20% or more (preferably 10% or more or not at all) and does not reduce or increase sugar consumption.

[0042] Example 1: Carbon Yield of Bacteria Escherichia coli, Corynebacterium glutamicum, and Streptomyces lividans, strain 1326 were prepared. A synthetic medium was used as the culture medium. The synthetic medium contained 1.8 g / L of TSB medium (pH 7.0) as the basal medium. The pH of the synthetic medium was adjusted with 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. Then, jar fermenter culture was performed. Bacteria obtained by preculture 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 to achieve a concentration of 50 g / L. An appropriate amount of antifoaming agent was also added to the medium. During the culture, CO in the exhaust gas was measured using an exhaust gas analyzer (OFF-GAS Jr., Biot). ２ The amount of E. coli was measured after 10 hours (logarithmic growth phase), the coryneform bacteria after 10 hours (logarithmic growth phase), and the actinomycetes after 40 hours (logarithmic growth phase) and 88 hours (stationary phase).

[0043] The medium containing the recovered bacteria was analyzed by HPLC to measure the carbon yield. The HPLC analysis conditions were as follows: Column: Aminex HPX-87H Eluent: 5 mM H ２ SO ４ Flow rate: 0.6 mL / min Temperature: 50°C Detector: UV detector and differential refractive index detector (RID)

[0044] The results are shown in Table 1.

[0045]

[0046] As shown in Table 1, the actinomycete strains examined were able to grow in logarithmic phase with CO ２ The carbon yield of ２ Although data is not shown in Table 1, it was revealed that CO ２ The carbon yield of bacteria (especially actinomycetes) was higher than that of bacteria in the logarithmic growth phase. ２ It was revealed that there are species and strains with high carbon yields.

[0047] CO ２ The carbon yield of the glucose-derived carbon is determined by the amount of CO ２ When bacteria are used for substance production, CO ２ The high carbon yield of CO2 can have a negative impact on substance production because the carbon derived from glucose is converted to CO2. ２ This is because it is consumed in the production of nutrients and can inhibit their utilization in material production.

[0048] Example 2: Preparation of enzyme-disrupted strains and CO ２ Example 2-1: Carbon yield of Δicd and glucose consumption. ２ There is an enzymatic reaction that produces α-ketoglutarate and CO from isocitrate (see Figure 1). ２ A strain (Δicd) lacking the enzyme (icd) that produces the enzyme was prepared, and the bacterial mass, glucose consumption, and CO ２ The carbon yield of the Δicd strain was measured. The strain was cultured under the same conditions as the wild-type strain (WT), except that the initial glucose concentration and added glucose concentration were 100 g / L, and the cells were collected. The ICD activity of the icd-disrupted strain was measured by measuring the increase in NADPH produced when isocitrate was converted to 2-ketosoglutarate by ICD, measured by absorbance at 340 nm. As a result, the ICD activity of the Δicd strain was significantly reduced.

[0049]

[0050] As shown in Table 2, in the stationary phase, the CO2 yield relative to carbon in the Δicd strain was reduced to about half that of the wild-type strain (WT).

[0051] Analysis of carbon yields also revealed that the Δicd strain had increased carbon yields of pyruvate, isocitrate or citrate, and ribulose during logarithmic growth, and increased carbon yields of ribulose and pyruvate during stationary growth.

[0052] Analysis of carbon yield further revealed that CO ２ In addition to the dramatic decrease in the carbon yield of CO2, the carbon yield of several unidentified metabolites accounted for 40.7% in total, indicating a significant improvement in the carbon utilization efficiency of the fungus. ２ The destruction of the enzyme responsible for the enzymatic reaction that produces CO ２ This caused a dramatic change in the carbon yield of the

[0053] This means that in bacteria where carbon source utilization is strongly restricted by carbon dioxide emission, this restriction can be released, and a state in which carbon sources can be used more effectively in metabolic reactions can be induced. Furthermore, this example demonstrated the effectiveness of gene disruption of an enzyme responsible for metabolic reactions that produce carbon dioxide.

[0054] Furthermore, as shown in FIG. 2, the Δicd strain showed a 2.6-fold increase in glucose consumption compared to the wild-type strain (WT).

[0055] Example 2-2: Carbon yield and glucose consumption of ΔpckA, ΔmaeA, and Δgnd ２As enzyme genes that produce phosphoenolpyruvate from oxaloacetate, a strain (ΔpckA) deficient in the enzyme (pckA) that produces phosphoenolpyruvate from oxaloacetate, a strain (ΔmaeA) deficient in the enzyme (maeA) that produces pyruvate from malate, and a strain (Δgnd) deficient in the enzyme (gnd) that produces ribulose-5-phosphate from 6-phosphogluconate were prepared. Since maeA has two homologs, a double-defective strain was prepared, and although gnd has three homologs, a strain deficient in only one of them was prepared (see Table 9). The prepared disrupted strains and the wild-type strain were aerobically cultured in a jar fermenter, and the bacterial mass, glucose consumption, and CO2 during the culture were measured. ２ The carbon yield of each strain was measured. The strain was cultured under the same conditions as the wild-type strain.

[0056]

[0057] As shown in Table 3, the CO ２ The carbon yield of the ΔpckA strain was 0.86 times lower, the ΔmaeA strain was 0.88 times lower, and the Δgnd strain was 0.86 times lower than that of the wild-type strain (parent strain). ２ The carbon species ratio of the ΔpckA strain was 0.82 times, the ΔmaeA strain was 0.86 times, and the Δgnd was 0.92 times compared to the wild-type strain, and in the stationary phase the ΔpckA strain was 0.97 times, the ΔmaeA strain was 0.90 times, and the Δgnd was 0.81 times.

[0058]

[0059] As shown in Table 4, the glucose consumption per unit cell of each gene-disrupted strain was increased by 1.3 times for the ΔpckA strain, 1.9 times for the ΔmaeA strain, and 1.2 times for the Δgnd strain compared to the wild-type strain (parent strain).

[0060] As shown in FIG. 5, the CO ２ The carbon yield of the strain was decreased compared to the wild-type strain, but the bacterial growth rate was not increased compared to the wild-type strain. Furthermore, the glucose consumption per bacterial cell increased. ２ Carbon can be expected to be used for metabolites.

[0061] Example 3: Secondary Metabolite Production by Enzyme Gene Disruption Strains Example 3-1: Undecylprodigiosin (Udp) Production by the Δicd Strain The wild-type strain and the Δicd strain were cultured, and the amount of undecylprodigiosin (Udp) produced was confirmed. Each strain was cultured in solid TSB medium (30°C for 48 hours) and then precultured in 10 mL of R2YE medium (see Tables 5 and 6) with shaking (28°C, 160 rpm, 48 hours). The entire volume was added to 50 mL of R2YE medium, and main culture was performed (28°C, 160 rpm, 72 hours). 48 and 72 hours after the start of main culture, 10 mL of culture was collected and centrifuged to recover the bacterial cells. 10 mL of methanol was added to treat the bacterial cells, and Udp was extracted. The absorbance (530 nm) of the supernatant containing the extracted Udp was measured, and the Udp concentration was calculated.

[0062]

[0063] As shown in Figure 3, the Δicd strain produced approximately five times more Udp than the wild-type strain (WT), demonstrating that the reduction in CO yield relative to carbon due to icd disruption is effective for substance production.

[0064] 3-2: Udp Production by the ΔpckA Strain The wild-type strain and the ΔpckA strain were cultured, and the amount of Udp produced was confirmed. Each strain was cultured in solid TSB medium (30°C for 48 hours) and then precultured in 10 mL of R2YE medium (see Tables 5 and 6) with shaking (28°C, 160 rpm, 48 hours). The entire volume was added to 50 mL of R2YE medium, and main culture was performed (28°C, 160 rpm, 72 hours). 48 and 72 hours after the start of main culture, 10 mL of the culture was collected and centrifuged to recover the bacterial cells. Five mL of methanol was added to treat the bacterial cells, and Udp was extracted. The absorbance (530 nm) of the supernatant containing the extracted Udp was measured, and the Udp concentration was calculated.

[0065] As shown in Figure 6, the ΔpckA strain produced approximately 2.1 times more Udp at 48 hours and approximately 1.3 times more Udp at 72 hours than the wild-type strain (WT). ２It was found that the decrease in carbon yield is effective for material production.

[0066] Example 4: Primary metabolite production by enzyme gene disruptant strains 4-1: Ribulose and α-ketoglutaric acid production by Δicd strain and ΔmaeA strain The wild-type strain, Δicd strain, and ΔmaeA strain were aerobically cultured in jar fermenters. After 88 hours of culture, the culture medium was collected and centrifuged at 15,000 rpm to obtain the supernatant. The resulting culture medium was filtered through a 0.2 μm filter and then subjected to HPLC analysis. The HPLC analysis conditions for ribulose and α-ketoglutaric acid are shown in Tables 7 and 8, respectively.

[0067]

[0068]

[0069] Ribulose production was 0.83 g / L for the wild-type strain, while it was 1.90 g / L for the Δicd strain and 1.24 g / L for the ΔmaeA strain, which was an approximately 2-fold and 1.5-fold increase in production, respectively. α-Ketoglutaric acid production was 0.63 g / L for the wild-type strain, while it was 32.0 g / L for the Δicd strain and 0.98 g / L for the ΔmaeA strain, which was an approximately 50.7-fold and 1.6-fold increase in production, respectively. This indicates that CO2 production due to icd disruption and maeA disruption is significantly reduced. ２ It was found that the decrease in carbon yield is effective for material production.

[0070] Udp is synthesized from malonyl-CoA and proline. Malonyl-CoA is produced from acetyl-CoA upstream of the icd enzyme, and proline is produced from α-ketoglutarate downstream of the icd enzyme via glutamate. In this culture, proline was added to the medium, which is thought to have compensated for the expected decrease in proline due to the destruction of the icd enzyme.

[0071] The gene disruptant strains used in this example are listed below. All experiments were carried out under aerobic conditions.

[0072] As described above, according to this example, a method for reducing carbon dioxide emissions associated with the cultivation of actinomycetes was found. This reduces the environmental load (especially CO２ In addition, according to this embodiment, it is expected that CO ２ By disrupting the enzyme responsible for the metabolic reaction that produces CO, the efficiency of carbon utilization within the bacterial cells was significantly improved, and the bacterial cells were successfully transformed into a state more suitable for substance production. ２ By disrupting the gene encoding the enzyme responsible for the metabolic reaction that produces the metabolite, it was possible to increase the production of a useful metabolite. This is expected to be effective for all microorganisms (especially bacteria), but is thought to be particularly effective for microorganisms (especially bacteria) that have a high carbon dioxide-to-carbon yield. This is thought to pave the way for genetically modified strains of such microorganisms (especially bacteria) that reduce environmental burden and / or improve metabolite production, culture methods for such strains, and metabolite production using these.

Claims

1. The enzyme produces carbon dioxide (CO) during at least the logarithmic or stationary phase of aerobic culture in a medium containing sufficient carbon sources and other nutrients. ２ A metabolic enzyme gene disruptant of actinomycete having a carbon yield of more than 50% by CO ２ At least one gene encoding an enzyme responsible for the metabolic reaction that produces CO ２ A metabolic enzyme gene disruptant in which the carbon yield of the actinomycete is reduced compared to a control actinomycete of the same species in which none of the genes is disrupted.

2. CO ２ The metabolic enzyme gene disruptant according to claim 1, wherein the enzyme responsible for the metabolic reaction that produces the compound 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.

3. The metabolic enzyme gene-disrupted strain according to claim 1 or 2, in which one or more selected from the group consisting of phosphogluconate dehydrogenase, pyruvate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, isocitrate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, and α-ketoglutarate dehydrogenase are disrupted.

4. A metabolic enzyme gene disruptant strain described in any one of claims 1 to 3, which, when aerobically cultured in the presence of a sufficient amount of carbon source, has the same growth ability as a control actinomycete or does not show a 50% or greater decrease in growth ability.

5. The metabolic enzyme gene-disrupted strain according to any one of claims 1 to 4, in which one or more genes consisting of at least isocitrate dehydrogenase, phosphogluconate dehydrogenase, phosphoenolpyruvate carboxykinase, and malate dehydrogenase are disrupted.

6. The metabolic enzyme gene disruptant described in any one of claims 1 to 5, wherein the actinomycete belongs to the genus Streptomyces.

7. A composition for use in producing a metabolic product, comprising the metabolic enzyme gene disruptant according to any one of claims 1 to 6.

8. A composition for use in producing a metabolic product, comprising the metabolic enzyme gene disruptant according to any one of claims 1 to 6.

9. A method for culturing a metabolic enzyme gene-disrupted strain of actinomycetes, wherein the metabolic enzyme gene-disrupted strain comprises a metabolic enzyme gene-disrupted strain described in any one of claims 1 to 6, and the method comprises culturing the metabolic enzyme gene-disrupted strain in a medium containing a sufficient amount of a carbon source and other nutrients under aerobic conditions.

10. The method according to claim 9, wherein the culturing comprises culturing the metabolic enzyme gene disruptant in logarithmic growth phase and culturing the metabolic enzyme gene disruptant in stationary phase.

11. A method for causing a metabolic enzyme gene-disrupted strain of an actinomycete to produce a metabolic product, comprising: culturing a metabolic enzyme gene-disrupted strain, wherein the metabolic enzyme gene-disrupted strain comprises the metabolic enzyme gene-disrupted strain described in any one of claims 1 to 6, the method comprising: culturing the metabolic enzyme gene-disrupted strain in a medium containing a sufficient amount of a carbon source and other nutrients under aerobic conditions to cause the metabolic enzyme gene-disrupted strain to produce the metabolic product; and recovering the metabolite.

12. The method of claim 11, wherein the metabolic product is derived from the citric acid cycle.

13. A method for selecting actinomycetes, comprising: providing an isolated actinomycete; measuring the carbon dioxide yield of said actinomycete during logarithmic growth phase or stationary phase in aerobic culture in a medium containing a sufficient amount of a carbon source and other nutrients; and selecting or obtaining an actinomycete having a carbon yield of more than 50% during either the logarithmic growth phase or the stationary phase.

14. A method for genetically modifying actinomycetes to obtain genetically modified actinomycetes, comprising: providing an isolated actinomycete; measuring the carbon dioxide yield of the actinomycete in logarithmic or stationary phase in aerobic culture in a medium containing a sufficient amount of a carbon source and other nutrients; selecting or obtaining actinomycetes having a carbon yield of more than 50% in either the logarithmic or stationary phase; and determining the carbon dioxide yield of the obtained actinomycete. ２ and obtaining a genetically modified actinomycete.

Images