AT528175A2 - Methanothermobacter strain and method using the same - Google Patents

Abstract

The present invention provides a strain of Methanothermobacter having a proline production rate lower than the proline production rate of Methanothermobacter marburgensis DSM 2133 and/or a glycine production rate higher than the glycine production rate of Methanothermobacter marburgensis DSM 2133. The strain preferably has an argininosuccinate lyase (ASL), wherein amino acid residue 51 is leucine, isoleucine, valine, or alanine. In particular, the strain Methanothermobacter marburgensis is deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under the deposit number DSM 34858. Method and use of cells of the strain for the industrial production of amino acids, in particular glycine.

Description

The field of the present invention relates to strains of the Archaea genus Methanothermobacter and their biotechnological

Uses. BACKGROUND

Methanogenic archaea are known for their ability to produce methane (CH) as the end product of their energy metabolism (see, e.g., Mand & Metcalf, 2019). Some of them are capable of autotrophic, hydrogenotrophic growth by reducing carbon dioxide (CO) with molecular hydrogen (H) to CH, and play a crucial role in the global carbon cycle (Lyu et al. 2018). According to their substrate utilization spectrum, methanogens can be divided into different metabolic groups: hydrogenotrophic (H,, formate, or simple alcohols), acetoclastic (acetate), methylotrophic (compounds containing a methyl group), H;-dependent methylotrophic (methylated compounds with H as an electron donor), and methoxydotrophic (methoxylated aromatic compounds) (Mayumi et al. 2016, Kurth et al. 2020). The biology of methanogenic archaea is also discussed in older publications such as Zeikus, 1977. A standardized

Taxonomy of Archaea is revealed in Rinke et al, 2021.

Rittmann et al, 2021, generally concerns the use of

Archaea in biotechnology.

Several studies discuss the use of methanogenic archaea in renewable energy production by reducing CO with H to CH (Pappenreiter et al. 2019, Rittmann et al. 2018, Mauerhofer et al. 2018, Abdel Azim et al. 2018, Abdel Azim et al. 2017, Rittmann 2015, Mauerhofer et al. 2021, Rittmann et al. 2012, Martin et al. 2013). Liu et al. 2021 address the effects of different amino acids and their configurations on methane yield and the biotransformation of intermediate metabolites during anaerobic digestion. WO 2012/110256 A1 discloses a process for the conversion of carbon dioxide and hydrogen to methane by methanogenic microorganisms. WO 2014/128300 A1 relates to a process and

System for producing methane using

Nitrogen concentrations in the liquid phase.

WO 2017/070726 A1 relates to a method for determining the culture status of microbial cultures, such as cultures

with hydrogenotrophic methanogenic microorganisms.

Hoffarth et al, 2019, concerns the effect of N on biological methanation in a continuous

Stirred tank reactor with Methanothermobacter marburgensis.

US 2011/281333 A1 relates to methane production from single-celled organisms, such as methanogens. The growth of methanogens involves the consumption of carbon dioxide to produce methane. Methods for enhancing growth are disclosed.

US 2018/0179559 A1 relates to a biological and chemical process in which chemoautotrophic microorganisms are used for the chemosynthetic fixation of carbon dioxide and/or other inorganic carbon sources in organic compounds and for the production of additional useful products. The microorganisms can be selected from many different bacterial

and archaea species are selected.

EP 2 192 170 A1 relates to an amino acid-producing microorganism and a process for producing amino acids. Disclosed is a microorganism (preferably selected from gamma-proteobacteria, coryneform bacteria or bacteria belonging to the genera Alicyclobacillus, Bacillus or the yeast Saccharomyces) which has the ability to produce an L-amino acid selected from the group consisting of L-lysine, L-threonine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine, and which has been modified such that the activity of

Pyruvate synthase or pyruvate:NADP'-oxidoreductase is increased. WO 2016/179545 A1 and US 2018/0163240 A1 disclose

Compositions and processes for the biological production of

Methionine.

US 2019/0194630 Al and US 2017/0130211 Al relate to compositions and methods for the biological production of

Amino acids in hydrogenotrophic microorganisms. In particular

Methanosarcina should be selected.

WO 2020/252335 A1 relates to processes and plants for the fermentative production of products. In particular, a process is disclosed which comprises: (a) feeding a gaseous mixture comprising CO and H, where x is 1 or 2, a nitrogen source, and optionally a sulfur source to a bioreactor containing hydrogenotrophic microorganisms under conditions such that the hydrogenotrophic microorganisms produce at least one fermentation product selected from an amino acid, an alcohol, aldehyde or ketone, a carboxylic acid, or a hydroxyl or keto acid; (b) removing a gas stream from the bioreactor, the gas stream comprising at least one compound selected from a sulfur-containing compound, a nitrogen-containing compound, H,, CO, and a hydrocarbon compound, where x is 1 or 2; (c) removing a liquid stream from the bioreactor comprising fermentation broth, hydrogenotrophic microorganisms and fermentation product; and (d) separating the hydrogenotrophic microorganisms from the liquid stream and returning the hydrogenotrophic microorganisms to the bioreactor. The hydrogenotrophic microorganisms may be

methanogenic archaea are selected.

WO 2023/247740 A1, WO 2023/247741 A1, and WO 2023/247742 A1 relate to methods for producing amino acids using methanogenic microorganisms in a bioreactor. Roughly speaking, the documents disclose a method for producing amino acids by fermentation in a bioreactor, wherein the bioreactor comprises methanogenic microorganisms in a fermentation broth, the method comprising at least the step of supplying a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source comprising nitrogen gas, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acids. Preferred methanogenic microorganisms are, for example, archaea selected from Methanothermobacter, Methanothermococcus

and Methanococcus.

WO 2023/247743 A1 relates to a process for the fermentative production of norvaline. In particular, the document discloses a process for the production of amino acids by fermentation in a bioreactor, wherein the bioreactor comprises methanogenic microorganisms in a fermentation broth, the process comprising at least the step of supplying a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acids

produce, with the amino acids including norvaline.

Taubner et al., 2023, provide a lipidomics and comparative metabolite excretion analysis of methanogenic archaea. The paper presents the lipidome and a comprehensive quantitative analysis of proteinogenic amino acid excretion as well as methane, water, and biomass production of three autotrophic, hydrogenotrophic methanogens: Methanothermobacter marburgensis, Methanothermococcus okinawensis, and Methanocaldococcus villosus at different temperatures and nutrient supplies. The patterns and production rates of excreted amino acids and the lipidome are unique for each tested methanogen and can be modulated by varying the incubation temperature and substrate concentration, respectively. According to the paper, the ability to scale amino acid production processes using a low-cost substrate (H2/CO2) will facilitate the use of industrial-scale methanogens.

Bring commercialization closer.

Consequently, there is a need for methanogens that are better adapted to industrial-scale production conditions, especially for amino acid production, and the specific

adapted to the needs of such environments.

It is therefore an object of the present invention to provide such methanogen strains and biotechnological processes for their use, in particular for the production of amino acids. These processes should be highly suitable for industrial amino acid production and/or overcome one or more disadvantages of prior art processes.

Overcome methanogenic strains.

The present invention relates to a strain of Methanothermobacter with a proline production rate that is lower than the proline production rate of Methanothermobacter marburgensis, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 2133 (Methanothermobacter marburgensis DSM 2133), and/or with a glycine production rate that is higher than the glycine production rate of Methanothermobacter marburgensis DSM 2133 (when both strains are cultivated under comparable conditions, preferably under the same conditions). The production rate can be calculated on a molar basis or a weight basis. Preferably, the

Production rate calculated on a weight basis (e.g. mg L”* h”).

The present invention further relates to a strain of Methanothermobacter with a proline content of the total secreted amino acids that is lower than the proline content of the total secreted amino acids of Methanothermobacter marburgensis DSM 2133, and/or a glycine content of the total secreted amino acids that is higher than the glycine content of the total secreted amino acids of Methanothermobacter marburgensis DSM 2133 (when both strains are cultivated under comparable conditions, preferably under the same conditions). The content can be calculated on a molar basis or on a weight basis.

Preferably, the proportion is calculated on a weight basis.

Another object of the present invention is a strain of Methanothermobacter which has an argininosuccinate lyase (ASL), wherein the amino acid residue 51 is leucine, isoleucine, valine or alanine, in particular leucine or

Isoleucine.

Another object of the present invention is a strain of Methanothermobacter marburgensis, which was deposited on November 21, 2023, with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under the deposit number DSM 34858 (herein also referred to as Methanothermobacter

marburgensis Arkeon).

a bioreactor containing the fermentation broth.

In a further aspect, the present invention relates to the use of such strains for producing amino acids (preferably glycine), in particular a (single) amino acid, preferably a (single) canonical amino acid such as glycine, in a purity of more than 50 wt.% (wt.%), preferably more than 60 wt.%, more preferably more than 70 wt.%, even further

preferably more than 80 wt%, in particular more than 90 wt%.

In yet another aspect, the present invention relates to a process for the fermentative production of amino acids in a bioreactor. The bioreactor contains cells of the strain according to the invention in a fermentation broth. This process comprises at least the steps of supplying a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acids; and harvesting at least a portion of the amino acids from the fermentation broth. This portion may comprise only a single amino acid or several amino acids (such as glycine). In embodiments, the amino acid may be present in a purity of more than 50 wt.%, preferably more than 60 wt.%, more preferably more than 70 wt.%, even more preferably more than 80

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% by weight, in particular more than 90 % by weight.

The inventors found that a strain of Methanothermobacter with a proline production rate lower than that of Methanothermobacter marburgensis DSM 2133 and/or with a glycine production rate higher than that of Methanothermobacter marburgensis DSM 2133 is well suited for industrial amino acid production. In particular, due to the lower proline production rate (see Fig. 1), it is possible to achieve the production of other amino acids in higher purity. The strain also forms an excellent basis for further shifting amino acid production to facilitate the production of a single

amino acid (such as glycine) at higher purities, e.g.

Glycine was observed (see Fig. 4). DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to all aspects of the invention mentioned above, unless

are expressly excluded.

Amino acids are used in a wide variety of fields: food and feed, agriculture, pharmaceuticals, and even packaging and housing. The biosynthesis of proteinogenic amino acids is an important branch of biotechnology (Becker and Wittmann 2012). The metabolic potential of archaea regarding amino acid production

However, it has been largely overlooked so far (Pfeifer et al. 2021).

The enzyme ASL (EC 4.3.2.1) catalyzes the degradation of argininosuccinate into fumarate and arginine. It is involved in arginine biosynthesis in all species. In archaea and bacteria, it is encoded by the argH gene. Functional conservation has been observed between archaeal ASL and the corresponding bacterial and eukaryotic genes (Cohen-Kupiec et al., 1999).

Several ASL sequences of Methanothermobacter have been deposited in public sequence databases, including UniProt with the following identifiers 026369.1 (Methanothermobacter thermautotrophicus) and D9PVR6.1 (Methanothermobacter

marburgensis).

Atypically, the ASL residue at position 51 (Methanothermobacter consensus sequence: .„.MLA*XEXII°X°*... (SEQ ID NO: 4); where each superscript number represents the position in the

Serine, aspartate or asparagine.

Strikingly, arginine production is metabolically closely linked to proline production. Without being bound to any particular theory, it is hypothesized that this unusual amino acid substitution in ASL forms, at least in part, the structural basis for the different phenotype of the inventive strain compared to Methanothermobacter marburgensis DSM 2133 (i.e., the lower proline production rate, which makes the inventive step a more favorable starting point for shifting amino acid production toward other amino acids at higher purities).

According to a preferred embodiment, the amino acid residue at position 51 of the ASL is leucine or

Isoleucine, especially leucine.

The strain according to the invention, DSM 34858, was found to be most closely related to Methanothermobacter marburgensis Marburg (DSMZ accession code 2133). However, apart from Methanothermobacter marburgensis, this amino acid substitution at position 51 of the ASL is also useful in other Methanothermobacter backgrounds. Accordingly, in a further preferred embodiment, the strain of Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermautotrophicus, Methanothermobacter crinale, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter sp. CaT2 (see, e.g., Kosaka et al., 2013), Methanothermobacter sp. DP (see, e.g., Prabhaharan et al., 2023), Methanothermobacter sp. KEPCO-1 (see e.g. Jeon, et al, 2020), Methanothermobacter sp. THM-1 (see e.g. Jeon, et

al, 2021) or Methanothermobacter tenebrarum.

The ASL protein and argH gene sequences found in strain DSM 34858 were as follows: > SEQ ID NO: 1

MNLRAGRLKRGMEDEAAEFTSSLTFDHHIFEADVDCNIAHTTMLAHEGIILEEVADKIIEALETLREEGVEALDMDP SVEDIHMAVENYVTARIGEEAGFMHTGKSRNDQVATDLRLALREKIKLISHELLDFIDRIAEMALEHTGTVMVGYTH LQHAQPTTLAHHLMAYAHSLKRDYERLQDTLKRIDINPLGSAAMTTTTFPINRELTTELLGFSDYMRNSMDAVSARD FIAEAIFDLSVLAVNLSRISEEMILWSTYEFGMIEIPDEFSSTSSIMPOKKNPDVAEIARAKTSTVQGALFSVLGIM KALPYTYNRDLQEITPHLWRATDTALSMIRVVRGMLLGIRVNRDRALELAGANFATATDLADILVRERGIPFRVAHR IVGRLVTRAIADGLGPSDIDSEYLDEVSLEVTGKRLELDQELVRRALDPLENVRMRMVPGGPAPEMVRAAIDDIRAF VESERTT

> SEQ ID NO: 2

TTGAACCTGAGGGETGGCAGATTAAAGAGGGGTATGGAGGATGAGGCAGEGGAGT TTACETCATECETCACATTCGA CCACCACATATTCGAGGEGGATGTTGACTGTAACATTGCACACACAACGATGETGGECCATGAGGGTATAATACTGG AGGAAGTGGCAGATAAGATCATAGAAGCECETTGAAACCCTCAGGGAGGAGGGTGTCGAGGECETTGACATGGACCCG TCGGTGGAGGATATCCACATGGETGTTGAGAACTACGTGACAGECAGGATAGGTGAGGAAGECGGGTTCATGCACAC AGGTAAATCCAGGAACGACCAGGTTGCAACTGACETTEGECETGGECETGAGGGAGAAGATAAAATTAATCAGECACG AACTCCTTGATTTTATTGATAGGATAGETGAAATGGCACTTGAGCACACAGGGACTGTCATGGTTGGETACACCECAC CTCCAGCATGECCAGECAACAACCETTGECECACFCACETCATGGECTATGECCACETECCTGAAAAGGGACTATGAGAG GETCECAGGACACCCTGAAGAGGATTGACATCAACCCGETGGGTTECGEGGECATGACAACCACAACATTECCGATAA ACAGGGAACTCACAACGGAACTTECTGGGTTTETETGATTACATGAGGAACTCCATGGACGEGGTGAGTGEAAGGGAC TTCATAGCAGAGGCAATATTTTGACCETCETCAGTGETGGEGGTGAACETCAGCAGGATECTCAGAGGAGATGATACTCTG GAGCACCTACGAGTTTGGCATGATAGAGATCCCTGATGAATTETCATCAACATCATCGATAATGECCCAGAAGAAGA ACCCETGACGTTGECGAGATAGECAGGGEAAAGACETECCACGGTTCAGGGGGEAETETETETGTACTTGGAATCATG AAGGECEETEEECTACACCETACAACAGGGACEETECCAGGAGATCACACECCACETETGGAGGGECACCGACAETGEALET GTCAATGATCCGTGTCGTGAGGGGGATGETECTGGGEAT CAGGGT TAACCGGGACAGGGCACT TGAACTTGCAGGGG CCAACTTTGCAACCGCAACAGACETTGEAGATATECTTGTCAGGGAGEGEGGEATACECETTCAGGGTGGECCACAGA ATAGTTGGAAGGCETGGTCACAAGGGECATTGECGATGGAETEGGGEEETETGACATAGAT TCAGAGTACCETTGATGA GGTGAGECTEGAGGTCACCGGGAAGAGACTTGAACTTGACCAGGAACTTGTGAGGAGGGEEETTGATECCACTTGAGA ATGTCAGGATGAGGATGGTTCCAGGTGGACCGGCEACETGAGATGGTCAGGGEGGECATTGATGATATCAGGGEETTT GTGGAGTCTGAGAGGACAACCTAA

Accordingly, in a further preferred embodiment, the strain has a (functional or active) ASL

which contains a sequence with at least 80%, preferably at least

85%, more preferably at least 90%, even more preferably at least 95% or even at least 97.5%, even more preferably at least

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98% or even at least 99% identity, in particular at least 99.5% or even at least 99.75% identity to the entire sequence identified by SEQ ID NO: 1, with the proviso that amino acid residue 51 is leucine,

Isoleucine, valine or alanine.

Alternatively or additionally, the strain preferably has an ASL (or argH) gene having a sequence of at least 70%, preferably at least 75%, more preferably at least 80%, still

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preferably at least 85% or even at least 90%, nor

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preferably at least 95% or even at least 97.5%

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Identity, in particular at least 99% or even at least 99.5% identity to the entire sequence identified by SEOQ ID NO: 2, with the proviso that the ASLGene encodes an ASL with the amino acid residue 51 leucine, isoleucine,

Valine or alanine encoded.

Even more preferred is a strain having an ASL comprising or having the sequence identified by SEO ID NO: 1 (preferably consisting of the sequence identified by SEO ID NO: 1

sequence).

According to a further preference, the strain has an ASL gene (on a chromosome or a plasmid) comprising or having the sequence identified by SEQ ID NO: 2 (preferably

consisting of the sequence identified by SEQ ID NO: 2).

Strain DSM 34858 was found to have the following 165 RNA: > SEQ ID NO: 3

GTTTGATCCTGGEGGAGGETACTGETATTGGGGT TEGAT TAAGECATGCAAGTCGAACGAACETTGTGTTEGTGGEG AACGGCETCAGTAACACGTGGATAACCETGECETTGGGACTGGGATAACCCCGGGAAACTGGGGATAAACCETGGATAGG TGATGCEGGECTGGAATGGTGETTCACCGAAACACCECTTEGGGGTGECCAAGGATGGGTETGEGGECGAT TAGGTAGT TGGTAGGGTAACGGECTACCAAGECCATCATCGGTACGGGTTGTGAGAGCAAGAGECCGGAGATGGAACCETGAGACA AGGTTCCAGGECCETACGGGGEGEAGEAGGEGEGAAACETECGEAATGEACGEAAGTGEGACGGGGGAACCCECCAAGTG CCACTCETTAACGGGGTGGETTTTCAGAAGTGTAAAAAGETTETGGAATAAGGGETGGGEAAGAECGGTGECAGECGE CGCEGGTAACACCGGCEAGETCAAGTGGTAGECGETTTTATTGGGECTAAAGEGTECGTAGECGGTETGATAAGTETET GGTGAAATCCCGCAGETTAACTGTGGGAATTGETGGAGATACTATCATGACTCGAGGTCGGGAGAGGETGGAGGTAC TCCCAGGGTAGGGGTGAAATCECTGTAATECTGGGAGGACCACETGTGGEGAAGGEGTECAGETGGAACGAACETGAC

GGTGAGGGACGAAAGCCAGGGGEGEGAACCCGAT TAGATACCCGGGTAGTECTGGECGTAAACGATGTGGACTTGGT GTTGGGATGGETTEGAGETGECECAGTGECGAAGGGAAGETGT TAAGTECACCGECTGGGAAGTACGGEEGEAAGEC TGAAACTTAAAGGAATTGGCGGGGGAGCACCACAACGEGTGGAGECTGEGGTTTAATTGGATTCAACGECGGACATC TCACCAGGGGEGACAGCAGTATGATGGECAGGTTGATGACETTGECTGACGAGETGAGAGGAGGTGCATGEECGECG TCAGCETCEGTACCGTGAGGEGTECTGTTAAGTCAGGEAACGAGEGAGACCCACGEEETTAGTTACCAGEGGGAECETT TGGGGTTGECGGGEACACTAAGGGGACCGECAGTGATAAACTGGAGGAAGGAGTGGACGACGGTAGGTECGTATGEC CCGAATECCCTGGGEAACACGEGGGEETACAATGGEECTGGACAATGGGT TECGACACTGAAAGGTGGAGGTAATECEC TAAACCAGGTCGTAGTTCGGATCGAGGGETGTAACTEGECETEGTGAAGETGGAATGEGTAGTAATCGEGTGTCATT ATCGECGEGGTGAATACGTEEETGETEETTGEACACACCGEEEGTCACGECACCCAAAAAGGGET TGGATGAGGECAC AGTATTTTGCTGTGGTECGAATECTGGGTTETTTGAGGAGGGEGAAGTCGTAACAAGGTAGECGTAGGGGAACCETGEGG CTGGATCACCETECTT

Therefore, in a further preferred embodiment, the 165 ribosomal RNA of the strain according to the invention has the sequence identified by SEO ID NO: 3.

The strain according to the invention (in particular the strain deposited as DSM 34858) can be cultivated under conditions known in the art (see, for example, Taubner et

al, 2023) or as disclosed in the example section.

According to a preferred embodiment of the method according to the invention, the method is a continuous process with or without cell retention, a fed-batch process, a batch process, a closed-batch process, a repetitive batch process, a repetitive fed-batch process, or a repetitive closed-batch process, preferably a continuous process, a fed-batch process, or a repetitive fed-batch process, in particular a continuous process. It is particularly preferred that the continuous process (culture) is a chemostat process (culture), in particular

with controlled pH (with or without cell retention).

In particular for continuous culture, the harvesting step may comprise removing a liquid stream from the bioreactor, the liquid stream comprising fermentation broth, methanogenic microorganisms (i.e. cells of the strain) and produced amino acids (e.g. in the supernatant of the fermentation broth), separating the methanogenic microorganisms from the liquid stream (e.g. by filtration) and recycling the

methanogenic microorganisms in the bioreactor. The

Harvested amino acids (which may be present in a liquid fraction of the fermentation broth) are then preferably further purified, e.g., by methods known in the art

Methods such as chromatography.

The strain according to the invention proved to be relatively robust during cultivation. It is therefore highly preferred that at least 50%, preferably at least 60%, further preferably at least 70%, even more preferably at least 80%, in particular at least 90% or even at least 95% of the methanogenic microorganisms (i.e., cells of the strain according to the invention) remain viable and/or intact before and during harvest. This applies in particular to the methanogenic microorganisms present in a liquid stream removed from the bioreactor (e.g., in continuous culture). The methanogenic microorganisms can then be reintroduced into

be returned to the bioreactor.

According to a further preferred embodiment, the total amino acid concentration in the supernatant of the fermentation broth (used in the harvesting step) is at least 1 μmol/L, preferably at least 5 μmol/L, more preferably at least 10 μmol/L or even at least 25 μmol/L, even more preferably at least 50 μmol/L or even at least 100 μmol/L, in particular at least 150 μmol/L. It is particularly preferred if the supernatant has a total amino acid concentration of at least 200 μmol/L, preferably at least 500 μmol/L, in particular at least 1000 μmol/L (or even higher

lower limits).

The biological fixation of molecular nitrogen (N,) is a key process in the global nitrogen cycle, where it is closely linked to the carbon cycle. With 16 mol ATP per mol N, fixed, it is one of the most expensive metabolic processes (Thamdrup 2012; Hu and Ribbe 2012). Certain strains of archaea, but also bacteria, are able to fix N, (Fernandez et al. 2019). This biological process is called diazotrophy. Diazotrophic growth was first reported in archaea in 1984, when it was shown that Methanosarcina barkeri (Murray and Zinder 1984) and Methanococcus thermolithotrophicus (Belay et al. 1984) in the

were able to fix N. The factors responsible for the reduction of N,

required enzymes are nitrogenases, which are found in the nif gene cluster

be coded (Raymond et al. 2004).

Jones & Stadtman, 1977, examines the effects of selenium and tungsten on the growth of Methanococcus vannielii. The document teaches that the microorganism's growth on formate is significantly stimulated by selenium and tungsten. Similarly, Dridi et al., 2012, refer to the tungsten-enhanced growth of Methanosphaera stadtmanae. Furthermore, Lobo & Zinder, 1988, addresses diazotrophy and nitrogenase activity in the archaeon Methanosarcina barkeri 227.

The strain according to the invention has been found to be capable of producing amino acids under nitrogen-fixing conditions. In particular, the nitrogen source may comprise nitrogen gas. (Alternatively or additionally, the nitrogen source may preferably comprise ammonia gas, ammonium and/or

nitrogen gas).

When the nitrogen source comprises nitrogen gas, a certain ammonium concentration range has been found to be particularly advantageous for energy production (under N-fixing conditions). Thus, it is preferred that the ammonium concentration in the fermentation broth is 0.1 mmol/L to 200 mmol/L, preferably 2 mmol/L to 100 mmol/L, more preferably 4 mmol/L to 40 mmol/L, even more preferably 5 mmol/L to 35 mmol/L, even more preferably 6 mmol/L to 30 mmol/L, in particular 7 mmol/L to 25 mmol/L, or even 10 mmol/L to 20 mmol/L. It will be appreciated by those skilled in the art that, particularly during continuous culture, the concentration in the broth may fluctuate (until a steady state is reached). However, it is preferred that the ammonium concentration remains in one of the aforementioned ranges (e.g. 0.1 mmol/L to 200 mmol/L or 10 mmol/L to 20 mmol/L) for at least 5 min, preferably at least 10 min, more preferably at least 20 min, even more preferably at least 1 h, in particular at least 5 h or even at least 10 h (or at least 20 h or at least 40 h).

Furthermore, it is preferred if the tungstate concentration (in particular the orthotungstate, i.e.

WO, ”-concentration) in the fermentation broth below 0.1 umol/L,

preferably below 0.01 umol/L, especially below 0.001 umol/L (especially if the fermentation broth is essentially tungstate-free). It was found that this

enables more efficient amino acid production.

In a preferred embodiment, an electron donor (or electron donor compound) suitable for the methanogenic microorganisms (i.e., cells of the strain according to the invention) is supplied to the bioreactor. In particular, hydrogen gas (i.e., molecular hydrogen or H2), acetate, a methyl compound, preferably selected from methylamines, methyl sulfides, and methanol, any other alcohol, preferably a secondary alcohol such as 2-propanol or 2-butanol, a methoxylated aromatic compound, and/or formate (as electron donor compounds) are supplied to the bioreactor. Hydrogen gas, acetate, methanol, or combinations thereof (e.g., methanol and acetate) are particularly preferred.

According to a further preferred embodiment, methane (produced by the methanogenic microorganisms) is extracted from the

Bioreactor harvested.

The amino acids produced by the methods and uses disclosed herein can be either the D- or the L-isomer, or both. The amino acid can be, for example, 2-aminobutyrate, alanine, beta-alanine, arginine, aspartate, carnitine, citrulline, cystine, dehydroalanine, glutamate, glutamine, glycine, hydroxyproline, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, pyroglutamate, pyrroproline, pyrrolysine, selenocysteine, selenomethionine, serine, homoserine, threonine, tryptophan, tyramine, tyrosine, or valine. The production of glycine (preferably in addition to other amino acids or as a single amino acid, e.g., in the

purities disclosed here) is particularly preferred.

The (harvested) amino acids preferably comprise at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, even more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least

18, in particular all of the 20 canonical amino acids (i.e. Asp,

Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, Trp, Ile, Phe, Leu, Lys, Cys and Pro). It is particularly preferred if the (harvested) amino acids comprise one or more of the following: essential amino acids (essential for human consumption), branched-chain amino acids (BCAAs) and glutamate, wherein preferably at least 50 mol%, preferably at least 60 mol%, in particular at least 70 mol% of the (produced or harvested) amino acids are essential amino acids, branched-chain amino acids (BCAAs) or glutamate

are.

According to a further preferred embodiment, the total amino acid production rate per volume (of the supernatant) of the fermentation broth is at least 0.01 µmol L*h, preferably at least 0.05 µmol L*h, more preferably at least 0.1 µmol L*h, even more preferably at least 0.5 µmol L*h, even more preferably at least 1.0 µmol L*h, in particular at least 5 µmol L*h or even at least 10 µmol L*h (or even higher, preferably at least 100 µmol L*h, more preferably at least 1000 µmol L*h, in particular at least 10000 µmol L*h).

According to yet another preferred embodiment, the total amino acid production rate per biomass is at least 0.1 umol g h, preferably at least 0.5 umol g h, more preferably at least 1.0 umol g h, even more preferably at least 5 umol g h, even more preferably at least 10 umol g h, in particular at least 50 umol g h or even at least 100 umol g h (or even higher, such as at least 1000 umol g h).

The strain of the invention can be natural (e.g., natural isolates or laboratory strains derived therefrom) or genetically manipulated. For example, genetic manipulation in methanogenic archaea, such as site-directed mutagenesis-based manipulation, selectable markers, transformation methods, and reporter genes, is available to the skilled person, see, for example, Sarmiento et al., 2011. Fink et al., 2023, report the use of suicide vector constructs for site-specific genomic deletion in Methanothermobacter. CRISPR-based gene editing and other

CRISPR-based genetic tools are available to the expert

also available, see e.g. Adalsteinsson et al, 2021

and Mougiakos et al, 2017.

A (defined) co-culture of methanogenic microorganisms in the bioreactor has also proven to be advantageous. Accordingly, the methanogenic microorganisms (in the bioreactor) preferably comprise at least two different species (of

one of which is from the strain according to the invention).

It is very preferred that the fermentation is started with the methanogenic microorganisms (i.e. cells of the strain according to the invention) in chemically defined fermentation medium

becomes.

In a further preferred embodiment, the

Fermentation carried out under anaerobic conditions.

When the process is a continuous process (continuous culture), it is preferred that the dilution rate D is 0.001 h⁻ to 1.5 h⁻, preferably 0.01 h⁻ to 0.5 h⁻, especially 0.0125 h⁻ to 0.1 h⁻.

Nitrogen-fixing conditions and/or carbon-fixing conditions have proven advantageous: Thus, according to a further preference, at least 50%, preferably at least 60%, further preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all nitrogen atoms of all nitrogen sources supplied to the bioreactor are supplied to the bioreactor in the form of nitrogen gas. In yet another preferred embodiment, at least 50%, preferably at least 60%, further preferably at least 70%, even more preferably at least 80%, even more preferably

Q

at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all carbon atoms of all carbon sources supplied to the bioreactor are in the form of carbon dioxide gas and/or

Carbon monoxide gas is fed into the bioreactor.

Typically, a sulfur source is also added to the bioreactor. This source preferably comprises cysteine (especially L-cysteine) and/or sulfide, in particular wherein the

Fermentation broth sulfide in a concentration of 0.001 - 150

mg/L, preferably 0.01 - 100 mg/L, in particular 1 to 80 mg/L and/or wherein the sulfide feed rate or cysteine feed rate is 0.0001 - 0.2 mol L"*h", preferably 0.001 - 0.05 mol L"* h"*.

A variety of different bioreactors can be used in the methods and uses disclosed herein. Liquid-phase bioreactors (e.g., stirred tank, fixed bed, single liquid phase, two liquid phases, hollow fiber membrane) are well known in the art. Multiphase bioreactors (e.g., bubble column bioreactor, trickle bed bioreactor (fixed or packed bed), fluidized bed bioreactor) can also be used. The bioreactor is typically made partly of (stainless) steel, plastic, and/or glass. Typically, the bioreactor contains at least one inlet allowing the entry of a gas or gas mixture and at least two outlets. One outlet allows the removal of a liquid stream containing the one or more fermentation products (i.e., amino acids), and the other outlet allows the removal of a gas stream (such as methane produced by the methanogenic microorganisms, i.e., cells of the strain of the invention).

In some embodiments, the bioreactor is a chemostat.

Herein, "UniProt" refers to Universal Protein Resource. UniProt is a comprehensive resource for protein sequence and annotation data. UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics, and Protein Information Resource (PIR). More than 100 people are involved in various tasks across the three institutes, including database curation, software development, and support.

involved. Website: http://www.uniprot.org/

Entries in the UniProt databases are identified by their deposition codes (herein, for example, as "UniProtDepositCode" or simply "UniProt" followed by the deposition code), typically a six-letter alphanumeric code (e.g., "D9PVR6"). Unless otherwise noted, the UniProt database status for all entries referenced herein is March 1, 2023 (UniProt/UniProtKB Release 2023 01).

"Percent (%) amino acid sequence identity" or "X% identical" (such as "70% identical") with respect to a reference polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after the sequences have been aligned, gaps introduced where appropriate, and no conservative substitutions considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR), or the pairwise "needle" sequence alignment application of the EMBOSS software package. One of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the compared sequences. However, for the purposes herein, % amino acid sequence identity values are calculated using the sequence alignment of the “Needle” computer program of the EMBOSS software package (5Publicly available from European

Molecular Biology Laboratory; Rice et al., 2000).

The Needle program can be accessed from the website http://www.ebi.ac.uk/Tools/psa/emboss_needle/ or downloaded for local installation as part of the EMBOSS package from http://emboss.sourceforge.net/. It runs on many widely used UNIX and UNIX-like systems.

Operating systems like Linux.

To align two protein sequences, the needle

Program preferably executed with the following parameters:

Command line: needle -auto -stdout -asequence SEQUENCE_ FILE _A -bsequence SEQUENCE FILE _B -datafile EBLOSUM62 gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 aformat3 pair -proteinl -protein2 (Align format: pair Report _file: stdout)

The percentage amino acid sequence identity of a particular amino acid sequence A to, with, or against a particular

Amino acid sequence B (which can alternatively be described as a specific

amino acid sequence A which has or comprises a certain percentage amino acid sequence identity to, with or against a certain amino acid sequence B) is defined as

calculated as follows: 100 x the fraction X/Y

where X is the number of amino acid residues determined to be identical by the Needle of the sequence alignment program in the alignment of A and B of that program, and where Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity from A to B will not be equal to the percent amino acid sequence identity from B to A. In cases where "the sequence of A is more than N% identical to the entire sequence of B," Y is the total sequence length of B (i.e., the total number of amino acid residues in B). Unless expressly stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the Needle computer program.

Q

"Percent (%) sequence identity" with respect to a reference nucleotide sequence is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the reference sequence after the sequences have been aligned and gaps have been introduced, if applicable, and no conservative substitutions have been considered as part of the sequence identity. Gaps result in a lack of identity. Alignment for the purpose of determining percent nucleotide sequence identity can be achieved in various ways that are within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR), or the pairwise "needle" sequence alignment application of the EMBOSS software package. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the compared sequences. However, for the purposes herein, % nucleotide sequence identity values are used below

Using the sequence alignment of the computer program “Needle”

the EMBOSS software package (Publicly available from the European Molecular Biology Laboratory; Rice et al, 2000).

The Needle program can be accessed from the website http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html or downloaded for local installation as part of the EMBOSS package from http://emboss.sourceforge.net/. It runs on many widely used UNIX or UNIX operating systems.

similar operating systems like Linux.

To align two nucleotide sequences, the needle

Program preferably executed with the following parameters:

Command line: needle -auto -stdout -asequence SEQUENCE_ FILE _A -bsequence SEQUENCE FILE _B -datafile EDNAFULL gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 aformat3 pair -snucleotidel -snucleotide2 (Align format: pair Report _file: stdout)

The percentage nucleotide sequence identity of a particular nucleotide sequence A to, with, or against a particular nucleotide sequence B (which may alternatively be formulated as a particular nucleotide sequence A having or comprising a particular percentage nucleotide sequence identity to, with, or against a particular nucleotide sequence B) is determined as

calculated as follows: 100 x the fraction X/Y

where X is the number of nucleotides evaluated as identical matches by the sequence alignment program needle in the alignment of A and B of this program, and where Y is the total number of nucleotides in B. It is understood that if the length of the nucleotide sequence A is not

Q

equal to the length of the nucleotide sequence B, the % nucleotide sequence identity of A to B is not equal to the % nucleotide sequence identity of B to A. In cases where "a sequence of A is at least N% identical to the entire sequence of B", Y is the entire length of B. Unless explicitly stated otherwise, all % nucleotide sequence identity values used herein are calculated as described in the immediately preceding paragraph using the Needle-

computer program.

For nucleotide sequences, "sequence similarity", "sequence identity", "sharing of a sequence" and similar terms also apply to the reverse complement of a sequence, i.e. the

Q

Expression "Sequence A is 80% identical to sequence B" applies

even if "Sequence A is 80% identical to the reverse

complement (or antisense sequence) of sequence B",

While the nucleotide sequences disclosed herein are listed as DNA sequences, it is understood that the reverse complement of the DNA sequences can be readily determined by one of ordinary skill in the art. It is also understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uridine nucleotide. Accordingly, whenever the present invention refers to a DNA sequence, the corresponding RNA sequence and the reverse complement of the DNA sequence are also to be understood as being within the scope of protection.

When reference is made herein to a "purity of an amino acid" (e.g., in wt.%), the purity refers to the relative purity, i.e., the relative amount of the amino acid among all amino acids present. For example, the purity of an amino acid X in wt.% can be calculated by dividing the total amount of X by the total amount of all amino acids present (on a weight basis). In other words, the presence of compounds other than amino acids does not affect the calculation of the

Amino acid purity as used herein.

The present invention further relates to the following

Embodiments:

Embodiment 1. A strain of Methanothermobacter with a proline production rate that is lower than the proline production rate of Methanothermobacter marburgensis, deposited with the DSMZ under the deposit number DSM 2133 (Methanothermobacter marburgensis DSM 2133) and/or with a glycine production rate that is higher than the glycine production rate of Methanothermobacter marburgensis DSM 2133.

Embodiment 2. Strain according to embodiment 1, wherein the strain has a proline content of the total secreted amino acids which is lower than the proline content of the total secreted amino acids of Methanothermobacter marburgensis DSM 2133, and/or wherein the strain has a glycine content of the total secreted amino acids which is higher than the glycine content of the total secreted

Amino acids of Methanothermobacter marburgensis DSM 2133.

Embodiment 3. A strain of Methanothermobacter having an ASL with an amino acid residue 51 which is leucine, isoleucine, valine or alanine, preferably the

Strain of embodiment 1 or 2.

Embodiment 4. Strain according to any one of embodiments 1 to 3, wherein amino acid residue 51 of ASL is leucine

or isoleucine.

Embodiment 5. Strain according to one of embodiments 1 to 4, wherein the strain of Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermautotrophicus, Methanothermobacter crinale, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter sp. CaT2, Methanothermobacter sp. DP, Methanothermobacter sp. KEPCO-1, Methanothermobacter sp. THM-1 or Methanothermobacter tenebrarum is; preferably Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermautotrophicus, Methanothermobacter crinale, Methanothermobacter thermoflexus, Methanothermobacter thermophilus or Methanothermobacter tenebrarum; in particular Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter

thermoautotrophicus or Methanothermobacter tenebrarum.

Embodiment 6. Strain according to any one of embodiments 1 to 5, wherein the strain has an ASL which has a sequence with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% or even at least 97.5%, even more preferably at least 98% or even at least 99% identity, in particular at least 99.5% or even at least 99.75% identity to the entire sequence represented by SEQ ID

NO: 1 identified sequence, provided that it

amino acid residue 51 is leucine, isoleucine, valine or alanine.

Embodiment 7. Strain according to any one of embodiments 1 to 6, wherein the strain has an ASL gene having a sequence with at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85% or even at least 90%, even more preferably at least 95% or even

Q Q

at least 97.5% identity, in particular at least 99% or even at least 99.5% identity to the entire sequence identified by SEO ID NO: 2, with the proviso that the ASL gene for an ASL with the amino acid residue 51 leucine, isoleucine,

Valine or alanine encoded.

Embodiment 8. Strain according to any one of embodiments 1 to

7, where the strain is Methanothermobacter marburgensis.

Embodiment 9. The strain of embodiment 8, wherein the strain has an ASL that encodes the sequence identified by SEO ID NO: 1

sequence includes.

Embodiment 10. Strain according to embodiment 8 or 9, wherein the strain has an ASL gene encoding the sequence identified by SEO ID NO: 2

identified sequence.

Embodiment 11. A strain of Methanothermobacter marburgens deposited with the DSMZ on November 21, 2023, under the accession number DSM 34858.

Embodiment 12. Strain according to any one of embodiments 1 to 11, wherein the 16S ribosomal RNA of the strain comprises the RNA sequence represented by SEQ ID

NO: 3 identified sequence.

Embodiment 13. A process for producing amino acids by fermentation in a bioreactor, wherein the bioreactor comprises cells of the strain according to any one of embodiments 1 to 12 in a fermentation broth, wherein the process comprises at least

the steps include

- supplying a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source and preferably a sulfur source to the bioreactor under conditions such that the cells

produce amino acids; and

- Harvesting at least part of the amino acids from the

Fermentation broth.

Embodiment 14. The method according to embodiment 13, wherein the part comprises a single amino acid, preferably a single canonical amino acid, in a purity of more than 50 wt.%, preferably more than 60 wt.%, more preferably more than 70 wt.%, even more preferably more than 80 wt.%, in particular more than 90

Q

% by weight.

Embodiment 15. The method according to embodiment 13, wherein said portion comprises at least two, preferably at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, even more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least 18, in particular all 20 canonical amino acids; wherein said portion preferably comprises at least arginine and/or aspartate

includes.

Embodiment 16. The method according to any one of embodiments 13 to 15, wherein the method is a continuous process with or without cell retention, a fed-batch process, a batch process, a closed-batch process, a repetitive batch process, a repetitive fed-batch process or a repetitive closed-batch process, preferably a continuous process, a fed-batch process or a repetitive fed-batch process, in particular a continuous process with or without

Cell retention.

Embodiment 17. The method of any one of embodiments 13 to 16, wherein the nitrogen source comprises ammonia gas, ammonium and/or nitrogen gas.

Embodiment 18. The method according to embodiment 17, wherein the nitrogen source comprises nitrogen gas; preferably wherein at least 50%, preferably at least 60%, further preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all nitrogen atoms supplied to the bioreactor of all nitrogen sources are supplied to the bioreactor in the form

of nitrogen gas.

Embodiment 19. The process according to any one of embodiments 13 to 18, wherein at least 50%, preferably at least 60%, further preferably at least 70%, even more preferably

Q

at least 80%, more preferably at least 90% or even

Q

more preferably at least 95%, in particular at least 99% or even at least 99.9% of all carbon atoms of all carbon sources supplied to the bioreactor in the form of carbon dioxide gas and/or carbon monoxide gas to the bioreactor

be supplied.

Embodiment 20. The method according to any one of embodiments 13 to 19, wherein the sulfur source is supplied to the bioreactor, wherein the sulfur source is preferably sulfide, cysteine or

cysteine and sulfide.

Embodiment 21. The process according to any one of embodiments 13 to 20, wherein hydrogen gas, acetate, a methyl compound, preferably selected from methylamines, methyl sulfides and methanol, any other alcohol, preferably a secondary alcohol such as 2-propanol or 2-butanol, a methoxylated aromatic compound and/or formate are fed to the bioreactor; wherein preferably hydrogen gas, acetate, methanol or combinations thereof are fed to the bioreactor

be supplied.

Embodiment 22. The method of embodiment 21, wherein harvesting comprises removing a liquid stream from the bioreactor, the liquid stream comprising fermentation broth, cells of the strain and produced amino acids, separating the cells of the strain from the liquid stream and recycling cells of the

strain back into the bioreactor.

Embodiment 23. The method of embodiment 22, further comprising the step of cleaning the produced

Amino acids from the fluid stream.

Embodiment 24. Use of the strain according to any one of embodiments 1 to 12 for the production of amino acids,

especially glycine.

Embodiment 25. Fermentation broth containing cells of the strain

according to any one of embodiments 1 to 12.

Embodiment 26. A bioreactor containing the fermentation broth

of embodiment 25.

The present invention is further illustrated by the following figures and examples, without being limited thereto.

be.

Figure 1: Amino acid production by Methanothermobacter marburgensis Arkeon (DSM 34858) was compared with amino acid production by Methanothermobacter marburgensis DSM 2133 (Marburg strain) under the same culture conditions. The (A) proline production rate and (B) its proportion of total amino acids ("AA") were significantly higher for DSM 34858.

lower.

Figure 2: Multiple-sequence alignment of argH, which encodes ASL, from several Methanothermobacter genomes (ArgH_Arkeon: argH from Methanothermobacter marburgensis DSM 34858, ArgH_DSMZ: argH from Methanothermobacter marburgensis DSM 2133; ArgH_CaT2: argH from Methanothermobacter sp. DSM 24414; ArgH_deltaH: Methanothermobacter thermautotrophicus Delta H; ArgH_wolfei: Methanothermobacter wolfei). The first two nucleotides of the codon encoding amino acid residue 51 of ASL are highly conserved among the previously known Methanothermobacter genomes. In contrast, the second nucleotide of the codon (highlighted) is mutated in the inventive strain DSM 34858, resulting in a change from proline to leucine at position 51 of ASL.

Figure 3: Multiple-sequence alignment of Methanothermobacter ASL sequences published in UniProt (UniProt accession numbers indicated in blue) and Methanothermobacter marburgensis DSM 34858 ("DSM 34858"). In general, position 51 (highlighted) is highly conserved. In contrast, in the inventive strain DSM 34858, position 51 of ASL is

Leucine.

Figure 4: The amino acid production by Methanothermobacter marburgensis Arkeon (DSM 34858) was compared with the amino acid production by Methanothermobacter marburgensis DSM 2133 (strain Marburg) under the same culture conditions

The (A) glycine production rate and (B) its proportion of

the total amino acids ("AA") was significantly higher for DSM 34858.

Examples

Example 1: Amino acid production and active secretion by

Methanothermobacter marburgensis Arkeon (DSM 34858)

Continuous culture of Methanothermobacter marburgensis Arkeon (deposited at the DSMZ under the accession number DSM 34858 on 21 November 2023) for amino acid production was

successfully established. MM medium was used.

MM Medium: NH Cl 1 2.1 g/L and KH 2PO 6.8 g/L in ddH 2O. 200x trace element (TE) solution to be added (e.g. up to a concentration of 5 ml per L MM medium): Titriplex I 9 g/L, add 800 ml H 2O and adjust to pH 6.5 with 5 mol/L NaOH solution, then add (up to the target concentration): MgCl 2 6H 2O 8 g/L, FeCl 4 4H 2O 2 g/L, CoCl 2 6H 2O 40 mg/L, NiCl 1 6H 2O 240 mg/L, NaMoO 2 2H 2O 40 mg/L, then adjust to pH 7.0 and a volume of 1 L with 1 mol/L NaOH and ddH 2O. The medium can also be used e.g. B. 3.6 g/L NaHCO23 as a carbon source. As a sulfur source, e.g. 2 ml 0.5 mol/L Na,S-9H,0 per liter can be used.

Anaerobic treatment and autoclaving are added.

The same medium was used as feed medium for the

continuous cultivation mode.

Experiments were conducted in 2 L bioreactors (Eppendorf AG, Hamburg, Germany) and in a 15 L bioreactor (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany).

To ensure anaerobic conditions in the reaction vessel, the entire system was flushed with a H,/CO,-, N,- or H,/CO,/N,- mixture for 10 minutes before inoculation.

Cultivation was carried out at 65°C and a stirrer speed of 100 to 1200 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and 100 to 1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). The pH was measured using a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz,

Switzerland) and kept constant at a value of 7.0.

The oxidation-reduction potential (ORP) was measured using a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol/L Na‚,S-9H‚ solution was used as the sulfur source and constantly fed to the bioreactor at, for example, 0.2 mL/h to 1.32 mL/h. The MM medium was applied using an analog peristaltic pump. The MM medium feed rate, the sodium Na‚,S:9H‚ feed rate, and the titration were recorded gravimetrically or adjusted by the pump speed. The bioreactor volume was kept constant by withdrawing culture suspension via a dip tube with a peristaltic pump controlled to a fixed bioreactor weight or by using a tube at a fixed height as a level control system. The withdrawn suspension was collected in a harvesting bottle, and its volume was recorded gravimetrically. All solutions were anaerobized by purging with N,, H,/CO,, or H,/CO,/N,. To maintain anaerobic conditions, all bottles were pressurized with N,. Pure H,/CO, (4:1) was used as the substrate. The CO, gas flow was controlled by the MX4/4 system (Eppendorf AG, Hamburg, Germany). The H, gas flow was controlled by the C100L unit (Sierra Instruments, Monterey, USA).

controlled.

Several runs of continuous cultures of M. marburgensis Arkeon were carried out under anaerobic conditions. The volume of one run ranged from 1.6 L to 10.29 L. The dilution rate (D) was varied between runs, specifically with values for D from 0.0125 h⁻¹ to 0.05 h⁻¹. Volume gas per volume liquid per minute (vvm) was also varied between runs, e.g., from 0.125 to 0.5. The agitation (rpm) was also varied between runs, e.g., from 375 to 1500. As a sulfur source, 0.5 mol/L Na⁻¹S was supplied at a rate of e.g., 0.2 ml/h to 1.32 ml/h. Typically, the ammonium concentration was maintained between 15 mmol/L and 35 mmol/L.

The production and secretion of the following amino acids (in combination) into the culture supernatant was typically observed: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, Lys.

Amino acid production rates were measured up to about 40 umol L”* h”*

(volumetric) and up to about 900 umol h7”!g” (specific per

biomass). Cys and Pro were not detected due to analytical limitations, but are expected to

are also produced and secreted.

In summary, a reliable production of

Amino acids observed in continuous culture.

Example 2: Genomic and physiological analysis of

Methanothermobacter marburgensis Arkeon (DSM 34858)

We found that Methanothermobacter marburgensis Arkeon (DSM 34858) was very similar to Methanothermobacter marburgensis DSM 2133 (strain Marburg) at the genomic level. Since M. marburgensis strain Marburg was the closest to this new strain, we compared the two strains at a detailed genomic level (SNPs, deletions, insertions) and in relation to

on amino acid secretion and specific growth rate. METHODS Genomic DNA extraction

As a first step, we harvested cells from M. marburgensis strain Arkeon. Therefore, we anaerobically centrifuged 12 ml of an overnight culture, which grew to an OD600 of approximately 0.4. The supernatant was discarded, and the pellet was resuspended in 300 μl of anaerobic sterile growth media. 5 μl of the cell wall-degrading enzyme PeiW [c = 5 μg/ml] was added to the cell suspension and incubated at 65°C for approximately 1 h until complete lysis occurred. We used this lysate of M. marburgensis for genomic DNA extraction using the Promega Wizard® Genomic DNA Purification Kit. Extracted genomic DNA was stored briefly at 4°C until shipping. Purity and yield were determined using the Qubit-

High sensitivity test measured. Nanopore sequencing

Native barcoding of genomic DNA NBE_ 9065 _v109 revAM protocol using kits SOL-LSK109 with native barcoding extensions 1-12 EXP-NDB104 and FLO-Min106 (R9.4.1) for flow cells. In brief, end repair for adapter attachment, ligation of native barcodes, ligation of sequencing adapters, PrimeFlow cell, and loading of DNA onto the flow cell. Standard

Sequencing operation.

Illumina sequencing

100 ng of high molecular weight DNA at a concentration of at least 10 ng/uL was sent to the Eurofins NGS sequencing facility in Konstanz, Germany. Library preparation and Illumina sequencing were performed by the

Eurofins' NGS sequencing facility. Genome assembly and alignment to reference genomes

A hybrid assembly was performed to generate the genome for M. marburgenis strain Arkeon. This included a high-depth set of Nanopore reads as well as data from an Illumina sequencing run. First, several different assemblers (Flye, Miniasm, Canu, and Raven) were used to create pure Nanopore assemblies, each using different subsets of the reads. Trycycler was used to circularize the genome by combining these results, and the Illumina reads were used to polish the final sequence (taking advantage of the higher accuracy of the Illumina platform). The final genome of M. marburgensis strain Arkeon had high identity (>99.9%) to both the original M. marburgenis DSM 2133 genome deposited at NCBI and the one recently collected at the University of Tübingen.

deposited M, marburgenis DSM 2133. Identification of SNPs, deletions and insertions

To identify potential variations in the strain of interest, the sequencing data were mapped to the genomes of M. marburgenis DSM 2133 (including the genome originally uploaded to NCBI as well as a recent genome from the University of Tübingen) using BowTie2, which was launched from the Geneious platform using default parameters. Variations, including SNPs, were identified using the integrated annotation tool in Geneious. A minimum variant frequency of 25% was used to allow for possible heterozygous effects.

Otherwise, standard parameters were sufficient. Preparation of growth media

One L of media consisted of 2.1 g ammonium chloride, 6.8 g potassium dihydrogen phosphate, 3.6 g sodium bicarbonate, 1 ml 0.025%

10% resazurin stock solution and 1 ml trace element solution. The pH of the medium was adjusted to 7.2 with 10 mol/L sodium hydroxide solution. One liter of trace element solution consisted of 90 g nitrilotriacetic acid, 40 g magnesium chloride hexahydrate, 10 g iron(II) chloride tetrahydrate, 0.2 g cobalt chloride hexahydrate, 1.2 g nickel chloride hexahydrate, and 0.2 g sodium molybdate dihydrate. The pH was adjusted to 7 with 1 mol/L sodium hydroxide solution. After weighing the chemicals, the medium was gassed with N2/CO2 (20% CO2 in N2) for 40 min. The medium was then transferred to the anaerobic chamber and reduced with 2 ml of 0.5 mol/L sodium sulfide nonahydrate solution. The reduced medium was dispensed in 25 ml volumes into 125 ml serum bottles. The bottles were sealed with blue butyl rubber stoppers (Chemglass Inc.) and crimped with aluminum caps. Subsequently, the headspace of the serum bottles was exchanged for H2/CO2 (20% CO2 in H2) in three gas-vacuum cycles. The final overpressure was set to 1.8 bar. In the final step, all finished media bottles were incubated for 20 min at 121 °C and 2 bar overpressure.

autoclaved. Phenotypic characterization approach

For both M. marburgensis strains, a preculture was prepared in the same medium as used for phenotypic characterization. Both strains were inoculated to the same optical density, based on the optical density of the preculture in the anaerobic chamber (5% H in N) at 6 p.m. After incubation at 65 °C overnight, the cultures were treated with H/CO (20% CO in H) to a volume of 1.8 bar overpressure and further incubated at 65 °C. This procedure was repeated three more times over the next 8 h. This allowed for a time-course experiment with exponential growth. Each sampling consisted of measuring the optical density at 600 nm and subsequent centrifugation of the samples for 4 min at 16,000 g at room temperature. While the pellet was discarded, the supernatant was submitted for amino acid quantification by HPLC-MS. A fifth sample was taken the next morning to obtain a sample from the late stationary growth phase for comparison purposes.

exponential growth phase.

RESULTS

Genome assembly

A new genome standard was assembled for the production strain M. marburgensis Arkeon (DSM 34858). The assembly of this genome was significant for several reasons. First, it provides a new standard for future analyses based on the Arkeon strain. Second, it provides evidence of genomic stability in the strain as well as information on likely mutations in the genome. This allows the unit to evaluate behavioral differences compared to M. marburgensis DSM 2133 (i.e., the one linked to the original genome stored at NCBI; NC_014408). Finally, to our knowledge, this is the first genome of an M. marburgensis strain assembled using hybrid sequencing, using both the Illumina and Oxford Nanopore Technology (ONTs) sequencing platforms.

to create a structurally and base-pair accurate genome.

We compared the Arkeon genome with the above-mentioned NCBI genome of M. marburgensis DSM 2133 and a genome of M. marburgensis DSM 2133 recently published by Casini et al. (2023) from the University of Tübingen. The Arkeon genome was 1,634,309 base pairs long, compared to 1,634,695 (NCBI) and 1,634,705 (Tübingen). Furthermore, pairwise agreement was achieved between all three genomes when aligned with Mauve. In other words, the genomes are highly identical, suggesting that our alignment

has a very high accuracy (Table 1).

Table 1: Comparison of the genomes. Arkeon refers to the genome of M. marburgensis Arkeon, NCBI refers to the genome associated with

Deposit NC_ 14408 is stored at NCBI, and Tübingen

refers to the genome published by Casini et al. (2023).

Genome % Identity Arkeon vs. NCBI 99.96

Arkeon vs. Tübingen 99.97

NCBI vs. Tübingen 99.99

Arkeon vs. Tübingen vs. NCBI 99.97

Finally, Prokka was used to name genes, including protein-coding, tRNA, and rRNA genes. Prokka identified 1,736 protein-coding genes, 38 tRNA genes, and 7 FrRNA genes. EggNOG 6.0 was used to improve functional annotations of the protein-coding genes and to obtain a good picture of the organism's metabolic capabilities.

Assessment of mutations

The genome alignments were examined to determine potentially interesting mutations that may have occurred in the Arkeon production strain. It appears that there are relatively few mutations, and of those identified, a significant proportion are likely due to small-scale assembly errors. In particular, so-called homopolymers (i.e., sections of the genome in which the same letter is repeated multiple times) pose a challenge for modern sequencing platforms, particularly with regard to their length. We identified 42 differences between the Tübingen genome and the Arkeon genome, of which 16 were homopolymer length changes and 26 were detected differences listed in Table 2, such as deletions and SNPs that resulted in silent or meaningful mutations in open reading frames. In addition, we identified 102 differences between the NCBI genome and ours, of which 10 were homopolymer length changes. We believe that the Arkeon genome as well as the Tübingen genome have higher assembly accuracies than the original NCBI genome, which was assembled over 10 years ago with

older technologies were assembled.

Table 2: SNPs and deletions in the Arkeon genome compared to

DSM 2133 (Tübingen sequencing)

a U hate Tyan] em Position in the Arkeon genome Length : acid : nucleotide change : change ; polymorphism type : effect frequency Notes change : : 8 : 8 NT N EEE BE 587% > N Occurs between genes - probably 454,963 18 * TCTTTCCATGGTTEGATE : ; Deletion : 67 9% between the TATA boxes, probably : : ; : 7 minimal effects on expression 491,708 1 GSX "SNP (Transition) 96.50% Silent ; ; : i Deletion 87.4% -> Single codon deletion, 15 AA from the end of the 1,494,011 3 : A-> : (TGC)3 -> (TGC)2 : GCA -> ; (Tandem repeat : Deletion 89% gene (respiratory chain complex I, subunit ; : : ; deletion) ; a 1) 427,099 1 : : 7 : ; Deletion © Frame Shift 95.90% Possible frameshift at 1/3 of hdrB, leading to an early stop codon ) : : ; ; ; 7A Potential frameshift in the last "1/4 of the ; ; ; { ; protein, changes the sequence and leads to a slightly early stop codon in the transglutaminase family protein 1,146,087 1 : : -C : : Deleti IF Shift 93.50 % ) ; ; : ; SIEHON : Fame SA A ; ; : i : 94.0% -> Potential frameshift in the first -1/4 of the protein, resulting in an early stop codon

leads

: : : ; ; Potential frameshift 1/2 way through 1,343,294 1 : : -T : ; Deletion : Frameshift 94.30% Protein, leading to early stop codon (DUF11; ; : ; ; domain-containing protein)

Potential frame shift in the last 1/4 of the

; ; ; i . ; . Proteins, resulting in an early stop codon 1,354,592 1 : : -A : ; Deleti UF Shift 90.90 % : : : ; CIEHON ; Fame SM 70 (Class | SAM-dependent DNA methyltransferase) : : 3 TAC-> i a ; . 903,826 1 : C->T 5 54T SNP (Transition) : None 99.80 % Silent 1,169,849 1 ; G->A i GGG-> | SNP (Transition) ; None 95.50 % Silent : : PL GGA :

SNP (Transition): Substitution 100.00% ORC1 type DNA replication protein Cdc6-1

282.089 1.3 G>R C->T : ve G ? SNP (Transition) : Substitution 100.00% cobalt ECF transporter T component CbiQ : : i CAC-> | u : nn - . . 389,110 1 : H->Y G->A ; TAC 1 SNP (transition): substitution 97.60% 3H domain-containing protein ET EA N a 507,380 1: L->P: A->G: CCA 1 SNP (transition); Substitution 92.80% MFS transporter : : i GTC-> SNP : in rn 577,283 105 V>L C->G So cte (transversion); Substitution 33.50% homoisocitrate dehydrogenase 652.060 1 PL CT ; CCA -> ; SNP (transition) ; Substitution 95.00% GTP-dependent dephospho-CoA kinase. ; ; ii CTA : ’ family protein : : i CCG-> | u : nn 2 .

652,762 1 P->L C->T 5 c1ie ; SNP (transition): Substitution 97.50% argininosuccinate lyase (ASL) TTETNUUNTENTTTTNNTENUUNUNUU EU ACEI> mem U coenzyme F420 hydrogenase/dehydrogenase, 661,870 1: T->A: T->C: ; SNP (T ti: Substituti 96.50% . . . N

; ; ai GCC (Transition) : UDSEEUMON ) beta-subunit C-terminal domain : : i AGG-> | N : nn nn . 833.823 1 ; R->K C->T © AG SNP (Transition) : Substitution 97.50 % ATP-dependent DNA helicase 965.067 1 ; E->K G->A : ; SNP (Transition) : Substitution 97.50 % hypothetical protein A EUTIN NUN GGT IS UT ee 1,027,633 1 i G->D : C->T : GAT i SNP (Transition) : Substitution 100.00 % hypothetical protein : : i GAC-> i LG nn N 1,182,802 1 1 D->N : C->T ; AAC ; SNP (Transition) : Substitution 99.80 % Transcription initiation factor IIB : : : -> : 8 _ 1,204,471 1° 1>M A->G ; ATA-> NP (Transition) : Substitution | 99.90% Formylmethanofuran dehydrogenase : ; ; ATG ; : Subunit B 1,253,160 1 H->Y G->A : CAT -> ; SNP (Transition) : Substitution 94.10 % Lactaldehyde dehydrogenase

; ; ; GATE SNP ; Sees al protein of the AA X end pentd ae 1,441,536 2° 1 1 M>R i A->C 5 ; . Substitution 97.50% © YP 8 PSP | ; : ; AGG2? (transversion) i ; | Family

Phenotypic comparison

The growth curve of DSM 2133 and the Arkeon strain did not vary except for time point 1 at 16.5 h. After that, the growth behavior was relatively similar. Since amino acid secretion in closed-batch experiments follows a linear correlation with the optical density, all samples were adjusted for the mean optical density per

Time set.

The amino acid production by DSM 34858 was compared with the amino acid production by DSM 2133. The proline production rate and its proportion of total amino acids

was significantly lower for DSM 34858 (see Fig. 1).

Conversely, the glycine production rate and its proportion of total amino acids were significantly higher for DSM 34858 (see Fig. 4).

While no putative genetic basis for this difference was immediately identified for glycine, this was different for proline, as an amino acid substitution was found in the enzyme ASL (involved in arginine biosynthesis, which is closely linked to proline biosynthesis). See Figures 2 and 3.

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Claims (10)

1. A strain of Methanothermobacter with a proline production rate lower than that of Methanothermobacter marburgensis, deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the deposit number DSM 2133 (Methanothermobacter marburgensis DSM 2133) and/or with a glycine production rate higher than that of Methanothermobacter marburgensis DSM 2133. 2. Strain according to claim 1, which expresses an argininosuccinate lyase (ASL) having an amino acid residue 51 selected from leucine, isoleucine, Valine and alanine. 3. Strain according to one of claims 1 to 2, wherein amino acid residue 51 of ASL is leucine or isoleucine. 4, strain according to one of claims 1 to 3, wherein the strain of Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermautotrophicus, Methanothermobacter crinale, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter sp. CaT2, Methanothermobacter sp. DP, Methanothermobacter sp. KEPCO-1, Methanothermobacter sp. THM-1 or Methanothermobacter tenebrarum is; preferably Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermautotrophicus, Methanothermobacter crinale, Methanothermobacter thermoflexus, Methanothermobacter thermophilus or Methanothermobacter tenebrarum; in particular Methanothermobacter marburgensis, Methanothermobacter defluvii, Methanothermobacter wolfeii, Methanothermobacter thermoautotrophicus or Methanothermobacter tenebrarum. 5. Strain according to any one of claims 1 to 4, wherein the strain has an ASL which has a sequence with at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably Q at least 95% or even at least 97.5%, more preferably Q at least 98% or even at least 99% identity, Q Q in particular at least 99.5% or even at least 99.75% Identity to the entire sequence identified by SEO ID NO: 1, with the proviso that amino acid residue 51 is leucine, isoleucine, valine or alanine. 6. Strain according to one of claims 1 to 5, wherein the strain has an ASL gene which comprises a sequence with at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85% or even at least 90%, even more preferably at least 95% or even at least 97.5% identity, in particular at least 99% or even at least 99.5% identity to the entire sequence identified by SEOQ ID NO: 2, with the proviso that the ASLGene encodes an ASL with the amino acid residue 51 leucine, isoleucine, Valine or alanine encoded. 7. Strain of Methanothermobacter marburgensis deposited with the DSMZ on November 21, 2023, under the accession number DSM 34858. 8. Strain according to any one of claims 1 to 7, wherein the 165 ribosomal RNA of the strain comprises the RNA identified by SEQ ID NO: 3 sequence. 9. A process for the production of amino acids by fermentation in a bioreactor, wherein the bioreactor contains cells of the strain according to any one of claims 1 to 8 in a fermentation broth wherein the method comprises at least the steps - supplying a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source and preferably a sulfur source to the bioreactor under conditions such that the cells produce amino acids; and - Harvesting at least part of the amino acids from the Fermentation broth. 10. Use of the strain according to any one of claims 1 to 8 for Production of amino acids, especially glycine.