Mutant cell strains and methods for protein or biomass production

Abstract

The present invention relates to mutant chemoautotrophic bacteria of the genus Xanthobacter, which contain a genetic modification that reduces the bacterial production of polyhydroxyalkanoates. Furthermore, the present invention relates to continuous culture methods for producing proteins or biomass using the mutant chemoautotrophic bacteria, which methods include feeding the cells with gases and minerals. The present invention also relates to products of these methods and the use of these products, for example in food or feed.

Description

Technical Field

[0001] The present invention relates to the production of proteins and / or other polymers using microorganisms. In particular, the present invention relates to novel bacterial strains and continuous culture methods for producing proteins or biomass using bacteria, in which gases and minerals are supplied to the cells. The present invention also relates to the products of these methods and the use of these products, for example, in food or feed.

Background Art

[0002] The increasing world population, climate change and water scarcity are increasingly posing threats to traditional agriculture and thus to the supply of sufficient food and feed. Therefore, alternative sources of organic molecules such as proteins are being studied. A possible alternative is single cell production, i.e., the production of proteins and / or other polymers using microorganisms. Chemoautotrophic microorganisms that can grow on minimal mineral media using hydrogen gas as an energy source and carbon dioxide as the sole carbon source have been described. For reviews of these microorganisms, see, for example, Shively et al. (1998) Annu Rev Microbiol 52:191. International Publication No. WO 2018 / 144965 describes various microorganisms and bioprocesses for converting gaseous substrates into high-protein biomass. Andersen et al. (1979) Biochim Biophys Acta 585:1-11 describe mutants of Alcaligenes eutrophus, a hydrogen bacterium that grows easily under heterotrophic and autotrophic conditions. Mutants with altered ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) activity were characterized. Ohmiya et al. (2003) J. Biosci. Bioeng. 95:549-561 review the application of microbial genes to resistant biomass utilization. Yu Jian et al. (2013) Int J Hydrogen Ener 38:8683-8690 describes carbon dioxide fixation by hydrogen-oxidizing bacterial isolates. A high energy efficiency of 50% was measured under moderate oxygen concentration (10 mol%).

[0003] However, various chemolithoautotrophic microorganisms have different characteristics regarding growth rate, yield, biomass composition, and characteristics related to use as food ingredients such as safety, taste, odor, texture, and technological and functional properties in cooking in human consumption. Not all chemolithoautotrophic microorganisms have a sufficient growth rate and provide a sufficient yield, and not all processes can be realistically upscaled to large-scale processes that are economically viable. For example, in order to have a sufficient output of functional proteins for food or feed applications, it is important to find suitable production organisms and suitable processes that can be carried out on a large scale. This need is addressed by the present invention. Under metabolic stress such as nitrogen limitation, bacteria may store energy in the form of storage polymers called polyhydroxyalkanoates (PHA) (Rehm and Steinbuchel, 1999 Int J Biol Macromol 25:3-19). When bacteria grow in large bioreactors in an industrial environment, gas exchange often becomes insufficient, leading to an increase in PHA production. PHA can be used as a bioplastic, but when growing bacteria for products other than PHA, its production is an undesirable result as it reduces the carbon yield. This problem is also addressed by the present invention.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0006] The inventors have found that chemosynthetic autotrophic bacteria, such as bacteria of the genus Xanthobacter, store energy in the form of PHA under specific conditions. Mutant strains containing gene disruption of the phaC1 gene hardly produced PHA under the same conditions, but were found to retain favorable characteristics and compatibility for methods for the production of biomass and / or protein. In a first main aspect, the present invention relates to a variant of the bacterial strain VTT-E-193585, which variant contains a genetic modification that reduces the bacterial production of polyhydroxyalkanoate (PHA) as compared to the VTT-E-193585 strain. In a further aspect, the present invention relates to a method for the production of biomass and / or protein, the method comprising culturing a mutant chemolithoautotrophic bacterial strain in continuous culture with hydrogen as an energy source and an inorganic carbon source, the inorganic carbon source comprising carbon dioxide, and the mutant chemolithoautotrophic strain comprising a gene disruption of one or more genes encoding PHA synthase. In a further major aspect, the present invention relates to bulk protein, biomass or non-protein cellular or chemical components obtainable or obtained by the method of the present invention, and food or feed products obtainable or obtained by the method of the present invention. In a further aspect, the present invention relates to a variant Xanthobacter strain comprising a gene disruption of one or more genes encoding PHA synthase. The present invention also relates to a method for the genetic modification of the bacterial strain VTT-E-193585, and to genetically modified variants of the VTT-E-193585 strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0007]

Figure 1

Figure 2

Figure 3

[0008] (Definitions) As used herein, for example in relation to strains, the term "isolated" means separated from its natural environment. Preferably, the isolated strain is pure, i.e., does not contain other strains. The term "variant", as used herein in relation to strains, refers to a strain derived from a reference strain, i.e., generated using the reference strain as a starting point and containing a genetic modification as compared to the reference strain. Genetic modifications include point mutations, as well as disruptions such as insertions or deletions of entire loci or fragments thereof. The variant preferably has less than 10, such as less than 5, such as 4, 3, 2, or 1, etc., genetic modifications as compared to the reference strain. Preferably, the genomic sequence of the variant strain is more than 90%, such as more than 95%, such as more than 99%, identical to the genomic sequence of the reference strain. As used herein, the term "chemoautotrophic" refers to the ability to grow in minimal mineral medium using hydrogen gas as an energy source and carbon dioxide as the sole carbon source. As used herein, the noun "culture" refers to a suspension of live cells in a liquid medium. The term "biomass" has its ordinary meaning in the field of bacterial fermentation and refers to cell material. As used herein, the term "continuous culture" refers to a culture process in which fresh medium is continuously added to the culture and the medium containing the bacterial culture is continuously removed at essentially the same rate.

[0009] (Aspects and Embodiments of the Invention) The VTT-E-193585 strain was isolated from the Baltic Sea coast in Naantali, Finland. This organism can grow under limited oxygen conditions, in minimal mineral medium, using hydrogen as an energy source and carbon dioxide as a carbon source, under appropriate bioreactor conditions. 16S sequencing and Illumina metagenomics sequencing indicate that this strain is most likely a member of the genus Xanthobacter, but is not a known species. The bacterial strain is very suitable for food and feed applications because the dried cell powder has a high protein content and contains all essential amino acids. It also contains more unsaturated fatty acids than saturated fatty acids and contains high levels of B-group vitamins. The levels of peptidoglycan and lipopolysaccharide that can cause allergies or toxicity are low. When a toxicity analysis was performed, no genotoxicity or cytotoxicity was observed for the strain. Furthermore, the strain is generally sensitive to antibiotics. The VTT-E-193585 strain (SoF1) was deposited on June 11, 2019, at the VTT Culture Collection of VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland, an international depository authority under the Budapest Treaty. Further information regarding the characteristics of the strain and the method for culturing the strain is provided in the examples of this specification and European Patent Application No. 19205786.7, which is incorporated herein by reference.

[0010] The present invention relates, inter alia, to variants of VTT-E-193585, in particular variants comprising a genetic modification that reduces the bacterial production of polyhydroxyalkanoic acid (PHA), and more generally to chemosynthetic autotrophic bacteria with reduced production of polyhydroxyalkanoic acid (PHA), such as strains comprising a gene disruption of one or more genes encoding PHA synthase. The inventors constructed a genetically modified variant of VTT-E-193585 that includes disruption of the phaC1 and / or phaC2 locus. Variants containing gene disruption of phaC1 were deposited on April 19, 2021, at the VTT Culture Collection of VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland, an international depositary authority under the Budapest Treaty. The accession number is VTT E-213595. Further information regarding the characteristics of the strain and the method for culturing the strain is provided in the examples of this document.

[0011] In a first main aspect, the present invention relates to a variant of the bacterial strain VTT-E-193585, which includes a genetic modification that reduces the bacterial production of polyhydroxyalkanoate (PHA) compared to the VTT-E-193585 strain. Thus, the present invention relates to a genetically modified variant, i.e., a derivative, of the bacterial strain VTT-E-193585. In other words, the VTT-E-193585 strain is further characterized by including a genetic modification. In one embodiment, the genetic modification reduces the bacterial PHA synthase activity compared to the VTT-E-193585 strain, and preferably the PHA synthase activity is reduced to less than 10%, less than 5%, etc., for example, less than 2%. In a further embodiment, the genetic modification reduces the bacterial PHB production to less than 10%, less than 5%, etc., for example, less than 2% under autotrophic growth conditions. This can be determined, for example, by measuring the PHB dry content as described in Example 5 of this document. In one embodiment, the variant includes a genetic modification that reduces the expression level and / or the activity of the phaC1 enzyme. In another embodiment, the variant includes a genetic modification that reduces the expression level and / or the activity of the phaC2 enzyme. In one embodiment, the genetic modification is a gene disruption such as insertion and / or deletion of a gene or a part thereof. In one embodiment, the variant includes gene disruptions of both phaC1 and phaC2. In another embodiment, the variant includes a gene disruption of phaC1 but does not include a gene disruption of phaC2. In one embodiment, the variant is a bacterial strain deposited under the number VTT-E-213595 in which the phaC1 gene is disrupted. In a preferred embodiment, the variant retains the ability to grow using hydrogen gas as an energy source and carbon dioxide as the sole carbon source. In one embodiment, when the strain is a variant of the VTT-E-193585 strain, the variant includes the 16S ribosomal RNA shown in SEQ ID NO: 1, or a 16S ribosomal RNA having up to 20, for example 1 to 10, 1 to 5, etc., for example 1, 2 or 3 nucleotide differences from SEQ ID NO: 1.

[0012] SEQ ID NO: 1. 16S ribosomal RNA sequence of the VTT-E-193585 strain:

[0013] In a further aspect, the present invention relates to a Variant Xanthobacter strain comprising a gene disruption of one or more genes encoding PHA synthase. Preferably, the strain is selected from the group consisting of X. agilis, X. aminoxidans, X. autotrophicus, X. flavus, X. tagetidis, X. viscosus, Xanthobacter sp. 126, Xanthobacter sp. 91 and the VTT-E-193585 strain. In one embodiment, the Variant Xanthobacter strain is a mutant X. autotrophicus strain in which the phaC gene (UniProtKB-A0A6C1KXK2) is disrupted. In another embodiment, the Variant Xanthobacter strain is a mutant X. tagetidis strain in which the phaC gene (UniProtKB-A0A3L7AJD5) is disrupted. In another embodiment, the Variant Xanthobacter strain is a variant of a Xanthobacter species in which the gene in the genome of the strain having the highest sequence identity with phaC1 (SEQ ID NO: 62) is disrupted. Methods for determining sequence identity are well known in the art. Preferably, the encoded protein has a sequence identity of more than 50%, more than 60%, such as more than 70%, more than 80%, such as more than 90%, more than 95% etc. with SEQ ID NO: 62. In another embodiment, the Variant Xanthobacter strain is a variant of X. agilis, X. aminoxidans, X. flavus, X. viscosus, Xanthobacter sp. 126 or Xanthobacter sp. 91 in which the gene in the genome of the strain encoding the protein having the highest sequence identity with phaC1 (SEQ ID NO: 62) is disrupted. For example, in one embodiment, the variant is a variant of X. agilis in which the X. agilis gene having the highest sequence identity with phaC1 (SEQ ID NO: 62) among all the genes in the X. agilis genome is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. tagetidis strain in which the gene described in NCBI ref.MBB6309058.1 is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. flavus strain in which the gene described in NCBI ref.MBP2147722.1 is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. autotrophicus strain in which the gene described in NCBI ref.WP_138398147.1 is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. tagetidis strain in which the gene described in NCBI ref.WP_210210858.1 is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. flavus strain in which the gene described in NCBI ref.WP_209489961.1 is disrupted. In one embodiment, the mutant Xanthobacter strain is a mutant X. autotrophicus Py2 strain in which the gene described in NCBI ref.ABS67253.1 is disrupted.

[0014] In a further aspect, the present invention relates to a culture comprising the mutant bacterial strain of the present invention. In a preferred embodiment, the volume of the culture is 100 mL or more, such as 1 L or more, such as 10 L or more, such as 1,000 L or more, 10,000 L or more, etc., such as 50,000 L or more, 100,000 L or more, etc., such as 200,000 L or more. In a further aspect, the present invention relates to a method for the production of biomass and / or protein, the method comprising culturing a mutant bacterial strain of the present invention. In one embodiment, the method is for the production of biomass. In another embodiment, the method is for the production of protein. In one embodiment, the method comprises culturing bacteria in continuous culture using hydrogen as an energy source and an inorganic carbon source, the inorganic carbon source comprising carbon dioxide. In a further embodiment, the method is for the production of biomass and comprises culturing bacteria in continuous culture using hydrogen as an energy source and an inorganic carbon source, the inorganic carbon source comprising carbon dioxide. Various further embodiments of the method are described hereinbelow.

[0015] In a further main aspect, the present invention is a method for the production of biomass and / or protein, the method comprising culturing a mutant chemoautotrophic bacterial strain in continuous culture with hydrogen as an energy source and an inorganic carbon source, the inorganic carbon source comprising carbon dioxide, and the mutant chemoautotrophic strain comprising a gene disruption of one or more genes encoding PHA synthase, preferably the gene having the highest sequence identity with phaC1 (SEQ ID NO: 62), and relates to a method for the production of biomass and / or protein. In one embodiment, the method is for the production of biomass. In another embodiment, the method is for the production of protein. Various further embodiments of the method are described hereinbelow. In one embodiment, the mutant chemoautotrophic strain used in the method is of the genus Xanthobacter, preferably a mutant of the VTT-E-193585 strain. In another embodiment, the mutant chemoautotrophic strain used in the method is of the species Cupriavidus necator.

[0016] According to the genomic sequence, the strain deposited under the number VTT-E-193585 uses the most likely Calvin-Benson-Bassham cycle for carbon fixation, in which carbon dioxide molecules are attached to the 5-carbon chain of ribulose 1,5-bisphosphate to form two molecules of glycerate 3-phosphate. Thereby, the strain can synthesize all other organic molecules necessary for growth. Energy from hydrogen most likely enters the cell via NAD + reducing hydrogenase and / or NiFeSe hydrogenase. Essentially, this is a redox reaction in which hydrogen (H2) is oxidized to H+ and NAD + is reduced to NADH. In addition to ATP, NADH is one of the main energy carriers in vivo. Alternatively, some other energy equivalents are reduced by other hydrogenase enzymes that use H2. The Calvin-Benson-Bassham cycle requires energy in the form of ATP and NADH / NADPH to fix CO2. This strain is most likely to produce ATP by oxidative phosphorylation, which consists of four protein complexes that generate a proton gradient across the membrane. The proton gradient is generated mainly using energy from NADH. The proton gradient drives the ATP synthase complex to produce ATP. According to the genomic sequence, the strain has a bacterial F-type ATP synthase.

[0017] If the method is specified to include culturing the strain with an inorganic carbon source, it can be understood that the inorganic carbon source is the main carbon source in the culture. Thus, a small amount of organic carbon source may be present in the culture, but the main metabolism and growth of the culture are based on the utilization of an inorganic carbon source, preferably carbon dioxide, as the carbon source. Preferably, the proportion of carbon supplied to the culture as an organic substance is less than 5%, such as less than 1%, such as less than 0.1%, of the total carbon supplied to the culture during the method. Preferably, no organic carbon source is supplied to the method.

[0018] Similarly, when it is specified that the method includes culturing stocks using hydrogen (H2) as an energy source, it can be understood that hydrogen is the main energy source in the culture. Thus, there may be other minor energy sources present in the culture such as ammonia that can be supplied as a nitrogen source, or minor amounts of organic compounds, but the main metabolism and growth of the culture is based on the utilization of hydrogen as an energy source. Throughout the method, hydrogen is preferably produced by water electrolysis, i.e., by decomposing water into hydrogen gas and oxygen gas with electricity. Thus, hydrogen gas and oxygen gas are supplied from a nearby electrolysis device to the bioreactor. Alternatively, electrodes can be placed inside the bioreactor to produce hydrogen and oxygen within the bioreactor rather than in a separate electrolysis device.

[0019] The inorganic carbon source containing carbon dioxide may include other inorganic carbon sources such as carbon monoxide. In one embodiment, only gaseous carbon sources are provided to the culture. In a preferred embodiment, carbon dioxide is the only inorganic carbon source provided to the culture and in fact the only carbon source. In one embodiment, only gases and minerals are supplied to the culture, and the level of carbon dioxide in the supplied gas is between 10% and 50%, such as between 15% and 45%, between 20% and 40%, etc., such as between 25% and 35%, between 26% and 30%, etc.

[0020] In another embodiment, gases and minerals are supplied to the culture, and the level of hydrogen (H2) in the supplied gas is between 30% and 80%, such as between 35% and 75%, between 40% and 70%, etc., such as between 45% and 65%, between 50% and 60%, etc.

[0021] In another embodiment, gases and minerals are supplied to the culture, and the level of oxygen (O2) in the supplied gas is between 10% and 25%, such as between 15% and 20%, between 16% and 18%, etc. In another embodiment, the level of oxygen provided is such that the level of dissolved oxygen in the culture is maintained between 5% and 10%.

[0022] In a preferred embodiment, only gas and minerals are supplied to the culture, a gas containing H2, CO2 and O2 is supplied, the percentage of H2 is between 40% and 70%, the percentage of CO2 is between 18% and 28%, and the percentage of O2 is between 12% and 22%.

[0023] Typically, the method of the present invention includes the addition of a nitrogen source. The nitrogen source may be provided, for example, in the form of an ammonium salt such as ammonium hydroxide, ammonium sulfate or ammonium chloride, ammonia, urea or nitrate, such as potassium nitrate. In other embodiments, nitrogen gas (N2) is provided as the nitrogen source. In a preferred embodiment, the nitrogen source is ammonium hydroxide or an ammonium salt such as ammonium sulfate. In one embodiment, the nitrogen source provided is ammonium hydroxide at a concentration between 100 mg / L and 10 g / L, between 250 mg / L and 4 g / L, for example between 0.5 g / L and 2 g / L, between 0.75 g / L and 1.5 g / L, etc.

[0024] Typically, the method of the present invention includes the addition of minerals such as minerals containing ammonium, phosphate, potassium, sodium, vanadium, iron, sulfate, magnesium, calcium, molybdenum, manganese, boron, zinc, cobalt, selenium, iodine, copper and / or nickel. Suitable mineral media are well-known techniques and are described, for example, in Thermophilic Bacteria, CRC Press, Boca Raton, FL, Jacob K. Kristjansson, ed., 1992, for example on page 87, Table 4.

[0025] In one embodiment, the minerals added include one or more of ammonia, ammonium (e.g., ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4)), nitrate (e.g., potassium nitrate (KNO3)), urea or an organic nitrogen source; phosphate (e.g., disodium phosphate (Na2HPO4), potassium phosphate (KH2PO4), phosphoric acid (H3PO4), potassium dithiophosphate (K3PS2O2), potassium orthophosphate (K3PO4), disodium phosphate (Na2HPO4·2H2O), dipotassium phosphate (K2HPO4) or monopotassium phosphate (KH2PO4)); sulfate; yeast extract; iron chelate (e.g., chelated with EDTA or citric acid); potassium (e.g., potassium phosphate (KH2PO4), potassium nitrate (KNO3), potassium iodide (KI), potassium bromide (KBr)); and other inorganic salts, minerals, and micronutrients (e.g., sodium chloride (NaCl), magnesium sulfate (MgSO4·7H2O) or magnesium chloride (MgCl2), calcium chloride (CaCl2), calcium sulfate (CaSO4) or calcium carbonate (CaCO3), manganese sulfate (MnSO4·7H2O) or manganese chloride (MnCl2), ferric chloride (FeCl2), ferrous sulfate (FeSO4·7H2O) or ferrous chloride (FeCl2·4H2O), sodium bicarbonate (NaHCO3) or sodium carbonate (Na2CO3), zinc sulfate (ZnSO4) or zinc chloride (ZnCl2), ammonium molybdate (NH4MoO4) or sodium molybdate (Na2MoO4·2H2O), cupric sulfate (CuSO4) or copper chloride (CuCl2·2H2O), cobalt chloride (CoCl2·6H2O) or cobalt sulfate (CoSO4), aluminum chloride (AlCl3·6H2O), lithium chloride (LiCl), boric acid (H3BO3), nickel chloride NiCl2·6H2O) or nickel sulfate (NiSO4), tin chloride (SnCl2·H2O), barium chloride (BaCl2·2H2O), copper selenate (CuSeO45H2O), sodium selenate (Na2SeO4) or sodium selenite (Na2SeO3), sodium metavanadate (NaVO3), chromium salts).

[0026] In a preferred embodiment, the method of the present invention includes the addition of one or more or all of NH4OH, KH2PO4, Na2HPO4·2H2O, NaVO3·H2O, FeSO4x7H2O, MgSO4·7H2O, CaSO4, Na2MoO4·2H2O, MnSO4·7H2O, ZnSO4·7H2O, H3BO3, CoSO4, CuSO4, NiSO4.

[0027] In one embodiment, the medium provided to the cells contains less than 1 g / L of chloride salts, less than 0.25 g / L of chloride salts, such as less than 0.1 g / L of chloride salts, less than 0.025 g / L of chloride salts, such as less than 0.01 g / L of chloride. In one embodiment, the chloride salt is not supplied to the culture. In another embodiment, vitamins are not supplied during the method, that is, the medium provided to the culture does not contain vitamins. In another embodiment, amino acids are not supplied during the method, that is, the medium provided to the culture does not contain amino acids. In another embodiment, organic compounds are not supplied during the method, that is, the medium provided to the culture does not contain organic compounds. In a specific embodiment, the pH of the bacterial culture is controlled at a specific level. In a specific embodiment, the pH is controlled within a range optimal for the maintenance and / or growth of the bacteria and / or the production of organic compounds. In one embodiment, the pH in the culture is maintained between 5.5 and 8.0, such as between 6.5 and 7.0, such as 6.8. In a specific embodiment, the temperature of the bacterial culture is controlled. In a specific embodiment, the temperature is controlled within a range optimal for the maintenance and / or growth of the bacteria and / or the production of organic compounds. In one embodiment, the culture is grown at a temperature between 25°C and 40°C, such as between 28°C and 32°C, 30°C, etc.

[0028] Typically, the method of the present invention is carried out in a bioreactor. The bioreactor is used for culturing cells and can be maintained at a specific stage of the cell growth curve. The use of a bioreactor is advantageous in many methods for culturing chemoautotrophic growth independently. In general, the control of growth conditions, including the control of dissolved carbon dioxide, oxygen, and other gases such as hydrogen, as well as other dissolved nutrients, trace elements, temperature, and pH, is promoted within the bioreactor. The nutrient medium and gases can be added to the bioreactor as a batch addition, or periodically, or in response to detected depletion or programmed set points, or continuously during the period in which the culture is growing and / or being maintained. In continuous culture methods, the nutrient medium and gases are continuously added to the bioreactor. Further, the bacterial-containing medium is continuously removed from the bioreactor.

[0029] In a preferred embodiment, the volume of the bacterial culture is 100 mL or more, 1 L or more, such as 10 L or more, 100 L or more, such as 1,000 L or more, 10,000 L or more, such as 50,000 L or more, 100,000 L or more, such as 200,000 L or more. In one embodiment, the productivity of the culture is more than 0.1 g per liter per hour, such as more than 0.2, such as more than 0.3, more than 0.4, etc., such as more than 0.5, more than 0.6 g, etc., such as more than 0.7, more than 0.8, etc., such as more than 0.9, more than 1 g, etc. of cell dry weight.

[0030] The bacteria can be inoculated directly from the cell bank or via a smaller-scale seed culture. Preferably, the supply of fresh medium to the culture and the removal of the used medium containing bacteria are carried out at the same rate so that the volume within the bioreactor remains the same. In one embodiment, after the initial phase of reaching an appropriate cell density, the bacteria grow in a steady state or pseudo-steady state and continuously maintain the logarithmic phase at an OD600 of more than 5, such as more than 10, such as more than 20, between 50 and 200, etc., such as between 50 and 100. In one embodiment of the method of the present invention, the bacterial strain has a growth rate of 0.001 - 0.12 h -1 , such as 0.01 - 0.12 h -1 , such as 0.04 - 0.12 h -1 . In another embodiment of the method of the present invention, the liquid supply rate in the continuous phase is 50 - 80% of the growth rate.

[0031] Xanthobacter is a genus of Gram-negative bacteria in the family Xanthobacteraceae. As described above, in one embodiment, the mutant chemoautotrophic strain used in the method of the present invention (i.e., a mutant comprising gene disruption of one or more genes encoding PHA synthase) belongs to the genus Xanthobacter. Preferably, the strain is selected from the group consisting of X. agilis, X. aminoxidans, X. autotrophicus, X. flavus, X. tagetidis, X. viscosus, Xanthobacter sp. 126, Xanthobacter sp. 91, and the VTT-E-193585 strain. In one embodiment, the mutant chemoautotrophic strain used in the method of the present invention is a strain that uses the Calvin Benson Bassham pathway to convert carbon dioxide into an organic compound essential for organisms, such as glucose. In one embodiment, the mutant chemoautotrophic strain used in the method of the present invention is a strain that uses NiFeSe-hydrogenase to convert hydrogen (H2) into cellular energy equivalents. In one embodiment, the mutant chemoautotrophic strain used in the method of the present invention uses NAD + reducing hydrogenase. In one embodiment, the mutant chemoautotrophic strain used in the method of the present invention is capable of nitrogen fixation.

[0032] In another embodiment, the mutant chemoautotrophic bacterial strain used in the method of the present invention comprises the 16S ribosomal RNA shown in SEQ ID NO: 1, or a 16S ribosomal RNA having a difference of up to 20, such as 1 to 10, 1 to 5, etc., for example 1, 2, or 3 nucleotides from SEQ ID NO: 1. In another embodiment, the mutant chemoautotrophic bacterial strain used in the method of the present invention contains a gene encoding a ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) large chain having the sequence shown in SEQ ID NO: 3, or a sequence having a sequence identity of more than 93%, for example more than 95%, more than 96%, for example more than 97%, more than 98%, for example more than 99% with the sequence shown in SEQ ID NO: 3. In another embodiment, the mutant chemoautotrophic bacterial strain used in the method of the present invention contains a gene encoding a ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) small chain having the sequence shown in SEQ ID NO: 5, or a sequence having a sequence identity of more than 83%, for example more than 86%, more than 90%, for example more than 95%, more than 96%, for example more than 97%, more than 98%, for example more than 99% with the sequence shown in SEQ ID NO: 5.

[0033] SEQ ID NO: 2: Nucleotide sequence of ribulose bisphosphate carboxylase large chain

[0034] Accession No. 3: Amino acid sequence of the large chain of ribulose bisphosphate carboxylase MGAEATVGQITDAKKRYAAGVLKYAQMGYWNGDYVPKDTDLLAVFRITPQAGVDPVEAAAAVAGESSTATWTVVWTDRLTAADVYRAKAYKVEPVPGQEGQYFCYIAYDLDLFEEGSIANLTASIIGNVFSFKPLKAARLEDMRLPVAYVKTFRGPPTGIVVERERLDKFGRPLLGATTKPKLGLSGKNYGRVVYEALKGGLDFVKDDENINSQPFMHWRDRFLYCMEAVNKAQAETGEVKGHYLNITAGTMEEMYRRAEFAKELGSVVVMVDLIIGWTAIQSMSNWCRENDMILHMHRAGHGTYTRQKSHGVSFRVIAKWLRLAGVDHLHTGTAVGKLEGDPMTVQGFYNVCRETTTQQDLTRGLFFEQDWGGIRKVMPVASGGIHAGQMHQLIDLFGEDVVLQFGGGTIGHPDGIQAGATANRVALETMILARNEGRDIRNEGPEILVEAAKWCRPLRAALDTWGEVTFNYASTDTSDYVPTASVA

[0035] Accession No. 4: Nucleotide sequence of the small chain of ribulose bisphosphate carboxylase: ATGCGCATCACCCAAGGCTCCTTCTCCTTCCTGCCGGACCTCACCGACACGCAGATCAAGGCCCAGGTGCAATATTGCCTGGACCAGGGCTGGGCGGTCTCGGTGGAGCACACCGACGATCCCCACCCGCGCAACACCTATTGGGAGATGTGGGGCCCGCCCATGTTCGATCTGCGCGACGCGGCCGGCGTCTTCGGCGAGATCGAAGCCTGCCGGGCCGCCAATCCCGAGCATTATGTGCGGGTGAACGCCTTCGATTCCAGCCGCGGATGGGAGACGATCCGCCTGTCCTTCATCGTTCAGCGGCCCACCGTGGAAGAGGGCTTCCGCCTCGACCGCACCGAAGGCAAGGGCCGCAACCAGAGCTACGCCATGCGCTACCGGGCGCAGTTCGCGCCGCGCTGA

[0036] SEQ ID NO: 5: Amino acid sequence of the small chain of ribulose bisphosphate carboxylase: MRITQGSFSFLPDLTDTQIKAQVQYCLDQGWAVSVEHTDDPHPRNTYWEMWGPPMFDLRDAAGVFGEIEACRAANPEHYVRVNAFDSSRGWETIRLSFIVQRPTVEEGFRLDRTEGKGRNQSYAMRYRAQFAPR

[0037] In another embodiment, the mutant chemoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 7, or a sequence identity exceeding 70% with the sequence shown in SEQ ID NO: 7, exceeding 80% identity, etc., for example, exceeding 90% identity, exceeding 95% identity, etc., for example, exceeding 96% identity, exceeding 97% identity, etc., for example, exceeding 98% sequence identity, exceeding 99% sequence identity, etc. and has NAD + It contains a gene encoding the reduced hydrogenase HoxS subunit alpha. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 9, or a sequence having a sequence identity of more than 77%, more than 80% identity, etc., such as more than 90% identity, more than 95% identity, etc., for example, more than 96% identity, more than 97% identity, etc., such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 9 and has NAD + It contains a gene encoding the NAD-reducing hydrogenase HoxS subunit beta. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 11, or a sequence having a sequence identity of more than 70%, more than 80% identity, etc., such as more than 90% identity, more than 95% identity, etc., for example, more than 96% identity, more than 97% identity, etc., such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 11 and has NAD + It contains a gene encoding the NAD-reducing hydrogenase HoxS subunit gamma. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 13, or a sequence having a sequence identity of more than 79%, more than 80% identity, etc., such as more than 90% identity, more than 95% identity, etc., for example, more than 96% identity, more than 97% identity, etc., such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 13 and has NAD + It contains a gene encoding the NAD-reducing hydrogenase HoxS subunit delta.

[0038] SEQ ID NO: 6: NAD + Nucleotide sequence of the NAD-reducing hydrogenase HoxS subunit alpha:

[0039] Accession number 7: NAD + Amino acid sequence of the reduced hydrogenase HoxS subunit alpha: MMPSEPHGAGMPPPREAAAVPTPQEVSAVVAEVVADAVASVGGARTRLMDIVQLAQQRLGHLSEETMAAIAARLAIPPVEVADMVSFYAFLNRAPKGRYHIRLSRSPISLMKGAEAVAAAFCQILGIAMGETSQDGDFTLEWTNDIGMADQEPAALVNGTVMTQLAPGDAAIIVGRLRAHHAPNALPLFPGAGVAGSGLPHARIRPSLVMPGQLLFREDHTTPGAGIKAALALTPDEVVQKVSAARLRGRGGAGFPTGLKWKLCRQSPATTRHVICNADEGEPGTFKDRVLLTQAPHLMFDGMTIAGYALGAREGVVYLRGEYAYLWEPLHAVLRERYGLGLAGANILGHAGFDFDIRIQLGAGAYICGEESALVESLEGKRGSPRDRPPFPTVRGHLQQPTAVDNVETFACAARILEDGVEAFAGIGTPESAGTKLLSVSGDCPRPGVYEVPFGLTVNALLDLVGAPDAAFVQMGGPSGQCVAPKDYGRRIAFEDLPTGGSVMVFGPGRDVLAMVREFADFFAGESCGWCTPCRVGTTLLKEELDKLLANRATLADIRALETLATTVSRTSRCGLGQTAPNPILSTMRNLPEAYEARLRPEDFLPWASLDEALKPAIVIQGRAPVPEEEA

[0040] Accession number 8: NAD + Nucleotide sequence of the reduced hydrogenase HoxS subunit beta:

[0041] Accession number 9: NAD + Amino acid sequence of the reduced hydrogenase HoxS subunit beta: MSRGSPDAGKDRTMSATDGTTAPRKIVIDPVTRVEGHGKVTIRLDEAGAVEDARFHIVEFRGFERFIQGRMYWEVPLIIQRLCGICPVSHHLAAAKAMDQVAGVDRVPPTAEKLRRLMHYGQVLQSNALHIFHLASPDLLFGFDAPAEQRNIIAVLQRYPEIGKWAIFIRKFGQEVIKATGGRKIHPTSAIPGGVNQNLAVEDRDALRAKVGEIISWCMAALDHHKAYVAENRALHDSFAAFPSAFMSLVGPDGGMDLYDGTLRVIDAEGAPLIEGAPPASYRDHLIEEVRPWSYLKFPHLRAFGRDDGWYRVGPLAQVNCAASIDTPRAEAARRDFMAEGGGKPVHATLAYHWARLIVLVHCAEKIEQLLFDDDLQGCDLRAEGTRRGEGVAWIEAPRGTLIHHYEVDENDQVRRANLIVSTTHNNEAMNRAVRQVAKTDLSGREITEGLLNHIEVAIRAFDPCLSCATHALGQMPLIVTLEDASGAEIARGVKE

[0042] Accession number 10: NAD + Nucleotide sequence of the reduced hydrogenase HoxS subunit gamma: ATGAGCGAGACCCCCTTCACCTTTACCGTGGACGGCATCGCGGTCCCGGCCACCCCCGGCCAGAGCGTCATCGAGGCGTGCGATGCGGCGGGCATCTATATCCCGCGCCTGTGCCACCACCCGGACCTGCCGCCGGCGGGCCATTGCCGGGTGTGCACCTGCATCATCGACGGGCGGCCGGCCAGCGCCTGCACCATGCCCGCCGCCAGGGGCATGGTGGTGGAGAACGAGACGCCCGCTTTGCTGGCGGAGCGGCGCACGCTGATCGAGATGCTGTTCGCGGAAGGCAACCATTTCTGCCAGTTCTGCGAGGCGAGCGGCGATTGCGAATTGCAGGCGCTGGGCTACCTGTTCGGCATGGTGGCCCCGCCCTTCCCCCATCTGTGGCCGAAGCGGCCGGTGGATGCCAGCCATCCGGATATCTATATCGACCACAATCGCTGCATCCTGTGCTCGCGCTGCGTGCGCGCCTCGCGCACCCTGGACGGCAAGTCCGTGTTCGGCTTCGAGGGGCGCGGCATCGAGATGCATCTGGCGGTGACCGGCGGGCACCTGGACGACAGCGCCATCGCCGCCGCCGACAGGGCGGTTGAGATGTGCCCGGTGGGCTGCATCGTCCTCAAGCGCACCGGCTACCGCACGCCCTATGGCCGGCGGCGCTACGACGCCGCGCCCATCGGCTCCGACATCACCGCCCGGCGCGGCGGCGCGAAGGACTGA

[0043] Sequence number 11: NAD + Amino acid sequence of the reduced hydrogenase HoxS subunit gamma: MSETPFTFTVDGIAVPATPGQSVIEACDAAGIYIPRLCHHPDLPPAGHCRVCTCIIDGRPASACTMPAARGMVVENETPALLAERRTLIEMLFAEGNHFCQFCEASGDCELQALGYLFGMVAPPFPHLWPKRPVDASHPDIYIDHNRCILCSRCVRASRTLDGKSVFGFEGRGIEMHLAVTGGHLDDSAIAAADRAVEMCPVGCIVLKRTGYRTPYGRRRYDAAPIGSDITARRGGAKD

[0044] Accession number 12: NAD + Nucleotide sequence of reduced hydrogenase HoxS subunit delta: ATGGCCAAGCCCAAACTCGCCACCTGCGCGCTGGCCGGCTGCTTCGGCTGCCACATGTCCTTCCTGGACATGGACGAGCGCATCGTCGAGCTCATCGACCTGGTGGACCTCGACGTCTCGCCCCTCGACGACAAGAAAAACTTCACCGGCATGGTGGAAATCGGCCTGGTGGAAGGCGGCTGCGCCGACGAGCGCCATGTGAAGGTGCTGCGCGAGTTCCGCGAGAAATCCCGCATCCTGGTGGCGGTGGGCGCCTGCGCCATCACCGGCGGCATCCCGGCATTGCGCAACCTCGCCGGCCTCGACGAATGCCTGAGGGAAGCCTACCTCACCGGCCCCACGGTGGAAGGCGGCGGGCTCATTCCCAACGACCCGGAGCTGCCGCTGCTGCTGGACAAGGTCTATCCGGTGCAGGACTTCGTGAAGATCGACCATTTCCTGCCCGGCTGCCCGCCCTCGGCCGACGCCATCTGGGCGGCTCTGAAGGCGCTGCTGACCGGCACCGAGCCGCATCTGCCCTACCCGCTTTTCAAGTACGAATGA

[0045] Accession number 13: NAD + Amino acid sequence of reduced hydrogenase HoxS subunit delta: MAKPKLATCALAGCFGCHMSFLDMDERIVELIDLVDLDVSPLDDKKNFTGMVEIGLVEGGCADERHVKVLREFREKSRILVAVGACAITGGIPALRNLAGLDECLREAYLTGPTVEGGGLIPNDPELPLLLDKVYPVQDFVKIDHFLPGCPPSADAIWAALKALLTGTEPHLPYPLFKYE

[0046] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 15, or a sequence identity exceeding 84% with the sequence shown in SEQ ID NO: 15, such as an identity exceeding 90%, an identity exceeding 95%, etc., such as an identity exceeding 96%, an identity exceeding 97%, etc., such as an identity exceeding 98%, an identity exceeding 99%, etc., and includes a gene encoding the NiFeSe hydrogenase large subunit having such a sequence. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 17, or a sequence identity exceeding 90% with the sequence shown in SEQ ID NO: 17, an identity exceeding 95%, etc., such as an identity exceeding 96%, an identity exceeding 97%, etc., such as an identity exceeding 98%, an identity exceeding 99%, etc., and includes a gene encoding the NiFeSe hydrogenase small subunit having such a sequence.

[0047] SEQ ID NO: 14: Nucleotide sequence of the periplasmic [NiFeSe] hydrogenase large subunit:

[0048] Accession number 15: Amino acid sequence of the large subunit of periplasmic [NiFeSe] hydrogenase: MSAETRRLVVGPFNRVEGDLEVRLDVQDGRVQQAFVSSPLFRGFERILEGRDPRDALVIAPRICGICSVSQSHAAALALAGLQGIAPTHDGRIATNLIVAAENVADHLTHFHVFFMPDFARAVYEDRPWFAQAARRFKANQGVSVRRALQTRATLLHVLGTLAGRWPHTLALQPGGVTRSADQHDRMRLLATLKAVRAALEETLFGAPLEEVAALDGAAAVEAWRANGPEGDFRLFLEIAADLELDRLGRAHDRFLSFGAYAQDEGRLYGAGTFEAGTAGGLDPNAITEDHAFARMEDRAAPHAPFDGSTFPDADDTEGYTWCKAPRLAGLPFETGAFARQVVAGHPLARDLVTREGGTVRSRVVGRLLETARTLIAMEGWVKELRPEGPWCAQGHLPQEGRAFGLTEAARGALGHWMVVEKGRIARYQIIAPTTWNFSPRDGAGLPGPLETALVGAPVRQGETTPVSVQHIVRSFDPCMVCTVH

[0049] Accession number 16: Nucleotide sequence of the small subunit of periplasmic [NiFeSe] hydrogenase ACGGGGGAGGAAGCCCGCGCCATCTTCGACGCCATCCTTGCCGGCGTTATCGTCCTCGACGCCCTGTGCGTGGAAGGCGCGCTGCTGCGCGGGCCGAACGGCACCGGGCGCTTCCATGTGCTGGCGGGCACGGACACCCCCACCATCGACTGGGCGCGGCAGCTCGCCGGCATGGCGCGCCACGTGGTGGCGGTGGGCACCTGCGCCGCCTATGGGGGCGTGACGGCGGCGGGCATCAACCCCACCGATGCCTGCGGCCTCCAGTTCGACGGACGCCGGAAGGGTGGGGCGCTGGGGGCGGACTTCCGCTCCCGCTCGGGGCTTCCGGTCATCAATGTGGCCGGCTGCCCCACCCATCCCAACTGGGTGACGGAAACCCTGATGCTGCTCGCCTGCGGCCTGCTGGGCGAGGCCGACCTCGACGTCTATGGCCGCCCGCGCTTCTATGCGGACCTGCTGGTGCATCACGGCTGCCCGCGCAACGAATACTATGAATACAAGGCGAGCGCCGAGAAGATGAGCGACCTCGGCTGCATGATGGAGCATCTGGGCTGCCTCGGCACCCAGGCCCACGCCGACTGCAACACGCGCCTTTGGAATGGCGAGGGCTCGTGCACCCGCGGCGGCTATGCCTGCATCAACTGCACGGCGCCGGAATTCGAGGAGCCGGGCCACGCCTTCCTGGAGACGCCCAAGATCGGCGGCATCCCCATCGGCCTGCCCACCGACATGCCCAAGGCCTGGTTCATCGCCTTGTCCTCCCTCGCCAAGGCGGCGACGCCGGAGCGGCTGCGCAAGAACGCGGTGTCCGACCATGTGGTCACGCCGCCCGCCGTCAAGGACATCAAGCGGCGATGA

[0050] Sequence number 17: Amino acid sequence of the small subunit of periplasmic [NiFeSe] hydrogenase MSTPFSVLWLQSGGCGGCTMSLLCAEAPDLATTLDAAGIGFLWHPALSEETGEEARAIFDAILAGVIVLDALCVEGALLRGPNGTGRFHVLAGTDTPTIDWARQLAGMARHVVAVGTCAAYGGVTAAGINPTDACGLQFDGRRKGGALGADFRSRSGLPVINVAGCPTHPNWVTETLMLLACGLLGEADLDVYGRPRFYADLLVHHGCPRNEYYEYKASAEKMSDLGCMMEHLGCLGTQAHADCNTRLWNGEGSCTRGGYACINCTAPEFEEPGHAFLETPKIGGIPIGLPTDMPKAWFIALSSLAKAATPERLRKNAVSDHVVTPPAVKDIKRR

[0051] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase gamma chain atpG_1 having a sequence shown in SEQ ID NO: 19, or a sequence having an identity of more than 70%, more than 80%, such as more than 90%, more than 95%, such as more than 96%, more than 97%, such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 19.

[0052] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase subunit alpha atpA_1 having a sequence shown in SEQ ID NO: 21, or a sequence having an identity of more than 78%, more than 80%, such as more than 90%, more than 95%, such as more than 96%, more than 97%, such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 21.

[0053] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit b atpF_1 having the sequence shown in SEQ ID NO: 23, or a sequence having an identity of more than 62%, for example more than 70%, more than 80%, etc., for example more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 23.

[0054] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit c, sodium ion-specific atpE_1 having the sequence shown in SEQ ID NO: 25, or a sequence having an identity of more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 25.

[0055] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit a atpB_1 having the sequence shown in SEQ ID NO: 27, or a sequence having an identity of more than 80%, for example more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 27.

[0056] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase epsilon chain atpC_1 having the sequence shown in SEQ ID NO: 29, or a sequence having an identity of more than 71%, more than 80%, etc., for example more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 29.

[0057] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase subunit beta atpD_1 having a sequence shown in SEQ ID NO: 31, or a sequence having an identity of more than 84%, such as more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 31.

[0058] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase subunit beta atpD_2 having a sequence shown in SEQ ID NO: 33, or a sequence having an identity of more than 97%, such as more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 33.

[0059] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase gamma chain atpG_2 having a sequence shown in SEQ ID NO: 35, or a sequence having an identity of more than 86%, such as more than 90%, more than 95%, etc., for example more than 96%, more than 97%, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 35.

[0060] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase subunit alpha atpA_2 having a sequence shown in SEQ ID NO: 37, or a sequence having an identity of more than 98%, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 37.

[0061] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit delta atpH having the sequence shown in SEQ ID NO: 39, or a sequence having more than 85% identity, such as more than 90% identity, more than 95% identity, etc., for example more than 96% identity, more than 97% identity, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 39.

[0062] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit b atpF_2 having the sequence shown in SEQ ID NO: 41, or a sequence having more than 87% identity, such as more than 90% identity, more than 95% identity, etc., for example more than 96% identity, more than 97% identity, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 41.

[0063] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit b' atpG_3 having the sequence shown in SEQ ID NO: 43, or a sequence having more than 81% identity, such as more than 90% identity, more than 95% identity, etc., for example more than 96% identity, more than 97% identity, etc., for example more than 98% sequence identity, more than 99% sequence identity, etc., with the sequence shown in SEQ ID NO: 43.

[0064] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention contains a gene encoding ATP synthase subunit c atpE_2 having the sequence shown in SEQ ID NO: 45, or a sequence having more than 98% sequence identity, more than 99% sequence identity, etc.

[0065] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase subunit a atpB_2 having the sequence shown in SEQ ID NO: 47, or a sequence having an identity of more than 92%, more than 95%, such as more than 96%, more than 97%, such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 47.

[0066] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention comprises a gene encoding ATP synthase protein I atpI having the sequence shown in SEQ ID NO: 49, or a sequence having an identity of more than 60%, such as more than 70%, more than 80%, such as more than 90%, more than 95%, such as more than 96%, more than 97%, such as more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 49.

[0067] SEQ ID NO: 18: Nucleotide sequence of ATP synthase gamma chain atpG_1 GTGACCGAGCGCCTGTCCGACGTCAACGCCCGCATCGCCTCGGTGCGGCAGCTCTCATCGGTCATCACGGCCATGCGGGGCATTGCGGCGGCGCGGGCGCGGGAGGCGCGGGGTCGGCTCGACGGCATCCGCGCCTATGCGCAGACCATCGCCGAGGCCATCGGCCATGTGCTCGCCGTGCTGCCCGAGGAGGCCCGCGCCCGGTCCTCCGGGCACCGGCATCGGGGCCATGCGGTCATCGCCCTGTGCGCGGAGCAGGGCTTTGCCGGCGTCTTCAACGAGCGGGTGCTGGACGAGGCCGCCCGGCTGCTGACCGGCGGGGCGGGGCCGGCCGAGCTGCTGCTGGTGGGCGACCGGGGCCTGATGGTGGCCCGCGAGCGGGGGCTCGATGTCTCCTGGTCGGTGCCCATGGTGGCCCATGCGGGCCAGGCCTCGGCGCTGGCGGACCGCATCAGCGAGGAGCTCTACCGGCGGATCGATGCGGGACGGGTGACGCGGGTGTCGGTGGTGCACGCCGAGCCCGCCGCGTCCGCCGCCATCGAGACGGTGGTGAAAGTGCTGGTGCCGTTCGACTTCGCCCGCTTCCCCCTGGCGCGGGTGGCATCCGCCCCGCTCATGACCATGCCGCCGCCGCGGCTGCTGGCCCAGCTGTCGGAGGAATATGTGTTCGCCGAGCTGTGCGAGGCGCTCACCTTGTCCTTCGCGGCGGAGAACGAGGCCCGCATGCGGGCCATGATCGCCGCCCGCGCCAATGTGGCCGATACCCTGGAGGGCCTCGTCGGCCGCGCCCGGCAGATGCGCCAGGAGGAGATCACCAACGAGATCATCGAGCTGGAAGGCGGCGCCGGCAGCGCCCGGCATGCGGATTGA

[0068] SEQ ID NO: 19: Amino acid sequence of the gamma chain of ATP synthase atpG_1 MTERLSDVNARIASVRQLSSVITAMRGIAAARAREARGRLDGIRAYAQTIAEAIGHVLAVLPEEARARSSGHRHRGHAVIALCAEQGFAGVFNERVLDEAARLLTGGAGPAELLLVGDRGLMVARERGLDVSWSVPMVAHAGQASALADRISEELYRRIDAGRVTRVSVVHAEPAASAAIETVVKVLVPFDFARFPLARVASAPLMTMPPPRLLAQLSEEYVFAELCEALTLSFAAENEARMRAMIAARANVADTLEGLVGRARQMRQEEITNEIIELEGGAGSARHAD

[0069] Sequence number 20: Nucleotide sequence of ATP synthase subunit alpha atpA_1

[0070] SEQ ID NO: 21: Amino acid sequence of ATP synthase subunit alpha atpA_1 MSTGAQASEDWLTRSRAALAGTRLSQQSQSVGRVEEMADGIARVSGLPDVRLDELLTFEGGQTGYALTLDRTEIAVVLLDDASGVEAGARVFGTGEVVKVPVGPGLLGRIVDPLGRPMDRSEPVVAQAHHPIERPAPAIIARDLVSQPVQTGTLVVDALFSLGRGQRELIIGDRATGKTAIAVDTIISQKHSDIVCIYVAVGQRAAAVERVVEAVRAHGAIERCIFVVASAAASPGLQWIAPFAGMTMAEYFRDNGQHALIIIDDLTKHAATHRELALLTHEPPGREAYPGDIFYVHARLLERAAKLSAELGGGSLTALPIAETDAGNLSAYIPTNLISITDGQIVLDSRLFAANQRPAVDVGLSVSRVGGKAQHPALRAVSGRIRLDYSQFLELEMFTRFGGITDTRVKAQITRGERIRALLTQPRFSTLRLQDEVALLAALAEGVFDTLAPGLMGAVRARIPAQLDAQVKDVASALAEGKVLEEGLHARLVAAVRAVAADVAATAKAGP

[0071] SEQ ID NO: 22: Nucleotide sequence of ATP synthase subunit b atpF_1 ATGCAGATCGACTGGTGGACGCTGGGCCTGCAGACGGTCAACGTCCTCGTTCTCATCTGGCTCCTGAGCCGCTTCCTGTTCAAGCCGGTGGCGCAGGTCATCGCGCAGCGCCGTGCCGAGATCGAGAAGCTGGTGGAGGATGCGCGCGCCGCCAAGGCCGCCGCCGAGGCCGAGCGGGACACGGCGAAGGCGGAGGAGGCGCGCCTTGCCGCCGAGCGCGGCGCCCGCATGGCGGCGGTCGCCAAGGAGGCGGAGGCGCAGAAGGCGGCATTGCTGGCCGCCGCCAAGACCGAGGCCGAGGCCCTGCACGCGGCCGCGGAAGCGGCCATCGTCCGGGCGCGGGCGAGCGAGGAGGAAGCCGCCGCCGACCGCGCCAGCCGCCTTGCCGTGGACATCGCCGCCAAGCTGCTGGACCGGCTGCCCGACGACGCCCGGGTCGCGGGCTTCATCGATGGCCTCGCCGAGGGGCTTGAAGCCCTGCCCGAGGCGAGCCGGGCGGTGATCGGCGTCGACGGCGCGCCAGTGCGCGTGACGGCCGCGCGCGCCCTTATGCCGGCGGAGGAGGAGGCCTGCCGCACGCGGCTCTCCCAGGCGCTGGGCCGTCCGGTGACGCTGGCCGTGACCATCGACCCCGCCCTCATCGCCGGCCTGGAGATGGAGACGCCCCACGCGGTGGTGCGCAATTCCTTCAAGGCCGATCTCGACCGCGTCACCGCGGCGCTCACCCATCATGGGACCTGA

[0072] SEQ ID NO: 23: Amino acid sequence of ATP synthase subunit b atpF_1 MQIDWWTLGLQTVNVLVLIWLLSRFLFKPVAQVIAQRRAEIEKLVEDARAAKAAAEAERDTAKAEEARLAAERGARMAAVAKEAEAQKAALLAAAKTEAEALHAAAEAAIVRARASEEEAAADRASRLAVDIAAKLLDRLPDDARVAGFIDGLAEGLEALPEASRAVIGVDGAPVRVTAARALMPAEEEACRTRLSQALGRPVTLAVTIDPALIAGLEMETPHAVVRNSFKADLDRVTAALTHHGT

[0073] Sequence number 24: Nucleotide sequence of ATP synthase subunit c, sodium ion-specific atpE_1 ATGACTGTCGAGATGGTCAGCATCTTCGCGGCGGCGCTCGCCGTCTCCTTCGGCGCCATCGGGCCGGCCCTGGGCGAGGGCCGGGCGGTGGCCGCGGCCATGGACGCCATCGCCCGCCAGCCGGAGGCGGCCGGAACCTTGTCGCGCACGCTCTTCGTCGGCCTCGCCATGATCGAGACCATGGCGATCTACTGCCTGGTGATCGCGCTCCTGGTGCTCTTCGCCAATCCGTTCGTGAAGTGA

[0074] Sequence number 25: Amino acid sequence of ATP synthase subunit c, sodium ion-specific atpE_1 MTVEMVSIFAAALAVSFGAIGPALGEGRAVAAAMDAIARQPEAAGTLSRTLFVGLAMIETMAIYCLVIALLVLFANPFVK

[0075] Sequence number 26: Nucleotide sequence of ATP synthase subunit a atpB_1 ATGGGCTCGCCGCTGATCCTCGAACCCCTGTTCCATATCGGGCCCGTGCCCATCACCGCGCCGGTGGTGGTCACCTGGCTCATCATGGCCGCCTTCATTGGGCTGGCGCGGCTCATCACCCGGAAGCTTTCCACCGATCCCACCCGGACCCAGGCGGCGGTGGAAACGGTGCTGACCGCCATCGATTCCCAGATCGCCGACACCATGCAGGCCGATCCCGCGCCTTATCGCGCGCTCATCGGCACCATCTTCCTTTATGTGCTGGTGGCCAACTGGTCCTCGCTCATCCCGGGCATCGAGCCGCCCACGGCGCATATCGAGACCGATGCGGCGCTCGCTTTCATCGTGTTCGCCGCCACCATCGGGTTCGGGTTGAAGACAAGGGGTGTGAAGGGCTATCTCGCCACCTTCGCCGAACCCTCCTGGGTGATGATCCCGCTCAATGTGGTGGAGCAGATCACCCGGACCTTCTCGCTCATCGTGCGCCTGTTCGGCAACATCATGAGCGGGGTGTTCGTGGTCGGCATCATCCTGTCCCTCGCCGGGCTGCTGGTGCCCATCCCCCTCATGGCGCTCGATCTCCTGACCGGCGCCGTGCAGGCCTACATCTTCGCGGTGCTGGCCTGCGTGTTCATCGGCGCGGCCATTGGCGAGGCGCCGGCAAAGCCCCAATCGAAGGAGCCAGGGAAAACATCATGA

[0076] Sequence number 27: Amino acid sequence of ATP synthase subunit a atpB_1 MGSPLILEPLFHIGPVPITAPVVVTWLIMAAFIGLARLITRKLSTDPTRTQAAVETVLTAIDSQIADTMQADPAPYRALIGTIFLYVLVANWSSLIPGIEPPTAHIETDAALAFIVFAATIGFGLKTRGVKGYLATFAEPSWVMIPLNVVEQITRTFSLIVRLFGNIMSGVFVVGIILSLAGLLVPIPLMALDLLTGAVQAYIFAVLACVFIGAAIGEAPAKPQSKEPGKTS

[0077] Sequence number 28: Nucleotide sequence of the epsilon chain of ATP synthase atpC_1 GTGAGCGCGCCGCTGCACCTCACCATCACCACGCCGGCCGCCGTTCTGGTGGACCGTGCCGACATCGTGGCCCTGCGTGCCGAGGACGAGAGCGGCAGCTTCGGCATCCTGCCCGGCCATGCGGATTTCCTGACCGTTCTGGAGGCCTGCGTGGTGCGCTTCAAGGATGGGGCCGACGGCGTGCATTATTGTGCTCTCAGTGGTGGCGTGCTGTCGGTCGAGGAGGGCCGGCGCATCGCCATCGCCTGCCGTCAGGGCACGGTGAGCGACGACCTGGTCGCCCTGGAAGGGGCGGTGGACGCCATGCGTTCGGCGGAGAGCGATGCCGACAAGCGGGCCCGGGTGGAGCAGATGCGCCTTCATGCCCACGCCGTGCGCCAGCTCCTGCACTATCTGCGGCCCGGCCGGGCCGGCGGCGTGGCGCCGGCCGCCGCGCCGGAGGAGGGGCCGTCATGA

[0078] Sequence number 29: Amino acid sequence of the epsilon chain of ATP synthase atpC_1 MSAPLHLTITTPAAVLVDRADIVALRAEDESGSFGILPGHADFLTVLEACVVRFKDGADGVHYCALSGGVLSVEEGRRIAIACRQGTVSDDLVALEGAVDAMRSAESDADKRARVEQMRLHAHAVRQLLHYLRPGRAGGVAPAAAPEEGPS

[0079] Accession No. 30: Nucleotide sequence of ATP synthase subunit beta atpD_1

[0080] SEQ ID NO: 31: Amino acid sequence of ATP synthase subunit beta atpD_1 MAAADEEAQSAAGPASGRVVAVRGAVIDIAFAQPPLPPLDDALLITDGRGGTVLVEVQSHMDRHTVRAIALQATTGLSRGLEAARVGGPVKVPVGDHVLGRLLDVTGAIGDKGGPLPADVPTRPIHHAPPSFAAQGGTSDLFRTGIKVIDLLAPLAQGGKAAMFGGAGVGKTVLVMELIHAMVASYKGISVFAGVGERSREGHEMLLDMTDSGVLDRTVLVYGQMNEPPGARWRVPMTALTIAEYFRDEKHQNVLLLMDNIFRFVQAGAEVSGLLGRPPSRVGYQPTLASEVAALQERITSVGEASVTAIEAVYVPADDFTDPAVTTIAAHVDSMVVLSRAMAAEGMYPAVDPISSSSVLLDPLIVGDEHARVANEVRRTIEHYRELQDVISLLGMEELGTEDRRIVERARRLQRFLTQPFTVTEAFTGVPGRSVAIADTIAGCRMILSGACDDWQESALYMVGTIDEARQKEEAARAKAGQGAPAGTAAETAEAAP

[0081] SEQ ID NO: 32: Nucleotide sequence of ATP synthase subunit beta atpD_2

[0082] Sequence number 33: Amino acid sequence of ATP synthase subunit beta atpD_2 MANKVGRITQIIGAVVDVQFDGHLPAILNAIETTNQGNRLVLEVAQHLGENTVRCIAMDATEGLVRGQEVADTDAPIQVPVGAATLGRIMNVIGEPVDELGPIEGEALRGIHQPAPSYAEQATEAEILVTGIKVVDLLAPYSKGGKVGLFGGAGVGKTVLIMELINNVAKAHGGYSVFAGVGERTREGNDLYHEMIESNVNKDPHENNGSAAGSKCALVYGQMNEPPGARARVALTGLTVAEHFRDQGQDVLFFVDNIFRFTQAGSEVSALLGRIPSAVGYQPTLATDMGQLQERITTTTKGSITSVQAIYVPADDLTDPAPAASFAHLDATTVLSRSIAEKGIYPAVDPLDSTSRMLSPAILGDEHYNTARQVQQTLQRYKALQDIIAILGMDELSEEDKLTVARARKIERFLSQPFHVAEVFTGSPGKLVDLADTIKGFKGLVDGKYDYLPEQAFYMVGTIEEAIEKGKKLAAEAA

[0083] Sequence number 34: Nucleotide sequence of ATP synthase gamma chain atpG_2 ATGGCGAGTCTGAAGGACCTGAGAAACCGCATTGCCTCGGTGAAGGCGACGCAGAAGATCACCAAGGCGATGCAGATGGTCGCCGCGGCGAAGCTGCGTCGCGCCCAGGCGGCGGCTGAAGCGGCCCGTCCCTATGCGGAACGCATGGAGACGGTGCTCGGAAATCTTGCCTCCGGCATGGTGGTGGGCGCGCAGGCGCCTGTTCTCATGACCGGGACGGGCAAGAGCGACACCCACCTGCTGCTGGTGTGCACCGGCGAGCGCGGCCTGTGCGGCGCCTTCAACTCGTCCATCGTGCGCTTCGCCCGCGAGCGGGCGCAGCTGCTGCTGGCCGAGGGCAAGAAGGTGAAAATCCTGTGCGTGGGCCGCAAGGGCCACGAGCAGCTGCGCCGCATCTACCCGGACAACATCATCGACGTGGTGGACCTGCGCGCGGTGCGCAACATCGGCTTCAAGGAGGCCGACGCCATCGCCCGCAAGGTGCTGGCCCTGCTCGATGAAGGCGCATTCGACGTCTGCACGCTCTTCTACTCCCACTTCAGGAGCGTGATCGCCCAGGTGCCGACGGCCCAGCAGCTCATTCCGGCCACCTTCGACGAGCGGCCGGCCGTCGCCGATGCGCCGGTCTATGAATATGAGCCGGAGGAGGAGGAGATCCTCGCCGAGCTGCTGCCGCGCAACGTGGCGGTGCAGATCTTCAAGGCCCTCCTCGAGAACCAGGCTTCTTTCTATGGCTCCCAGATGAGCGCCATGGACAACGCCACGCGCAATGCGGGCGAGATGATCAAGAAGCAGACGCTCACCTACAACCGTACCCGCCAGGCCATGATCACGAAGGAACTCATCGAGATCATCTCCGGCGCCGAGGCCGTCTGA

[0084] SEQ ID NO: 35: Amino acid sequence of the gamma chain of ATP synthase atpG_2 MASLKDLRNRIASVKATQKITKAMQMVAAAKLRRAQAAAEAARPYAERMETVLGNLASGMVVGAQAPVLMTGTGKSDTHLLLVCTGERGLCGAFNSSIVRFARERAQLLLAEGKKVKILCVGRKGHEQLRRIYPDNIIDVVDLRAVRNIGFKEADAIARKVLALLDEGAFDVCTLFYSHFRSVIAQVPTAQQLIPATFDERPAVADAPVYEYEPEEEEILAELLPRNVAVQIFKALLENQASFYGSQMSAMDNATRNAGEMIKKQTLTYNRTRQAMITKELIEIISGAEAV

[0085] Sequence number 36: Nucleotide sequence of ATP synthase subunit alpha atpA_2

[0086] SEQ ID NO: 37: Amino acid sequence of ATP synthase subunit alpha atpA_2 MDIRAAEISAILKEQIQNFGQEAEVSEVGQVLSVGDGIARVYGLDNVQAGEMVEFENGTRGMALNLELDNVGIVIFGSDREIKEGQTVKRTGAIVDAPVGKGLLGRVVDALGNPIDGKGPIMFTERRRVDVKAPGIIPRKSVHEPMQTGLKAIDALIPIGRGQRELIIGDRQTGKTAVALDSILNQKPINQGDDEKAKLYCVYVAVGQKRSTVAQFVKVLEEHGALEYSIVVAATASDAAPMQFLAPFTGTAMGEYFRDNGMHALIIHDDLSKQAVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKLNDEHGAGSLTALPVIETQANDVSAYIPTNVISITDGQIFLESDLFYQGIRPAVNVGLSVSRVGSSAQIKAMKQVAGKIKGELAQYRELAAFAQFGSDLDAATQKLLNRGARLTELLKQSQFSPLKVEEQVAVIYAGTNGYLDPLPVSKVREFEQGLLLSLRSQHPEILDAIRTSKELSKDTAEKLTKAIDAFAKSFS

[0087] SEQ ID NO: 38: Nucleotide sequence of ATP synthase subunit delta pH GTGGCGGAAACGATCGTGTCAGGCATGGCGGGACGCTATGCGACCGCGCTGTTCGAGCTGGCGGACGAAGCCGGTGCCATCGATTCCGTCCAGGCGGATCTTGATCGCCTGTCCGGCCTTCTGGCCGAGAGCGCGGATCTGGCGCGGCTGGTCAAGAGCCCGGTCTTCACCGCCGAGCAGCAGCTCGGCGCGATGGCGGCCATTCTCGATCAAGCAGGCATTTCCGGCCTTGCGGGCAAATTCGTGAAGCTGGTGGCGCAGAACCGCCGCCTGTTCGCACTGCCGCGCATGATTGCCGAATACGCCGTCCTGGTGGCCCGGAAGAAGGGCGAGACCTCGGCGAGCGTGACCGTTGCCACCCCCCTGAGCGATGAGCATCTGGCCACGCTCAAGGCGGCCCTGGCTGAAAAGACCGGCAAGGACGTGAAGCTCGACGTCACCGTCGATCCGTCCATCCTCGGTGGTCTCATCGTGAAGCTCGGCTCGCGCATGGTCGATGCTTCCCTGAAGACCAAACTCAATTCTATCCGGCATGCGATGAAAGAGGTCCGCTGA

[0088] SEQ ID NO: 39: Amino acid sequence of ATP synthase subunit delta atpH MAETIVSGMAGRYATALFELADEAGAIDSVQADLDRLSGLLAESADLARLVKSPVFTAEQQLGAMAAILDQAGISGLAGKFVKLVAQNRRLFALPRMIAEYAVLVARKKGETSASVTVATPLSDEHLATLKAALAEKTGKDVKLDVTVDPSILGGLIVKLGSRMVDASLKTKLNSIRHAMKEVR

[0089] SEQ ID NO: 40: Nucleotide sequence of ATP synthase subunit b atpF_2 ATGACCGAAATGGAACTGGCTGAGCTCTGGGTCGCCATCGCCTTCCTGGTTTTCGTAGGCCTCCTGATCTATGCGGGCGCCCACCGCGCCATCGTCTCCGCCCTGGATTCCCGCGGCTCGCGCATCGCCTCGGAACTGGAGGAGGCCCGTCGGCTCAAGGAAGAGGCCCAGAAGCTGGTGGCCGAATTCAAGCGCAAGCAGCGCGAGGCCGAGGCCGAGGCCGAATCCATCGTCACCGGCGCCAAGGCCGAGGCCGAGCGCCTCGCCGCCGAGGCCAAGGCGAAGATCGAGGATTTCGTCACCCGCCGCACCAAGATGGCCGAGGACAAGATCGCCCAGGCCGAGCATCAGGCTCTGGCGGACGTGAAGTCCATCGCCGCCGAGGCGGCGGCCAAGGCGGCCGAGGTGATCCTCGGCGCCCAGGCCACCGGCGCGGTGGCGGAGCGTCTGCTGTCGGGCGCCATCTCCGAGGTCAAGACCAAGCTCAACTGA

[0090] SEQ ID NO: 41: Amino acid sequence of ATP synthase subunit b atpF_2 MTEMELAELWVAIAFLVFVGLLIYAGAHRAIVSALDSRGSRIASELEEARRLKEEAQKLVAEFKRKQREAEAEAESIVTGAKAEAERLAAEAKAKIEDFVTRRTKMAEDKIAQAEHQALADVKSIAAEAAAKAAEVILGAQATGAVAERLLSGAISEVKTKLN

[0091] SEQ ID NO: 42: Nucleotide sequence of ATP synthase subunit b' atpG_3 ATGATGATTGCATGGAAGCGGACCTTCGCAGTCGTGACCTTCGGGGCCGCCCTGATGGCCATGCCCGTCGCGGGCGTGGTCGCAGCTGAGACTTCTCCCGCTCCGGCGGCAGTGGCGCAGGCCGATCATGCGGTGCCCACCGAGGCGGCCGGCCAGGGCACCGCCGATGCGGCCCATGCCGCCGCGCCGGGCGAGGCCGCCCATGGTGGCGCGGCCAAGCACGAAACCCATTTCCCGCCCTTCGACGGCACCACCTTCGCCTCCCAGTTGCTGTGGCTCGCCGTCACCTTCGGCCTGCTTTACTACCTCATGAGCAAGGTCACGCTGCCGCGCATCGGCCGCATCCTGGAAGAGCGCCACGACCGCATCGCCGATGATCTGGAGGAAGCCTCCAAGCATCGCGCCGAGAGCGAGGCCGCCCAGCGGGCCTATGAGAAGGCGCTGAGCGAGGCCCGCGCGAAGGCCCATTCCATCGCCGCGGAAACCCGCGACCGCCTTGCCGCCCACGCCGACACCAACCGCAAGGCGCTGGAGAGCGAGCTCACCGCCAAGCTGCAGGCGGCCGAGGAGCGCATCGCCACCACCAAGAGCGAAGCCCTCACCCATGTGCGCGGCATCGCGGTGGACGCCACCCAATCCATCGTCTCCACCCTCATCGGTGTCGCGCCCGCGGCGGCCGACGTGGAAAAAGCGGTGGACGGCGCCCTGTCCCAGCACGGCCAGGCCTGA

[0092] Sequence number 43: Amino acid sequence of ATP synthase subunit b’ atpG_3 MMIAWKRTFAVVTFGAALMAMPVAGVVAAETSPAPAAVAQADHAVPTEAAGQGTADAAHAAAPGEAAHGGAAKHETHFPPFDGTTFASQLLWLAVTFGLLYYLMSKVTLPRIGRILEERHDRIADDLEEASKHRAESEAAQRAYEKALSEARAKAHSIAAETRDRLAAHADTNRKALESELTAKLQAAEERIATTKSEALTHVRGIAVDATQSIVSTLIGVAPAAADVEKAVDGALSQHGQA

[0093] Sequence number 44: Nucleotide sequence of ATP synthase subunit c atpE_2 ATGGAAGCGGAAGCTGGAAAGTTCATCGGTGCCGGCCTCGCCTGCCTCGGCATGGGTCTCGCTGGCGTCGGCGTCGGTAACATCTTCGGTAACTTCCTCTCCGGCGCCCTGCGCAACCCGTCCGCTGCCGACGGCCAGTTCGCCCGCGCCTTCATCGGCGCCGCCCTCGCGGAAGGTCTCGGCATCTTCTCGCTGGTCGTTGCGCTCGTCCTGCTGTTCGTGGCCTGA

[0094] Sequence number 45: Amino acid sequence of ATP synthase subunit c atpE_2 MEAEAGKFIGAGLACLGMGLAGVGVGNIFGNFLSGALRNPSAADGQFARAFIGAALAEGLGIFSLVVALVLLFVA

[0095] Sequence number 46: Nucleotide sequence of ATP synthase subunit a atpB_2 SEQ ID NO: 46: ATGACCGTCGATCCGATCCACCAGTTCGAGATCAAGCGCTACGTGGATCTGCTGAACGTCGGCGGTGTCCAGTTCTCCTTCACCAACGCAACGGTGTTCATGATTGGCATCGTCCTGGTGATTTTCTTCTTCCTGACTTTCGCGACACGCGGTCGCACCCTTGTGCCGGGCCGGATGCAGTCGGCGGCGGAGCTGAGCTACGAGTTCATCGCCAAGATGGTGCGCGACGCGGCCGGCAGCGAGGGAATGGTGTTCTTTCCCTTCGTCTTCTCGCTCTTCATGTTCGTGCTGGTGGCGAACGTATTGGGGCTCATCCCCTACACCTTCACGGTGACCGCCCACCTCATCGTCACCGCCGCCCTGGCGGCGACGGTGATCCTCACCGTCATCATCTACGGCTTCGTGCGGCACGGCACCCACTTCCTGCACCTGTTCGTGCCGTCGGGCGTGCCGGGCTTCCTCCTGCCCTTCCTCGTGGTGATCGAGGTGGTGTCGTTCCTGTCGCGGCCCATCAGCCTCTCGCTGCGTCTGTTCGCCAACATGCTGGCGGGCCACATCGCCCTCAAGGTGTTCGCCTTCTTCGTCGTGGGACTGGCCTCGGCCGGCGCGATCGGCTGGTTCGGCGCCACCCTGCCCTTCTTCATGATCGTGGCGCTCACCGCGCTGGAGCTGCTGGTGGCGGTGCTGCAGGCCTACGTGTTCGCGGTGCTGACCTCGATCTACCTCAACGACGCCATCCATCCCGGCCACTGA

[0096] Sequence number 47: Amino acid sequence of ATP synthase subunit a atpB_2 MTVDPIHQFEIKRYVDLLNVGGVQFSFTNATVFMIGIVLVIFFFLTFATRGRTLVPGRMQSAAELSYEFIAKMVRDAAGSEGMVFFPFVFSLFMFVLVANVLGLIPYTFTVTAHLIVTAALAATVILTVIIYGFVRHGTHFLHLFVPSGVPGFLLPFLVVIEVVSFLSRPISLSLRLFANMLAGHIALKVFAFFVVGLASAGAIGWFGATLPFFMIVALTALELLVAVLQAYVFAVLTSIYLNDAIHPGH

[0097] Sequence number 48: Nucleotide sequence of ATP synthase protein I, atpI ATGTCCGAGCCGAATGATCCATCCCGCAGGGACGGTGCGAAGGCGAAAGACGAGACGCAGGACTCCCGGCCCGGTGAGGCGGATCTTGCTCGGCGCCTCGATGCGCTCGGCACCTCCATCGGTCAGGTCAAGTCCAGAAGCGGGGAGCCCGCGGCGACGCCGCGCAAGGACACCTCCTCGGCCTCCGGCGCGGCCCTGGCGTTTCGGCTGGGCGCCGAGTTTGTTTCAGGCGTGCTGGTGGGCTCGCTCATCGGCTACGGGTTGGATTATGCGTTTGCGATTTCGCCCTGGGGGCTGATCGCCTTCACGCTGATCGGCTTTGCCGCCGGCGTCCTGAACATGCTGCGCGTGGCGAACAGCGATGCCAAGCGCCACAGCGCGGACAGGTGA

[0098] Sequence number 49: Amino acid sequence of ATP synthase protein I, atpI MSEPNDPSRRDGAKAKDETQDSRPGEADLARRLDALGTSIGQVKSRSGEPAATPRKDTSSASGAALAFRLGAEFVSGVLVGSLIGYGLDYAFAISPWGLIAFTLIGFAAGVLNMLRVANSDAKRHSADR

[0099] In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 51, or a sequence having an identity of more than 60%, for example, more than 70%, more than 92%, etc., more than 95%, for example, more than 96%, more than 97%, etc., for example, more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 51, and contains a gene encoding nitrogenase molybdenum-iron protein alpha chain nifD_1. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 53, or a sequence having an identity of more than 60%, for example, more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 53, and contains a gene encoding nitrogenase molybdenum-iron protein alpha chain nifD_2. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 55, or a sequence having an identity of more than 87%, for example, more than 90%, etc., more than 95%, for example, more than 96%, more than 97%, etc., for example, more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 55, and contains a gene encoding nitrogenase molybdenum-iron protein beta chain nifK_1. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 57, or a sequence having an identity of more than 95%, for example, more than 96%, more than 97%, etc., for example, more than 98% sequence identity, more than 99% sequence identity, etc. with the sequence shown in SEQ ID NO: 57, and contains a gene encoding nitrogenase molybdenum-iron protein beta chain nifK_2. In another embodiment, the mutant chemolithoautotrophic bacterial strain used in the method of the present invention has a sequence shown in SEQ ID NO: 59, or a sequence having a sequence identity of more than 98.5% with the sequence shown in SEQ ID NO: 59, and contains a gene encoding nitrogenase iron protein nifH.

[0100] Accession number 50: Nucleotide sequence of the alpha chain nifD_1 of nitrogenase molybdenum-iron protein

[0101] Accession No. 51: Amino acid sequence of the alpha chain nifD_1 of molybdenum-iron protein of nitrogenase MSSLSATIQQVFNEPGCAKNQNKSEAEKKKGCTKQLQPGGAAGGCAFDGAKIALQPLTDVAHLVHGPIACEGNSWDNRGAKSSGSNIWRTGFTTDINETDVVFGGEKRLFKSIKEIIEKYDPPAVFVYQTCVPAMIGDDIDAVCKAAREKFGKPVIPINSPGFVGPKNLGNKLAGEALLDHVIGTEEPDYTTAYDINIIGEYNLSGELWQVKPLLDELGIRILACISGDGKYKDVASSHRAKAAMMVCSKAMINVARKMEERYDIPFFEGSFYGIEDSSDSLREIARMLIEKGADPELMDRTEALIEREEKKAWDAIAAYKPRFKDKKVLLITGGVKSWSVVAALQEAGLELVGTSVKKSTKEDKERIKELMGQDAHMIDDMTPREMYKMLKDAKADIMLSGGRSQFIALKAAMPWLDINQERHHAYMGYVGMVKLVEEIDKALYNPVWEQVRKPAPWENPEDTWQARALAEMEAEAAALAADPVRAEEVRRSKKICNCKSVDLGTIEDAIKAHALTTVEGVREHTNASGGCGACSGRIEEIFEAVGVVAAPPPAEAAPSPQEIAPDPLAAEEKRRAKKACGCKEVAVGTIEDAIRAKGLRNIAEVRAATDANTGCGNCQERVEGILDRVLAEAASELQAAE

[0102] Accession No. 52: Nucleotide sequence of the alpha chain nifD_2 of molybdenum-iron protein of nitrogenase

[0103] Accession No. 53: Amino acid sequence of the alpha chain nifD_2 of the molybdenum-iron protein of nitrogenase MSVAQSQSVAEIKARNKELIEEVLKVYPEKTAKRRAKHLNVHEAGKSDCGVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSWAARRNYYIGTTGIDTFVTMQFTSDFQEKDIVFGGDKKLAKIMDEIQELFPLNNGITVQSECPIGLIGDDIEAVSKQKSKEYEGKTIVPVRCEGFRGVSQSLGHHIANDAIRDWVFDKIAPDAEPRFEPTPYDVAIIGDYNIGGDAWSSRILLEEMGLRVIAQWSGDGSLAELEATPKAKLNVLHCYRSMNYISRHMEEKYGIPWCEYNFFGPSKIAESLRKIASYFDDKIKEGAERVIAKYQPLMDAVIAKYRPRLEGKTVMLYVGGLRPRHVIGAYEDLGMEVVGTGYEFAHNDDYQRTAQHYVKDGTIIYDDVTGYEFEKFVEKIQPDLVGSGIKEKYVFQKMGVPFRQMHSWDYSGPYHGYDGFAIFARDMDMAINSPVWKMTQAPWKSVPKPTMLAAE

[0104] Accession No. 54: Nucleotide sequence of the beta chain nifK_1 of the molybdenum-iron protein of nitrogenase

[0105] Accession number 55: Amino acid sequence of the beta chain nifK_1 of the molybdenum-iron protein of nitrogenase MATVSVSKKACAVNPLKMSQPVGGALAFMGVRKAMPLLHGSQGCTSFGLVLFVRHFKEAIPMQTTAMSEVATVLGGLENVEQAILNIYNRTKPEIIGICSTGVTETKGDDVDGYIKLIRDKYPQLADFPLVYVSTPDFKDAFQDGWEKTVAKMVEALVKPAADKQKDKTRVNVLPGCHLTPGDLDEMRTIFEDFGLTPYFLPDLAGSLDGHIPEDFSPTTIGGIGIDEIATMGEAAHTICIGAQMRRAGEAMEKKTGIPFKLFERLCGLEANDAFIMHLSQISGRPVPVKYRRQRGQLVDAMLDGHFHLGGRKVAMGAEPDLLYDVGSFLHEMGAHILSAVTTTQSPVLARLPAEEVLIGDLEDLETQAKARGCDLLLTHSHGRQAAERLHIPFYRIGIPMFDRLGAGHLLSVGYRGTRDLIFHLANLVIADHEENHEPTPDTWATGHGEHAAAPTSH

[0106] Accession number 56: Nucleotide sequence of the beta chain nifK_2 of the molybdenum-iron protein of nitrogenase

[0107] SEQ ID NO: 57: Amino acid sequence of the beta chain nifK_2 of nitrogenase molybdenum-iron protein MPQNADNVLDHFELFRGPEYQQMLANKKKMFENPRDPAEVERVREWAKTPEYKELNFAREALTVNPAKACQPLGAVFVAVGFESTIPFVHGSQGCVAYYRSHLSRHFKEPSSCVSSSMTEDAAVFGGLNNMIDGLANTYNMYKPKMIAVSTTCMAEVIGDDLNAFIKTAKEKGSVPAEYDVPFAHTPAFVGSHVTGYDNALKGILEHFWDGKAGTAPKLERVPNEKINFIGGFDGYTVGNTREVKRIFEAFGADYTILADNSEVFDTPTDGEFRMYDGGTTLEDAANAVHAKATISMQEYCTEKTLPMIAGHGQDVVALNHPVGVGGTDKFLMEIARLTGKEIPEELTRERGRLVDAIADSSAHIHGKKFAIYGDPDLCLGLAAFLLELGAEPTHVLATNGTKKWAEKVQELFDSSPFGANCKVYPGKDLWHMRSLLFVEPVDFIIGNTYGKYLERDTGTPLIRIGFPVFDRHHHHRRPVWGYQGGMNVLITILDKIFDEIDRNTNVPAKTDYSFDIIR

[0108] SEQ ID NO: 58: Nucleotide sequence of nitrogenase iron protein nifH GTGGAGTCCGGTGGTCCTGAGCCGGGCGTGGGCTGCGCCGGCCGCGGCGTGATCACCTCCATCAACTTCCTGGAGGAGAACGGCGCCTACGAGGACATCGACTATGTGTCCTACGACGTGCTGGGCGACGTGGTGTGCGGCGGCTTCGCCATGCCCATCCGCGAGAACAAGGCGCAGGAAATCTACATCGTGATGTCCGGCGAGATGATGGCCATGTATGCGGCCAACAACATCTCCAAGGGCATCCTGAAGTATGCCAATTCCGGCGGCGTGCGCCTGGGCGGGCTGGTCTGCAACGAGCGCCAGACCGACAAGGAGCTGGAGCTGGCGGAGGCTCTGGCGAAGAAGCTCGGCACCGAGCTGATCTACTTCGTGCCGCGCGACAACATCGTGCAGCATGCCGAGCTGCGCCGCATGACAGTGATCGAGTATGCGCCCGATTCCGCCCAGGCCCAGCACTACCGGAACCTGGCCGAGAAGGTGCACGCCAACAAGGGCAACGGCATCATCCCGACCCCGATCACCATGGACGAGCTGGAAGACATGCTCATGGAGCACGGCATCATGAAGGCCGTGGACGAGAGCCAGATCGGCAAGACCGCCGCCGAGCTCGCCGTCTGA

[0109] SEQ ID NO: 59: Amino acid sequence of nitrogenase iron protein nifH MESGGPEPGVGCAGRGVITSINFLEENGAYEDIDYVSYDVLGDVVCGGFAMPIRENKAQEIYIVMSGEMMAMYAANNISKGILKYANSGGVRLGGLVCNERQTDKELELAEALAKKLGTELIYFVPRDNIVQHAELRRMTVIEYAPDSAQAQHYRNLAEKVHANKGNGIIPTPITMDELEDMLMEHGIMKAVDESQIGKTAAELAV

[0110] (Genetic modification of VTT-E-193585 and its variants, e.g., strain VTT-E-213595) As described above, in a further main aspect, the present invention relates to a general method for genetic modification of the bacterial strain VTT-E-193585 or its variants, and to genetically modified variants of the VTT-E-193585 strain. These methods are illustrated in Example 5 of this document. Thus, in one aspect, the present invention is a method for genetic modification of the VTT-E-193585 bacterial strain, comprising: a) providing bacteria of the VTT-E-193585 strain, or a genetically modified or mutant strain generated using a VTT-E-193585 bacterial strain such as VTT-E-213595; b) introducing a nucleic acid construct into said bacteria, wherein said nucleic acid construct comprises: i) a sequence encoding a selectable marker; ii) optionally, a further sequence to be integrated into the bacterial genome; iii) flanking sequences enabling homologous recombination with the bacterial genome; and and c) selecting a genetically modified strain in which the nucleic acid construct has been integrated into the bacterial genome based on the selectable marker. The present invention relates to a method for genetic modification of the VTT-E-193585 bacterial strain, comprising the above steps. In one embodiment, the nucleic acid construct is a plasmid. In some embodiments, the selectable marker is a gene providing antibiotic resistance such as kanamycin resistance or tetracycline resistance, and step c) is performed by growing the bacteria in the presence of the antibiotic. In another embodiment, the selectable marker is a gene encoding a fluorescent protein, and step c) is performed based on fluorescence. The flanking sequences are typically completely identical to sequences in the bacterial genome to enable site-specific integration of the nucleic acid construct. In one embodiment, the method is used to disrupt genes by insertion. Thus, the flanking sequences are selected such that upon integration of the nucleic acid construct, the endogenous gene in the bacterial genome is disrupted and thus inactivated. In another embodiment, the method is used to insert genes such as heterologous genes or mutant genes, or multiple copies of a gene, into the bacterial genome. In a further aspect, the present invention relates to a variant of the VTT-E-193585 bacterial strain comprising a genetic modification, wherein the genetic modification comprises disruption of a bacterial gene by a selectable marker that provides antibiotic resistance such as kanamycin resistance or tetracycline resistance. Such variants are exemplified in this document, for example in Example 5. The present invention also relates to a culture comprising such a variant and a method for biomass production, the method comprising culturing such a variant. The method may have any of the further features described above in this specification.

[0111] (Downstream processing) In one embodiment, the method of the present invention comprises a further step of recovering the biomass produced during the culture. The biomass can be recovered, for example, by sedimentation (sedimentation based on gravity), filtration, centrifugation or aggregation. Aggregation may require the addition of a flocculant. Centrifugation can be carried out, for example, using a continuous flow centrifuge. In one embodiment, the recovered biomass is then dried. Drying can be carried out using well-known methods including, for example, centrifugation, drum drying, evaporation, freeze-drying, heating, spray drying, vacuum drying and / or vacuum filtration. The dried biomass can then be used in products such as food or feed products, or as feed or food ingredients. In another embodiment, the cells of the recovered biomass are lysed. The lysate can, in some embodiments, be separated into an insoluble fraction and a soluble fraction, either or both of which can then be concentrated or dried and then used in products such as food or feed products.

[0112] In one embodiment, biomass is recovered, protein is isolated from the biomass, and a fraction containing a protein fraction and non-protein components is obtained. Thus, in one embodiment, the method is for the production of protein and includes the steps of culturing the VTT-E-213595 strain, subsequently recovering the biomass, and a further step of isolating the protein from the biomass. In another embodiment, the method is for the production of protein and involves culturing a mutant chemosynthetic autotrophic bacterial strain, such as a strain of the genus Xanthobacter, in continuous culture using hydrogen as an energy source and an inorganic carbon source, where the inorganic carbon source includes carbon dioxide, followed by the steps of recovering the biomass and a further step of isolating the protein from the biomass. Depending on the method of protein isolation, the resulting fraction may be more or less pure. Thus, the term "protein fraction" means a fraction in which the protein is concentrated. The protein fraction may still contain a significant amount of other components, and a significant amount of protein may end up in the "fraction containing non-protein components". Protein isolation can be carried out using any suitable method. For example, in one embodiment, the protein is isolated by mechanically disrupting the cells and separating the protein from the cell debris by one or more filtration steps, such as continuous filtration through a plurality of filters with decreasing pore sizes. Mechanical disruption can be performed using any suitable method, such as ball milling, sonication, homogenization, high-pressure homogenization, mechanical shearing, etc. The resulting filtered protein fraction is concentrated in protein but also contains further other smaller components. The protein may optionally be further purified from this fraction using any suitable method. In another embodiment, the protein fraction is isolated by performing ethanol extraction followed by one or more filtration steps. Such methods are known, for example, from the preparation of soybean proteins (see, for example, Chapter 5 "Soybean Protein Concentrates" of "Technology of production of edible flours and protein products from soybeans" by Berk FAO Agricultural Services Bulletin No. 97 (1992)). The resulting protein fraction is protein-enriched but still contains other components. The protein may optionally be further purified from this fraction using any suitable method. In one embodiment, the method of the present invention includes a further step of hydrolyzing the protein fraction obtained from the method of the present invention to obtain amino acids and small peptides.

[0113] In one embodiment of the method of the present invention, the method includes a further step of manufacturing a food or feed product from the biomass, the protein fraction, or the fraction containing non-protein components. The further step may simply include incorporating the biomass, the protein fraction, or the fraction containing non-protein components into the food or feed product by adding it during the manufacture of the food or feed product. In other embodiments, further purification or modification of the biomass or its fraction is carried out during the process of incorporation into the food or feed product. In a further aspect, the present invention relates to products such as biomass, protein, or non-protein components obtained or obtainable by the method according to the present invention. In one embodiment, the product obtained from the method of the present invention contains more than 40% protein, for example, protein between 40% and 99%, for example, protein between 40% and 90%, for example, protein between 40% and 60%. In certain embodiments, the product comprises between 25% and 75% protein, between 0% and 20% lipid and between 5% and 40% carbohydrate. In further embodiments, the product comprises between 40% and 60% protein, between 0% and 15% lipid and between 10% and 25% carbohydrate. In still further embodiments, the product obtained from the method of the present invention comprises between 45% and 55% protein, between 5% and 10% lipid and between 10% and 20% carbohydrate.

[0114] As described above, a further aspect of the present invention relates to food or feed products obtainable or obtained by the method according to the present invention. As used herein, the terms "food" and "feed" are intended to include not only conventional food and feed products such as processed foods, but also food and feed supplements, such as protein bars, powders or shakes, meat substitutes, food ingredients, probiotics, prebiotics, nutraceuticals and the like. In certain embodiments, the biomass, the protein fraction or the fraction containing non-protein components is utilized in the manufacture of vegetarian or vegan foods. In a further aspect, the present invention relates to the manufacture of pharmaceuticals, bioactive compounds, nutraceuticals, antioxidants and / or vitamins using the bacterium of the VTT-E-193585 strain or variants thereof, such as the variants described herein, for example the VTT E-213595 strain. Bioactive compounds, nutraceuticals, antioxidants, vitamins can be extracted from the biomass using methods known in the art. Accordingly, in a further embodiment, the present invention relates to a method for the production of the biomass described herein, which comprises a further step of isolating, for example extracting, compounds such as bioactive compounds, nutraceuticals, antioxidants, vitamins from the biomass or culture broth. In a further embodiment, the present invention relates to the use of these compounds as pharmaceuticals or nutraceuticals. In one embodiment, the extracted compound is beta-carotene (provitamin A). In another embodiment, the extracted compound is coenzyme Q10. In another embodiment, the extracted compound is in the form of heme iron, such as cytochrome C. In another embodiment, the extracted compound is vitamin B12. The present invention will be further described using the following non-limiting examples.

Example

[0115] Example 1 Isolation of a Bacterial Strain Capable of Chemosynthetic Autotrophic Growth A 50 mL sample containing soil and seawater was collected from the coast of the Baltic Sea in Naantali, Finland, into a sterile Falcon tube. In a sterile Erlenmeyer flask, a portion of the soil sample was mixed with 10 mL of mineral medium. The medium consisted of 1 g / L NH4OH, 0.23 g / L KH2PO4, 0.29 g / L Na2HPO4·2H2O, 0.005 g / L NaVO3·H2O, 0.2 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L CaSO4, 0.00015 g / L Na2MoO4·2H2O, 0.005 g / L MnSO4, 0.0005 g / L ZnSO4·7H2O, 0.0015 g / L H3BO3, 0.001 g / L CoSO4, 0.00005 g / L CuSO4, and 0.0001 g / L NiSO4 prepared in tap water. The suspension of soil and medium was incubated in a shaking incubator at +30 °C in a sealed steel box continuously flushed with a gas mixture: 150 mL / min of N2, 18 mL / min of H2, 3 mL / min of O2, and 6 mL / min of CO2. 1 mL of the suspension was taken and added aseptically to 9 mL of medium in an Erlenmeyer flask, and then returned to the incubation box to refresh the culture at 7-day intervals. After the fourth dilution, no visible soil remained in the suspension. To grow biomass for bioreactor culture, the volume of the cell suspension was increased to 100 mL. The optical density (OD 600) was 1.53 when inoculated into 190 mL of mineral medium in a 15 vessel 200 mL parallel bioreactor system (Medicel Explorer, Medicel Oy, Finland). The culture conditions were agitation at 800 rpm, a temperature of +30 °C, the pH was set to 6.8 and controlled with 1 M NaOH. A gas mixture consisting of 14 mL / min of H2, 3 mL / min of O2 and 6 mL / min of CO2 was supplied through a sparger. The headspace of the reactor was flushed with air at 300 mL / min. Mineral medium was supplied to the continuous culture at 6 mL / h, and the cell suspension was removed from the reactor via a capillary while maintaining the volume constant at 200 mL. The cell suspension removed from the reactor was stored at +4 °C. Samples were automatically taken from the bioreactor daily and the absorbance at 600 nm was measured to monitor growth. After 498 h of bioreactor culture, samples were aseptically removed, the suspension was diluted and plated onto agar mineral medium plates containing the above minerals and 2% bacteriological agar. The plates were incubated under the same conditions as described above for the Erlenmeyer flasks. Colonies were then picked from the agar plates and streaked onto fresh agar plates to isolate one organism in one colony. This was repeated twice. Single colonies were picked and suspended in 200 μL of medium in a 96 well microtiter plate. The suspension was incubated at a temperature of +30 °C, shaken at 625 rpm in an EnzyScreen airtight box and continuously flushed with 150 mL / min of N2, 18 mL / min of H2, 3 mL / min of O2 and 6 mL / min of CO2. The suspension from one well was transferred to an Erlenmeyer flask and replenished with fresh medium. The volume was increased until sufficient biomass was present to perform bioreactor culture. The organism was deposited in the VTT culture collection as VTT-E-193585.

[0116] 16S rRNA sequencing of the sample demonstrated that the sample contained only one organism. The same sample was used for Illumina NextSeq sequencing to obtain 1×150 bp metagenomic shotgun sequences. De novo assembly was performed on the metagenomic sequences consisting of 101 contigs using Unicycler (Wick et al, 2017 PLoS computational biology 13:e1005595). The total genome length was 4,846,739 bp and the GC content was 67.9%. Gene prediction and functional annotation were performed using Prokka (Seemann, 2014 Bioinformatics 30:2068). Genome annotation generated 4,429 genes. Roary pan-genome alignment (Page et al, 2015 Bioinformatics 31:3691) grouped VTT-E-193585 among Xanthobacter species. Therefore, this strain was identified as Xanthobacter sp. and the closest genome was Xanthobacter tagetidis. Calculations based on alignments of average nucleotide identity considering only orthologous fragments (OrthoANI) (Lee et al, 2016 Int J Syst Evol Microbiol 66:1100) gave the best match of 80.4% to Xanthobacter tagetidis (ATCC 700314; GCF_003667445.1), although the proposed species boundary cut-off is 95 - 96% (see, for example, Chun et al., 2018 Int J Syst Evol Microbiol, 68:461 - 466). Xanthobacter autotrophicus Py2 gave a 79.6% identity, while the identity in the case of Xanthobacter sp. 91 was 79.0%. Therefore, it could be concluded that the isolated bacterial strain deposited as VTT-E-193585 belongs to the phylum: Proteobacteria; class: Alphaproteobacteria; and order: Rhizobiales. The most likely family is the Xanthobacteraceae and the genus is Xanthobacter. The VTT-E-193585 bacterial strain could not be clearly assigned to any known species.

[0117] A search for putative antimicrobial resistance genes was conducted. Using the ABRicate (https: / / github.com / tseemann / abricate) tool, the genome was searched against the Arg-Annot, NCBI, ResFinder, ecOH, Megares, and VFDB databases using blastn or blastp. A threshold of 50% was set for both identity and coverage at both the nucleotide and protein levels. Only two putative antimicrobial resistance genes were identified. These two genes do not contain amino acid changes associated with antibiotic resistance and thus no resistant phenotype is expected.

[0118] Example 2 Pilot culture and analysis of the isolated bacterial strain The isolated bacterial strain deposited as VTT-E-193585 was cultured in a conventional 200-liter stirred tank bioreactor (MPF-U, Marubishi Ltd, Japan). Mixing was performed using a Rushton type impeller rotating at 400 rpm. The temperature during the culture was maintained at +30 °C. The pH was maintained at 6.8 ± 0.2 by adding 8 M NaOH or 3.6 M H3PO4 by software control. The culture medium contained 1 g / L NH4OH, 0.23 g / L KH2PO4, 0.29 g / L Na2HPO4·2H2O, 0.005 g / L NaVO3·H2O, 0.2 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L CaSO4, 0.00015 g / L Na2MoO4·2H2O, 0.005 g / L MnSO4, 0.0005 g / L ZnSO4·7H2O, 0.0015 g / L H3BO3, 0.001 g / L CoSO4, 0.00005 g / L CuSO4 and 0.0001 g / L NiSO4 prepared in tap water. A mixture containing 1.8 - 10.5 L / min of hydrogen gas, 0.6 - 2.5 L / min of oxygen gas and 1.8 - 5 L / min of carbon dioxide gas was constantly supplied as the main source of energy and carbon. By adjusting the composition of the gas mixture, the dissolved oxygen level was maintained at 7.2 ± 0.5%. The inoculation material for the culture was prepared as described in Example 1. Samples were taken manually and the cell density was analyzed as optical density by measuring the absorbance at 600 nm (Ultrospec 2100 pro UV / visible spectrophotometer, Biochrom Ltd., UK), and the cell dry weight (CDW) was measured by drying overnight in an oven at 105 °C to monitor the growth. The optical density was also monitored using an in situ absorbance probe (Trucell 2, Finesse Ltd., USA). The growth curve of the culture is shown in Figure 1. The maximum growth rate in the batch phase was 0.06 h -1 -1. The maximum cell density was 4.5 g_CDW / L at 92 h. After 92 h of culture, 0.01 h -1The supply of the fresh culture medium as described above was started at the dilution rate of . During the continuous supply, the cell density was 2.9 g_CDW / L on average. The culture broth was always collected in a cooled (+10 °C) tank and then supplied from there in 300-liter batches to a continuous centrifuge (BTPX-205, Alfa-Laval AB, Sweden). The cell-containing slurry collected from the separator was supplied to an atmospheric pressure double drum dryer (Buflovak 6x8 ADDD, Hebeler process solutions Llc., USA) and heated with steam at 4 bar and a drum rotating at 3.5 rpm. As a result, dry cell powder with a dry matter content of about 96% was obtained. The analysis results of the dry cell powder are shown in Table 1 for the general composition, Table 2 for the amino acid composition, Table 3 for the fatty acid composition, and Table 4 for the vitamin content. The analysis demonstrates that the dry cell powder has a high protein content containing all essential amino acids. It also contains more unsaturated fatty acids than saturated fatty acids and contains many B-group vitamins. The peptidoglycan content was 0.002 mg / g_CDW and the lipopolysaccharide content was 0.01 mg / g_CDW. It would be beneficial for these concentrations to be as low as possible. In comparison, in a commercially available lactic acid bacteria preparation analyzed simultaneously, the peptidoglycan content was 0.244 mg / g_DW and the lipopolysaccharide content was 0.015 mg / g_DW. Cytotoxicity and genotoxicity assays were performed using the supernatant samples of the culture. No cytotoxicity was observed against the HepG2 or HeLa229 human cell lines. No genotoxicity was observed against the Escherichia coli WP2 trp- or CM871 uvrA recA lexA strains.

[0119]

Table 1

[0120]

Table 2

[0121] [Table 3] Table 3. Fatty acid composition of the dried cell powder of the isolated bacterial strain deposited as VTT-E-193585.

[0122] [Table 4] Table 4. Vitamin content of the dried cell powder of the isolated bacterial strain deposited as VTT-E-193585.

[0123] Example 3. Cultivation of the isolated bacterial strain with different nitrogen sources. The isolated bacterial strain deposited as VTT-E-193585 was cultured in a 15-vessel parallel bioreactor system at a volume of 200 mL (Medicel Explorer, Medicel Oy, Finland). Mixing was carried out using a Rushton-type impeller rotating at 800 rpm. The temperature during cultivation was maintained at +30 °C. The pH was maintained at 6.8 by adding 1 M NaOH. The culture medium was prepared in tap water and contained 0.23 g / L KH2PO4, 0.29 g / L Na2HPO4·2H2O, 0.005 g / L NaVO3·H2O, 0.2 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L CaSO4, 0.00015 g / L Na2MoO4·2H2O, 0.005 g / L MnSO4, 0.0005 g / L ZnSO4·7H2O, 0.0015 g / L H3BO3, 0.001 g / L CoSO4, 0.00005 g / L CuSO4 and 0.0001 g / L NiSO4. Furthermore, the nitrogen source was varied in the cultures such that four cultures contained 18.7 mM NH4OH, four cultures contained 9.34 mM urea (OC(NH2)2), four cultures contained 18.7 mM potassium nitrate (KNO3), and three cultures were left without a nitrogen source in the medium. A mixture containing 22 mL / min of hydrogen gas, 3.2 mL / min of air, and 6.4 mL / min of carbon dioxide gas was constantly supplied as the main source of energy and carbon. Thus, nitrogen gas was also supplied to all the cultivations using air. Growth was monitored by automatically sampling the samples and analyzing the cell density as optical density by measuring the absorbance at 600 nm (Ultrospec 2100 pro UV / visible spectrophotometer, Biochrom Ltd., UK). The growth curves of the cultures are shown in Figure 2. Growth with ammonia and urea was comparable. Growth with nitrate or nitrogen gas was clearly slower than growth with ammonia or urea. Towards the end of the cultivation, growth with nitrate was better than growth with nitrogen gas as the sole nitrogen source. Nevertheless, there was growth even in the cultures where nitrogen gas was the sole nitrogen source, demonstrating that the isolated bacterial strain deposited as VTT-E-193585 is capable of nitrogen fixation.

[0124] Example 4 Characterization of Antibiotic Sensitivity For the isolated bacterial strain deposited as VTT-E-193585, the antibiotic susceptibilities to gentamicin, kanamycin, streptomycin, tetracycline, ampicillin, ciprofloxacin, colistin and fosfomycin were determined according to the CLSI M07-A111 standard (Clinical and laboratory standards institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 11th ed. CLSI standard M07, 2018). For ampicillin, ciprofloxacin and colistin, a manually prepared microdilution plate was used, for gentamicin, kanamycin, streptomycin and tetracycline, VetMIC Lact-1 plates (SVA National Veterinary Institute, Uppsala, Sweden) were used with the broth microdilution method, and for fosfomycin, the agar dilution method was used with cation-adjusted Mueller-Hinton broth medium (LabM, LAB114, with cations Mg 2+ and Ca 2+ added separately), and analyzed under aerobic conditions at +35 ± 2 °C for 48 ± 1 hours. Escherichia coli ATCC 25922 was used as a quality control strain and incubated under aerobic conditions at +35 ± 2 °C for 18 ± 2 hours. The results of the antibiotic susceptibilities of the strains are shown in Table 5. The isolated bacterial strain was generally found to be sensitive to antibiotics. For gentamicin, kanamycin, streptomycin and tetracycline, the minimum inhibitory concentration (MIC) values of VTT-E-193585 were lower than or equivalent to those of Escherichia coli ATCC 25922, but for ampicillin, ciprofloxacin, colistin and fosfomycin, the MIC values were higher for VTT-E-193585.

[0125]

Table 5

[0126] Example 5 Construction of phaC knockout strain Medium composition per liter of liquid DSM81-LO4 (DSM) KH2PO4 2.3 g Na2HPO4·2H2O 2.9 g Na2SO4 5.45 g (NH4)2SO4 * 1.19 g MgSO4·7H2O 0.5 g CaSO4·2H2O 11.7 mg MnSO4·H2O 4.4 mg NaVO3 5 mg NaHCO3 0.5 g Ferric ammonium citrate (III) 5 mg ZnSO4·7H2O 0.5 mg H3BO3 1.5 mg CoCl2·6H2O 1 mg CuCl2·2H2O 50 μg NiCl2·6H2O 0.1 mg Na2MoO4·2H2O 0.15 mg Riboflavin 0.5 mg Thiamine-HCl·2H2O 2.5 mg Nicotinic acid 2.5 mg Pyridoxine-HCl 2.5 mg Ca-pantothenate 2.5 mg Biotin 5 μg Folic acid 10 μg Vitamin B 12 50 μg *Changed for GC-MS analysis

[0127] Lysogenic broth (LB) Yeast extract 5 g Tryptone 10 g 10 g of NaCl 15 g of agar (for plates)

[0128] Super Optimal Broth (SOB) 20 g of tryptone 5 g of yeast extract 0.58 g of NaCl 0.18 g of KCl 0.95 g of MgCl₂ 1.20 g of MgSO₄

[0129] Super Optimal Broth containing ethanol (SOBE) 5.8 mL of ethanol / 1 L of SOB

[0130] Tryptic Soy Agar (TSA) 40 g of BBL™ Trypticase™ Soy Agar (BD)

[0131] Cultivation method All SoF1 cultures under heterotrophic growth conditions were cultured at 30 °C with shaking at 220 rpm in 10 mL volume of Super Optimal Broth containing 100 mM ethanol (SOBE). All SoF1 cultures under autotrophic conditions were cultured in DSM81 - LO4 medium (DSM) at the same temperature and volume as the heterotrophic growth conditions with shaking at 136 rpm. The gas composition under autotrophic conditions was as follows: 44% CO₂, 26% N₂, 22% H₂, 7% O₂, and 1% of other gases.

[0132] Escherichia coli strains The Escherichia coli (E. coli) strains used and their related characteristics are summarized in Table 6. All strains were grown on Lysogeny Broth (LB). Antibiotics were used at the following concentrations: 100 μg / mL of ampicillin (AMP), 50 μg / mL of kanamycin (KAN), and 10 μg / mL of tetracycline (TET).

[0133]

Table 6

[0134] Plasmid construction By sequencing the bacterial genome of the VTT-E-193585 strain described in Example 1, genes phaC1 (encoding the proteins shown in SEQ ID NO: 60 and SEQ ID NO: 62) and phaC2 (encoding the proteins shown in SEQ ID NO: 61 and SEQ ID NO: 63) having homology with the phaC gene found in other Xanthobacter spp. encoding polyhydroxyalkanoate (PHA) synthase were identified.

[0135] Two plasmids targeting the deletion of the phaC1 and phaC2 genes in the genome of SoF1 were constructed (Table 7). The left (LHA) and right (RHA) homologous arms of 1000 bps adjacent to phaC1 and phaC2 were amplified from the genomic DNA of SoF1 containing oligos (8 oligos). Both plasmids were constructed from pUC57 by Gibson assembly. The kanamycin resistance gene (kan), tetracycline resistance gene (tet) and mobilizable region (mob) sequences were the same as those used in the plasmids described in Van den Bergh et al. 1993 J Bacteriol 175:6097-6104.

[0136]

Table 7

[0137] Knockout strain construction The plasmids were transferred to SoF1 by conjugation or electroporation. The antibiotic concentrations used for the selection of the modified SoF1 strain were 20 μg / mL KAN and 10 μg / mL TET.

[0138] For conjugation, the liquid culture (LC) of SoF1 was grown under autotrophic conditions as described above for 2 - 3 days until an OD of 0.7 - 1 was reached. The overnight (O / N) LC of plasmid - containing and plasmid - free Escherichia coli (E.coli) S17 - 1 and JM109(DE3) was grown at 37°C with shaking at 220 rpm. The next day, fresh LC was inoculated from the O / N culture and grown until the exponential phase (OD 0.3 - 0.6). The E.coli cells were centrifuged at 5900 rpm for 30 seconds, washed, and resuspended in 1 volume of 0.9% NaCl. The E.coli and SoF1 cells were mixed at an OD ratio of 1:15 and 1 mL of DSM medium was added. Plasmid - free S17 - 1 mixed with SoF1 was used as a negative control, and JM109(DE3) with an S17 - 1 - containing plasmid was used as a positive control. The mixture was vacuum - filtered through a 0.22 - μm GV Durapore® membrane filter (MilliporeSigma, USA). The filter was placed on a pre - warmed TSA plate with the cells facing away from the agar and incubated O / N under autotrophic conditions. The next day, the filter was washed with 1 mL of 0.9% NaCl, vortexed, centrifuged at 4000 rpm for 1 minute, the filter was removed, and the cells were resuspended. The cells were seeded onto selective plates with serial dilutions and grown under appropriate conditions. The conjugate JM109(DE3) control was seeded onto a TSA plate containing 40 μg / mL of nalidixic acid for selection and incubated at 37°C O / N. The conjugate SoF1 culture was seeded onto a DSM plate containing KAN and incubated under autotrophic conditions for approximately 1 week. After colonies appeared on the SoF1 plates, they were re - applied to fresh DSM KAN plates and incubated for an additional 1 week under autotrophic conditions. Single colonies from these plates were tapped onto TSA plates and grown at 30°C under heterotrophic conditions. If growth on the TSA plate resulted in the growth of E.coli, the colonies were re - applied to selective DSM plates.

[0139] For electroporation, the LC of SoF1 was cultured under autotrophic conditions for 2 - 3 days until it reached an OD of 0.7 - 1.5. The cells were transformed into Falcon tubes and cooled on ice for 15 - 30 minutes. The cells were centrifuged at 4°C and 4000 rpm for 5 - 10 minutes, the supernatant was discarded, and the pellet was resuspended in 1 volume of ice-cold double-distilled H2O. Centrifugation was repeated and the supernatant was discarded. Washing was repeated with 1 volume of ice-cold 10% glycerol. The cells were resuspended in ice-cold 10% glycerol to a concentration of approximately 2·10 10 cells / mL. The cells were used immediately for electroporation. 40 μL of cells were mixed with 1 μL of plasmid and incubated on ice for 10 minutes. The cells were transformed into an electroporation cuvette on ice. The cuvette was subjected to a single electrical pulse of 2.5 kV with a capacitance of 25 μV and a resistance of 400 Ω or 600 Ω. 1 mL of pre-warmed (30°C) SOBE was added immediately, the solution was transferred to a Falcon tube, and incubated O / N under autotrophic conditions. After incubation, the cells were seeded onto TSA selection plates containing multiple dilutions and incubated under heterotrophic conditions.

[0140] After transformation by conjugation or electroporation, successful transformants were screened by colony PCR, and dimethyl sulfoxide with a final concentration of 3% was added to the standard PCR mixture. The constructed SoF1 strains are summarized in Table 8.

[0141]

Table 8

[0142] PHB content analysis The PHB contents of the wild-type (WT) strain SoF1 and the knockout strain SoF1-2.0 were analyzed by gas chromatography-mass spectrometry (GC-MS). Both strains were grown under the above autotrophic and heterotrophic conditions in a 5 mL culture volume with various nitrogen concentrations (18.0, 13.5, 9.0, and 4.5 mM nitrogen). Cultures grown under autotrophic conditions were inoculated to obtain a starting OD of 0.1. Analyses were performed as early as 1 week after inoculation. 1-3 mL of each sample was centrifuged, and the pellet was stored at -20 °C. The pellet was thawed, washed twice with redistilled H2O, and freeze-dried for 24-48 h. 10 mg of each sample was subjected to methanolysis by heating in a solution containing 1 mL of chloroform, 150 μL of sulfuric acid, 20 μL of internal standard (3-hydroxybutyric acid), and 830 μL of methanol at 100 °C for 140 min. 3-Hydroxybutyric acid was treated in the same manner as the reference sample. After cooling the sample to room temperature, water-soluble particles were removed with 0.5 mL of water. The chloroform phase was analyzed using a gas chromatography system (7890, Agilent) and an HP-FFAP column (19091 F-102, Agilent).

[0143] Results According to GC-MS, almost no PHB was produced in the SoF1-2.0 strain, but in WT SoF1, when grown under autotrophic conditions, the PHB dry content was 15-30% (Table 9). When grown under heterotrophic conditions, almost no PHB was produced in either SoF1 or SoF1-2.0.

[0144] [Table 9] Table 9. PHB contents of WT SoF1 and SoF1-2.0 grown under autotrophic conditions at different nitrogen concentrations under autotrophic conditions and under heterotrophic conditions (SOBE) as determined by GC-MS.

[0145] The autotrophic growth curves of SoF1 and SoF1-2.0 are shown in Figure 3. SoF1-2.0 has a slightly lower growth rate compared to SoF1.

[0146] phaC1 DNA sequence (SEQ ID NO: 60):

[0147] phaC2 DNA sequence (SEQ ID NO: 61):

[0148] phaC1 amino acid sequence (SEQ ID NO: 62): MSAAEETSTHAELRLPQDGVEHDVAAAEAAVDRSAGEPSGTQASAAPAEAAPSSAPVSAAAGETQPQDDTPPQDPSSLSDGPGLSPPVTQPGAASGDFGGPEAMGDAFMPPVPEEPMEGMAPAPAISAAPASVPSAGWPEDAPSALLDAVDASGDLPAAAEGAATAPEPIFRELPMTAAPAAPAIANILEPVAEALSALGAVAVSRPQVQREFRAAPEHPPMPRMAPPQAAPEPAPAVPPKPEAAKPEAAKPEAAKPEAAKPEVAKPDAAAPDAAKSSGKAERPSGAGDGSGTSGVNMEAFSRNLARLVEEGGKAMAAYLSPREQGKTDDLADDIADAMKTVGQVVEYWVADPQRTVEAQSRLMGGYLSVWANTLKRLAGEEATPVAAPDPKDARFKDAGWNDSPMFDALKQAYLVTSDWAQNMVDEAKGLDPHTKHKAEFLVRQIANAISPSNFVLTNPELIRETLHSSGENLVKGMQNLTADLMAGQGTLKIRQTDLSAFEVGRNLATTPGKVIFENELMQLIQYEPTTETVKKTPVLIVPPWINKFYILDLTAEKSLIKWLVSQGLTVFTISWVNPDGRLAAKGFDDYMRDGIMAALDAVAVASGERRAHAVGYCVGGTLLATTLAYMAATGDDRIASATFLTTQIDFTHAGDLKVFVDESQLATIERKMKEMGYLEGSKMASAFNMLRSNDLIWPYVVNNYMKGKAPFPFDLLFWNSDSTRMPAANHSYYLRNCYLTNNIARGLAELAGLKIDVTKVSIPVYSLATREDHIAPANSVYIGANLLSGPVRYVLAGSGHIAGVVNPPAKMKYQYWADGPVGPSYEAWLAGAQEHKGSWWPDWFNWFSFNHPEEVPARAIGGGRLAPIEDAPGRYVKERS

[0149] phaC2 amino acid sequence (SEQ ID NO: 63): MEARKMPVSPPSSATILPLPVSAAPPSTAPAASLPATASSSTNKASPSAFPAAWARSFALPGLPAFLCPDEEDFEEGSGPAAFHAVDRAAAALVARTTQGLSPAALTLAYMDWAMHLAAAPGKQAELAVKATRKAARFWAYVLASTLDRTQAPCIAPLVGDERFSAPAWQDWPYRFWYQAFLLNQQWWHNATHGVPGVAPHNQDVVAFAARQVLDMFSPSNSPLTNPEVVKKARQTLGANFVQGARNFMEDQSRKTTGRPPVGAEAFTPGKEVAITPGEVIYRNHLIELIQYRATTPDVHAEPILIVPAWIMKYYILDLSPDNSLIRYLVDKGHTVFCISWRNVNAEDRDLGFEDYRKMGIMAALDAVNAVVPNQKVHAVGYCLGGTLLSIAAAAMARVVDDRLGSVTLFAAQTDFTEPGELQLFVDPSELYALESLMWDQGYLGARQMAGAFEMLRSNDLVWSRMVRDYLMGERAPMNDLMAWNADATRMPYRMHSQYLRNLFLDNELAVGRYMVEGRPVSLQNIRVPLFVVGTERDHVAPWKSVYKIHQLTDTDVTFVLASGGHNAGIVSEPGHKHRHYRIHDTKLGEMHVSPEEWMEANRSQDGSWWPAWEAWLAGQSSGRIGLPPLGAPGYEVLGPAPGTYVMQR

Claims

1. A variant of the bacterial strain VTT-E-193585, comprising a genetic modification that reduces the bacterial production of polyhydroxyalkanoic acid (PHA) compared to the VTT-E-193585 strain. The variant comprising a genetic modification that is a gene disruption that reduces the expression level of phaC1 and / or the activity of the phaC1 enzyme.

2. The variant according to claim 1, wherein the genetic modification reduces the bacterial PHA synthase activity to less than 10% compared to the VTT-E-193585 strain.

3. The variant according to claim 1 or 2, wherein the variant is a bacterial strain deposited under the number VTT-E-213595.

4. The variant according to claim 1 or 2, which retains the ability to grow using hydrogen gas as an energy source and carbon dioxide as the sole carbon source.

5. A culture comprising the bacteria of the variant according to claim 1 or 2.

6. A method for biomass production, the method comprising culturing the bacteria of the variant according to claim 1 or 2.

7. The method according to claim 6, comprising culturing the bacteria of the variant in continuous culture with hydrogen as an energy source and an inorganic carbon source, wherein the inorganic carbon source comprises carbon dioxide.

8. The method according to claim 7, wherein the dissolved oxygen in the culture is maintained between 5% and 10%.

9. The method according to claim 7, wherein ammonium, urea, nitrate and / or nitrogen gas is used as a nitrogen source.

10. The method according to claim 7, wherein the pH in the culture is maintained between 5.5 and 8.

0.

11. The method according to claim 7, wherein the culture is grown at a temperature between 25°C and 40°C.

12. The method according to claim 7, further comprising the step of recovering the biomass produced during the culture.

13. The method according to claim 12, further comprising the step of drying the recovered biomass.

14. A method for protein production, comprising the method according to claim 12 and further comprising the step of isolating the protein from the biomass, wherein the method results in a fraction containing a protein fraction and a non-protein component.

15. The method according to claim 14, further comprising the step of manufacturing a food or feed product from the biomass, from the protein fraction, or from the fraction containing the non-protein component.

16. A method for genetic modification of the VTT-E-193585 bacterial strain, comprising: a) providing bacteria of the VTT-E-193585 strain, or a genetically modified strain or mutant strain generated using the VTT-E-193585 bacterial strain; b) introducing a nucleic acid construct into the bacteria, wherein the nucleic acid construct comprises: a sequence encoding a selectable marker; and adjacent sequences enabling homologous recombination with the bacterial genome; and c) selecting a genetically modified strain based on the selectable marker.

17. The method according to claim 16, wherein the nucleic acid construct further comprises additional sequences to be integrated into the bacterial genome.

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