AT527241A2 - ACETOGENIC FERMENTATION OF CARBON MONOXIDE GAS - Google Patents

Abstract

The invention relates to a method for conditioning and thus mutating an acetogen for acetogenic fermentation of CO gas ("CO feed gas") to thereby obtain a mutated acetogen for acetogenic fermentation of CO gas, the method comprising cultivating a non-mutated strain of an acetogen to be conditioned and thus mutated for acetogenic fermentation of CO feed gas in a fermentation medium without yeast extract and vitamins for at least three consecutive growth cycles on CO gas ("CO treatment gas") as the main or sole carbon source, the CO concentration in the CO treatment gas being gradually increased over the three growth cycles, thereby producing a conditioned acetogen for acetogenic fermentation of CO feed gas.

Description

[0002] The present invention relates to the acetogenic gas fermentation, in particular of carbon monoxide (CO), which is typically contained in synthesis gas or industrial exhaust gases in varying amounts, using acetogens (acetogenic bacteria) such as Thermoanaerobacter kivui (T. kivun). The invention finds particular application in the industrial fermentation of the gaseous C1 substrates CO and CO»

as well as H2 and any mixtures thereof in bioreactors.

[0003] The invention provides a process for conditioning acetogens such as T. kivui for the acetogenic fermentation of CO-containing gases such as biomass gasification, waste gasification, blast furnace gas and other industrial exhaust gases containing varying amounts of CO, so that they are able to utilize not only co2 and m2 but also non-constant gas streams with high CO content. The invention extends to conditioned acetogens such as T. kivui produced by carrying out the process. The invention also provides mutant strains of acetogens such as T. kivur, optionally for use in carrying out the acetogenic fermentation of CO gas. The invention further provides a process for the acetogenic fermentation of CO gas. The invention also provides for the use of mutated strains of acetogens, such as 7. kivui, in carrying out acetogenic fermentation

of CO gas.

[0004] BACKGROUND OF THE INVENTION

[0005] ACETOGENESIS is, generally speaking, a process in which

Microorganisms known as acetogens, carbon dioxide (CO2) or carbon monoxide

(CO) to acetate along the Wood-Ljungdahl pathway, typically via acetyl-

CoA.

[0006] Along this pathway, the CO, in the case of CO, is first oxidized to CO» by a CO dehydrogenase in a biological water-gas shift reaction. The CO» is then bound to a methyl group by a bifunctional CO dehydrogenase/acetyl-CoA synthetase and fused with another molecule of enzyme-bound CO to form acetyl-CoA. CO can serve as the sole carbon and energy source,

while CO» as a carbon source requires H2 to provide energy.

[0007] The acetogenic ability of acetogens finds useful application in the treatment of CO» and/or CO2-containing gases such as syngas (synthesis gas) or industrial exhaust gases such as blast furnace gas or flue gases from combustion processes in order to produce useful products such as acetic acid and ethanol from these gases. This

The process is called acetogenic gas fermentation.

[0008] Acetogens are naturally able to metabolise CO via the Wood-Ljungdahl pathway (or reductive acetyl-CoA pathway) to grow autotrophically and produce acetyl-CoA. However, such growth is often inhibited by the presence of high CO concentrations due to CO toxicity. This limits the biosynthetic potential of acetogens in industrial applications. Industrial applications may contain gases with high CO content, and this CO content may also fluctuate over time, e.g. in non-

constant starting material (biomass, waste) and/or with throughput fluctuations.

[0009] The challenge posed by CO toxicity in the use of acetogens for

the treatment of gases with high CO concentration, was developed by inventors in the

No. 11, 1680-1699.

[0010] In the applicant's experience, although there has been moderate success in conditioning acetogens for CO metabolism, there is an unmet need to do this in a way that achieves microorganism growth rates that could be commercially attractive to industry (e.g. higher growth rates to make the microbial catalyst more economical). Another unmet need is to condition acetogens for CO metabolism without the need for expensive yeast extracts, vitamins and other costly additives. Such a yeast extract-containing medium is referred to in the present invention as a "complex medium" or "undefined medium", in contrast to a chemically defined medium without yeast and

nutrients.

[0011] T. kivul is a thermophilic anaerobic acetogen known to exhibit autotrophic growth using CO2/hydrogen (H2) as a carbon and energy substrate. T. kivui was originally thought to grow non-autotrophically in a high CO2 concentration or, more specifically, in a 100% substrate, but was later found to be susceptible to conditioning that promotes CO2 metabolism and autotrophic growth in a high CO2 substrate.

concentration or even in a 100% CO substrate, albeit with

unattractively low growth rates in both chemically defined media and in

complex media.

[0012] The present invention finds application in improving the growth rate of acetogens such as T. kivui in substrates with high CO

concentration and in particular in a substrate with 100% CO. [0013] SUMMARY OF THE INVENTION

[0014] ACCORDING TO A FIRST, BROAD ASPECT OF THE INVENTION, there is provided a METHOD FOR conditioning and thereby mutating an acetogen for the acetogenic fermentation of carbon monoxide (CO) gas to be fermented (i.e. CO feed gas), the method comprising culturing a non-mutated strain of the acetogen in a fermentation medium without yeast extract and vitamins for at least three consecutive growth cycles with CO gas (i.e. CO treatment gas) other than CO feed gas as the main or sole carbon source, wherein the CO concentration in the CO treatment gas is gradually increased over the three growth cycles. Thus, the CO concentration in the CO treatment gas is increased over the

successive growth cycles.

[0015] When reference is made here to "main or sole carbon source", this should be understood to mean that on a molar basis at least 50% of the biomass growth of the acetogen and/or the production of the fermentation product referred to here

is taken through which acetogens originate from the metabolism of CO.

[0016] A conditioned acetogen prepared in this way can, compared to the acetogen subjected to conditioning (i.e. the non-mutated

acetogens), have at least one mutation resulting from conditioning.

or primary carbon source.

[0018] The acetogen subjected to conditioning and the conditioned acetogen may in particular be selected from T7. kivui and mutants thereof, but the invention extends to the acetogen subjected to conditioning and the conditioned acetogen, which may be any thermophilic acetogen. Most desirable thermophilic acetogens to which the invention extends are those which have growth characteristics, optimal and/or robust growth conditions and other growth characteristics close to those of T7. kivu.

This may include optimum growth at a temperature of 55-70°C.

[0019] Both the conditioning process described here and the process for fermenting CO feed gas described here can benefit from the sensible heat of the CO gas, whether as CO feed gas or as CO treatment gas, with respect to the growth of the acetogen. In other words, the feed gas or the treatment gas can provide at least part of the heat required to achieve an optimal growth temperature for the acetogen subjected to conditioning.

is required or the conditioned acetogen is required.

[0020] THE FIRST BROAD ASPECT OF THE INVENTION EXTENDS TO the use of the conditioned acetogen thus obtained in a process for fermenting a CO feed gas and for producing a fermentation product, which is described in more detail below with reference to the fourth aspect of the invention.

The process can be part of a CO feed gas fermentation process that

in addition to the method, upstream and downstream operations are included,

as described in more detail herein.

[0021] The fermentation product can be in solid, liquid or gaseous form. In a preferred embodiment, it is a liquid or gaseous

Alcohol or acid, mono- or divalent.

[0022] The CO feed gas may be any CO-containing gas, including CO-containing industrial gases, e.g. a CO-containing industrial exhaust gas or a synthesis gas (e.g.

produced in a gasification process of e.g. biomass or waste).

[0023] The fermentation product may primarily comprise acetic acid, but the invention extends the meaning of the term "fermentation product" to downstream products produced by the fermentation of the CO feed gas or by further processing of products directly produced by the fermentation, e.g. including mono- or dihydric alcohols or lactic acid and/or

additional or other products, as more fully described below.

[0024] Carrying out the process may comprise the use of a reactor. In other words, the process may be carried out using a reactor. Such a reactor may, for example, consist of an industrial-scale fermenter or stirred tank, an airlift reactor, a bubble column reactor or

a loop reactor (plug flow).

[0025] The gas fermentation process can be carried out continuously, i.e. as part of a continuous process. Such continuous operation of the process can enable the continuous provision of fresh CO feed gas for the

conditioned acetogens used for fermentation, while

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the fermentation product which (in a preferred embodiment extracellularly) is

the conditioned acetogen is continuously collected.

[0026] The continuous collection of the fermentation product produced from the conditioned acetogen may comprise the continuous extraction or separation of the fermentation product from the fermentation medium. Such extraction or separation may, for example, involve the use of membrane processes, evaporation

or solvent extraction, typically in situ.

[0027] In the context of a continuous process which includes carrying out the process using a reactor, such a process may therefore comprise feeding fresh CO feed gas into the reactor and continuously

Include collection of the fermentation product produced in the reactor.

[0028] Such continuous recovery can either be carried out in situ in the reactor or it can involve the removal of fermentation product-rich fermentation medium from the reactor, the recovery of fermentation product from the fermentation product-rich fermentation medium to produce recovered fermentation product and fermentation product-poor or -free fermentation medium and the continuous recycling of the fermentation product

poor or free fermentation medium to the reactor.

[0029] The process can be carried out to remove all the CO from the CO feed gas undergoing fermentation. Therefore, the invention can be used to obtain a target product, but also for the biological purification of a gas stream,

e.g. an exhaust gas.

such as methane, which may be present in the feed gas.

[0031] IN PARTICULAR, ACCORDING TO THE FIRST ASPECT OF THE INVENTION, A METHOD IS PROVIDED FOR CONDITIONING AN ACETOGEN, SUCH AS Thermoanaerobacter kivui (T. kivul), FOR THE ACETOGENIC FERMENTATION OF CARBON MONITOR (CO) GAS (i.e. CO-

feed gas), the method comprising the following

successively culturing the acetogen on CO gas (CO treatment gas) whose CO concentration is progressively increased in respective growth cycles (i.e. using respective successive gaseous substrates of CO treatment gas with increased CO concentration), whereby the CO concentrations in the CO treatment gases are gradually increased from about 30% v/v to 100% v/v ("CO-

growth step"),

thereby producing a conditioned acetogen for the acetogenic fermentation of CO feed gas

is generated.

[0032] The method may comprise a prior step of culturing the acetogen for at least one growth cycle on (i.e. using a gaseous substrate of) high CO content synthesis gas (“syngas”) (“syngas growth step”)

include.

[0033] The method may also comprise a previous step of culturing the

acetogens for at least one growth cycle (i.e. using a

gaseous substrate comprising a hydrogen (H2) and carbon dioxide (CO2) gas, the

is essentially free of CO2 ("CO2-free growth step").

[0034] Preferably, the method comprises the sequential performance of firstly the CO2-free growth step ("first growth stage"), secondly the syngas growth step ("second growth stage") and thirdly the CO2 growth step

("third growth stage").

[0035] The first of the growth steps can be carried out with CO2-free gas, and the growth step can be carried out with a non-mutated strain of the acetogen. Thus, if the CO2-free growth step is the first growth step, then the CO2-free growth step can be carried out with a non-mutated strain of the

acetogens.

[0036] In this specification, the term "growth cycle" refers to a single division cycle to form a new generation of an acetogen, such as T. kivui, from a previous generation, such that in one growth cycle two cells arise from a single cell, so that the cell population of acetogens doubles. Such a "growth cycle" may also be referred to as a "passage" of a

culture that encompasses several generations of exponential growth.

[0037] Furthermore, the term "CO gas" in this specification means any gas containing or consisting of CO, including 100% v/v CO and gas with lower CO concentrations, unless it is explicitly stated in the text that 100% v/v CO or gas with a lower CO concentration is used, in which case the term has a corresponding, more restricted meaning. In this sense, the

CO gases to which the invention applies, in particular with regard to

high CO content, typically in combination with CO» and H2.

[0038] In this specification, the term "acetogen" also includes any mesophilic or thermophilic bacteria, such as T. kivui, capable of producing acetate as the end product of the anaerobic fermentation of carbon dioxide gas, usually in the presence of

hydrogen gas.

[0039] The temperature range of the medium in which such mesophilic and thermophilic bacteria are used for fermentation can be 15 to 44°C, in particular 15 to 37°C, and 37 to 85°C, in particular 45 to 85°C. Thermophilic bacteria are preferred because they use the sensible heat in the feed gas

, for example if it comes from a thermal process.

[0040] It is known to those skilled in the art that acetogenic bacteria (i.e. acetogens) are a diverse group of bacteria capable of producing acetate as a metabolic end product. The present invention has particular application with respect to thermophilic bacteria (i.e. thermophilic acetogens). Some examples of such thermophilic acetogenic bacteria to which the present invention relates (i.e. which are prepared by the inventive method for conditioning

of an acetogen for the metabolism of CO), are

Moorella thermoacetica DSM 521; Moorella thermoacetica ATCGC 39073; Moorella thermoacetica ATCC 49707; Moorella thermoacetica DSM 6867; Moorella thermoacetica ATCC 31490; Moorella thermoacetica ATCC 35608; Moorella thermoacetica DSM 2955; Moorella thermoacetica DSM 12993; Moorella thermoacetica DSM 12797; Moorella thermoacetica DSM 11768; Moorella thermoacetica ATCC 33924; Moorella thermoacetica DSM 103132; Moorella thermoacetica DSM 103284; Moorella thermoacetica DSM 21394; Moorella thermoacetica Y72; Moorella thermoautotrophica DSM 7417; Moorella glyerini DSM 11254 Moorella stamsii DSM 26217 Moorella perchloratireducens An10 Moorella mulderi DSM 14980 Moorella humiferrea DSM 23265

Thermoanaerobacter kivui LKT-1 DSM 2030

Thermoacetogenium phaeum DSM 12270.

[0041] Those skilled in the art will know that acetogenic bacteria are named for their ability to produce acetate, which is their primary metabolic end product. Acetate is a versatile chemical that has numerous industrial and commercial applications, such as in the production of

solvents, plastics and food additives.

[0042] There are several other products that can be produced from acetate, either by continued fermentation or further processing, to which the present

Invention extends to fermentation products. Such products may be

monohydric alcohols: ethanol; n-propanol; isopropanol; n-butanol; 2-butanol; isobutanol; pentanol; hexanol; heptanol; octanol;

dihydric alcohols:

malic acid;

13

[0043] Other compounds that can produce acetogens and that fall into the range of fermentation products include acetone, hydrogen, carbohydrates, proteins

and hydrocarbons.

[0044] The fermentation products can be intracellular or extracellular products

and can be gaseous, liquid or solid.

[0045] In a preferred embodiment of the invention, the fermentation products are produced as extracellular products. Optically pure products in

Cases of enantiomers are preferred.

[0046] The fermentation products can be used as chemical building blocks, animal feed,

Food or fuel may be useful.

[0047] "Unmutated" as used herein means unmutated with respect to the mutations imparted by the performance of the conditioning process of the invention and embodied by the mutated acetogens of the invention. When reference is made to a "non-mutated" acetogen, this reference can therefore include acetogens with mutations resulting from treatments other than the conditioning process of the invention. Genetically modified microorganisms also fall under the term "non-mutated" and such microorganisms can therefore also be subjected to conditioning according to the conditioning process of the invention. A

Genetic post-conditioning is also not excluded.

[0048] The (non-mutated) strain of acetogen subjected to conditioning

may be a wild-type strain of the acetogen.

[0049] The acetogen may in particular be T. kivui. In this case, the 7th kivui strain subjected to conditioning according to the method of the first aspect of the invention may be a wild-type T. kivuri LKT-1 strain,

especially DSM 2030 (ATCC 33488).

[0050] The first growth stage may preferably comprise at least three successive growth cycles, wherein after completion of each

growth cycle a fresh gaseous substrate of H» and CO»2 gas is provided.

[0051] The second growth stage may preferably comprise at least two successive growth cycles, wherein after each growth cycle a

fresh gaseous substrate is provided from syngas with high CO content.

[0052] The third growth stage may preferably comprise successive growth of the acetogen, e.g. 7. kivui, from the second growth stage for at least one growth cycle on each of five CO treatment gases with increasing CO concentration (i.e. using respective successive gaseous substrates of CO treatment gas), wherein the CO concentrations in the respective CO treatment gases (i.e. in the successive gaseous substrates of CO treatment gas) are 30% v/v, 40% vv, 50% v/v, 60% v/v and 100% respectively.

v/v are selected or preferred.

[0053] In the third growth stage, after each growth cycle, a fresh

CO treatment gas (i.e. a gaseous substrate) must be provided.

to be used in a subsequent growth cycle.

[0055] The high CO content synthesis gas may be a synthesis gas containing a larger volume fraction of CO gas. Thus, the high CO content syngas

contain more CO by volume than any other gas.

[0056] For example, the high CO syngas may contain 50% v/v or more CO. In one embodiment of the invention, the high CO syngas may contain about 52% v/v CO. Typically, the high CO syngas may

Content may contain H> and CO-> in addition to CO.

[0057] The first, second and third growth steps may each comprise culturing the acetogen in a liquid growth medium (i.e., fermentation medium).

The liquid growth medium can, for example, be a synthetic serum medium.

[0058] The first, second and third growth stages and thus the growth cycles may comprise the cultivation of the acetogen in a chemically defined minimal growth medium (i.e. fermentation medium), i.e. a growth medium which excludes growth stimulants and in particular does not contain

Contains vitamins, complex nutrients, yeast extract and/or peptone.

[0059] The method may also comprise the isolation of a strain of conditioned

Acetogens from the third growth stage.

German single nucleotide polymorphism)

[0061] If the acetogen is T. kivui, the conditioned T. kivui and thus the isolated strain of the conditioned T. kivur may have at least one mutation, deletion and/or duplication compared to the wild type 7. kivui (DSM 2030), so

that it is mutated 7. kivui compared to the wild type T. kivui (DSM 2030).

[0062] The mutations may in particular include, but are not limited to, SNP mutations. More specifically, the mutations may include, in addition to SNP mutations, at least one deletion and optionally, but preferably, at least one

duplication.

[0063] SNP mutations that may be involved may include one or more,

typically all, be of -

cbiQ, cobalt ECF transporter T component;

moC, DNA-directed RNA polymerase subunit beta’;

dapA, 4-hydroxy-tetrahydrodipicolinate synthase;

phoU, phosphate signaling complex protein;

hypF, carbamoyltransferase; and

acsV, corrinoid activation/regeneration protein.

[0064] Even more specifically, the SNP mutations that can be included can be identified, in particular with reference to the NCBI genome with accession number

NZ_CP009170.1, one or more, typically all, of

cbiQ, cobalt ECF transporter T component at position 341,995;

mMoC, DNA-directed RNA polymerase subunit beta’ at positions 1,969,972 and 1,970,146; and

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980.

[0065] In addition, the SNP mutations that can be included can be described, in particular, with reference to the NCBI genome with accession number

NZ_CP009170.1, one or more, typically all, of

phoU, phosphate signaling complex protein at position 1,459,688;

hypF, carbamoyltransferase, at position 136,300; and

acsV, corrinoid activation/regeneration protein, at position 1,903,679.

[0066] In particular, the mutated 7th kivui sequence may be derived from a clonal strain of T. kivui designated CO-1, which has a nucleotide sequence of one,

typically all of the following elements

cbiQ, cobalt ECF transporter T component as set forth in SEQ ID NO: 1;

mMoC, DNA-directed RNA polymerase subunit beta’, as set forth in SEQ ID NO: 2; and

dapA, 4-hydroxy-tetrahydrodipicolinate synthase, as shown in SEQ ID NO: 3.

[0067] In addition, the mutated 7th kivui sequence may be derived from a clonal strain of T. kivui designated CO-1, which comprises a further nucleotide sequence of

one, typically all, of

phoU, phosphate signaling complex protein as set forth in SEQ ID NO: 4;

hypF, carbamoyltransferase, as set forth in SEQ ID NO: 5; and

acsV, corrinoid activation/regeneration protein, as set forth in SEQ ID NO: 6.

[0068] The nucleotide sequence may have at least 80%, 85%, 90%, 95% or more identity with the nucleotide sequences according to SEQ ID NOs: 1 to 6 or be a sequence which, under stringent conditions, nucleotides are identical to the reverse complement of the

Nucleotide sequences according to SEQ ID NOs: 1 to 6.

[0069] Deletions that may be included may be complete or

partial deletions of one or more of the following sequences:

N-acetyltransferase of the GNAT family;

DUF881 domain-containing protein; and

hypothetical protein.

[0070] More specifically, deletions, in particular with reference to the NCBI genome with accession number NZ_CP009170.1, may be complete or partial deletions

of one or more of the following substances

N-acetyltransferase of the GNAT family (partial deletion of position 233,431 to 234,484);

235,260); and

hypothetical protein (partial deletion from position 235,425 to 235,869).

[0071] In particular, the mutated 7. kivui sequence may be derived from a clonal strain of T. kivui designated CO-1 comprising a nucleotide sequence of SEQ ID NO: 7. The nucleotide sequence may have at least 80%, 85%, 90%, 95% or more identity with the nucleotide sequence according to SEQ ID NO: 7 or be a sequence which, under stringent conditions, is nucleotide-reversed to the complement of

nucleotide sequence according to SEQ ID NO: 7.

[0072] Both duplications that may be included may be, in particular with reference to the NCBI genome with accession number NZ_CP009170.1, one or both

the following act

at least one, optionally two, duplications of at least one of the regions

between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735; and

the region from 1,917,220 to 1,932,727.

[0073] The duplication in the region from 1,917,220 to 1,932,727 may be hydrogen-dependent CO2 reductase (HDCR). Such a duplication may be in the region from

1,921,445 to 1,922,281, particularly at position 1,921,445.

1,900,304.

[0075] The duplication(s) in the range 1,910,027 to 1,935,735 may include at least one, preferably all, of acetyl-CoA decarbonylase/synthase complex subunit alpha (ACDS), IpdA, methylene-THF reductase, methylene-THF dehydrogenase, formate-tetrahydrofolate ligase, formate/nitrite family transporter protein, hydrogen-dependent CO2 reductase (HDCR), and carbon monoxide dehydrogenase accessory protein CooC. These duplications can be located at positions 1,910,148 to 1,911,086, 1,911,897 to 1,913,282, 1,913,298 to 1,914,839, 1,914,852 to 1,916,379, 1,917,220 to 1,918,899, 1,919,741 to 1,920,616,

1,921,445 to 1,927,301, and 1,934,883 to 1,935,659.

[0076] In particular, the mutated 7th kivui sequence with duplication(s) may be derived from a clonal strain of T. kivui designated CO-1 having a nucleotide sequence of SEQ ID NO: 8. The nucleotide sequence may have at least 80%, 85%, 90%, 95% or more identity with the nucleotide sequence according to SEQ ID NO: 8 or be a sequence which, under stringent conditions, is identical to the

reverse complement of the nucleotide sequence according to SEQ ID NO: 8.

SEQ ID NO: 9.

[0078] Hybridization can be performed under stringent conditions, which include hybridization in 6x sodium chloride/sodium citrate (SSC) at 40 to 45 °C, followed by

from a wash in 0.1 to 0.2x SSC at about 60 °C to about 65 °C.

[0079] The doubling in the range 1,917,220 to 1,932,727 may be due to conditioning in syngas with high CO content. One or both of the doublings in the ranges 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735

can come from conditioning in 100% CO.

[0080] Typically, in conditioning with 100% CO following conditioning with high CO syngas, the duplication in the region 1,917,220 to 1,932,727 may be replaced by one or both of the duplications in the regions 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735. Therefore, the duplications in the regions between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 may be

Excluding duplication in the region from 1,917,220 to 1,932,727.

[0081] The duplication(s) in the region from 1,917,220 to 1,932,727 may have a size of 15,507 bp. Duplication(s) in the region from 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 may have a size of 60,101 bp and 25,708 bp, respectively, or a

Individual size of 85,809 bp.

[0082] In a specific embodiment of the invention, the SNP mutations, deletions and duplications in the genome, in particular with reference to the NCBI genome with accession number NZ_CP009170.1, may comprise one or more, and

typically all of -

which originate from the second growth phase in CO2-rich synthesis gas substrate:

SNP mutations of:

cbiQ, cobalt ECF Transporter T component at position 341,995;

rp0C, DNA-directed RNA polymerase subunit beta' at the

Positions 1,969,972 and 1,970,146;

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position

818,980;

doubling of:

the region between 1,917,220 and 1,932,727;

from the third growth stage in 100% CO substrate:

24 / 682

SNP mutations of:

cbiQ, cobalt ECF-T component at position 341,995

(typically from the second growth phase);

rp0C, DNA-directed RNA polymerase subunit beta' at positions 1,969,972 and 1,970,146 (typically from the second

growth stage left over)

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position

818,980 (r typically from the second growth phase);

phoU, phosphate signaling complex protein at position 1,459,688.

Deletions:

N-acetyltransferase of the GNAT family (partial deletion of

Position 233,431 to 234,484);

DUF881 domain-containing protein (complete deletion of

Position 234,523 to 235,260);

hypothetical protein (partial deletion from position 235.425 to

235,869);

Duplication of:

Regions between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 (typically replacement of the duplication of the region between

1,917,220 and 1,932,727, which comes from the second growth stage).

[0083] The method may comprise cloning the 100% CO produced by the method.

population and the isolation of individual clones from it.

[0084] In addition, the SNP mutations, deletions and duplications in the genome that typically result from conditioning with 100% CO (i.e., the third growth stage), in particular with reference to the NCBI genome with

accession number NZ_CP009170.1, one or both of the following mutations

hypF, carbamoyltransferase, at position 136,300; and

acsV, corrinoid activation/regeneration protein, at position 1,903,679.

[0085] THE INVENTION EXTENDS, AS A SECOND ASPECT, to a conditioned and therefore mutated acetogen, such as T. kivui, which is produced according to the method of

first aspect of the invention.

[0086] The mutant acetogen may be a mutant strain of acetogen isolated from the acetogen produced according to the process of the first aspect of the invention. The mutant acetogen may be prepared as described above with reference to the

first aspect of the invention.

[0087] According to a third aspect of the invention, a mutated strain of an acetogen, such as T. kivui, is provided which, compared to a wild type of the acetogen, such as T. kivui (DSM 2030), has at least one SNP mutation, at least

a deletion and possibly at least one duplication in the genome.

[0088] The at least one SNP mutation, at least one deletion and at least one duplication can be obtained from the conditioning of a wild type of the acetogen according to

the conditioning process of the first aspect of the invention.

[0089] SNP mutations that may be involved may include one or more,

typically all, be of -

cbiQ;

mOoC;

dapA;

phoU;

hypF; and

acsV.

[0090] More specifically, SNP mutations that can be included can

or several, typically all, of -

cbiQ, cobalt ECF transporter T component;

mMoC, DNA-directed RNA polymerase subunit beta’;

dapA, 4-hydroxy-tetrahydrodipicolinate synthase;

phoU, phosphate signaling complex protein;

hypF, carbamoyltransferase; and

acsV, corrinoid activation/regeneration protein.

[0091] More specifically, the SNP mutations that may be included, particularly with reference to the NCBI genome with accession number NZ_CP009170.1, may be one or more, typically all, of -

cbiQ, cobalt ECF transporter T component at position 341,995;

moC, DNA-directed RNA polymerase subunit beta’ at positions 1,969,972 and 1,970,146; and

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980.

[0092] In addition, the SNP mutations that can be included can be described in particular with reference to the NCBI genome with accession number

NZ_CP009170.1, one or more, typically all, of -

phoU, phosphate signaling complex protein at position 1,459,688; hypF, carbamoyltransferase, at position 136,300; and

acsV, corrinoid activation/regeneration protein, at position 1,903,679.

[0093] In particular, the mutated 7th kivui strain may be a clonal strain of T. kivui designated CO-1, which has a nucleotide sequence of one, typically all, of

cbiQ, cobalt ECF transporter T component as set forth in SEQ ID NO: 1;

mMoC, DNA-directed RNA polymerase subunit beta’, as set forth in SEQ ID NO: 2; and

dapA, 4-hydroxy-tetrahydrodipicolinate synthase, as set forth in SEQ ID NO: 3.

[0094] In addition, the mutated T. kivui strain may be a clonal 7. kivui strain designated CO-1 which contains an additional nucleotide sequence of one, typically all, of the following elements

phoU, phosphate signaling complex protein as set forth in SEQ ID NO: 4; hypF, carbamoyltransferase as set forth in SEQ ID NO: 5; and

acsV, corrinoid activation/regeneration protein, as set forth in SEQ ID NO: 6.

[0095] The nucleotide sequence may be at least 80%, 85%, 90%, 95% or more

Identity with the nucleotide sequences according to SEQ ID NO: 1 to 6 or a

Sequence that under stringent conditions binds to the reverse complement of the

Nucleotide sequences according to SEQ ID NO: 1 to 6.

[0096] Deletions that may be included may be complete or partial deletions of one or more of the following sequences: N-acetyltransferase of the GNAT family; DUF881 domain-containing protein; and

hypothetical protein.

[0097] More specifically, deletions that may be included, in particular with reference to the NCBI genome with accession number NZ_CP009170.1,

complete or partial deletions of one or more of the following proteins

N-acetyltransferase of the GNAT family (partial deletion from position 233,431 to 234,484);

DUF881 domain-containing protein (complete deletion from position 234,523 to 235,260); and

hypothetical protein (partial deletion of position 235,425 to 235,869).

[0098] In particular, the mutant 7. kivui strain may be a clonal strain of T. kivu[ designated CO-1, which comprises a nucleotide sequence of SEQ ID NO: 7. The nucleotide sequence may have at least 80%, 85%, 90%, 95% or more identity with the

nucleotide sequence according to SEQ ID NO: 7 or be a sequence which is

stringent conditions with the reverse complement of the nucleotide sequence according to SEQ

ID NO: 7 hybridized.

[0099] Duplications that may be included, particularly with respect to the NCBI genome with accession number NZ_CP009170.1, may be one or both of the following:

be

at least one, optionally two, duplications of at least one of the regions

between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735; and

the region from 1,917,220 to 1,932,727.

[0100] The duplication(s) in the regions between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 can be excluded from the duplication of the region of

1,917,220 to 1,932,727.

[0101] The duplication(s) in the region from 1,917,220 to 1,932,727 may have a size of 15,507 bp. The duplication(s) in the region from 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 may have a size of 60,101 bp and 25,708 bp, respectively, or a

Individual size of 85,809 bp.

1,921,445 to 1,922,281, particularly at position 1,921,445.

[0103] The duplication(s) in the range 1,841,579 to 1,901,680 may be one, preferably both, of electron branching hydrogenase (EBH) and energy conserving hydrogenase-2 complex (Ech-hydrogenase). Such duplications may be at positions 1,883,375 to 1,887,553 and 1,893,535 to

1,900,304.

[0104] The duplication(s) in the region from 1,910,027 to 1,935,735 may be at least one, preferably all, of the following substances: acetylCoA decarbonylase/synthase complex subunit alpha (ACDS), IpdA, methylene-THF reductase, methylene-THF dehydrogenase, formate-tetrahydrofolate ligase, formate/nitrite family transporter protein, hydrogen-dependent CO2 reductase (HDCR) and carbon monoxide dehydrogenase accessory protein CooC. These duplications can be found at positions 1,910,148 to 1,911,086, 1,911,897 to 1,913,282, 1,913,298 to 1,914,839, 1,914,852 to 1,916,379, 1,917,220 to 1,918,899, 1,919,741 to 1,920,616,

1,921,445 to 1,927,301, and 1,934,883 to 1,935,659.

[0105] In particular, the mutated T. kivui strain with duplication(s) may be a clonal

strain of T. kivui called CO-1, which has a nucleotide sequence of SEQ ID

nucleotide sequence according to SEQ ID NO: 8.

[0106] The mutant 7. kivui strain with duplication(s) may be a clonal strain of 7. kivui designated CO-1 having additional or alternative duplication(s) and a nucleotide sequence as set forth in SEQ ID NO: 9. The nucleotide sequence may have at least 80%, 85%, 90%, 95% or more identity with the nucleotide sequence as set forth in SEQ ID NO: 9 or be a sequence which, under stringent conditions, identifies with the

reverse complement of the nucleotide sequence according to SEQ ID NO: 9.

[0107] Hybridization can be performed under stringent conditions, which include hybridization in 6x sodium chloride/sodium citrate (SSC) at 40 to 45 °C, followed by

from a wash in 0.1 to 0.2x SSC at about 60 °C to about 65 °C.

[0108] IN PARTICULAR ACCORDING TO A THIRD ASPECT OF THE INVENTION, there is provided a mutant strain of an acetogen such as T. kivui which, compared to a wild type of the acetogen such as T. kivui (DSM 2030), in particular with reference to the NCBI genome with accession number NZ_CP009170.1, has one or more of the following:

properties

partial deletion of the N-acetyltransferase of the GNAT family;

SNP mutation of cbiQ, a component of the cobalt ECF transporter T; complete deletion of the DUF881 domain-containing protein; partial deletion of a hypothetical protein;

SNP mutation of dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980;

SNP mutation of phoU, phosphate signaling complex protein at position 1,459,688;

SNP mutation of m0C, DNA-directed RNA polymerase subunit beta’ at positions 1969972 and 1970146;

SNP mutation of hypF, carbamoyltransferase, at position 136,300; and

SNP mutation of acsV, corrinoid activation/regeneration protein, at position

1,903,679.

[0109] In particular, the mutated strain may comprise one or more of the following

elements included

N-acetyltransferase of the GNAT family (partial deletion of position 233,431 to 234,484);

cbiQ, cobalt ECF transporter T component at position 341,995 (SNP mutation);

DUF881 domain-containing protein (complete deletion from position 234,523 to 235,260);

hypothetical protein (partial deletion of position 235,425 to 235,869) (SNP mutation);

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980 (SNP mutation);

phoU, phosphate signaling complex protein at position 1,459,688 (SNP mutation);

mMoC, DNA-directed RNA polymerase subunit beta’ at positions 1969972 and 1970146 (SNP mutation);

hypF, carbamoyltransferase, at position 136,300 (SNP mutation); and

acsV, corrinoid activation/regeneration protein, at position 1,903,679 (SNP mutation).

[0110] The mutated strain may contain at least -

cbiQ, cobalt ECF transporter T component, at position 341,995 (SNP mutation);

DUF881 domain-containing protein (complete deletion from position 234,523 to 235,260);

hypothetical protein (partial deletion of position 235,425 to 235,869) (SNP mutation);

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980 (SNP mutation);

and

phoU, phosphate signaling complex protein at position 1,459,688 (SNP mutation).

[0111] Preferably, the mutated strain also comprises -

hypF, carbamoyltransferase, at position 136,300 (SNP mutation); and

acsV, corrinoid activation/regeneration protein, at position 1,903,679 (SNP-

mutation)

[0112] Preferably, the mutated strain further comprises -

N-acetyltransferase of the GNAT family (partial deletion of position 233,431 to 234,484);

DUF881 domain-containing protein (complete deletion from position 234,523 to 235,260); and

hypothetical protein (partial deletion of position 235,425 to 235,869) (SNP mutation).

[0113] Most preferably, the mutant strain comprises all -

N-acetyltransferase of the GNAT family (partial deletion of position 233,431 to 234,484);

cbiQ, cobalt ECF transporter T component at position 341,995 (SNP mutation);

DUF881 domain-containing protein (complete deletion from position 234,523 to 235,260);

hypothetical protein (partial deletion of position 235,425 to 235,869) (SNP mutation);

dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980 (SNP mutation); phoU, phosphate signaling complex protein at position 1,459,688 (SNP mutation);

mMoC, DNA-directed RNA polymerase subunit beta’ at positions 1969972 and 1970146 (SNP mutation);

hypF, carbamoyltransferase, at position 136,300 (SNP mutation); and

acsV, corrinoid activation/regeneration protein, at position 1,903,679 (SNP mutation).

[0114] The acetogen may in particular be 7. kivui and therefore the mutant strain may be a mutant strain of T. kivui which, compared to the wild type T. kivui (DSM

2030) has mutated.

[0115] The mutant strain may have been mutated by conditioning the wild type of T. kivur by carrying out the method of the first aspect of the invention

became.

[0116] The mutated strain can be used in carrying out the acetogenic

Fermentation of CO feed gas.

[0117] According to a fourth aspect of the invention there is provided a process for the acetogenic fermentation of CO gas to be fermented (i.e. CO feed gas), the process including growing a mutant strain of an acetogen, such as T. kivul, according to the second or third aspect of the invention on CO gas to be fermented (i.e. CO feed gas) as the main or sole carbon source, thereby producing a

fermentation product is produced.

[0118] The cultivation of the mutant strain can facilitate the cultivation of the acetogen in

a liquid growth medium (i.e. fermentation medium).

Contains vitamins, nutrients and yeast extracts.

[0119] The CO feed gas substrate may consist of 100% v/v CO. Alternatively, the CO feed gas substrate may comprise less than 100% v/v CO, for example in a range

from 50% v/v CO up to 100% v/v CO.

[0120] The process of the fourth aspect of the invention is further characterized by the features of the gas fermentation process described with reference to the first

comprehensive aspect of the invention.

[0121] According to a fifth aspect of the invention there is provided the use of a mutated acetogen, such as T. kivui, or a mutated strain of an acetogen, such as T. kivui, according to the second or third aspect of the invention in the acetogenic

Fermentation of CO gas to be fermented.

[0122] The use can be carried out according to the method of the fourth aspect of the invention. The CO gas can also be used as described with reference to the fourth aspect of the

invention must be described.

[0123] According to a sixth aspect of the invention there is provided a method of rendering a thermophilic acetogen capable of metabolising 100% CO gas, which method comprises introducing one or more genetic modifications according to the second or third aspect of the invention into the thermophilic

acetogens.

[0124] EXPERIMENTAL

[0125] THE INVENTION WILL NOW BE DESCRIBED IN MORE DETAIL WITH RESPECT TO EXPERIMENTAL DATA COLLECTED WHEN CARRYING OUT A COMPARATIVE CONDITIONING OF T. kivul AND A COMPARATIVE ACETOGENIC GAS FERMENTATION USING SO-CONDITIONED T. kivui. The invention is also applicable to other acetogens, in particular thermophiles, and the experimental data presented with respect to T. kivu[ are therefore only given as a non-limiting example.

shown.

[0126] More specifically, 7. kivui (DSM 2030) was conditioned in serum bottles with a culture volume of 20 ml according to the method of the first aspect of the invention and alternative methods. The conditioned strains were then tested for their ability to undergo acetogenic gas fermentation in various substrates by

Cultivation in a DasGip ® DASbox ® mini bioreactor system (supplied by

Eppendorf AG, Hamburg, Germany). The genome properties and the

Performance was determined under the conditions and procedures described below

analyzed. [0127] Growth media and cultivation of the organisms [0128] Two different variants of a synthetic growth medium (i.e.

Fermentation medium) were used for serum bottles and bioreactors, respectively.

[0129] More specifically, the media for serum bottles and bioreactors were modified by Moon et al. (Moon, J., Jain, S., Müller, V., Basen, M., 2020. Homoacetogenic Conversion of Mannitol by the Thermophilic Acetogenic Bacterium Thermoanaerobacter

kivui Requires External CO». Front. Microbiol. 11, 571736).

[0130] The specific compositions of the respective growth media are given in

Table 1 shows the results.

Table 1: Composition of growth media

40 / 682

41 / 682

[0131] The agar medium was prepared according to the recipe for the serum bottle medium, with additional 2 g/L yeast extract and 10 g/L Nobleagar (supplied by Carl-Roth,

Schoemperlenstraße 3-5, D-76185 Karlsruhe).

[0132] Cultivation of T. kivui (DSM 2030) for adaptation (conditioning)

was carried out at 66°C in 125 mL serum bottles with 20 mL culture volume.

[0133] The serum bottles were incubated in a water bath shaker at medium shaking speed, while the bioreactors were placed in a static water bath

at 66 °C.

[0134] The gases for the serum bottles were mixed using Brooks 4800 series mass flow controllers, except for CO, which was added separately, and the

Concentration was adjusted via the pressure component.

[0135] For the bioreactors, premixed gases were used, which were supplied by Messer

Austria GmbH, Gumpoldskirchen, Austria.

[0136] For the serum bottles, 1 ml of the cell culture was routinely added to each of the

adaptation steps (conditioning) described below.

[0137] The bioreactors were filled to an ODeoo of 0.1 from 500 mL serum bottles

inoculated with 100 mL culture volume.

[0138] Bioreactor cultivations [0139] The bioreactor cultivations described below were carried out as above

mentioned, in a DASbox ® mini bioreactor system.

[0140] Bioreactors with a maximum volume of 250 mL and a

Working volume of 200 ml at a temperature of 66 °C is used.

[0141] The pH was initially adjusted to 6.4, monitored with an EasyFerm Plus K8 120 pH electrode (Hamilton, Reno, NV, USA) and adjusted by adding 5 M KOH with

an MP8 multipump module (Eppendorf AG, Hamburg, Germany).

[0142] The bioreactor was purged with 0.05 vvm (0.6 sLh-1) gas purchased premixed from Messer Austria GmbH, Gumpoldskirchen, Austria. To improve

of gas transfer into the liquid phase, microspargers made of sintered metal with a

43 / 682

pore size of 10 um (Sartorius Stedim Biotech GmbH, Göttingen, Germany)

used.

[0143] Before inoculation, the reactor medium was incubated for at least 3 hours with

flushed with the corresponding gas.

[0144] In continuous cultivation, in contrast to batch cultivation, the anaerobic growth medium was continuously added to the growth medium in the bioreactor at the specified dilution rate using the MP8 pump module. In order to keep the reaction volume constant, the culture broth was continuously removed using peristaltic pumps (Ismatec SA, Glattburg, Germany) and a dip tube,

The culture volume was kept at about 200 mL.

[0145] The feeding bottles were kept anaerobically by using N» and a

An overpressure of 0.1 bar was exerted on the head space by the pressure reducer.

[0146] Steady-state conditions were achieved after at least 4 volume changes

and the OD600 was measured regularly.

[0147] To determine the dry biomass, the culture broth was centrifuged for 5 minutes at 10,000 g and washed twice with 0.9% NaCl before being poured into a

glass tubes containing RO (reverse osmosis) water.

44 / 682

[0148] The supernatant of the culture broth was used for HPLC analysis.

[0149] HPLC analysis

[0150] The substrates and products in the liquid medium were analyzed with an Aminex HPX87H column (300 x 7.8 mm) from Bio-Rad, Hercules, CA, USA, using an Ultimate 3000 system from Thermo Scientific, Waltham, MA, USA. 4 mM H2SO4 was used as the mobile phase and the column was incubated at 60°C with a flow rate

of 0.6 ml min-1 for 20 min.

[0151] A 10 μl sample was injected onto the column. Detection was performed using a refractive index detector (Refractomax 520, Thermo Fisher Scientific, Waltham, MA, USA) and a diode array detector (Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA). Chromeleon 7.2.6 was used to control, monitor and evaluate the analysis.

Chromatography Data System (Thermo Fisher Scientific, Waltham, MA, USA) was used.

[0152] 540 μl of the culture supernatant were mixed with 60 μl of 40 mM H2SO4 and centrifuged for 5 min at 14,000 rpm (21,913 g) at 4°C. The remaining supernatant was used for

used for further analysis. Standards with defined acetate and

45 / 682

Glucose concentrations were treated in the same way. For quantification

A five-point calibration was used.

[0153] Genome sequencing [0154] Cells were grown to late log phase (OD600 » 1) by

Centrifugation and frozen at -80°C.

[0155] CO adaptation (conditioning)

[0156] Wild type T. kivui (DSM2030) was adapted (conditioned) for CO metabolism according to the method of the first aspect of the invention by inoculating T. kivui for three growth cycles in the first growth stage, two growth cycles in the second growth stage and five growth cycles in the third growth stage in each case

30% v/v CO, 40% v/v CO, 50% v/v CO, 60% v/v CO and 100% v/v CO respectively.

[0157] Conditioning was carried out without the addition of growth stimulants, i.e. free

of vitamins or yeast extract.

46 / 682

[0158] For comparison purposes, after three passages (growth cycles) with H2/CO2, the strain was cultured twice with two different H2/CO2/CO mixtures, which are referred to according to the invention as "syngas with low CO content" (30% CO, 9% CO2, 58% Ho, 3% N2) and "syngas with high CO content" (52% CO, 21% CO2, 24% Ho, 3% N2), and then transferred to corresponding serum bottles with 30%

CO in the headspace.

[0159] More specifically, as shown in Figure 1, the wild type of T. kivur was grown three times in the H2/CO2 gas phase only starting from a mixotrophic culture for subsequent clone isolation and gene sequencing. The strain from the same vessel was cultured in two different synthetic syngas mixtures, namely syngas with low CO2 content and syngas with high

CO content.

[0160] Surprisingly, it was found that after the second transfer to the high CO syngas mixture, growth to 30% CO was already facilitated, while the transfer of low CO syngas did not result in growth to 30% CO (data not shown). The CO concentration could be increased from 30 to 60% in 10% steps for each transfer of T. kivui grown on the high CO syngas, but continuous growth was not achieved.

was observed.

[0161] With a strain from the 60% culture, transfer and growth on

100 % CO gas phase in a single step. Therefore, the adaptation in

A total of ten transfers were carried out, namely three to H2/CO» and seven to CO-containing

Gas, five of which are CO alone.

[0162] Surprisingly, a strong predisposition of the wild-type strain to grow on pure CO was observed in this stepwise conditioning method. This

was not expected before the experiments.

[0163] The H2/CO», CO-poor and CO-rich syngas strains as well as the strain grown 100% on CO were plated and individual colonies were

characterization and gene sequencing.

[0164] It was found that only the culture grown at high CO was able to grow at 30% CO and that it could subsequently be transferred to growth at 100% CO in four further transfers. In this context, reference is also made to Figure 1, which schematically shows a flow diagram

the adaptation (conditioning) steps applied.

[0165] As the following analysis shows (see in particular Table 4), the T. kivui strain identified as CO-1, a clonal strain obtained from T. kivui obtained by carrying out the method of the first aspect of the invention

was surprisingly found to be able to produce H» and CO at

48 / 682

and even more so their combination.

[0166] The adaptation resulted in four different populations adapted to H2/CO2, low and high CO syngas and 100% CO. The adaptation was determined by an increase in OD600 compared to control cultures with the same H2/CO2 content in the headspace but using N2 instead of CO. In this context, reference is made to Figure 2 which shows the optical densities of the high and low CO syngas lines and the pure CO line.

from Figure 1 shows.

[0167] As can be seen from Figure 2, increasing the CO concentration in the headspace of the serum bottles enabled dense growth of T. kivur on 100% CO in a total of seven runs (H2/CO»> —> high CO syngas — stepwise

Increase of 10% from 30% to 60% and then a jump to 100% CO in the headspace).

[0168] From the H2/CO»- and CO-adapted cultures, the populations were plated and individual colonies harvested to obtain genetically uniform strains.

receive.

49 / 682

[0169] The colony from the H2/CO»2-adapted population is designated G1, and three clonal strains from the CO-adapted population are designated CO-1, CO-2 and CO-

3. Each was subjected to genome analysis.

[0170] Genome analysis [0171] The adapted (conditioned) populations (low CO syngas

content, syngas with high CO content, 100% CO) were directly subjected to genome

subjected to mutation analysis.

[0172] From the pure CO population, colonies were also isolated on a medium containing Noble agar (10 g/L) and tested for their ability to grow on 100% CO. Three different clonal strains were obtained and used for

genome sequencing, designated CO-1, CO-2, and CO-3, respectively.

[0173] The sequencing data were used to perform SNP analyses. Table 2 shows a summary of the different mutations identified in the strains obtained from the low CO, high CO and 100% CO populations. Table 3 shows a

Summary of the various mutations found in the clonal strains CO-1, CO-

2 and CO-3 were identified, with only mutations with a frequency of >90%

are specified and silent mutations have been filtered out.

Table 2: Mutations in low CO syngas, high CO syngas and strains

with 100% CO conditioning.

low-CO2 syngas population

Mutation type Locus Tag Product Position Ref. Alt. Freq. | FCwr p Missense TKV_RS00550 | Membrane protein 110469 |C G 24% 1|3.3 (P75A) Intergenic - 136048 |CT GC |- 11% 11.5 Cobalt ECF insertion TKV_RS01675 | Transporter T 341995 |T C 69% 11.0 (X207fs) component cbiQ ATP-binding missense TKV_RS01680 | Cassette domain 342421 C T 60% 1|3.0 (P92S) containing protein 1S200/1S605 family accessory missense TKV_RS13215 432209 |G C 13**% | protein TnpB- (G434A)

related protein

Missense TKV_RS03470 | Amino acid permease | 704424 |T A 20% 1|3.3 (T575S) Intergenic - 704604 |A AT |- 18% 15.6 Intergenic - 759157 |CA GC |- 12% 11.2 4-HydroxyMissense TKV_RS04105 | Tetrahydrodipicolinate- |818980 IC G 27% |13.8 (A93P) Synthase Missense TKV_RS04285 | Sensor histidine kinase | 851211 C T 17% 13.7 (D329N) Penicillin-binding insert TKV_RS04390 873559 |T TA 46% 1|3.1 Protein 2 (M576fs) Missense TKV_RS06290 | Aspartate kinase 1240652 |C G 37% 1|10.4 (G342A) 1-Deoxy-D-xyluloseMissense TKV_RS06405 | 5-phosphate- 1264984 |G C 14% |(Q358E) reductoisomerase type | Glyceraldehyde-3Missense TKV_RS07960 | phosphate- 1559965 |T C 100% |1.0 (N35D) dehydrogenase CRISPR-associated missense TKV_RS08460 1670432 |G C 50% 1|18.5 CARF protein Csx1 (H215D) [FeFe] hydrogenase, missense TKV_RS09720 1922486 |C A 17% 15.8 Group A (G440V )

DNA-guided RNA

Missense TKV_RS09895 | Polymerase- 1950405 |G A 50% 1|32.3 (S168F) subunit alpha DNA-guided RNAMissense TKV_RS$S10075 | Polymerase 1976392 |C G 23% 13.0 (G113R) subunit beta DNA repair protein missense TKV_RS10195 1995865 |C A 52% 1|7.8 RadA (R238l) carbamoyl phosphate synthase (glutamine hydrolyzing) large subunit phosphoesterase of the missense TKV_RS11995 2376288 |A T 59% 1|2.0 DHH family (M82K) high CO syngas Population Mutation type Locus Tag Product Position | Ref. Alt. Freq. | FCwr p protein of the nonsense TKV_RS00500 | sporulation family in the | 100921 C T 78% |(Q84X) Stage 0 ATPase of the AAA missense TKV_RS00610 124880 |A G 12% |Family (T350A)

DUF881 domain deletion TKV_RS01125 234579 |CT C 80% 1|6.0 containing protein (V228fs)

Cobalt-ECF insertion TKV_RS01675 | Transporter T- 341995 |T C 92% 11.3 (X207fs) component cbiQ ATP-binding missense TKV_RS01680 | Cassette domain 342421 C T 73% 1|3.6 (P92S) containing protein ATP-binding missense TKV_RS01680 | Cassette domain 342746 |A C 12% |(H200P) containing protein missense TKV_RS02635 | Flagellar motor protein |535038 I|G A 11% |(E269K) Flagellar motor missense TKV_RS02640 535038 |G A 11% |Protein MotB (M1)) TATAGCTTT deletion TKV_RS03600 | hypothetical protein | 729103 T 31% |AGA (1150fs)

Intergenic - 759157 |CA CC |NA 12% 11.2 Penicillin-binding insert TKV_RS04390 873559 IT TA 10% 1|0.7 Protein 2 (M576fs)

Intergenic - 1134756 |G T J- 30% 1|1.6

Type-IlMissense TKV_RS05675 | Secretion system 1136321 IC T 12% |(S518L) ATPase GspE PolyamineMissense TKV_RS06070 | Aminopropyltransferase | 1197002 |A G 13% |(T54A) e Protein of the YlzJ-like family (M1I) Missense TKV_RS06280 1239704 |C A 14% | ProlipoproteinMissense TKV_RS07855 | Diacylglyceryl- 1539981 |C T 61% 14.8 (G200R) Transferase Glyceraldehyde-3Missense TKV_RS07960 | phosphate- 1559965 |T C 100% |1.0 (N35D) dehydrogenase type | missense TKV_RS07975 | acylphosphatase 1562892 |T A 14% |(N53Y) metalloregulator transcription factor missense TKV_RS09310 1847332 |C G 41% |of the ArsR/SmtB- (W65C) family Intergenic - 1869603 |C T |- 67% |DNA-guided RNA missense TKV_RS10070 | polymerase- 1969972 |C T 52% |(G1002E)

subunit beta

DNA-guided RNA

Missense TKV_RS10070 | Polymerase- 1970146 |G T 12% |(A944D) Subunit beta Transcription termination Missense TKV_RS10100 | on/antitermination- 1979737 |T A 13% |(K28J) Protein NusG Intergenic - 2268120 |G GC |- 52% 1|5.7 Phosphoesterase of the Missense TKV_RS11995 2376288 |A T 77% 1|2.5 DHH family (M82K) 100% CO population Mutation Locus Tag Product Position | Ref. Alt. Freq. | FCwr styp Chromosome replicati Missense TKV_RS00005 | onsinitiator protein 107 G A 63% |(G3R) DnaA protein of the nonsense TKV_RS00500 | sporulation family in stage 0 | 100921 C T 82% |(Q84X) carbamoyltransferase missense TKV_RS00680 136300 |G C 23% |hypF (G22A) DUF881 domain deletion TKV_RS01125 234579 |CT C 99% containing protein (V228fs)

Cobalt ECF insertion TKV_RS01675 | Transporter T- 341995 |T C 99% 1|1.4 (X207fs) Component cbiQ ATP-binding missense TKV_RS01680 | Cassette domain 342421 C T 17% 10.8 (P92S)

containing protein

ATP-binding CT | Insert TKV_RS01680 | Cassette domain 342777 IC 37% 1|6.3 A |(K215fs) containing protein

DUF1648 domain missense TKV_RS01745 355062 |G C 24% |containing protein (V94L) TATAGCTTT deletion TKV_RS03600 |hypothetical protein |729103 T 39% |AGA (1150fs) 4-hydroxymissense TKV_RS04105 |tetrahydrodipicolinate |818980 |C G 82% |41.7 (A93P) synthase penicillin-binding insertion TKV_RS04390 873559 IT TA 48% |3.2 protein 2 (M576fs) missense TKV_RS05625 | Ribonuclease J 1125479 IG A 20% |(M511) Type II Missense TKV_RS05675 | Secretion system 1136321 IC T 17% |(S518L)

ATPase GspE

Missense TKV_RS05745 | TIM ton protein 1146134 |G 42% |(S269T) PhosphatMissense TKV_RS07415 | Signal complex protein | 1459688 |C 87% |(D215N) PhoU UDP-NAcetylmuramoyl-LMissense TKV_RS07535 | Alanyl-D-Glutamate- 1483593 |C 17% |(G334C) 2,6-diaminopimelate ligase type | Glyceraldehyde-3Missense Y phosphate- 1559965 |T 100% |1.0 (N35D) Dehydrogenase PhosphocarrierMissense TKV_RS08245 | Protein of the HPr- 1625737 IC 23% |(G67R) family Intergenic - 1676464 |G - 11% |Metalloregulator transcription factor missense TKV_RS09310 1847332 IC 43% |of the ArsR/SmtB- (W65C) family Intergenic - 1869603 |C - 53% |-

nickel-dependent

Missense TKV_RS09610 | Hydrogenase large 1899612 |G T 62% |(H109N) subunit intergenic - 1900320 |G GC |- 14% |DNA-guided RNAMissense TKV_RS10070 | Polymerase- 1969972 |C T 74% |(G1002E) subunit beta DNA-guided RNAMissense TKV_RS10070 | Polymerase- 1970146 |G T 26% |(A944D) subunit beta Intergenic - 2268120 |G GC |- 31% 1|3.4 Phosphoesterase of the missense TKV_RS11995 2376288 |A T 31% 11.0 DHH family (M82K)

Table 3: Mutations in CO-1, CO-2 and CO-3. A frequency cutoff of 10% was used and

Silent mutations were filtered out.

CO-1

Locus Tag Product Position | Ref. | Alt. | Mutation type Freq. | FCwr Carbamoyltransferases

TKV_RS00680 136300 |G IC | Missense (G22A) 100% | e hypF cobalt ECF

TKV_RS01675 | Transporter T- 341995 IT C | Insertion (X207fs) 100% | 1.4

component cbiQ

4-HydroxyTKV_RS04105 | Tetrahydrodipicolinate |818980 IC G | Missense (A93P) 100% | 50.9

synthase

TKV_RS05745 | TIM ton protein |1146134 |IG |C | Missense (S269T) 100% | -

Protein of the YlzJTKV_RS06280 1239704 IC A |Missense (M1l) 100% | similar family

PhosphatTKV_RS07415 | Signal complex- 1459688 |C 1|T | Missense (D215N) 100% | -

protein PhoU

Type | GlyceraldehydeTKV_RS07960 | 3-phosphate- 1559965 |T C | Missense (N35D) 100% | 1.0

dehydrogenase

Stalk domainTKV_RS08050 1576833 |C G |Missense (E349D) 100% | containing protein

Corrinoid activation

TKV_RS09640 1903679 |C 1|G | Missense (G411A) 100% | /regeneration protein

n AcsV

DNA-guided TKV_RS10070 | RNA polymerase- 1970146 |G IT | Missense (A944D) 100% | -

subunit beta

Intergenic - 2268120 |G IC |- 100% | 11.0

CO-2

Locus Tag Product Position | Ref. | Alt. | Mutation Type Freq. | FCwr Cobalt-ECFTKV_RS01675 | Transporter T- 341995 IT C | Insertion (X207fs) 100% | 1.4

component cbiQ

4-HydroxyTKV_RS04105 | Tetrahydrodipicolinate |818980 IC G | Missense (A93P) 99% | 50.5

synthase

TKV_RS04565 | Acyl-CoA ligase 903470 |G |T |Nonsense (G383X) 99% |-

deacetylase of

TKV_RS05260 1055168 |G [|A | Missense (T193M) 100% | PIG-L family

TKV_RS05625 | Ribonuclease J 1125479 |G [|A [| Missense (M51l) 100% | Phosphate-

TKV_RS07415 | Signal complex- 1459688 |C 1|T | Missense (D215N) 100% | -

protein PhoU

Intergenic - 1528260 |G |A |- 100% | -

Type | GlyceraldehydeTKV_RS07960 | 3-phosphate- 1559965 |T C |Missense (N35D) 100% | 1.0

dehydrogenase

DNA-guided TKV_RS10070 | RNA polymerase- 1970146 |G IT | Missense (A944D) 100% | -

subunit beta

Intergenic - 2268120 |G IC |- 100% | 11.0

CO-3

Locus Tag Product Position | Ref. | Alt. | Mutation Type Freq. | FCwr

Intergenic - 303329 |T A |- 100% | 8.073501

ECF-Transporter S-

TKV_RS01555 322778 |TG |T | Deletion (G128fs) 97% | component cobalt ECF

TKV_RS01675 | Transporter T- 341995 IT C | Insertion (X207fs) 100% | 1.399329

component cbiQ

Flagellar ExportTKV_RS02415 486030 |G [|T |Missense (A116S) 100% | Chaperone FIliS

4-hydroxy-

TKV_RS04105 | Tetrahydrodipicolinate |818980 IC G | Missense (A93P) 100% | 50.9 synthase

TKV_RS05625 | Ribonuclease J 1125479 |G [|A [| Missense (M51l) 100% | -

Protein of the YlzJTKV_RS06280 1239704 IC A |Missense (M1l) 100% | similar family

PhosphatTKV_RS07415 | Signal complex- 1459688 |C 1|T | Missense (D215N) 100% | -

protein PhoU

Type | GlyceraldehydeTKV_RS07960 | 3-phosphate- 1559965 |T C | Missense (N35D) 100% | 1

dehydrogenase

DNA-guided TKV_RS10070 | RNA polymerase- 1970146 |G IT | Missense (A944D) 100% | -

subunit beta

[0174] To complement the point mutation analysis, the Illumina read coverage was analyzed for the different mutants developed (low, high syngas and pure CO populations as well as clonal CO-1, CO-2 and CO-3 strains). The comparison with the wild type read coverage shows an uneven distribution of reads along the genome, with clear differences at two specific loci (Figure 3). The first locus (Figure 3A) has a deletion of approximately 2.5 kb (positions 233,431 to 235,869) in the pure CO populations as well as in the derived clones (CO-1, CO-2, CO-3). This deletion is not present in the low and high syngas populations. It inactivates/delets two genes with unknown functions as well as a N-acetyltransferase of the GNAT family involved in the

post-translational gene regulation.

[0175] The second locus (Figure 3B) shows an increased distribution of reads over two large genomic regions in close proximity (region 1: positions 1,841,579 to 1,901,680, 60,101 bp; region 2: positions 1,910,027 to 1,935,735, 25,708 bp). In these regions, the read coverage is approximately twice as high as in the rest of the genome (mean read coverage + SD in CO-1 NGS data: region 1: 1686.1 + 253.4 reads; region 2: 1642.1 + 218.9 reads; whole genome: 718.0 + 217.6 reads). This suggests that these two regions have been duplicated in the genome. This pattern is also evident in the pure CO population dataset, but not in the low

Synthesis gas content. In the data set with high synthesis gas content, only a part of the

which gives the derived clones the ability to grow with 100% CO.

[0176] Figure 4 shows the final optical densities and acetate titres of the

adapted populations cultured in triplicate in serum bottles.

A clear increase in OD can be seen with higher energy availability in the gas phase (same coloured bars with asterisks represent two-sample t-tests with assumed unequal variances: * represents p<=0.05 ** represents p<=1*10-2 *** represents p<=1*10-3). Thereafter, there is a significant difference in the optical densities between all gas compositions. This difference is not evident in the acetate titers, where the only significant difference is between low CO syngas and

content and H2/CO» was determined.

[0177] Cultures grown with CO exhibited higher overall variability and significantly higher optical densities than the other gas mixtures. The final OD600 grown on 100% CO was determined to be 1.14 + 0.14, which is 2.43x higher than the final OD600 reported in Weghoff et al. (2016) as 0.47. Similarly, the acetate titer of 5.44 + 1.10 g/L is 2.1-fold higher than the

reported acetate titer of 2.65 g/L at 70% CO (100% CO is not given).

[0178] The cell and acetate titers of the acquired strain are remarkably high, and the growth rate on carbon monoxide is also significant for acetogenic gas fermentation (Table 4). E. limosum and C. carboxidivorans are known to be fast growing strains on CO (both of which grow on complex medium). C. autoethanogenum, which also grows fastest in continuous cultures, is used in industrial applications and has numerous generations of adaptive evolution.

in bioreactors.

Table 4: Comparison of growth rates of different acetogens on CO or its derivatives (* =

calculated using data provided by the inventors)

Strain Source Carbon Source | Synthetic | Culture Conditions | Max. Medium Growth Rate [h”]

T. kivut CO1 presence | 100% CO X bioreactor, batch- 0.25 +/- 0.03

de culture

Invention T. kivut CO1 Presence | Syngas with X Bioreactor, Batch- 0.20 +/- 0.03

the high CO culture

invention | salary

T. kivul presence | Syngas with X bioreactor, 0.18 de high CO- continuous culture

invention | salary

T. kivui (Weghoff | 100% CO serum bottles 0.021 and Müller, 2016)

E. limosum (Jin et 66% CO serum bottles 0.11 al., 2022)*

E. limosum (Kanget | 44% CO serum bottles 0.089 al., Syngas 2020)°

E. limosum (Pregnon | 200 mM X serum bottles 0.076 et al., MeOH 2022)*

H. pseudoflava ( Grenz et | 40% CO |. 100% CO serum bottles 0.100 + 5.5 x 107*

carboxidivorans | et al.,

2020)” C. (de Lima | 60% CO X bioreactor, 0.12 autoethanogenu | et al., continuous culture m 2022)®

Weghoff, M.C., Müller, V., 2016. CO Metabolism in the Thermophilic Acetogen Thermoanaerobacter kivui. Appl.

Environ. Microbiol. 82, 2312-2319

Jin, S., Kang, S., Bae, J., Lee, H., Cho, B.-K., 2022. Development of CO gas conversion system using high CO tolerance

biocatalyst. Chem. Eng. J. 449, 137678

Kang, S., Song, Y., Jin, S., Shin, J., Bae, J., Kim, D.R., Lee, J.-K., Kim, S.C., Cho, S., Cho, B. -K., 2020. Adaptive

Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide. Front. Microbiol. 11, 402

Pregnon, G., Minton, N.P., Soucaille, P., 2022. Genome Sequence of Eubacterium limosum B2 and Evolution for Growth

on a Mineral Medium with Methanol and CO: as Sole Carbon Sources. Microorganisms 10, 1790

Grenz, S., Baumann, P.T., Rückert, C., Nebel, B.A., Siebert, D., Schwentner, A., Eikmanns, B.J., Hauer, B., Kalinowski, J., Takors, R., Blombach, B., 2019. Exploiting Hydrogenophaga pseudoflava for aerobic syngas-based production of

chemicals. Metab. Closely. 55, 220-230

Zhu, H.-F., Liu, Z.-Y., Zhou, X., Yi, J.-H., Lun, Z.-M., Wang, S.-N., Tang, W.- Z., Li, F.-L., 2020. Energy Conservation and

Carbon Flux Distribution During Fermentation of CO or H2/CO2 by Clostridium ljungdahlii. Front. Microbiol. 11, 416

Lanzillo, F., Ruggiero, G., Raganati, F., Russo, M.E., Marzocchella, A., 2020. Batch Syngas Fermentation by Clostridium

carboxydivorans for production of acids and alcohols

is inexpensive and can be recycled, continuous processing is advantageous.

[0180] Batch fermentation

[0181] T. kivui was grown in duplicate in two different batches. T. kivui was grown with both 100% CO and high CO syngas (52% CO, 21% CO», 24% Ho) to demonstrate the high potential of the strain to grow in CO.

and demonstrate the ability to utilize H2 and CO together.

[0182] Figure 5 shows the acetate titres and optical densities measured during the batch process at 100% CO. The culture showed a lag phase of about 12 hours, followed by two distinct growth phases (Figure 4 and Table

5) The titres achieved in this experiment are in comparison to syngas fermentation

significantly higher, and an OD600 of 4.85 (= 2.03 gCDM L-1) (CDM =

cell dry mass) and an acetate titer of 32.48 g/L were observed.

[0183] It is also noteworthy that after 40 hours the cells catalyzed a water-gas shift reaction in which no growth occurred (Figure 6). The CO uptake of the cells is approximately constant over the entire experimental period. After cessation of growth and acetate production, the cells catalytically converted CO into H2 and CO», as shown by the negative uptake rates (i.e. production rates) for H2 and CO».

after 40 hours of cultivation (Figure 5).

Table 5: Parameters of growth phases in batches with high CO syngas and 100% CO

in comparison. W Average [h'] Growth phase | Period [h-h] | Syngas (070) Syngas (070) 1 6-13 0.20 0.18 | 0.32 0.21 2 13-19 0.13 0.25 | 0.68 0.46 3 20-38 0.03 0.05 | 0.53 0.69 [0184] Figure 7 shows the growth and acetate production in syngas with high

CO content. The peak OD600 was 2.89 (= 1.28 gCDM L-1) and the acetate titer averaged 19.9 g/L. These values correspond to 63.1 and 61.2% of the titers achieved by growth with 100% CO, respectively, which could indicate that the titers achieved

function of the energy content in the gas substrate, and also shows how acetate production and

growth are directly linked.

[0185] The uptake rates of the batch process (Figure 8) with synthesis gas show the same gas shift reaction, but only in one reactor. The

The mechanism behind this and why only one reactor seemed to catalyze is unknown.

[0186] Continuous process

[0187] Since syngas is a more likely scenario for the production of acetate from process vapors, a continuous process with different

production parameters.

[0188] Two highly productive continuous processes were established with 7. kivur on syngas. One was carried out to achieve a certain growth rate (high dilution rate) and the other to maximize the acetate titer

(high titer). Both achieved approximately the same productivities (Table 6).

Table 6: Key parameters of the continuous process with T. kivul, adjusted to 100% CO.

High dilution rate High titer

input

Dilution rate [1/h] 0.179 0.074

Gassing rate [vvm] 0.068 0.066 Stirrer speed [rpm] 1200 900 pH 6.4 6.4 Output

CDM [g/L] 0.41 0.51 ODseoo 1.24 1.78 Cacetate [9/L] 5.73 12.94 Facetate [9/L/h] 1.022 0.953 H2 UR [mmol/L/h] 22.1101 0.9782 CO UR [mmo//L/h] 60.4105 59.4268 CO2 UR [mmol//L/h] -2.0783 -5.7367

UR = absorption rate; vvm = vessel volume per minute. Rom = stirrer speed, revolutions

per minute. [0189] DISCUSSION [0190] The following statements discuss relevant features of the invention and

further characterize the invention.

[0191] Conditioning

[0192] Differentiation from Weghoff and Müller (2016):

to use.

[0194] Only after this initial conditioning step was the strain subjected to strong selection pressure by growing in the presence of CO as the sole carbon and energy source (with no other sources provided via yeast extract). A stepwise increase in the CO content surprisingly allowed the cells to adapt to the use of CO as the sole carbon and energy source (instead of H2/CO2 used by the wild type). At 60% CO in the gas phase, the inventors again subjected the cells to strong selection pressure by increasing the CO concentration to 100% within one passage.

increased.

[0195] The inventors also used a detour via synthesis gas to deliver T. kivui to the

growth on pure CO.

[0196] Moreover, the inventors surprisingly found that they applied a sufficiently strong selection pressure to force adaptation. For example, the inventors found that transferring T. kivui grown on syngas with low CO to 30% pure CO was unsuccessful. The inventors only succeeded in adapting when they applied a strong selection pressure

by successively applying H2/CO>» to syngas with high CO content, syngas with

7217682

100% pure CO).

[0197] Significantly, the inventors also relied on the use of a chemically

defined medium without vitamins and yeast extract. [0198] Differentiation from other studies with syngas:

[0199] In other studies known to the inventors, syngas alone was used to adapt acetogens to growth on syngas (e.g. C. autoethanogenum, 142

generations, Ingelman et al., 2023), E. imosum, 400 generations, Jin et al., 2022).

[0200] The approach that the inventors followed and which surprisingly led to the results discussed here was to use synthesis gas first and then CO. This not only resulted in cells that can grow well in 100% CO, but

also to higher specific growth rates on synthesis gas.

[0201] General

[0202] Surprisingly, a much faster adaptation of T. kivui (>31 generations) to growth on pure CO and synthesis gas at higher growth rates (Uu= 0.2-0.24 h-1) was observed compared to C. autoethanogenum (142 generations,

U = 0.12 h-1) and E. limosum (400 generations, u = 0.11 h-1).

[0203] The inventors are of the opinion that the method according to the invention can be combined with UV or chemical mutagenesis (e.g. by means of EMS) in order to

to increase the rate of mutagenesis. EMS stands for ethyl methanesulfonate, a chemical mutagen,

used to increase the overall mutation rate in the genome of an organism.

[0204] In addition, the process according to the invention can be applied in continuous gas fermentations to obtain superior phenotypes (e.g. educt or

product tolerance) when appropriate selection pressure is applied.

[0205] Subculturing the mutated 7th kivui strain on glucose over 40 generations

did not lead to a reversion of the phenotype.

[0206] The adapted strains proved to be robust with respect to CO growth, i.e. when re-exposed to gases with low CO concentration and then again to gases with high CO content, they were still able to achieve high growth rates

so that the adjustments were considered long-lasting/permanent.

[0207] In further experiments it was found that the process and the strains are robust against contaminants such as those found in synthesis gas from biomass and

Waste gasification may result in dust, tar and sulphur-containing compounds.

[0208] It has been found that the method described in this specification with

Pure and mixed cultures work in a bioreactor, with all strains being anaerobic.

[0209] For downstream processing, the methods known from the state of the art can be used, such as centrifugation to obtain biomass in

Case of intracellular products. For extracellular products, in-situ methods are particularly

advantageous. Compounds with a low boiling point such as acetone can be evaporated in thermophilic fermentations. Membrane processes such as electrodialysis or dialysis are also suitable for product extraction. Extraction with

Solvents, e.g. ionic liquids, can be used.

source

1..768mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

atgctaatagatagttactcttacacaaacaggatgtacaatgtacat cctgttgaaaaacttttatttgectttttgacaatgattttatgttttaaatttgatgectatacaaatat tgcagtaattattttgatgtttgtggtaactgtatttaaagecgaaaattccagcaaaagtgtatattaaaCc ttatgttaattccttattcttttctcataataagcatattgacacttgtaataaatgttgtagaggataaa agtattgcattagtaagttttaatatttttggaataaccttagggattaccgcaaaaggagttagcactge tattattttattcttcagagetttagcagtggtetettgettatattttettgetettactacteccotgtag ttgacatcataaatgtcttaaagaaatttaaatttccatcattatttttggagetgettcagettatttat cgatttatatttgttttaatacaagcageccgatgatatctacatatctcaggattcaagactgggatatge cacattaaagaatggetataggtetttaggacttttgatatecttetttttattaagtecttacaaagatt cacaggatttatacatagctttagaagcaagatgttataatggggagcetaaaagttgttagcaaagattat aaattttgttataaaaatgtaatatttattattttaatagaactaattttaataagegcagcaatgttttt aagaaggtga

source

1..3555mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

ttgtttgagttgaaaaactttgaagetattaagatcggtttggcatca cctgaaaaaataagagaatggtcaaggggggaagtaaaaaagectgaaactataaactacaggacgttaaa accagaaaaagatgggettttetgtgagegaatttttggacctacaaaagactgggaatgecactgtggta agtataaaagagttagatacaaaggtgttgtetgtgataggtgeggtgttgaggttacacgttectaaagtt agaagagaaagaatggggcatattgaacttgetgecccagtttecccatatatggtattttaaaggtatacce cagccgcatgggactccttetegacatgteteccaagagetttagaaaaagttttatactttgettcctatg tagtaattgacccaggtgacactcctcttaccaaaaagcagttattgtcagagaaagagtacagagaatat ttggacaaatatggtaataggtttagagcaggaatgggagcagaagetataaaagagttgttgcaagagat tgaccttgaaaaattgagcaaagaactgaaagcggagataagagaatctacaggecaaaaaagggtaaggg ctataagaagattagaagtagtgcaggettttatagattecgggtaacagaccggagtggatgattttagag gttattcctgttataccacctgatttaaggectatggtgcagttggatggeggacgetttgetacttcaga cctcaatgacttatatagaagagttataaacagaaacaatagattaaaaaggettttagacctecggtgete ctgacatcatagttagaaatgagaagagaatgettcaagaagcagtagacgetttaatagataatggaaga agaggaagacctgtcacagggcecaggcaacagacctttaaagtetetttcagacatgetaaaaggtaaaca gggaagattccgtcaaaatettttgggtaaaagggtagactattcaggtegttetgttatagttgtaggac ctgaactcaaagtttatcagtgtggtttgccaaaagaaatggcecattagagettttaagecttttgtgatg aaaaaattagttgatgaaggtttggcacagcacataaaaagegetaagagaatggttgaaaaagtaaagec tgaagtgtgggatgtattggaggaagttataaaagaacatecctgttettttaaatagagetccgacactte acagactcggtatccaagetttgaaccagtgetggtagaaggaagagetataaagetcatectettgtg tgtacggettataacgcagactttgacggtgaccagatggetgttcacataccgetgtecagtggaagetca agccgaagcaaggtttttaatgttggcagcaaacaacatatattaaagecgcaagatggaaaacctgtcatga cacctacgcaagacatggttttgggatgttattatttaactgcagatgaggatggggtattgggtgaaggt aaatatttctccagcccagaagaagetattatggcataccaattaggatatattcacatccacgcaaaaat aaaagtaaaaatgacaagagaaattgacggggtgaaaaggtecgaagataattgagacgacggtcggtaaaa tcatttttaacgaggetatacctcaaaatttgggetatgtegacaggacaaacccagaaaccgettttgac cttgaagtcaatgatttagtcgataaatctaaattgggaaaaattettgatagagtectatagattacacgg acctacaaagacagcagaaactcttgataaaattaaagaattaggatttagatattctacaaaagcageta ttacagtaagcgtatectgacatggtaatacctaaagaaaaggaacagetgetaaaagaagcagatgaaatg 7717682

7717682

894 DNAPAT source1..894mol_typegenomic DNA organismThermoanaerobacter kivui atgcectgtttttaaaggttegggegtegecattgtcacccecttttaac gaaaaaaggtgttaaatttcgaaaagetggegagettatagaatggcatataaaagaaggaactgatgetat

DNA

PAT source1..894mol_typegenomic DNA organismThermoanaerobacter kivui atgcectgtttttaaaggttegggegtegecattgtcacccecttttaac gaaaaaaggtgttaaatttcgaaaagetggegagettatagaatggcatataaaagaaggaactgatgetat

source1..894mol_typegenomic DNA organismThermoanaerobacter kivui

source1..894mol_typegenomic DNA organismThermoanaerobacter kivui

source

1..894mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

Thermoanaerobacter kivui

atgcectgtttttaaaggttegggegtegecattgtcacccecttttaac gaaaaaaggtgttaaatttcgaaaagetggegagettatagaatggcatataaaagaaggaactgatgetat

654 DNAPAT source1..654mol_typegenomic DNA organismThermoanaerobacter kivui gtgaatcgtacccattttgaaaaggaattagaggaacttcattatgac gtattgaaaatgggaagtecttgtagaagaagecgatagetaatgecattgettetttagtgaaccatgatac agagttggcetcaaaaagttatagatgacgacgacagaatagacaaaatggaagtagaaattgacaataagt gtgcecaaaatcatggtgacacagcagecgattgcaagggatttgagaatagttttgacgggeccttaaaatt gttacagacttggaaagaatggcagaccatgcagtggatatagcaaaaactactttgaggattgetcacca aaaatatataaagecctttgatagatattcccagaatggetgatattgtgagggaaatggtaaaaatgtcac tggattcatatgttagacaggacttagaccttgcaaagacaatcggtgaaaaggatgatatagtecgatget ctttataagcaaatcttcagagaactcttaacttatatgatggaagatccaagaaacatcgaccaggcaaCc acaatttttgtttgtagcecgcgatatctagaaagaattgetgaccatgetactaatatatgegaatgggtaa tttacttggatactggagagcatataaatttaaattaa

DNA

PAT source1..654mol_typegenomic DNA organismThermoanaerobacter kivui gtgaatcgtacccattttgaaaaggaattagaggaacttcattatgac gtattgaaaatgggaagtecttgtagaagaagecgatagetaatgecattgettetttagtgaaccatgatac agagttggcetcaaaaagttatagatgacgacgacagaatagacaaaatggaagtagaaattgacaataagt gtgcecaaaatcatggtgacacagcagecgattgcaagggatttgagaatagttttgacgggeccttaaaatt gttacagacttggaaagaatggcagaccatgcagtggatatagcaaaaactactttgaggattgetcacca aaaatatataaagecctttgatagatattcccagaatggetgatattgtgagggaaatggtaaaaaatgtcac tggattcatatgttagacaggacttagaccttgcaaagacaatcggtgaaaaggatgatatagtecgatget ctttataagcaaatcttcagagaactcttaacttatatgatggaagatccaagaaacatcgaccaggcaaCc acaatttttgtttgtagcecgcgatatctagaaagaattgetgaccatgetactaatatatgegaatgggtaa tttacttggatactggagagcatataaatttaaattaa

source1..654mol_typegenomic DNA organismThermoanaerobacter kivui

source1..654mol_typegenomic DNA organismThermoanaerobacter kivui

source

1..654mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

gtgaatcgtacccattttgaaaaggaattagaggaacttcattatgac gtattgaaaatgggaagtecttgtagaagaagecgatagetaatgecattgettetttagtgaaccatgatac agagttggcetcaaaaagttatagatgacgacgacagaatagacaaaatggaagtagaaattgacaataagt gtgcecaaaatcatggtgacacagcagecgattgcaagggatttgagaatagttttgacgggeccttaaaatt gttacagacttggaaagaatggcagaccatgcagtggatatagcaaaaactactttgaggattgetcacca aaaatatataaagecctttgatagatattcccagaatggetgatattgtgagggaaatggtaaaaatgtcac tggattcatatgttagacaggacttagaccttgcaaagacaatcggtgaaaaggatgatatagtecgatget ctttataagcaaatcttcagagaactcttaacttatatgatggaagatccaagaaacatcgaccaggcaaCc acaatttttgtttgtagcecgcgatatctagaaagaattgetgaccatgetactaatatatgegaatgggtaa tttacttggatactggagagcatataaatttaaattaa

source

1..2256mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

atggttagaggaacagtacgaaaaatacagacaaaacaaatagaaata tctggtattgtacaggecgtaggttttegecetttgtatttacacttgcaaagaagtatcatttaggagg catagtctataacacctettcaggtgtttatatagaagtagaaggggaggaggaaaatgtcaacagtttta taaaagaattaaaaaataatcctcctccacttgetgttatagatgaggtaaaagtaaaagatttagaagtt aagggttatgaggatttcgaaattatatcgagtcaagaaaatgatggatttgtacctgtatectecctgatat gggggtatgegacgactgtttaagagaaatgagagacccaaatgacagaagatataggtatccttttataa attgcactaattgtggtccaaggttttctataatagaggatatteccttatgacagacccaaaacttctatg aaggtatttcctatgtgcgaaaaatgegaaaaagagtacaatgaccccaatgacaggagatttcacgecgca gecggtagecctgttttgactgegggectagttggaatttgtaggagetgattgtcagcaagatgaaataa aatgtgctatagattecccttaaaaaggcaaaattgtegetattaagggaatagggggttttcacttagca gtaaatgeccttagegatgaagetgtetetacettgagaagtecgaaaaaagaggtatggaaagectttecge tgtcatgatgaaagacatagaacaggtagaaaagtattgtgttgtaagtgaagaagaaaaaagettett taagccagagaaggcecgattgtgettttaaagaaaaaaaggagaaaaacttgegaagggeattgetgatgge ttagatactttgggagtaatacttccatatgetecctatacattatecttttaatggaccaaatagactttcc tattgtgatgacaagtgggaatgtcagtgaagagcecactgtgcaaagacaatgaagaggetataaaaaatt taaaagacatagcagatgtttttecttttaaacaatagggatatagtaaatagaatagatgattcagttact tcttttaatgegggttatgagaggattataagaagggegagaggttatgetecctcaaccectattttgettaa aaaagatactaaggtcaatgtgetggeggtaggaggtttttacaaaaatactttttgetttaccaaaggge attatgccttcataagtcatcacataggagatttggacaatgaaaagacttttgagtattacaaagaagaa atagaaaaatataaacggetttttaaagtgacacctgaggtagtagetcatgacatgcataaaggatatect ttcgacgcagtatgetttattgtgtggtttgectettgttgaggttcaacaccatcatgetcatategeta getgtatggeggagtacaacattacagataaagtaatagggatagectatgacggtactggatatggtact gacggtaatatatggggtgcagaattttttatatgcecgacttaaaagattttataagagtagggecatetcga gtataaaccgetteccaggeggtgaactggcaataaaaaagatatacagaacagetttgggatttatttcag aagatataggaagttatggagattttttggaaaggtttgaccaaaaagaggttgagataattttgaaacaa attaagagaaaaattaatactccttatgtgtceccagcatgggcaggttttttgatgetgettettetetact tggattaagagatgaggtgetetttgaaggacaaggtgetatggagetggaaagtttgatagttgacactg acagcttttatgaatttgaaatttectcataaagatggetatataataaacacagatataatattacagcaa

source

1..1938mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

atggaaaaatgtacagtaatttttaaaccctctaataaaagtataagt gtaacaaaaaggcactaatttactcgaggcagcaagaaaagcaggggtttttattgatgegecttgtggtgg gaaaggacgatgtggaaaatgtaggtaaaaatcatttectggagattataaagaggaaataacttetectgt taaccagtgaggataggcaaaaaggcattagacttgegtettacetttgtagaaggegatatgattgtt gaggtagaggaatctaagcaagtacaaaatatecttagtggaaggtataaagectggagatgaaaaaaatga aagagttcttaaagcaagcaagettttagaggaagagggcataaaaaaagagtgtggtgtattcaatattg atatagaacttcctgageccttctattgatgataatgtggcagattttgaaaggttaaaaagagagetaaaa gtaaagtataacatagaaaattttttttgttcattgaatgttttaaagaatatgectaaaatattgaggga aaataattttaaagtaagagetacatttttaaaaggtaaaaacggttgtgaattaataaggattaaggete gaaataaagaaaccagcacatatggaatatgtgtggatataggtacgacaacagtagcagttaatttaatt gatttggaaagcaataagatcatagetgetgettecatcaggecaacettcaaatgeagtatggtggtgacgt aataagccgaataatctatgecteccacagagggtggtttggaaaaaataaagtetgecagtagtggatggga ctataaacaggttgatagacgaactggtgaagaaagctaacattgaaaagcaagatattattatctcaaca tttgcagggaataccacaatgacacatecttttettgggtgtagaacctgegtatataagggtggagectta tatacctgcatttagggaatgecctgaattaaaagcaaaggatataggaattgacataaatcccgaageta aagtaatagttattccaaatgttgcaagttatgttggtggggatattgtatcaggtgttttggetacaggt atttggaaagaagaaaaaaatgtattgtttatggatttgggtacaaatggagagattgtttttggcaataa agaccttatgetaacctgtgettgetetgeaggacetgecatttgaagetggagagattagttgtggaatga gagcaatgccaggagcaattgatgaaatttcaattgataggaagacattggaacctaagattagggtaata ggcaatgttaaacctaaaggcatatgtggetcaggtttaatagaccttetggcecagagatgttectttcagg

source

1..2439mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

acttttagatattgaggatatatggcatgaattagaaaccgtttcgga tatttatccttttaatacttttgagtgggtgettaattggtggaaatattttggttatgggaaaaaattat ggatattggtgatttatgaggaaagcactecctattggtattgegecttttatgataacttattttgaaaag ggacttttggcaaaaaggataaagtttataggttccaacaacagecgattatettgattttattgtgaggecg aggatatgaaaatcaattttatacaageccttgtaagttttttggaaaggcatatagatgettttacagttt tggatttagagcatattcctgaaaagagtgagtttttecccttttatcatgggtagcaatectttattacgat tatgacgttgaagatatatgcccttatgtggagttgectcaaagetgggaggaatatttgaatagettaga aggcaaattcaggagaaatctcaattatgaaacgaggagattttttaaagagtacgatgggatgttttectt gtgtaaaagatatagatggacttgatgaggetatggacaatttaattgttettcaccaaaggegatggega gaaaggcacatgccaggtgetttttattcaaaaagaatgagggaattccacaaggatgtggcacgagetat gettttaaaaggegetttaagtetttttgaattaaaagacggcaggaagacagttgecagecttttaagtt atcatgtaggtggtaagaggtattactatataagtggttatgaccttgattacagcaaaagaagecgtgggg acaataactcttggacttgctataaaacaatctattgaagaaggagacagtgtgtttgattttttaagagg ggatgaaaggtataaaaaaaattggaattectttaaaaaagaagaacatgagatttgttgeggcaaagectt caatagcaggaaagcetgttttgetattatatcattgecggaaaataaaattattaaaaaaatcaaagacagg tttagcgaataaccaaacctgtcttaatatataaaagaaggtaagaggetttattggtttacgggetttge gtaaaaaaaageggattgggtegetgtacttaggaactactatgttatttgaagtttgtatgtetatttta

32765 DNAPAT source1..32765mol_typegenomic DNA organismThermoanaerobacter kivui ttcagactgttgacaaaatatcgggaacccgttatgatgagaagggtt ccttgtttttttaacggtacaaagcaaaagaggagaggagaagaaagatgttatcaaagaaacaggatgeac agacatcaaatagaatttgtaagcatagatcagttagttccaaaagatcaccttttaaggaagatagaaag agttatagattttagtttcatatatgatttagtaaaagagaaatattccgaagatcacggcagaccaagca tagacccagtagtactcataaaaatacttttcattcaatatectttttggtataccatcgatgaggaggaca atagcagaaataaaaacaaatgtagecgtatagatggtttttagggtatgggetgacagaagaaatacctca tttttcaacatttagtcagaactacataagaagattcaaggggacggatatatttgaaaaaatatttacga

DNA

PAT source1..32765mol_typegenomic DNA organismThermoanaerobacter kivui ttcagactgttgacaaaatatcgggaacccgttatgatgagaagggtt ccttgtttttttaacggtacaaagcaaaagaggagaggagaagaaagatgttatcaaagaaacaggatgeac agacatcaaatagaatttgtaagcatagatcagttagttccaaaagatcaccttttaaggaagatagaaag agttatagattttagtttcatatatgatttagtaaaagagaaatattccgaagatcacggcagaccaagca tagacccagtagtactcataaaaatacttttcattcaatatectttttggtataccatcgatgaggaggaca atagcagaaataaaaacaaatgtagecgtatagatggtttttagggtatgggetgacagaagaaatacctca tttttcaacatttagtcagaactacataagaagattcaaggggacggatatatttgaaaaaatatttacga

source1..32765mol_typegenomic DNA organismThermoanaerobacter kivui

source1..32765mol_typegenomic DNA organismThermoanaerobacter kivui

source

1..32765mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

ttcagactgttgacaaaatatcgggaacccgttatgatgagaagggtt ccttgtttttttaacggtacaaagcaaaagaggagaggagaagaaagatgttatcaaagaaacaggatgeac agacatcaaatagaatttgtaagcatagatcagttagttccaaaagatcaccttttaaggaagatagaaag agttatagattttagtttcatatatgatttagtaaaagagaaatattccgaagatcacggcagaccaagca tagacccagtagtactcataaaaatacttttcattcaatatectttttggtataccatcgatgaggaggaca atagcagaaataaaaacaaatgtagecgtatagatggtttttagggtatgggetgacagaagaaatacctca tttttcaacatttagtcagaactacataagaagattcaaggggacggatatatttgaaaaaatatttacga

25709 DNAPAT source1..25709mol_typegenomic DNA

DNA

PAT source1..25709mol_typegenomic DNA

source1..25709mol_typegenomic DNA

source1..25709mol_typegenomic DNA

source

1..25709mol_typegenomic DNA

mol_typegenomic DNA

Thermoanaerobacter kivui

attgcaaaagcaagacaagttggaaatccacattccttacaattecttc tttggaagcattttatatatatccatacctgttaaagecatecttaaatatecgeccctttcagaattagtaa aattagttcaacattgaattgataaagtttttcaaaacttcaacagaacgtggatgtecttaaaattacacc attcgaaccagetgetaatacaccaaccgeteccacaaatetecattgetattectetatettetaaactte cccactcaggagcgtettettetgttgecagttgattecoctttacttteccagetttcaaaggaaataggtgaa attattggcatctgaagagtecgtategttetgateccaaggetgcaagttttattetgtecagggtggttat aacatattcataaccataacccaccgcagatatacctggattcatcatgatcttttctggtttaacaccaa getgcatcactaaaatattcatectgttttgcaaggtttaggtcaactgagetttcagecactacgatgtta ccataagcaaggcctacagecgcacectacttetttgtaattagettetactgetgaaagaaaagcataatt tttatttgcaaggacttcagaaatttttgtaaaaatecttaccattettttcgttagattcacaaccageta tcacaagtggaacttcaacactatcagceccactttcttagetatttetgecacattectetggagttetateec tttacatcaggatttgetcccacaaatecttaaaactataaaatectggattgtaattttttacatagaactg tgeccatgcagcaggatecctetgetacatetttaataagattcattageccctcaggecaacecetgteggag ctacgtcccagacttccataccaattecttgggggatttecctgtatecgecgtcaaaagtgtaaaaaaggaagt actgattcacctccaatctttaaatccttacccattaccacttcttttatttggectttgaatgtttgtcg tggtttttgataagecatttttttcgectccatctactatttttttgttgttattcttggaagtaaaattt tatctaaaatatctctataggetttaaccgetgecattatetttggtaaatttagcageggaatgecagca ttatcatattcatagatgccattatcaagaggaacaaagectgcaaaaggaatgttataactttttatata ttctaaaagetettcactcattececttececttggggactetgttgactataaggaagagetettttacatcaa gettcaattecctctatcagtgtetttactecgatatgcagcatectatecetettttagaacattcactgaca gcaaacatcaaatcaacttttttagtagttcttecttgaaaggtgetcaageccagettecattgtegactac taaaaacgaataagaatctgacagtttgtcagttacttctcttaaaactccatttacaaaacaataacate ctagcccctcaggectteccattacaagcaagtcataaccattgeecttecgacaatageectgetgeaccatg aaattcaaataatcagcctttgtcattecccgcaggaagtttttcatcatetectaacttectetetgatttg acccaaagtaaattccacatctaaacctaaaacattattcaaatttgaattcggatectgcatcaaccgcaa gaaccggacccaatttcttttctaccaaataatcaattgttaaaccagcaagagtagtettacctgtteca ccttttcctgctaatgcaataacatataccattttttcactccactttatacattgtgtatcgactgecc atgtatatcatgaattgettccatgacagectcactaaacgtaggatgagaatgtacaacttetgetactt cagatactttcaaaccacacgttatagccacggcaatttcatgcacaaggteccgtecgcattaggteectata atatgagcacctaagattttgtcgttatcttgatccgcaacaattttcacaaaaccatctaatttccctga ggectgtectttaccaagacccctgaaataaaaccttecctatgetaatattgtatecccottttgcaaagett gttettcagttaagectacagcacctatttecgggetcagtgaaaattgtatgeggtacagecgtataatec ataatgetttetttgeccattatattectetactgcacaaataccttcaaaagaagecacatgagetaattg aatacttgggattacatcaccgatggcatatataccatctacatttgtttgcatecttttcattgactacta ttctacctttttcggtatttactcccactectttctaaccctatttcttcaatatttgettttecttccaaca gcaacaagcattacttcggettttatgattttaccatecttctaaagagacttectacctecgttttcattaat tttgacttccgttatctttgacttggtataaagtttaattttecttecttecttaaaaattttectectatttectt tgettatetcaaaatectetgtacttaatgetegateccaacatetcaactagtgtaacgtcagtaccaage cccttataaaacatcgcaaattcgcaaccaataacccctgetecccactattagtatgetettaggaatata tcttaaatctaaaatectcatcacttgtcaacacccttttttcatcaaaaggggaaaagaggaateccgecttg gtgatgaacccgttgcaataattattgcatcagatttaattectectettetgetecatttttttatgact ttaacttctctattgttaagaatttcaccttttccttttattattttaacaccatttttattaaatagcaa gtetattecctttatttaatetttcaataatecttattttttcgttcaattattgegtcaatatetgetttga tttectccttctaaaattatcccataagattttgetttttttattatttccaaaacctcacttgaagaaaga agagccttggtgggaatgcaccctttattcaggcatgteccacctaatttateccttttectattactgttac atctgcaccgagetttgcagettttataceccgetacatatecctecctggacetgcacctataattgtaatte tctttttagacattcatatcgtccttccttacaaattgcacatgtcaagtagaattggaacattectettet gcaccaatagccattatatgaacaccatcacacagtttttecttectttcacttggtttatgaactectgetge aattttcaaaccttctttaaccttattttctgcaccttttaatctectctataagagcatcaggaacatgta

2397824 DNAPAT source1..2397824mol_typegenomic DNA organismThermoanaerobacter kivui attaacttatcaataattaacttataccagataacatactatgccacc ctttttcgtaagttatccacatttcttattgaatgtecttaggaggtcatacaatgtatggagatcatecgte aagtatgggagagaatagtggaggtcatcaaaagtaaacttacccccaccagttacaacacttggcetagtt catataaagccactagcaatcattgataatgtactttttttaagcactccaaacagttttacaaaaaatat aataaatggaagatatataaacataattcacgacgetgcatcaaaagecactaacaaactctataacataa aaatactgtctgaagacgaagaagaatacagagaaatcaaggaatcagtagaaaaagaaatattaaacgaa tcaacaaccctaagtaccttaaatcctaaatatacttttgacacctttgtggtagggaacagtaacaaact tgetcacgecgettgtettgecagtagecacaagetecctgcaaaagettacaatececctatttatttacggcg gtgtaggacttggaaaaactcacctaatgcacgeccataggecactttatcaacaaacatcacagtggttat aaaataatgtacgttacctcagaaacgtttacaaatgaattggtaaattcaataaaagacgacaaaaatga agaatttagaaacaaatacagaaatattgacgtacttctaatagacgatattcaatttatagctaaaaaag

DNA

PAT source1..2397824mol_typegenomic DNA organismThermoanaerobacter kivui attaacttatcaataattaacttataccagataacatactatgccacc ctttttcgtaagttatccacatttcttattgaatgtecttaggaggtcatacaatgtatggagatcatecgte aagtatgggagagaatagtggaggtcatcaaaagtaaacttacccccaccagttacaacacttggcetagtt catataaagccactagcaatcattgataatgtactttttttaagcactccaaacagttttacaaaaaatat aataaatggaagatatataaacataattcacgacgetgcatcaaaagecactaacaaactctataacataa aaatactgtctgaagacgaagaagaatacagagaaatcaaggaatcagtagaaaaagaaatattaaacgaa tcaacaaccctaagtaccttaaatcctaaatatacttttgacacctttgtggtagggaacagtaacaaact tgetcacgecgettgtettgecagtagecacaagetecctgcaaaagettacaatececctatttatttacggcg gtgtaggacttggaaaaactcacctaatgcacgeccataggecactttatcaacaaacatcacagtggttat aaaataatgtacgttacctcagaaacgtttacaaatgaattggtaaattcaataaaagacgacaaaaatga agaatttagaaacaaatacagaaatattgacgtacttctaatagacgatattcaatttatagctaaaaaag

source1..2397824mol_typegenomic DNA organismThermoanaerobacter kivui

source1..2397824mol_typegenomic DNA organismThermoanaerobacter kivui

source

1..2397824mol_typegenomic DNA organismThermoanaerobacter kivui

mol_typegenomic DNA organism

organism

Thermoanaerobacter kivui

attaacttatcaataattaacttataccagataacatactatgccacc ctttttcgtaagttatccacatttcttattgaatgtecttaggaggtcatacaatgtatggagatcatecgte aagtatgggagagaatagtggaggtcatcaaaagtaaacttacccccaccagttacaacacttggcetagtt catataaagccactagcaatcattgataatgtactttttttaagcactccaaacagttttacaaaaaatat aataaatggaagatatataaacataattcacgacgetgcatcaaaagecactaacaaactcttaacataa aaatactgtctgaagacgaagaagaatacagagaaatcaaggaatcagtagaaaaagaaatattaaacgaa tcaacaaccctaagtaccttaaatcctaaatatacttttgacacctttgtggtagggaacagtaacaaact tgetcacgecgettgtettgecagtagecacaagetecctgcaaaagettacaatececctatttatttacggcg gtgtaggacttggaaaaactcacctaatgcacgeccataggecactttatcaacaaacatcacagtggttat aaaataatgtacgttacctcagaaacgtttacaaatgaattggtaaattcaataaaagacgacaaaaatga agaatttagaaacaaatacagaaatattgacgtacttctaatagacgatattcaatttatagctaaaaaag

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Claims (11)

1. A method for conditioning and thus mutating an acetogen for the acetogenic fermentation of CO gas ("CO treatment gas") to thereby obtain a mutated acetogen for the acetogenic fermentation of CO gas, the method comprising culturing a non-mutated strain of an acetogen to be conditioned and thus mutated for the acetogenic fermentation of CO treatment gas in a fermentation medium without yeast extract and vitamins for at least three consecutive growth cycles on CO gas ("CO treatment gas") as the main or sole carbon source, wherein the CO concentration in the CO treatment gas is gradually increased over the three growth cycles, thereby obtaining a conditioned acetogen for the acetogenic fermentation of CO feed gas. 2. A method according to claim 1, which includes the successive cultivation of the acetogen with CO treatment gas, the CO concentration of which is gradually increased in the respective growth cycles, wherein the CO concentration in the CO treatment gas is gradually from approximately 30% v/v to 100% v/v. 3. Process according to claim 2, which comprises a previous step of culturing the acetogen for at least one growth cycle with synthesis gas with high CO content ("Syngas"). 4. A method according to claim 3, which comprises a previous step of culturing the Acetogens for at least one growth cycle with a hydrogen (H») and Carbon dioxide (CO2) gas which is essentially free of CO. 5. A process according to any one of claims 1 to 4, comprising, in a first growth stage, culturing the non-mutated strain of the acetogen for at least a first growth cycle with a gas of hydrogen (H2) and carbon dioxide (CO2) which is substantially free of CO; in a second growth stage, cultivating the acetogen from the first growth stage for at least a second growth cycle with synthesis gas with a high CO content (“syngas”); and in a third growth stage, successively cultivating the acetogen from the second growth stage with CO treatment gas, wherein the CO concentration in the CO treatment gas is gradually increased from about 30% v/v to 100% in at least three growth cycles v/v is increased. 6. The method of claim 5, wherein the first growth stage comprises at least three successive growth cycles, wherein after completion of each growth cycle a fresh gaseous substrate of H» and CO»2 gas is provided. 7. The method according to claim 5, wherein the second growth stage comprises at least two successive growth cycles, wherein after each growth cycle a fresh gaseous substrate is provided from syngas with high CO content. 8. The method of claim 5, wherein the third growth stage comprises successive growth of the acetogen from the second growth stage for at least one growth cycle on each of five CO treatment gases, each with 663 / 682 increasing CO concentration, whereby the CO concentrations in the respective CO treatment gases are 30% v/v, 40% v/v, 50% v/v, 60% v/v and 100% v/v respectively. 9. The method of claim 5, wherein the non-mutated strain of the acetogen, subjected to conditioning is a wild-type strain of the acetogen. 10. A process according to any one of claims 1 to 9, wherein the acetogen subjected to conditioning is a thermophilic acetogen. 11. A process according to any one of claims 1 to 10, wherein the acetogen which is conditioning is the wild-type strain of T. kivui (DSM 2030). 12. Isolated mutant strain of an acetogen, produced by the process one of claims 1 to 11. 13. An isolated mutant strain of T. kivui adapted for growth on syngas with high CO content, optionally produced by the method according to any one of claims 1 to 11, which, based on a wild type of T. kivui (DSM 2030), comprises at least one SNP mutation, preferably all SNP mutations, selected from at least cbiQ, cobalt ECF transporter T component at position 341,995; rp0C, DNA-directed RNA polymerase subunit beta' at positions 1,969,972 and 1,970,146; and dapA, 4-hydroxy-tetrahydrodipicolinate synthase at position 818,980. 664 / 682 a duplication of 15,507 bp in the region from 1,917,220 to 1,932,727. 15. Isolated, mutated strain of T. kivu[ adapted to growth on 100% CO gas, optionally produced by the method according to one of claims 1 to 11, which in addition to the SNP mutations according to claim 13, in comparison to a wild type of T. kivui (DSM 2030) comprises at least one SNP mutation, preferably all SNP mutations, selected from at least phoU, phosphate signaling complex protein at position 1,459,688; hypF, carbamoyltransferase, at position 136,300; and acsV, corrinoid activation/regeneration protein, at position 1,903,679. 16. An isolated mutant strain of T. kivui adapted for growth on 100% CO gas, optionally produced by the process according to any one of claims 1 to 11, which in addition to the SNP mutations according to claims 13 and 15, comprises the following compared to a wild type of T. kivui (DSM 2030): at least one, possibly two, duplications of at least one of the Regions between 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735. 17. An isolated mutant strain of T. kivui! according to claim 16, wherein the Duplication(s) in the region from 1,841,579 to 1,901,680 and 1,910,027 to 1,935,735 a size(s) of 60,101 bp or 25,708 bp or a single size of 85,809 bp. 18. An isolated strain of T. kivui adapted to growth on 100% CO gas, optionally produced by the method according to any one of claims 1 to 11, which, in addition to the SNP mutations according to claims 13 and 15 and the duplication(s) according to claims 16 and 17, comprises at least one partial or complete deletion, preferably all partial or complete deletions, which are selected at least, based on a wild type of T. kivui (DSM 2030), from - N-acetyltransferase of the GNAT family, partial deletion of position 233,431 to 234,484; DUF881 domain-containing protein, complete deletion of position 234,523 to 235,260; and hypothetical protein, partial deletion from position 235,425 to 235,869. 19. A process for the acetogenic fermentation of CO gas to be fermented ("CO feed gas") to produce a fermentation product, the process including growing a mutant strain of an acetogen according to any one of claims 13 to 18 on CO feed gas as the main or sole carbon source, thereby producing a fermentation product is produced. 20. The method according to claim 19, wherein the CO feed gas is a CO-containing industrial exhaust gas. 21. The method according to claim 19, wherein the CO feed gas is a synthesis gas from a gasification process. 666 / 682 22. The method according to claim 19, wherein the fermentation product is acetic acid or a mono- or dihydric alcohol or acid. 23. The method of claim 19, wherein the fermentation product is lactic acid. 24. The process of claim 19, wherein the fermentation is carried out in a reactor selected from a stirred tank reactor, a air lift bubble column reactor or a loop fermentation reactor. 25. The process of claim 26, wherein the fermentation is carried out continuously and comprises continuously feeding fresh CO feed gas into the reactor while continuously withdrawing fermentation product-rich fermentation medium from the reactor and collecting fermentation product from the fermentation product-rich fermentation medium to produce collected fermentation product and fermentation product-poor or free fermentation medium which is then is continuously returned to the reactor. 26. The method of claim 25, wherein the collection of the fermentation product from fermentation product-rich fermentation medium for the production of fermentation product-poor or free fermentation medium is carried out using a membrane process, an evaporation process or a solvent extraction process. 27. The method of claim 19, wherein the fermentation of the CO feed gas removes substantially all of the CO from the CO feed gas by substantially the entire CO content of the CO feed gas is metabolized. 668 / 682