Sporulation-deficient bacterial cells and methods for their use

Abstract

The present invention relates to sporulation-deficient bacterial cells and methods of their use for production of volatile compounds, in particular acetone, butanone, and / or isopropanol. Also provided are nucleic acid constructs, vectors and host cells useful in such methods.

Description

[0001] P6952PC00

[0002] Sporulation-deficient bacterial cells and methods for their use

[0003] Technical field

[0004] The present invention relates to sporulation-deficient bacterial cells and methods of their use for production of volatile compounds, in particular acetone, butanone, and / or isopropanol. Also provided are nucleic acid constructs, vectors and host cells useful in such methods.

[0005] Background

[0006] With the increasing concerns regarding the environmental impact of fossil fuels, there has been a steady push towards the development of more sustainable industrial production processes. Chemical industry is considered to be a major contributor to greenhouse gas emissions, as most modern bulk chemical productions remain energy- intensive and heavily reliant on petrochemical feedstocks. In comparison, bio-based production presents an attractive platform for sustainable production of bulk chemicals, as it allows for the utilisation of renewable feedstocks. As such, the establishment of more bio-based chemical production processes would greatly benefit the transition towards a more sustainable economy.

[0007] One of the major challenges of bio-based approaches lies in their economic viability, as most biochemical productions struggle to compete with the higher yields and lower productions costs of their conventional petrochemical-based counterparts. For sustainable biochemical production to endure on the global market, it is therefore necessary to improve their competitiveness.

[0008] Thermophilic fermentation holds a lot of potential for more optimised biochemical production, as high-temperature bio-based production carry several advantages. This includes reduced risk of contamination and lowered cooling costs. Notably, the application of higher temperatures also allows for continuous recovery of volatile products through evaporation, thus easing the downstream process while avoiding issues with end-product inhibition (Najar and Thakur 2020). The development and optimisation of thermophilic production strains would therefore be a major advantage to the budding biochemical industry. P6952PC00

[0009] Summary

[0010] The present inventors have surprisingly found that microbial cell factories, in particular engineered bacterial cells and more specifically thermophilic bacterial cells, can produce bulk volatile chemicals, such as acetone, isopropanol and / or butanone, with increased efficiency, titers and / or yields, when such cells, besides having been engineered for production of these compounds, also are modified to be sporulationdeficient.

[0011] Previously, the inventors have developed bacterial cells (WO 2022 / 049125) modified to produced acetone, butanone and / or isopropanol, and they have now surprisingly found that inactivating sporulation in such bacterial cells results in increased titers of said compounds.

[0012] Thus, with the present disclosure, the inventors provide improved microbial platforms for production of various compounds, and especially of volatile bulk chemicals, in particular acetone, butanone and / or isopropanol. In particular, the inventors have developed sporulation-deficient strains of the thermophilic bacterial species Parageobacillus thermoglucosidasius yielding increased amounts and / or increased titers of bulk chemicals, such as acetone, butanone and / or isopropanol.

[0013] In addition to improving the titers of the produced volatile compounds, the sporedeficiency also provides for a reduced sterilization / decontamination burden of fermentation, bioreactor and other processing equipment, because the sporulationdeficient bacterial cells do not produce spores that are of risk of contamination future work. By taking advantage of thermophilic bacterial cells for production of volatile compounds, these compounds can be easily and continuously be removed from the fermentation, such as from the cultivation broth, which, in addition to rendering the process more labour- and cost-efficient, also solves the problem of product inhibition and negative effects on cell growth associated to the toxicity of the product(s).

[0014] Provided herein is a sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising; i) a first enzyme selected from an acetyl-CoA acetyltransferase and an enzyme of EC number 2.3.3.20; P6952PC00 ii) a second enzyme selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, an acyl CoA:acetate / 3-ketoacid CoA-transferase, and an acyl-CoA thioesterase II, and iii) an acetoacetate decarboxylase (EC 4.1.1.4), whereby said sporulation-deficient bacterial is capable of converting acetyl-CoA to acetone, thereby producing acetone, and / or whereby said sporulation-deficient bacterial is capable of converting acetyl-CoA and propionyl-CoA to butanone, thereby producing butanone; and iv) optionally an isopropanol dehydrogenase (EC 1.1.1.80), whereby said sporulation-deficient bacterial cell is capable of converting acetone to isopropanol, thereby producing isopropanol, whereby said sporulation-deficient bacterial cell is capable of producing acetone, butanone, and / or isopropanol, wherein said sporulation-deficient bacterial cell is capable of producing an increased amount and / or increased titer of acetone, butanone, and / or isopropanol compared to a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell.

[0015] Also provided herein is a method of producing acetone, butanone, and / or isopropanol, comprising: i. providing a sporulation-deficient bacterial cell described herein; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth comprising acetone, butanone, and / or isopropanol produced by said sporulation-deficient bacterial cell; and iii. optionally recovering the cultivation broth or the acetone, butanone, and / or isopropanol.

[0016] Provided herein is also a method for increasing the titer of acetone, butanone, and / or isopropanol produced by a sporulation-proficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising: i. providing said sporulation-proficient bacterial cell; ii. inactivating sporulation in said bacterial cell, thereby obtaining a sporulationdeficient bacterial cell capable of producing acetone, butanone, and / or isopropanol; P6952PC00 iii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth; whereby said sporulation-deficient bacterial cell produces acetone, butanone, and / or isopropanol with an increased titer compared to the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, such as the sporulation-proficient bacterial cell provided in step i., in the same conditions; and iv. optionally recovering the cultivation broth, the acetone, butanone, and / or isopropanol.

[0017] Herein provided is an off-gas obtained or obtainable by a method described herein. Herein provided is also, a cultivation broth obtained or obtainable by a method described herein. When said method is a method for producing acetone, butanone and / or isopropanol, said off-gas and / or cultivation broth preferably comprises acetone, butanone and / or isopropanol, respectively.

[0018] Provided is also acetone, butanone and / or isopropanol obtained or obtainable by a method described herein.

[0019] Description of Drawings

[0020] Figure 1 : Structure of the spoOA deletion performed in both the acetone and butanone production strains.

[0021] Figure 2: In both the acetone and butanone production strain, increased product titers were observed, when the sporulation key regulator spoOA was deleted.

[0022] Figure 3: Growth of nutrient-starved cultures of P. thermoglucosidasius STC and P. thermoglucosidasius CTC with and without AspoOA, before and after heat treatment. Prior to heat treatment, all cultures swiftly regrew when inoculated into fresh SPY medium. However, following heat treatment, the AspoOA strains failed to recover, suggesting that they lack the ability to produce the heat-resistant spores that allow the wild type strains to readily regrow.

[0023] Figure 4: Concentrations of acetone, as measured for cultures of P. thermoglucosidasius STC carrying knockouts of spoOA, spoOB, spoOF or sigF. P6952PC00

[0024] Compared to that of the parent strain STC, the deletion of spoOA and spoOB yielded a notable increase in acetone production, while deletion of either of spoOF and sigF resulted in decreased acetone production. Medium contained 7.5 g / L acetate and 2 g / L yeast extract, pH was 6.5.

[0025] Detailed description

[0026] Definitions

[0027] The terms sporulation-deficient cell or sporulation-deficient bacterial cell herein refers to a cell, in particular a bacterial cell, in which sporulation is hampered, impaired, inactivated, inhibited, suppressed, or annihilated, either partially or totally. Sporulation deficiency can be achieved by introducing in the bacterial cell a modification resulting in decreased sporulation compared to a cell which does not comprise said modification. The skilled person knows how to test whether a bacterial cell is sporulation-deficient. For instance, microscopy analysis can be employed, or a heat-resistance assay can be used, such as described in Example 2. The terms “sporulation-deficient cell” and “sporulation-deficient bacterial cell” also encompasses a cell having up to 25 % sporulation capacity / ability / activity of a corresponding sporulation-proficient bacterial cell that the sporulation-deficient bacterial cell is otherwise identical to, such as up to 20 %, such as up to 15 %, such as up to 10 %, such as up to 5 % sporulation capacity, ability or activity of a corresponding sporulation-proficient bacterial cell that the sporulation-deficient bacterial cell is otherwise identical to. The terms “sporulation- supressed”, “sporulation-inactivated” and “sporulation-deficient” may be used interchangeably herein.

[0028] With reference to microorganisms, in particular to bacteria, the terms thermophile or herein refers to microorganisms, in particular bacteria, that thrive best, or at least are capable of growing, at temperatures above 42°C.

[0029] The term functional variant is herein applied to functional variants of enzymes, i.e. modified versions of an enzyme, or homologous enzymes originating from a different species, which retain some or all the catalytic activity of the original enzyme. Functional variants may have been modified by introducing mutations which confer e.g. increased activity, a change in intracellular localisation, increased thermostability, prolonged halflife, among others, but retain the ability to perform the same enzymatic reaction as the enzymes they are derived from, albeit possibly to a different extent. Preferably the P6952PC00 mutation(s) introduced in the functional variant are mutations in the gene encoding the corresponding enzyme, for example a mutation in the promoter of the gene or in the coding sequence encoding the enzyme.

[0030] The term homologue herein refers to a variant of a polynucleotide encoding the same polypeptide, i.e. amino acid sequence. Skilled person knows that different codons may encode the same amino acid, and it therefore follows that two different polynucleotides may encode the same polypeptide.

[0031] The term "identity" with respect to a polynucleotide (or polypeptide) are defined herein as the percentage of nucleic acids (or amino acids) in the candidate sequence that are identical with the residues of a corresponding native nucleic acids (or amino acids), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and considering any conservative substitutions according to the NCIIIB rules (https: / / iubmb.qmul.ac.uk / misc / naseq.html; NC-llIB, Eur J Biochem (1985) 150: 1-5) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity. Methods and computer programs for the alignments are well known in the art. Generally, a given homology between two sequences implies that the identity between these sequences is at least equal to the homology; for example, if two sequences are 70% homologous to one another, they cannot be less than 70% identical to one another - but could be sharing 80% identity. Throughout this disclosure, a sequence (amino acid sequence or nucleic acid sequence) sharing at least 70% identity to another sequence means that the sequence shares at least 70% identity to said sequence, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity. P6952PC00

[0032] The term "acetyl-CoA acetyltransferase" or “thiolase” herein refers to an enzyme that catalyzes either the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA), or the conversion of one acetyl-CoA and one propionyl-CoA leading to 3-ketovaleryl-CoA. In particular the term refers to acetyl-CoA acetyltransferases of EC number 2.3.1.9. These particular enzymes have a substrate preference for acetyl-CoA or propionyl-CoA and therefore preferably catalyse the reaction in the forward direction. The skilled person will know how to determine whether a mutant enzyme has thiolase activity. For example, the potential thiolase can be incubated with acetoacetyl-CoA and CoA, and absorbance at 303 nm can be monitored. A decrease in the absorbance at 303 nm indicates that the potential thiolase can perform said reaction and has thiolase activity.

[0033] The term acyl-CoA:acyl-CoA alkyltransferase refers to an enzyme of EC number 2.3.3.20. It catalyses the conversion of two molecules of acyl-CoA into one molecule of either 3-oxoalkanoate (if the substrate is two acetyl-CoA molecules) or 2-alkyl-3- oxoalkanoate (if the substrate comprises at least one acyl-CoA other than acetyl-CoA), such as (2R)-2-alkyl-3-oxoalkanoate, in the presence of water, thereby generating two molecules of coenzyme A (CoA). The reaction is a head-to-head non-decarboxylative Claisen condensation. The skilled person will know how to determine whether a mutant enzyme has acyl-CoA:acyl-CoA alkyltransferase activity. For example, the potential acyl-CoA:acyl-CoA alkyltransferase can be incubated with acetyl-CoA (or acetyl-CoA and propionyl-CoA), with subsequent addition of 5,5’-dithio-bis-(2-nitrobenzoic acid), which reacts with a free thiol group of the released CoASH. The absorbance of the product can be monitored at 412 nm. Formation of the product indicates that the potential acyl-CoA:acyl-CoA alkyltransferase retains acyl-CoA:acyl-CoA alkyltransferase activity.

[0034] The term acetoacetate decarboxylase herein refers to is an enzyme of EC number 4.1.1.4. Acetoacetate decarboxylases are involved in both the ketone body production pathway in humans and other mammals, and solventogenesis in bacteria. They catalyse the decarboxylation of acetoacetate, yielding acetone and carbon dioxide. The skilled person will know how to determine whether a mutant enzyme has acetoacetate decarboxylase activity. For example, the potential acetoacetate decarboxylase can be incubated with lithium acetoacetate. The accompanying release of CO2 which ensues P6952PC00 can be monitored, e.g. manometrically. Release of CO2 indicates that the tested enzyme has acetoacetate decarboxylase activity.

[0035] The term “acetate CoA-transferase” (EC 2.8.3.8) herein refers to an enzyme that can catalyze the chemical reaction: acyl-CoA + an acetate fatty acid + an acetyl-CoA. The activity of variants of acetate CoA transferase can be measured by methods known in the art, for example by incubating this enzyme with acetyl-CoA and lithium acetoacetate, and following the acetoacetyl-CoA formation by measuring absorbance at 313 nm.

[0036] The term “3-oxoacid CoA-transferase” (EC 2.8.3.5) herein refers to an enzyme that can catalyze the chemical reaction: 3-ketovaleryl-CoA + fatty acid 3-oxopentanoate + acyl-CoA. The activity of variants of acetate CoA transferase can be tested by methods known in the art, for example by incubating this enzyme with acetyl-CoA and lithium 3- oxopentanoate, and following the 3-ketovaleryl-CoA formation by measuring absorbance at 304 nm.

[0037] The term “acyl CoA:acetate / 3-ketoacid CoA-transferase” (EC 2.8.3.1) herein refers to an enzyme that can catalyze the chemical reaction: 3-ketoacyl-CoA + fatty acid 3- ketoacid + acyl-CoA. Other names include propionate CoA-transferase, acetyl- CoA:propanoate CoA-transferase, propionate coenzyme A-transferase, propionate- CoA:lactoyl-CoA transferase, propionyl CoA:acetate CoA transferase, and propionyl- CoA transferase. The activity of variants of acetate CoA transferase can be measured by methods known in the art, for example by incubating this enzyme with acetyl-CoA and lithium 3-oxopentanoate, and following the 3-ketovaleryl-CoA formation by measuring absorbance at 304 nm.

[0038] The term “acyl-CoA thioesterase II” (EC 3.1.2.-) herein refers to an enzyme that can catalyze the chemical reaction: acyl-CoA + H2O — > fatty acid + CoA. The skilled person will know how to determine whether a mutant enzyme has acyl-CoA thioesterase II activity. For example, the potential acyl-CoA thioesterase II can be incubated with acetoacetyl-CoA in the presence of 5,5’-dithiobis(2-nitrobenzoic acid). The release of free thiol groups of CoA will result in formation of 5-thio-2-nitrobenzoate, which can be quantified by measuring the absorbance at 412 nm. P6952PC00

[0039] The terms “isopropanol dehydrogenase” or “isopropanol dehydrogenase (NADP+)” (EC 1.1.1.80) herein refers to an enzyme that can catalyze the conversion of propan-2-ol to acetone and acetone to propan-2-ol. The activity of (mutated) variants of isopropanol dehydrogenase can be measured by incubating this enzyme with acetone and NAD(P)H, and following the NAD(P)H oxidation by measuring absorbance at 340 nm.

[0040] The term titer or titer of a compound refers herein to the produced concentration of a compound, for example the produced concentration of acetone, isopropanol or butanone. When the compound is produced by a cell, the term refers to the total concentration produced by the cell, i.e. the total amount of the compound divided by the volume of the cultivation medium. This means that, particularly for volatile compounds, the titer includes the portion of the compound which may have evaporated from the cultivation medium, and it is thus determined by collecting the produced compound from the fermentation broth or cultivation broth and from potential off-gas from the fermenter / bioreactor.

[0041] Bacterial cell

[0042] The present inventors have surprisingly found that microbial cell factories, in particular engineered bacterial cells and more specifically engineered, thermophilic bacterial cells, can produce bulk volatile chemicals such as acetone, isopropanol and / or butanone with increased efficiency, increased titers and / or increased yield, when such cells, besides having been engineered for production of these chemicals, also are modified to be sporulation-deficient. The inventors have previously developed engineered bacterial cells (WO 2022 / 049125) modified to produced acetone, butanone and / or isopropanol, and surprisingly found that inactivating sporulation in such bacterial cells results in increased titers and / or yield of said volatile compounds.

[0043] The cells employed in the context of the present disclosure are bacterial cells, more specifically thermophilic bacterial cells. In particular, bacterial cells which have an optimal growth temperature of 42°C or more are of interest. The term “cell” will herein generally, unless specified otherwise, be construed to designate a bacterial cell or multiple bacterial cells, more specifically a thermophilic bacterial cell or thermophilic bacterial cells, i.e. a cell or multiple cells which are capable of growing at temperatures of 42°C or more. Preferably, the bacterial cell is acetogenic, i.e. is capable of producing P6952PC00 acetone. Section “Volatile compounds” details how a bacterial cell which is not acetogenic or is mildly acetogenic can be engineered to become an acetone producer.

[0044] The present invention takes advantage of bacterial cells, preferably thermophilic, for bioproduction of volatile compounds, in particular acetone, butanone and isopropanol. Because such cells thrive at higher temperatures than conventional, i.e. non- thermophilic, cells, recovery of the volatile products can be facilitated, as these are typically present in the off-gases produced during cultivation of the thermophilic cell. Not only does this reduce the production costs, it is also generally expected to be beneficial for the longevity of the producer as the end product (acetone, butanone and isopropanol) is typically toxic for the producing cell.

[0045] The cells described herein have been engineered to produce volatile compounds, i.e. acetone, butanone and / or isopropanol. The cells described therein preferably do not occur naturally. In some embodiments, the cell is a non-natural cell or an engineered cell, which has been modified either to express a heterologous pathway, i.e. a pathway which is not present in the parent cell, or to express a modified native pathway, as detailed herein below, as described herein below in the section “Volatile compounds”. In some embodiments, the bacterial cell is thermophilic, i.e. a cell of a thermophilic bacterium. In some embodiments, the bacterial cell is an isolated bacterial cell, such as an isolated non-sporulating bacterial cell, for example an isolated non-sporulating thermophilic bacterial cell.

[0046] In some embodiments, the bacterial cell comprises a functional native ethanol pathway. In other words, the cell may be non-sporulating and naturally capable of producing ethanol, in addition to being capable of producing acetone, butanone and / or isopropanol. For example, in some embodiments the bacterial cell is a P. thermoglucosidasius cell and comprises a functional AdhE encoded by the polynucleotide having KEGG locus tag AOT13_03315.

[0047] In some embodiments, the thermophilic cell belongs to a genus selected from Parageobacillus, Geobacillus, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Acetivibrio, Heyndrickxia, Thermoclostridium, Anoxybacillus, Moorella, and Clostridium. In other embodiments, the thermophilic cell belongs to a genus selected from Parageobacillus, Geobacillus, P6952PC00

[0048] Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Acetivibrio, Heyndrickxia, Thermoclostridium, Anoxybacillus, and Clostridium. In specific embodiments, the cell is a Parageobacillus cell, a Geobacillus cell, a Bacillus cell, or a Clostridium cell. In other specific embodiments, the cell is a Parageobacillus cell, a Geobacillus cell, or a Bacillus cell.

[0049] With respect to taxonomy, the bacterial cell in preferred embodiments belongs to the phylum Bacillota. In some embodiments, the bacterial cell belongs to the class Bacilli. In other embodiments, the bacterial cell belongs to the order Bacillales. In preferred embodiments, the bacterial cell belongs to the family Anoxybacillaceae or Bacillaceae', preferably Anoxybacillaceae. In other embodiments, the bacterial cell belongs to a genus selected from Anoxybacillus, Bacillus, Geobacillus, Heyndrickxia and Parageobacillus. In preferred embodiments, the cell is a Parageobacillus cell.

[0050] In some embodiments, the cell belongs to a species selected from Parageobacillus thermoglucosidasius, Parageobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Acetivibrio thermocellus, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Heyndrickxia coagulans, Bacillus subtilis, Bacillus licheniformis, Bacillus smithii, Bacillus methanolicus, Anoxybacillus flavithermus, Bacillus flavothermus, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Moorella thermoacetica, Moorella thermoautotrophica, and Neomoorella thermoacetica. In other embodiments, the cell belongs to a species selected from Parageobacillus thermoglucosidasius, Parageobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Acetivibrio thermocellus, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Heyndrickxia coagulans, Bacillus subtilis, Bacillus licheniformis, Bacillus P6952PC00 smithii, Bacillus methanolicus, Anoxybacillus flavithermus, Bacillus flavothermus, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, and Anoxybacillus gonensis. In other embodiments, the cell belongs to a species selected from Anoxybacillus gonensis, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, Anoxybacillus flavithermus, Bacillus flavothermus, Bacillus licheniformis, Bacillus methanolicus, Bacillus smithii, Bacillus subtilis, Geobacillus kaustophilus, Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoleovorans, Heyndrickxia coagulans, Parageobacillus thermoglucosidasius, and Parageobacillus toebii. In specific embodiments, the cell is a Parageobacillus thermoglucosidasius cell or a Bacillus subtilis cell. In specific embodiments, the cell is a Parageobacillus thermoglucosidasius cell or a Geobacillus thermoglucosidasius cell; preferably the cell is a Parageobacillus thermoglucosidasius cell. In specific embodiments, the cell is a Parageobacillus thermoglucosidasius DSM2542 cell. In other embodiments, the cell is a Bacillus subtilis cell. In other embodiments, the cell is a Acetivibrio thermocellus cell.

[0051] In some embodiments, the thermophilic bacterial cell has an optimal growth temperature between 42 and 80°C, or is capable of growing at a temperature between 42 and 80°C, such as between 50 and 75°C, for example at 60°C. For example, the cell has an optimal growth temperature of 42°C or more, such as 43°C or more, such as 44°C or more, such as 45°C or more, such as 46°C or more, such as 47°C or more, such as 48°C or more, such as 49°C or more, such as 50°C or more, such as 51 °C or more, 52°C or more, 53°C or more, 54°C or more, 55°C or more, 56°C or more, 57°C or more, 58°C or more, 59°C or more, for example 60°C or more. In some embodiments, the thermophilic bacterial cell is capable of growing at a temperature of between 42 and 80°C, such as between 50 and 75°C, for example at 60°C. For example, the thermophilic bacterial cell is capable of growing at a temperature of 42°C or more, such as 43°C or more, such as 44°C or more, such as 45°C or more, such as 46°C or more, such as 47°C or more, such as 48°C or more, such as 49°C or more, such as 50°C or more, such as 51°C or more, 52°C or more, 53°C or more, 54°C or more, 55°C or more, 56°C or more, 57°C or more, 58°C or more, 59°C or more, for example 60°C or more.

[0052] In particular, the thermophilic bacterial cell is preferably able to grow at temperatures where at least part of the volatile compound, such as acetone, butanone and / or P6952PC00 isopropanol, that the cell produces evaporates, thereby facilitating recovery of the produced compound. Thus, in some embodiments, the cell is able to grow at temperatures equal to or greater than the boiling point of the produced volatile compound, for example acetone, butanone and / or isopropanol. In some embodiments, the cell is capable of growing at a temperature of 56°C (boiling point of acetone) or more.

[0053] Thus, the thermophilic bacterial cell of the present disclosure may comprise one or more modifications for impairing their sporulation. Such modifications are detailed in the sections herein below. i-deficient

[0054] Herein is disclosed a sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising; i) a first enzyme selected from an acetyl-CoA acetyltransferase and an enzyme of EC number 2.3.3.20; ii) a second enzyme selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, an acyl CoA:acetate / 3-ketoacid CoA-transferase, and an acyl-CoA thioesterase II, and iii) an acetoacetate decarboxylase (EC 4.1.1 .4), whereby said sporulation-deficient bacterial is capable of converting acetyl-CoA to acetone, thereby producing acetone, and / or whereby said sporulation-deficient bacterial is capable of converting acetyl-CoA and propionyl-CoA to butanone, thereby producing butanone; and iv) optionally an isopropanol dehydrogenase (EC 1.1.1.80), whereby said sporulation-deficient bacterial cell is capable of converting acetone to isopropanol, thereby producing isopropanol, whereby said sporulation-deficient bacterial cell is capable of producing acetone, butanone, and / or isopropanol, and wherein said sporulation-deficient bacterial cell is capable of producing an increased amount and / or increased titer of acetone, butanone, and / or isopropanol compared to a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell. P6952PC00

[0055] In preferred embodiments, said sporulation-deficient bacterial cell comprises a modification for inactivating sporulation. For instance, the modification may be a mutation in a polynucleotide involved in sporulation or homologues thereof; and / or in a polynucleotide encoding a polypeptide involved in sporulation, or functional variants thereof. In particular, the bacterial cell described herein may have a mutation in a polynucleotide encoding a stage 0 sporulation protein (SpoO), more specifically a mutation in a polynucleotide encoding stage 0 sporulation protein A (SpoOA, such as SEQ ID NO: 65), and / or in a polynucleotide encoding stage 0 sporulation protein B (SpoOB, such as SEQ ID NO: 67), or in homologues thereof encoding functional variants of SpoOA and / or SpoOB, and having an amino acid sequence at least 70% identical to SpoOA or SpoOB (such as SEQ ID NO: 65 or SEQ ID NO: 67, respectively). Preferably, the bacterial cell does not comprise a mutation in a polynucleotide encoding stage 0 sporulation protein F (SpoOF, such as SEQ ID NO: 69).

[0056] In some embodiments, the modification may be a loss-of-function modification. The modification may be one or more mutations, such as loss-of-function mutations. For instance, the modification is a mutation in one or more polynucleotides involved in sporulation, which results in a loss of function of the polynucleotides themselves, which may be directly involved in regulating sporulation, thereby reducing the ability of the cell to sporulate; or the mutation may result in a loss of function of the polypeptides encoded by the polynucleotides, which polypeptides may be involved in regulating sporulation, thereby reducing the ability of the cell to sporulate. A reduced ability to sporulate means that the cell is partially or totally unable to sporulate, when compared to a cell which does not comprise the loss-of-function mutation.

[0057] The modification may thus be a loss-of-function mutation such as a substitution in a polynucleotide resulting in loss-of-function of said polynucleotide or of the polypeptide it encodes, wherein the polynucleotide or the polypeptide are as described herein below. The loss-of-function mutation may be a total or partial deletion of said polynucleotide, resulting either in the encoded polypeptide being absent from the cell, or being present in a truncated form which has reduced function compared to the untruncated form. The loss-of-function mutation may be an insertion in said polynucleotide, resulting either in an early termination of transcription, or having the effect that the encoded polypeptide is shorter or longer than the wild-type form, or resulting in a modified polypeptide which P6952PC00 lost some or all of its activity compared to the wild-type form. In some embodiments, the mutation is an indel.

[0058] In some embodiments, the modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein A, for example a mutation resulting in a partial or total loss of function of spoOA or SpoOA, such as a substitution in, a deletion of or an insertion in spoOA, resulting in a loss-of-function of SpoOA. In some embodiments, the polynucleotide is spoOA as set forth in SEQ ID NO: 64, or a homologue thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. SEQ ID NO: 64 encodes SpoOA as set forth in SEQ ID NO: 65. Thus in some embodiments, the polypeptide is SpoOA as set forth in SEQ ID NO: 65, or a functional variant thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. In some embodiments, the bacterial cell is a P. thermoglucosidasius cell, and the polynucleotide is a homologue of SEQ ID NO: 64 having at least 70% identity thereto. Preferably, said homologue encodes a functional variant of SpoOA having at least 70% identity to SEQ ID NO: 65, which functional variant retains at least part of SpoOA function in the context of sporulation. In some embodiments, the bacterial cell is a P. thermoglucosidasius cell and the polypeptide is SpoOA or a functional variant thereof having at least 70% identity to SEQ ID NO: 65. P6952PC00

[0059] The term “inactivation of a polynucleotide” as used herein in the context of a modification shall be construed as referring to any modification which results in the polypeptide encoded by the inactivated polynucleotide having reduced activity and / or function compared to the activity and / or function of the polypeptide encoded by the same polynucleotide without inactivation. In other words, inactivation of a polynucleotide results in loss-of-function of the polypeptide encoded thereby. The skilled person knows how to achieve this, for instance by mutating or deleting the polynucleotide, so that the encoded polypeptide is produced in lower amounts, thereby overall displaying reduced activity and / or function. For example, a gene deletion can be a type of inactivation of a polynucleotide.

[0060] Inactivation of a polypeptide, such as inactivation of SpoOA or SpoOB, may be obtained by inactivating the polynucleotide encoding the polypeptide. Inactivation of a polypeptide may be obtained by a loss-of-function modification within the polypeptide, for example by an amino acid substitution, insertion or deletion resulting in reduced activity and / or function compared to the activity and / or function of the corresponding polypeptide without said loss-of-function modification.

[0061] In other embodiments, the bacterial cell is a P. thermoglucosidasius cell, and the polynucleotide is a homologue or orthologue of SEQ ID NO: 64 having at least 70% identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOA. In some embodiments, the bacterial cell is a P. thermoglucosidasius cell and the polypeptide is a functional variant of SpoOA having at least 70% identity to SEQ ID NO: 65.

[0062] In other embodiments, the bacterial cell is a B. subtilis cell, and the polynucleotide is a homologue or orthologue of SEQ ID NO: 64 having at least 70% identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOA, which carries out a similar function in the B. subtilis cell as SpoOA does in P. thermoglucosidasius in the context of sporulation. In some embodiments, the bacterial cell is a B. subtilis cell and the polypeptide is a functional variant of SpoOA having at least 70% identity to SEQ ID NO: 65.

[0063] In other embodiments, the bacterial cell is a A. thermocellus cell, and the polynucleotide is a homologue or orthologue of SEQ ID NO: 64 having at least 70% P6952PC00 identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOA, which carries out a similar function in the A. thermocellus cell as SpoOA does in P. thermoglucosidasius in the context of sporulation. In some embodiments, the bacterial cell is a A. thermocellus cell and the polypeptide is a functional variant of SpoOA having at least 70% identity to SEQ ID NO: 65.

[0064] In some embodiments, the bacterial cell is a P. thermoglucosidasius cell and the spoOA gene has been deleted, i.e. at least the coding sequence as set forth in SEQ ID NO: 64 or a homologue thereof has been deleted. In some embodiments, the sequence upstream the sequence encoding SpoOA (SEQ ID NO: 65) has been modified to prevent transcription, whereby transcription of spoOA as set forth in SEQ ID NO: 64 or a homologue thereof is partially or totally inhibited.

[0065] In some embodiments, the modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein B, for example a mutation resulting in a partial or total loss of function of spoOB or SpoOB, such as a substitution in, a deletion of or an insertion in spoOB, resulting in a loss-of-function of SpoOB. In some embodiments, the polynucleotide comprises or consists of spoOB as set forth in SEQ ID NO: 66, or a homologue thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. SEQ ID NO: 66 encodes SpoOB as set forth in SEQ ID NO: 67. Thus in some embodiments, the polypeptide is SpoOB as set forth in SEQ ID NO: 67, or a functional variant thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least P6952PC00

[0066] 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. In some embodiments, the thermophilic bacterial cell is a P. thermoglucosidasius cell, and the polynucleotide comprises or consists of a homologue of SEQ ID NO: 66 having at least 70% identity thereto. Preferably, said homologue encodes a functional variant of SpoOB having at least 70% identity to SEQ ID NO: 67, which functional variant retains at least part of SpoOB function in the context of sporulation. In some embodiments, the cell is a P. thermoglucosidasius cell and the polypeptide is SpoOB or a functional variant thereof having at least 70% identity to SEQ ID NO: 67.

[0067] In other embodiments, the thermophilic bacterial cell is a P. thermoglucosidasius cell, and the polynucleotide comprises or consists of a homologue or orthologue of SEQ ID NO: 66 having at least 70% identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOB. In some embodiments, the cell is a P. thermoglucosidasius cell and the polypeptide is a functional variant of SpoOB having at least 70% identity to SEQ ID NO: 67.

[0068] In other embodiments, the thermophilic bacterial cell is a B. subtilis cell, and the polynucleotide comprises or consists of a homologue or orthologue of SEQ ID NO: 66 having at least 70% identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOA, which carries out a similar function in the B. subtilis cell as SpoOB does in P. thermoglucosidasius in the context of sporulation. In some embodiments, the cell is a B. subtilis cell and the polypeptide is a functional variant of SpoOB having at least 70% identity to SEQ ID NO: 67.

[0069] In other embodiments, the thermophilic bacterial cell is a A. thermocellus cell, and the polynucleotide comprises or consists of a homologue or orthologue of SEQ ID NO: 66 having at least 70% identity thereto; preferably, said homologue or orthologue encodes a functional variant of SpoOB, which carries out a similar function in the A. thermocellus cell as SpoOB does in P. thermoglucosidasius in the context of sporulation. In some embodiments, the cell is a A. thermocellus cell and the polypeptide is a functional variant of SpoOB having at least 70% identity to SEQ ID NO: 67.

[0070] In some embodiments, the thermophilic bacterial cell is a P. thermoglucosidasius cell and the spoOB gene has been deleted, i.e. at least the coding sequence as set forth in P6952PC00

[0071] SEQ ID NO: 66 or a homologue thereof has been deleted. In some embodiments, the sequence upstream the sequence encoding SpoOB (SEQ ID NO: 67) has been modified to prevent transcription, whereby transcription of spoOB as set forth in SEQ ID NO: 66 or a homologue thereof is partially or totally inhibited.

[0072] In some embodiments, the cell comprises two modifications, i.e. a modification resulting in total or partial loss of function of SpoOA or of a functional variant thereof as described herein above, and a modification resulting in total or partial loss of function of SpoOB or a functional variant as described herein above.

[0073] In some embodiments, the cell comprises a modification for inactivating sporulation which is a modification of one or more polynucleotides regulating the expression of one or more polypeptides involved in sporulation, for example of SpoOA and / or SpoOB or functional variants thereof as described herein above. Such polynucleotides may regulate the transcription of the polynucleotides encoding the polypeptides involved in sporulation, or they may regulate transcription of the polynucleotides in the polypeptides involved in sporulation. In some embodiments the modification is a modification of one or more polypeptides involved in sporulation, such as a modification of SpoOA and / or SpoOB or functional variants thereof.

[0074] In some embodiments, the modification for inactivating sporulation is not a modification of a polynucleotide encoding a stage 0 sporulation protein F, such as SpoOF of P. thermoglucosidasius as set forth in SEQ ID NO: 69 or a functional variant thereof having at least 70% identity thereto, encoded by spoOF of SEQ ID NO: 68 or a homologue thereof having at least 70% identity thereto. In other words, the thermophilic bacterial cell preferably does not comprise a modification which results in loss of function of SpoOF or of a homologue, orthologue or functional variant thereof, preferably having at least 70% identity to SEQ ID NO: 69. SpoOF or its functional variant, homologue or orthologue preferably retains at least some of its activity, preferably all of its activity, in the cell.

[0075] The skilled person knows how to test whether a cell is sporulation-deficient. For instance, microscopy analysis can be employed, or a heat-resistance assay can be used, such as described in Example 2. P6952PC00

[0076] The cell of the present disclosure is capable of producing one or more volatile compounds, preferably acetone, butanone and / or isopropanol. The skilled person will know how to adapt the conditions under which the cell is incubated in order to obtain production of one or more specific compounds. The cells described herein may be capable of synthesising acetyl-CoA endogenously, or they may be provided with acetyl- CoA, acetic acid and / or acetate, whereby they can, when also expressing the enzymes detailed herein below, namely a first enzyme as described in “First enzyme”, a second enzyme as described in “Second enzyme” and an acetoacetate decarboxylase as described in “Acetoacetate decarboxylase”, produce acetone, and optionally isopropanol when also expressing an isopropanol dehydrogenase, as described in “Isopropanol dehydrogenase”. The cells expressing the first enzyme, the second enzyme and the acetoacetate decarboxylase may be capable of producing butanone, when the right substrates are available, either because the cell can produce them, or because they are provided, e.g. as part of the cultivation medium. These substrates are then acetyl-CoA, acetic acid, acetate, propionic acid and / or propionate.

[0077] The cell is thus versatile: by changing the incubation conditions, one or more of acetone, butanone and isopropanol can be obtained.

[0078] Volatile compounds

[0079] Besides the sporulation-deficient phenotype discussed in detail herein above, the cells disclosed herein comprise several additional modifications, i.e. either naturally express certain enzymes necessary for the production of one or more volatile compounds, in particular acetone, butanone and / or isopropanol, or have been engineered to express said enzymes.

[0080] The bacterial cell is thus thermophilic, sporulation-deficient and capable of producing acetone, butanone and / or isopropanol. The sporulation-deficient bacterial cell comprises, such as expresses: i) a first enzyme selected from acetyl-CoA acetyltransferase and an enzyme of EC number 2.3.3.20; ii) a second enzyme selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, an acyl CoA:acetate / 3-ketoacid CoA-transferase, and an acyl-CoA thioesterase II; and iii) an acetoacetate decarboxylase (EC 4.1.1.4). P6952PC00

[0081] Optionally, the bacterial cell may also express, such as overexpress, an alcohol dehydrogenase, preferably an isopropanol dehydrogenase (EC 1.1.1.80).

[0082] Specifically, in embodiments where the volatile compound to be produced is acetone, the cell is capable of converting acetyl-CoA to acetone via the following steps:

[0083] 1) conversion of acetyl-CoA to acetoacetyl-CoA;

[0084] 2) conversion of acetoacetyl-CoA to acetoacetate;

[0085] 3) conversion of acetoacetate to acetone.

[0086] In embodiments where the volatile compound to be produced is butanone, the cell is capable of converting propionyl-CoA and acetyl-CoA to butanone via the following steps:

[0087] 1) conversion of propionyl-CoA and acetyl-CoA to 3-ketovaleryl-CoA;

[0088] 2) conversion of 3-ketovaleryl-CoA to 3-oxopentanoate;

[0089] 3) conversion of 3-oxopentanoate to butanone.

[0090] In some embodiments, the cell is capable of converting acetyl-CoA to acetone and propionyl-CoA and acetyl-CoA to butanone via the steps described in the two preceding paragraphs. In other words, the cell may be able to co-produce both acetone and butanone in embodiments, when the cell is cultivated in the presence of acetyl- CoA and propionyl-CoA. The cell may be able to synthesise propionyl-CoA, for example when provided with propionic acid / propionate in the cultivation medium, or propionyl-CoA synthesized by the cell from other substrates or can be provided to the cell, e.g. if the cell has been engineered to be able to utilise extracellular propionyl- CoA, which could be provided in the cultivation medium, cultivation broth or fermentation broth.

[0091] In embodiments where the volatile compound to be produced is isopropanol, the cell is capable of producing acetone as described herein, and is further capable of converting acetone to isopropanol. This involves the following steps:

[0092] 1) conversion of acetyl-CoA to acetoacetyl-CoA;

[0093] 2) conversion of acetoacetyl-CoA to acetoacetate;

[0094] 3) conversion of acetoacetate to acetone;

[0095] 4) conversion of acetone to isopropanol. P6952PC00

[0096] Steps 1) to 3) above can be performed by the same enzymes independently of which volatile compound is to be produced. Enzymes capable of catalysing the reactions of steps 1) to 3) are described herein below, in particular in the sections “First enzyme”, “Second enzyme” and “Acetoacetate decarboxylase”, respectively. Production of isopropanol according to the present disclosure requires that the bacterial cell expresses a further enzyme, preferably an alcohol dehydrogenase, such as an isopropanol dehydrogenase, which is not required for production of acetone or butanone, as detailed herein below.

[0097] It follows thus, that acetone, butanone and isopropanol may all be produced by the same cell, provided that said cell is cultivated in the presence of the necessary substrates for production of all of said compounds. In particular, at least a part of the acetone produced by acetone-producing and / or butanone-producing cells may be converted into isopropanol by one or more endogenous, i.e. native, alcohol dehydrogenases of said cells. Expression, such as overexpression, of a native or nonnative isopropanol dehydrogenase can increase the titer of produced isopropanol by for acetone-producing and / or butanone-producing cells. For cells that do not naturally convert acetone or butanone to isopropanol, the introduction of a further enzyme, preferably an alcohol dehydrogenase, as described herein below in “Isopropanol dehydrogenase”, may result in isopropanol production by an acetone-producing or from a butanone-producing cell.

[0098] In some embodiments, the bacterial cell further comprises a modification resulting in inactivation of a lactate dehydrogenase, such as L-lactate dehydrogenase. The modification resulting in inactivation of the lactate dehydrogenase may for example be a mutation in a polynucleotide encoding a lactate dehydrogenase, such as a loss-of- function mutation. In some embodiments, the modification resulting in inactivation of a lactate dehydrogenase is an inactivation of a polynucleotide encoding Ldh of locus tag AOT13_RS05985 in the Gene database of the database National Center for Biotechnology (NCBI, Gene ID: 29237965, entry discontinued on 31 July 2020) for P. thermoglucosidasius DSM2542 or a functional variant thereof having at least 70% identity thereto, for example a mutation resulting in a partial or total loss of function of the polynucleotide having NCBI locus tag AOT13_RS05985, such as a substitution in, a deletion of or an insertion in the polynucleotide having NCBI locus tag AOT13_RS05985 or a homologue thereof having at least 70% identity thereto, P6952PC00 resulting in a loss-of-function of Ldh or a functional variant thereof having at least 70% identity thereto. The loss-of-function mutation may be a total or partial deletion of said polynucleotide resulting either in the encoded polypeptide being absent from the cell, or being present in a truncated form which has reduced function compared to the untruncated form. The loss-of-function mutation may be an insertion in said polynucleotide, resulting e.g. either in an early termination of transcription, in decreased translation, having the effect that the encoded polypeptide is shorter or longer than the wild-type form, or resulting in a modified polypeptide which lost some or all of its activity compared to the wild-type form. In some embodiments, the mutation is an indel.

[0099] First enzyme

[0100] The above modifications, in particular the presence of an acetyl-CoA acetyltransferase or an enzyme of EC number 2.3.3.20, allow the sporulation-deficient bacterial cell to convert acetyl-CoA to acetone. In some embodiments, the first enzyme is either an acetyl-CoA acetyltransferase, also termed thiolase, or an acyl-CoA:acyl-CoA alkyltransferase .

[0101] Thiolases can catalyse either the conversion of: i. two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA), or ii. one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA and coenzyme A (CoA).

[0102] Which reaction actually occurs in the cell will depend on which substrates are present in the cultivation medium, cultivation broth or fermentation broth, or on the metabolism of the particular cell used, as the skilled person well knows. If acetyl-CoA is present, then reaction i. will occur. If both acetyl-CoA and propionyl-CoA are present, then reaction ii. or both reactions will occur. Supplementing the cultivation medium or cultivation broth with acetic acid and / or propionic acid / propionate may increase the titer. The sporulation-deficient bacterial cell may also have been engineered to be capable of synthesising propionyl-CoA and / or acetyl-CoA, or to synthesise propionyl- CoA and / or acetyl-CoA in greater amounts than in a corresponding non-engineered sporulation-deficient bacterial cell. P6952PC00

[0103] Reaction i. is required for production of acetone according to the present disclosure. Reaction ii. is required for the production of butanone according to the present disclosure.

[0104] In some embodiments the first enzyme is an acetyl-CoA acetyltransferase of EC number 2.3.1.9. These enzymes have a substrate preference for acetyl-CoA or propionyl-CoA and therefore preferably catalyse the reaction in the forward direction.

[0105] In some embodiments, the first enzyme is an enzyme of EC number 2.3.3.20, i.e. an acyl-CoA:acyl-CoA alkyltransferase. This enzyme catalyses the conversion of two molecules of acyl-CoA into one molecule of either 3-oxoalkanoate or 2-alkyl-3- oxoalkanoate, such as (2R)-2-alkyl-3-oxoalkanoate, depending on the substrate, as defined herein, in the presence of water, thereby generating two molecules of coenzyme A (CoA). As detailed herein above, the specific product of the reaction catalysed by acyl-CoA:acyl-CoA alkyltransferase depends on the substrate.

[0106] The first enzyme may be an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Tfu_1520 (SEQ ID NO: 5), Tfu_0436 (SEQ ID NO: 6), Slip_0880 (SEQ ID NO: 7), Tfu_2394 (SEQ ID NO: 8), Slip_1236 (SEQ ID NO: 9), Caur_1540 (SEQ ID NO: 10), Tfu_0253 (SEQ ID NO: 11), CHY_1604 (SEQ ID NO: 14), CHY_1288 (SEQ ID NO: 15), Slip_2085 (SEQ ID NO: 16), Slip_0465 (SEQ ID NO: 17), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), CHY_1355 (SEQ ID NO: 18), and functional variants thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. In some embodiments, the first enzyme is CHY_1288 (SEQ ID NO: 15), CHY_1355 (SEQ ID NO: 18), Caur_1540 (SEQ ID NO: 10), GHH_c20420 (SEQ ID NO: 1), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), or Slip_0880 (SEQ ID NO: 7), or a functional P6952PC00 variant thereof having at least 70% identity thereto, preferably wherein the first enzyme is Caur_1461 (SEQ ID NO: 3), Rxy2 (SEQ ID NO: 60), Slip_0880 (SEQ ID NO: 7), or Dde1 (SEQ ID NO: 59). Preferably, the first enzyme is an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Slip_0880 (SEQ ID NO: 7), and Dde1 (SEQ ID NO: 59), and functional variants thereof having at least 70% identity thereto.

[0107] In one embodiment, the first enzyme is GHH_c20420 (SEQ ID NO: 1), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_0499 (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_0479 (SEQ ID NO: 4), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Tfu_1520 (SEQ ID NO: 5), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Tfu_0436 (SEQ ID NO: 6), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_0880 (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Tfu_2394 (SEQ ID NO: 8), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_1236 (SEQ ID NO: 9), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Caur_1540 (SEQ ID NO: 10), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Tfu_0253 (SEQ ID NO: 11), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is CHY_1604 (SEQ ID NO: 14), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is CHY_1288 (SEQ ID NO: 15), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_2085 (SEQ ID NO: 16), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Slip_0465 (SEQ ID NO: 17), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Dde1 (SEQ ID NO: 59) or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Rxy2 (SEQ ID NO: 60) or a functional variant thereof P6952PC00 having at least 70% identity thereto. In another embodiment, the first enzyme is CHY_1355 (SEQ ID NO: 18), or a functional variant thereof having at least 70% identity thereto.

[0108] Alternatively, the first enzyme is an enzyme of EC number 2.3.3.20 selected from the acyl-CoA:acyl-CoA alkyltransferase SVA_3859 (SEQ ID NO: 12) and the acyl- CoA:acyl-CoA alkyltransferase Despr_2661 (SEQ ID NO: 13). In one embodiment, the first enzyme is SVA_3859 (SEQ ID NO: 12), or a functional variant thereof having at least 70% identity thereto. In another embodiment, the first enzyme is Despr_2661 (SEQ ID NO: 13), or a functional variant thereof having at least 70% identity thereto.

[0109] In some embodiments, the first enzyme is Slip_0880 (SEQ ID NO: 7), Caur_1461 (SEQ ID NO: 3), GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Slip_0479 (SEQ ID NO: 4), or a functional variant thereof having at least 70% identity thereto.

[0110] Functional variants of the above enzymes are modified versions of said enzyme which still retain at least some of the activity of the original enzyme. In the case of thermostable enzymes, the functional variant preferably also is thermostable. In some embodiments, the functional variant harbours mutations compared to the original enzyme, which preferably are not located within the active site of the enzyme. The skilled person knows how to determine if a variant of the first enzyme is functional or not. For example, potential thiolases can be incubated with acetoacetyl-CoA and CoA, and absorbance at 303 nm can be monitored. A decrease in the absorbance at 303 nm indicates that the potential thiolase can perform said reaction and has thiolase activity - it can thus be considered a functional variant. Potential acyl-CoA:acyl-CoA alkyltransferase can be incubated with acetyl-CoA (or acetyl-CoA and propionyl-CoA), with subsequent addition of 5,5’-dithio-bis-(2-nitrobenzoic acid), which reacts with a free thiol group of the released CoASH. The absorbance of the product can be monitored at 412 nm. Formation of the product indicates that the potential acyl- CoA:acyl-CoA alkyltransferase retains acyl-CoA:acyl-CoA alkyltransferase activity.

[0111] Enzymes of this type contain thiolase N-terminal domain (Pfam accession number PF00108), thiolase C-terminal domain (PF02803) and beta-ketoacyl synthase domain (PF00109), which contain the active center and participate in oligomerization of functional enzyme (Mathieu et al., 1997). Thus, functional variants of such enzymes P6952PC00 preferably comprise said domains. Functional variants may have been engineered as is otherwise known in the art.

[0112] Second enzyme

[0113] The sporulation-deficient bacterial cells described herein and employed in the methods disclosed herein further express a second enzyme, which is selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA- transferase, and an acyl-CoA thioesterase II. This can be achieved by further engineering the cell, if the cell does not naturally express these enzymes.

[0114] Acetate CoA-transferases (EC 2.8.3.8) catalyse the conversion of acetate and an acyl- CoA to acetyl-CoA and a fatty acid. 3-oxoacid CoA-transferases (EC 2.8.3.5) catalyse the conversion of 3-oxoacyl-CoA and a succinate to 3-oxoacid and a 3-succinyl-CoA, or the conversion of 3-ketovaleryl-CoA + acetate to 3-oxopentanoate + acetyl-CoA.

[0115] Acyl CoA:acetate / 3-ketoacid CoA-transferases (EC 2.8.3.1) catalyse the conversion of acetyl-CoA and propanoate to acetate and propanoyl-CoA. Acyl-CoA thioesterases II (EC 3.1.2.-) catalyse the reaction of hydrolysis of acyl-CoA into fatty acid and CoASH.

[0116] Some of the above enzymes can catalyse different reactions. The type of reaction that actually occurs in the sporulation-deficient bacterial cell will depend on which substrates are present in the cultivation medium, cultivation broth, fermentation broth or on how the cell has been engineered, as the skilled person well knows.

[0117] More specifically, the second enzyme is selected from: Tle2, Dde2 (EC 2.8.3.5) (SEQ ID NO: 21), Ghh2 (EC 2.8.3.5), Tme (EC 2.8.3.8), Pth (EC 2.8.3.1) (SEQ ID NO: 26), Rma (EC 3.1.2.-) (SEQ ID NO: 27), and functional variants thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20) or functional variants thereof having P6952PC00 at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto; wherein Ghh2 comprises or consists of Ghh2 subunit A (SEQ ID NO: 22) and Ghh2 subunit B (SEQ ID NO: 23) or functional variants thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto; and wherein Tme comprises or consists of Tme subunit A (SEQ ID NO: 24) and Tme subunit B (SEQ ID NO: 25) or functional variants thereof having at least 70% identity thereto, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.

[0118] Preferably, the second enzyme is Tle2 or Dde2 (SEQ ID NO: 21) or a functional variant thereof having at least 70% identity thereto, wherein Tle2 consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto. P6952PC00

[0119] In some embodiments, the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto. This enzyme comprises or consists of two subunits, subunit A as set forth in SEQ ID NO: 19, and subunit B as set forth in SEQ ID NO: 20. Subunit A has an EC number 2.8.3.8, and subunit B has an EC number 2.8.3.9. In some embodiments, the second enzyme is Tle2 which consists of Tle2 subunit A and Tle2 subunit B, as set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some embodiments, the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto. In some embodiments, the second enzyme is a functional variant of Tle2 consisting of Tle2 subunit A and of a functional variant of Tle2 subunit B having at least 70% identity to SEQ ID NO: 20. In other embodiments, the second enzyme is a functional variant of Tle2 consisting of Tle2 subunit B and of a functional variant of Tle2 subunit A having at least 70% identity to SEQ ID NO: 19. In some embodiments, the second enzyme is a functional variant of Tle2 consisting of a functional variant of Tle2 subunit A having at least 70% identity to SEQ ID NO: 19 and of a functional variant of Tle2 subunit B having at least 70% identity to SEQ ID NO: 20.

[0120] In some embodiments, the second enzyme is an enzyme having an EC number 2.8.3.5. In some embodiments, the second enzyme is Dde2 (SEQ ID NO: 21) or a functional variant thereof having at least 70% identity thereto.

[0121] In some embodiments, the second enzyme is Ghh2 or a functional variant thereof having at least 70% identity. This enzyme comprises or consists of two subunits, subunit A as set forth in SEQ ID NO: 22, and subunit B as set forth in SEQ ID NO: 23. Both subunits have EC number 2.8.3.5. In some embodiments, the second enzyme is Ghh2 which consists of Ghh2 subunit A and Ghh2 subunit B, as set forth in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. In some embodiments, the second enzyme is Ghh2 or a functional variant thereof having at least 70% identity thereto. In some embodiments, the second enzyme is a functional variant of Ghh2 consisting of Ghh2 subunit A and of a functional variant of Ghh2 subunit B having at least 70% identity to SEQ ID NO: 23. In other embodiments, the second enzyme is a functional variant of Ghh2 consisting of Ghh2 subunit B and of a functional variant of Ghh2 subunit A having at least 70% identity to SEQ ID NO: 22. In some embodiments, the second enzyme is a functional variant of Ghh2 consisting of a functional variant of Ghh2 subunit A having at least 70% identity to SEQ ID NO: 22, and of a functional variant of Ghh2 subunit B having at least 70% identity to SEQ ID NO: 23. P6952PC00

[0122] In some embodiments, the second enzyme is Tme or a functional variant thereof having at least 70% identity thereto. This enzyme of EC number 2.8.3.8 comprises or consists of two subunits, subunit A as set forth in SEQ ID NO: 24, and subunit B as set forth in SEQ ID NO: 25. In some embodiments, the second enzyme is Tme which consists of Tme subunit A and Tme subunit B, as set forth in SEQ ID NO: 24 and SEQ ID NO: 25, respectively. In some embodiments, the second enzyme is a functional variant of T me consisting of T me subunit A and of a functional variant of T me subunit B having at least 70% identity to SEQ ID NO: 25. In other embodiments, the second enzyme is a functional variant of Tme consisting of Tme subunit B and of a functional variant of Tme subunit A having at least 70% identity to SEQ ID NO: 24. In some embodiments, the second enzyme is a functional variant of T me consisting of a functional variant of Tme subunit A having at least 70% identity to SEQ ID NO: 24 and of a functional variant of Tme subunit B having at least 70% identity to SEQ I D NO: 25.

[0123] In some embodiments, the second enzyme is an enzyme having an EC number 2.8.3.1. In some embodiments, the second enzyme is Pth (SEQ ID NO: 26) or a functional variant thereof having at least 70% identity thereto.

[0124] In some embodiments, the second enzyme is an enzyme having an EC number 3.1.2.-. In some embodiments, the second enzyme is Rma (SEQ ID NO: 27) or a functional variant thereof having at least 70% identity thereto.

[0125] In preferred embodiments, the second enzyme is Tle2 consisting of Tle2 subunit A and of Tle2 subunit B (SEQ ID NO: 19 and SEQ ID NO: 20, respectively), or a functional variant thereof having at least 70% identity thereto.

[0126] Functional variants of the above enzymes are modified versions of said enzyme which still retain at least some of the activity of the original enzyme. In the case of thermostable enzymes, the functional variant preferably also is thermostable. In some embodiments, the functional variant harbours mutations compared to the original enzyme, which preferably are not located within the active site of the enzyme. The skilled person knows how to determine if a variant of the second enzyme is functional or not. For example, potential acetate CoA-transferases of EC number 2.8.3.8 can be incubated with acetyl-CoA and lithium acetoacetate, and absorbance at 313 nm can be P6952PC00 monitored to follow the formation of acetoacetyl-CoA. Potential 3-oxoacid CoA- transferases of EC number 2.8.3.5 can be tested as described herein above. Potential acyl CoA:acetate / 3-ketoacid CoA-transferases (EC 2.8.3.1) can be tested as described herein above. Potential acyl-CoA thioesterases II (EC 3.1.2.-) can be tested as described herein above.

[0127] Enzymes of this type contain coenzyme A transferase domain (Pfam accession number PF01144) and acetyl-CoA hydrolase / transferase C-terminal domain (PF13336), which contain the active center and participate in oligomerization of functional enzyme. Thus, functional variants of such enzymes preferably comprise said domains. Functional variants may have been engineered as is otherwise known in the art.

[0128] Acetoacetate decarboxylase

[0129] The sporulation-deficient bacterial cells of the present disclosure and employed in the present methods further express an acetoacetate decarboxylase. This enzyme has an EC number of 4.1.1.4, and catalyses the decarboxylation of acetoacetate, yielding acetone and carbon dioxide; the enzyme can also catalyse decarboxylation of said 3- oxopentanoate to butanone; it can also participate in the conversion of acetoacetyl- CoA to acetone or of 3-ketovaleryl-CoA to butanone. The expression of this enzyme in the sporulation-deficient bacterial cells disclosed herein thus allows conversion of acetate to acetone, where the acetate is either provided to the cell, e.g. in the cultivation medium, or is produced by the cell. When the cell is incubated in conditions where 3-oxopentanoate is produced, e.g. if the cultivation medium, cultivation broth or fermentation broth comprises propionic acid, the enzyme can catalyse the decarboxylation of said 3-oxopentanoate to butanone.

[0130] The acetoacetate decarboxylase is preferably a thermostable acetoacetate decarboxylase. In preferred embodiments, the acetoacetate decarboxylase is not native to a thermophilic microorganism, in particular the acetoacetate decarboxylase may be native to a Clostridium species such as Clostridium acetobutylicum. The acetoacetate decarboxylase Cac, as set forth in SEQ ID NO: 28, may be particularly advantageous in the present context.

[0131] Thus in some embodiments, the acetoacetate decarboxylase is Cac as set forth in

[0132] SEQ ID NO: 28, or a functional variant having at least 70% identity thereto, such as at P6952PC00 least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.

[0133] Functional variants of the above enzymes are modified versions of said enzymes which still retain at least some of the activity of the original enzymes. In the case of thermostable enzymes, the functional variant preferably also is thermostable. In some embodiments, the functional variant harbours mutations compared to the original enzyme, which preferably are not located within the active site of the enzyme. The skilled person knows how to determine if a variant of an acetoacetate decarboxylase is functional or not. For example, the potential acetoacetate decarboxylase can be incubated with lithium acetoacetate. The accompanying release of CO2 which ensues can be monitored, e.g. manometrically. Release of CO2 indicates that the tested enzyme has acetoacetate decarboxylase activity.

[0134] Enzymes of this type contain acetoacetate decarboxylase domain (Pfam accession number PF06314), which contain the active center and participate in oligomerization of functional enzyme. Amino acid residues Lys 115, Lys 116, Arg 29, Glu 61, Glu 76 in the active site are necessary for the activity of the enzyme (Ho et al., 2009). Thus, functional variants of such enzymes preferably comprise said domains and / or residues. Functional variants may have been engineered as is otherwise known in the art.

[0135] Isopropanol dehydrogenase

[0136] Also provided herein are sporulation-deficient bacterial cells and methods for the production of isopropanol using said cells. For this, the cell, in addition to the above enzymes, i.e. in addition to the first enzyme, the second enzyme and the acetoacetate decarboxylase, may further express an isopropanol dehydrogenase. This enzyme (EC 1.1.1.80) catalyses the conversion of acetone to propan-2-ol. Thus a sporulationdeficient bacterial cell capable of producing acetone as described herein can be further modified to express an isopropanol dehydrogenase which can then convert the produced acetone, or at least part thereof, to isopropanol. Alternatively, the cell may P6952PC00 convert at least part of the produced acetone to isopropanol by means of at least one endogenous, i.e. one native, alcohol dehydrogenase having at least some isopropanol dehydrogenase-activity.

[0137] In some embodiments, the isopropanol dehydrogenase is Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto.

[0138] Combinations of enzymes

[0139] In some embodiments, the bacterial cell can produce at least acetone and isopropanol, and expresses Cac (SEQ ID NO: 28) and Tbr (SEQ ID NO: 29) or functional variants thereof and one of the following combinations of first and second enzymes: i) Dde1 (SEQ ID NO: 59) and Dde2 (SEQ ID NO: 21), or functional variants thereof; or ii) Caur_1461 (SEQ ID NO: 3) and Tle2, or functional variants thereof; or iii) Slip_0880 (SEQ ID NO: 7) and Tle2, or functional variants thereof; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19 and Tle2 subunit B (EC 2.8.3.9) as set forth in SEQ ID NO: 20 or functional variants thereof.

[0140] In some embodiments, the bacterial cell can produce at least butanone and isopropanol, and expresses Cac (SEQ ID NO: 28) and Tbr (SEQ ID NO: 29) or functional variants thereof and one of the following combinations of first and second enzymes: i) Caur_1461 (SEQ ID NO: 3) and Tle2, or functional variants thereof; or ii) GHH_c20420 (SEQ ID NO: 1) and Tle2, or functional variants thereof; or iii) Slip_0499 (SEQ ID NO: 2) and Tle2, or functional variants thereof; or iv) Slip_0479 (SEQ ID NO: 4) and Tle2, or functional variants; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19 and Tle2 subunit B (EC 2.8.3.9) as set forth in SEQ ID NO: 20 or functional variants thereof.

[0141] In some embodiments: the first enzyme is Slip_0880 (SEQ ID NO: 7) or a functional variant thereof having at least 70% identity thereto; the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (SEQ ID NO: 19) and of P6952PC00 tle2 subunit B (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto and having acetate CoA transferase, 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA-transferase or acyl-CoA thioesterase II activity; and the sporulation-deficient bacterial cell further expresses an acetatoacetate decarboxylase which is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. The cell may further express Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto and having isopropanol dehydrogenase activity.

[0142] In some embodiments: the first enzyme is Caur_1461 (SEQ ID NO: 3) or a functional variant thereof having at least 70% identity thereto; the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (SEQ ID NO: 19) and of tle2 subunit B (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto and having acetate CoA transferase, 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA-transferase or acyl-CoA thioesterase II activity; and the sporulation-deficient bacterial cell further expresses an acetatoacetate decarboxylase which is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. The cell may further express Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto and having isopropanol dehydrogenase activity.

[0143] In some embodiments: the first enzyme is GHH_c20420 (SEQ ID NO: 1) or a functional variant thereof having at least 70% identity thereto; the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (SEQ ID NO: 19) and of tle2 subunit B (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto and having acetate CoA transferase, 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA-transferase or acyl-CoA thioesterase II activity; and the sporulation-deficient bacterial cell further expresses an acetatoacetate decarboxylase which is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. The cell may further express Tbr (SEQ ID NO: 29) or a P6952PC00 functional variant thereof having at least 70% identity thereto and having isopropanol dehydrogenase activity.

[0144] In some embodiments: the first enzyme is Slip_0499 (SEQ ID NO: 2) or a functional variant thereof having at least 70% identity thereto; the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (SEQ ID NO: 19) and of tle2 subunit B (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto and having acetate CoA transferase, 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA-transferase or acyl-CoA thioesterase II activity; and the sporulation-deficient bacterial cell further expresses an acetatoacetate decarboxylase which is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. The cell may further express Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto and having isopropanol dehydrogenase activity.

[0145] In some embodiments: the first enzyme is Slip_0479 (SEQ ID NO: 4) or a functional variant thereof having at least 70% identity thereto; the second enzyme is Tle2 or a functional variant thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (SEQ ID NO: 19) and of tle2 subunit B (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto and having acetate CoA transferase, 3-oxoacid CoA transferase, acyl CoA:acetate / 3-ketoacid CoA-transferase or acyl-CoA thioesterase II activity; and the sporulation-deficient bacterial cell further expresses an acetatoacetate decarboxylase which is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. The cell may further express Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto and having isopropanol dehydrogenase activity.

[0146] In some embodiments, the bacterial cell comprises Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto, wherein the bacterial cell further comprises: P6952PC00 i) Dde1 (SEQ ID NO: 59) and Dde2 (SEQ ID NO: 21), or functional variants thereof having at least 70% identity thereto, whereby at least acetone is produced; ii) Caur_1461 (SEQ ID NO: 3) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least acetone and / or butanone is produced; iii) Slip_0880 (SEQ ID NO: 7) and Tle2; or functional variants thereof having at least 70% identity thereto, whereby at least acetone is produced; iv) GHH_c20420 (SEQ ID NO: 1) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; v) Slip_0499 (SEQ ID NO: 2) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; or vi) Slip_0479 (SEQ ID NO: 4) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), or functional variants thereof having at least 70% identity thereto

[0147] As detailed herein above, the cell of the present disclosure is sporulation-deficient. For instance, the bacterial cell comprises a modification for inactivation of sporulation, such as a modification of the gene encoding SpoOA and / or SpoOB.

[0148] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOA (SEQ ID NO: 65), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto. P6952PC00

[0149] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOB (SEQ ID NO: 67), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0150] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0151] Loss-of-function SpoOA and loss-of-function SpoOB may be obtained by mutating the polynucleotide encoding SpoOA and SpoOB, respectively, such as inactivating the polynucleotide encoding the polynucleotide and / or inactivating the encoded polypeptide, here SpoOA and SpoOB, respectively. For instance, a loss-of-function modification may be a partial or total deletion of the gene encoding the polypeptide. The loss-of-function modification may also be obtained by a modification of or mutation in the promoter region for the gene encoding the polypeptide.

[0152] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOA (SEQ ID NO: 64) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: iii. Slip_0880 (SEQ ID NO: 7); or iv. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0153] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOB (SEQ ID NO: P6952PC00

[0154] 66) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: iii. Slip_0880 (SEQ ID NO: 7); or iv. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0155] In some embodiments, the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOA (SEQ ID NO: 64) and spoOB (SEQ ID NO: 66) or homologues thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: iii. Slip_0880 (SEQ ID NO: 7); or iv. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0156] Polynucleotides

[0157] The cell in some embodiments comprises one or more polynucleotides encoding the first enzyme, the second enzyme, the acetoacetate decarboxylase and optionally the isopropanol dehydrogenase. These may be codon optimised for expression in the bacterial cell.

[0158] The polynucleotides may be comprised within one or more vectors, such as one or more plasmids, or they may be integrated in the genome of the bacterial cell, for example under the control of an inducible promoter or a constitutive promoter.

[0159] In some embodiments, the thermophilic, sporulation-deficient cell is a non-natural cell.

[0160] Titers

[0161] The sporulation-deficient bacterial cell described herein produces increased amounts of volatile compounds, such as acetone, butanone and / or isopropanol, compared to a reference cell, i.e. a cell which is not sporulation-deficient but is otherwise identical to the sporulation-deficient bacterial cell, at least when cultivated in the same conditions. The sporulation-deficient bacterial cell disclosed herein thus produces acetone, butanone and / or isopropanol (depending on which enzymes they express, as detailed herein above) with an increased titer compared to the reference cell, preferably when P6952PC00 the reference cell and the sporulation-deficient bacterial cell are cultivated in the same conditions.

[0162] A sporulation-proficient bacterial cell, or a reference cell, shall be construed herein as a cell which does not comprise any of the modifications described in the section “Sporulation-deficient” herein above. The sporulation-proficient bacterial cell may still exhibit some sporulation deficiency compared to a corresponding wild type bacterial cell. In other words, the term “sporulation-proficient” herein refers to a cell which does not comprise any of the modifications described in the section “Sporulation-deficient” herein, and is capable of sporulating more than the sporulation-deficient bacterial cell.

[0163] A sporulation-proficient bacterial cell in the present context may thus be fully sporulation-proficient, i.e. have a sporulation comparable, similar or identical to the sporulation observed in a corresponding wild type bacterial cell, or it may be partly sporulation-proficient, i.e. have reduced sporulation compared to the sporulation of a corresponding wild type bacterial cell, but increased sporulation compared to the sporulation-deficient bacterial cells described herein.

[0164] In some embodiments, the sporulation-deficient bacterial cells are capable of producing acetone, and the sporulation-deficient bacterial cells described herein can produce acetone with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.37 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at P6952PC00 least 150 g / L, such as at least 250 g / L, or more. Thus in some embodiments the sporulation-deficient bacterial cells described herein produce acetone with a titer which is improved by at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0165] In some embodiments, the sporulation-deficient bacterial cells are capable of producing butanone, and the sporulation-deficient bacterial cells described herein can produce butanone with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.3 g / L, such as at least 0.4 g / L, such as at least 0.5 g / L, such as at least 0.55 g / L, such as at least 0.560 g / L, such as at least 0.7 g / L, such as at least 0.73 g / L, such as at least 0.75 g / L, such as at least 1.0 g / L, such as at least 2.0 g / L, such as at least 3.0 g / L, such as at least 4.0 g / L, such as at least 5.0 g / L, such as at least 7.5 g / L, such as at least 10.0 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more. Thus in some embodiments the sporulation-deficient bacterial cells described herein produce butanone with a titer which is improved by at least 30%, such as at least 31%, such as at least 32%, such as at least 33%, such as at least 34%, such as at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0166] In some embodiments, the sporulation-deficient bacterial cells are capable of producing isopropanol, and the sporulation-deficient bacterial cells described herein can produce isopropanol with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, P6952PC00 such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1 .4 g / L, such as at least 1 .5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more. Thus in some embodiments the sporulation-deficient bacterial cells described herein produce isopropanol with a titer which is increased by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 31%, such as at least 32%, such as at least 33%, such as at least 34%, such as at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0167] Preferably, the sporulation-deficient cell can produce at least acetone and isopropanol, and expresses Cac (SEQ ID NO: 28) and Tbr (SEQ ID NO: 29) or functional variants thereof having at least 70% identity thereto and one of the following combinations of first and second enzymes: i. Dde1 (SEQ ID NO: 59) and Dde2 (SEQ ID NO: 21); or functional variants thereof having at least 70% identity thereto; or ii. Caur_1461 (SEQ ID NO: 3) and Tle2; or functional variants thereof having at least 70% identity thereto; or iii. Slip_0880 (SEQ ID NO: 7) and Tle2; or functional variants thereof having at least 70% identity thereto; P6952PC00 wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20) or functional variants thereof having at least 70% identity thereto. In addition the sporulation-deficient bacterial cell has one or more modifications as described in the section “Sporulation-deficient” herein above, such as a modification of SpoOA, SpoOB, spoOA or spoOB, or functional variants or homologues thereof, respectively. Such sporulation-deficient bacterial cell can yield an increased titer of acetone and / or of isopropanol, compared to a sporulation-proficient bacterial cell expressing the same enzymes leading to production of acetone and isopropanol.

[0168] In some embodiments, the sporulation-deficient bacterial cell can produce at least butanone and isopropanol, and express Cac (SEQ ID NO: 28) and Tbr (SEQ ID NO: 29) or functional variants thereof having at least 70% identity thereto and one of the following combinations of first and second enzymes: i) Caur_1461 (SEQ ID NO: 3) and Tle2; or functional variants thereof having at least 70% identity thereto; or ii) GHH_c20420 (SEQ ID NO: 1) and Tle2; or functional variants thereof having at least 70% identity thereto; or iii) Slip_0499 (SEQ ID NO: 2) and Tle2; or functional variants thereof having at least 70% identity thereto; or iv) Slip_0479 (SEQ ID NO: 4) and Tle2; or functional variants thereof having at least 70% identity thereto; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), or functional variants thereof having at least 70% identity thereto. In addition the sporulation-deficient bacterial cell has one or more modifications as described in the section “Sporulation-deficient” herein above, such as a modification of SpoOA, SpoOB, spoOA or spoOB, or functional variants or homologues thereof, respectively. Such sporulation-deficient bacterial cell can yield an increased titer of butanone and / or of isopropanol, compared to a sporulation-proficient bacterial cell expressing the same enzymes leading to production of butanone and isopropanol.

[0169] Method for producing volatile compounds with an increased titer

[0170] The sporulation-deficient bacterial cells described herein above can advantageously be used to produce volatile compounds, in particular acetone, butanone and / or P6952PC00 isopropanol, with an increased titer(s) compared to cells which are sporulationproficient but otherwise identical to above-mentioned sporulation-deficient bacterial cells.

[0171] Thus, herein is provided a method for producing a volatile compound, comprising the steps of: i. providing a sporulation-deficient bacterial cell as described herein above, said sporulation-deficient bacterial cell being capable of producing said volatile compound; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth comprising said volatile compound; and iii. optionally recovering the cultivation broth and / or said volatile compound.

[0172] Herein is thus provided a method for producing acetone, butanone, and / or isopropanol, comprising the steps of: i. providing a sporulation-deficient bacterial cell as described herein above; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth comprising acetone, butanone, and / or isopropanol; and iii. optionally recovering the cultivation broth and / or the acetone, butanone, and / or isopropanol.

[0173] Also provided is a method for increasing the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell capable of producing acetone, butanone and / or isopropanol, said method comprising the steps of: i. providing said sporulation-proficient bacterial cell; ii. inactivating sporulation in said bacterial cell, thereby obtaining a sporulationdeficient bacterial cell capable of producing acetone, butanone and / or isopropanol; iii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth; whereby said sporulation-deficient bacterial cell produces acetone, butanone and / or isopropanol with an increased titer compared to the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell P6952PC00 otherwise identical to said sporulation-deficient bacterial cell, such as the sporulation-proficient bacterial cell provided in step i., in the same conditions; and iv. optionally recovering the cultivation broth and / or the acetone, butanone and / or isopropanol.

[0174] As explained in detail herein above, the terms “sporulation-proficient” and “sporulationdeficient” must be construed relative to one another. In other words, the sporulationproficient cell may display partial impairment of sporulation, but said impairment can be amplified or increased by introducing in the cell the modifications described herein above in the section “Sporulation-deficient”. In particular, the cell may be modified by partially or totally inactivating SpoOA and / or SpoOB, or functional variants thereof.

[0175] Preferably, the thermophilic bacterial cell comprises a modification for inactivation of its sporulation. In some embodiments, said modification is a mutation in a polynucleotide involved in sporulation or homologues thereof, and / or in a polynucleotide encoding a polypeptide involved in sporulation, or functional variants thereof. In some embodiments, the bacterial cell has a mutation in a polynucleotide encoding a stage 0 sporulation protein (SpoO), more specifically a mutation in a polynucleotide encoding stage 0 sporulation protein A (SpoOA, such as SEQ ID NO: 65), and / or in a polynucleotide encoding stage 0 sporulation protein B (SpoOB, such as SEQ ID NO: 67), or in homologues thereof encoding functional variants of SpoOA and / or SpoOB, and having an amino acid sequence at least 70% identical to SpoOA or SpoOB (such as SEQ ID NO: 65 or SEQ ID NO: 67, respectively). Preferably, the thermophilic bacterial cell does not comprise a mutation in a polynucleotide encoding stage 0 sporulation protein F (SpoOF, such as SEQ ID NO: 69).

[0176] The bacterial cell in which sporulation should be inactivated as described hereinis however capable of producing a volatile compound, preferably acetone, butanone and / or isopropanol, as described herein above, in particular in the section “Volatile compounds”.

[0177] As described previously, thermophilic cells naturally producing or engineered to produce acetone, butanone and / or isopropanol, are particularly suitable for the present methods. When modified to impair or further impair sporulation, such bacterial cells produce acetone, butanone and / or isopropanol with an increased titer compared to a P6952PC00 corresponding bacterial cell without impaired sporulation or further impaired sporulation. Which enzymes engineered cells may express in order to produce these compounds has been described in detail in the section “Volatile compounds”.

[0178] The cell may be cultivated in a reactor, for example a bioreactor or a fermenter, as is known in the art. Cultivation may comprise or consist of a continuous fermentation. The skilled person will have no difficulty determining how to best cultivate the present sporulation-deficient bacterial cell based on the cell’s genotype and / or phenotype.

[0179] In the context of the present disclosure, the cell is preferably cultivated at “high” temperatures, i.e. temperatures above the conventional 37°C normally employed for bacterial cultivations, which temperatures can still be tolerated by thermophilic cells. The advantage of performing the cultivation at higher temperatures is that this allows facilitated recovery of the produced volatile compounds. Preferably, the cell is cultivated at a temperature of between 42 and 80°C, such as between 50 and 75°C, for example at 60°C. In some embodiments, cultivation is performed at a temperature of 42°C or more, such as 43°C or more, such as 44°C or more, such as 45°C or more, such as 46°C or more, such as 47°C or more, such as 48°C or more, such as 49°C or more, such as 50°C or more, such as 51 °C or more, 52°C or more, 53°C or more, 54°C or more, 55°C or more, 56°C or more, 57°C or more, 58°C or more, 59°C or more, for example 60°C or more. In some embodiments, the method is performed at a temperature of between 42 and 80°C, such as between 50 and 75°C, for example at 60°C. For example, the method is performed at a temperature of 42°C or more, such as 43°C or more, such as 44°C or more, such as 45°C or more, such as 46°C or more, such as 47°C or more, such as 48°C or more, such as 49°C or more, such as 50°C or more, such as 51 °C or more, 52°C or more, 53°C or more, 54°C or more, 55°C or more, 56°C or more, 57°C or more, 58°C or more, 59°C or more, for example 60°C or more.

[0180] The acetone, butanone and / or isopropanol may be recovered as known in the art. For example, the compounds may be recovered from the off-gas which is produced during cultivation of the cells, which may form as a result of e.g. condensation. P6952PC00

[0181] Cultivation medium

[0182] The cultivation medium, fermentation broth, or cultivation broth, may comprise a fermentable substrate, such as a fermentable carbon source, as known in the art. In some embodiments, the cultivation medium comprises a substrate comprising carbohydrates. In particular, pentose or hexose sugars can be used as substrate, such as glucose, xylose, or a mixture thereof, or the cultivation medium may comprise or consist of a biomass hydrolysate, for example a lignocellulosic hydrolysate. In the present context the term "lignocellulosic hydrolysate" is intended to designate a lignocellulosic biomass which preferably has been subjected to a pre-treatment step whereby lignocellulosic material has been at least partially separated into cellulose, hemicellulose and lignin. The lignocellulosic material may typically be derived from plant material, such as straw, hay, garden refuse, comminuted wood, fruit hulls and seed hulls.

[0183] The pre-treatment method most often used is acid hydrolysis, where the lignocellulosic material is subjected to an acid such as sulphuric acid whereby the sugar polymers cellulose and hemicellulose are partly or completely hydrolysed to their constituent sugar monomers. Another type of lignocellulose hydrolysis is steam explosion, a process comprising heating of the lignocellulosic material by steam injection to a temperature of 190-230°C. A third method is wet oxidation wherein the material is treated with oxygen at 150-185°C. The pre-treatments can be followed by enzymatic hydrolysis to complete the release of sugar monomers. This pre-treatment step results in the hydrolysis of cellulose into glucose or cellobiose, while hemicellulose is transformed into the pentoses xylose and arabinose and the hexoses glucose, galactose and mannose. The pre-treatment step may in certain embodiments be supplemented with treatment resulting in further hydrolysis of the cellulose and hemicellulose. The purpose of such an additional hydrolysis treatment is to hydrolyse oligosaccharide and possibly polysaccharide species produced during the acid hydrolysis, wet oxidation, or steam explosion of cellulose and / or hemicellulose origin to form fermentable sugars (e.g. glucose, xylose and possibly other monosaccharides). Such further treatments may be either chemical or enzymatic. Chemical hydrolysis is typically achieved by treatment with an acid, such as treatment with aqueous sulphuric acid, at a temperature in the range of about 100-150°C. Enzymatic hydrolysis is typically performed by treatment with one or more appropriate enzymes such as cellulases, glucosidases and hemicellulases including xylanases. P6952PC00

[0184] Treatment of biomass to extract fermentable sugars can be carried out physically, chemically or biologically. Lignocellulose consists of cellulose, hemicellulose (a mixture of homo- and heteropolymers of xylose, arabinose, mannose, etc.), pectins and lignin organized in complex microstructures, which evolved to resist attacks by microorganisms and insects. Hence it can be relatively resistant to enzymatic decomposition and different methods of deconstruction are often combined. Pretreatment is usually performed in order to make cellulose fibers more accessible to respective enzymes (cellulases), hydrolyze hemicelluloses and / or remove lignin. Typical process involves treatment with diluted acid or base at temperatures between 100°C and 220°C. Due to its amorphous structure, hemicellulose is more readily hydrolyzed in this step, and up to 90% of its sugars can be recovered. However, this method also yields furfural and other products which may inhibit microorganisms’ growth. Thus, enzyme cocktails collectively known as hemicellulases are sometimes used. On the other hand, cellulose is organized in microcrystalline fibers and is not easily hydrolyzed, but the degradation of cell wall matrix during pretreatment makes it more accessible for enzymes. Cellulases include: 1) endo-glucanases which act in the middle of cellulose molecule; 2) cellobiohydrolases which release cellobiose from the ends of cellulose; 3) p-D-glucosidases which hydrolyze cellobiose into glucose. As described above, enzymatic hydrolysis can be performed as a separate step (separate hydrolysis and fermentation, SHF) or simultaneously with fermentation (SSF). Recently, a complementary method has been proposed for complete solubilization of lignocellulose using biomass-derived y-valerolactone.

[0185] Another attractive method is consolidated bioprocessing (CBP), which combines enzyme production, saccharification and fermentation in one step. This can be done by designing the producing cell of the present disclosure to express a heterologous metabolic pathway to degrade and utilize biomass.

[0186] Alternatively, the sporulation-deficient bacterial cell of the present disclosure may be cultivated together with another cell which is capable of degrading and utilising biomass, particularly at higher temperatures as described herein above. Using this setup, one microorganism, for example A. thermocellus, degrades the biomass and provides the necessary substrates for the other microorganism, which can produce the volatile compounds as described above. P6952PC00

[0187] In some embodiments, the cultivation medium comprises glucose, xylose, or a mixture thereof. For example, the cultivation medium may comprise between 0.1% and between 20% (w / vol) glucose, xylose, or mixture thereof. For example, the cultivation medium comprises between 0.1% and 15% (w / vol) glucose, xylose, or mixture thereof, such as between 0.5% and 15% (w / vol) glucose, xylose, or mixture thereof, such as between 1% and 10% (w / vol) glucose, xylose, or mixture thereof, such as between 2% and 10% (w / vol) glucose, xylose, or mixture thereof, such as between 5% and 10% (w / vol) glucose, xylose, or mixture thereof, such as between 5% and 7.5% (w / vol) glucose, xylose, or mixture thereof. In some embodiments, the cultivation medium comprises at least 0.1% (w / vol) glucose, xylose, or mixture thereof, such as at least 0.25% (w / vol), such as at least 0.5%(w / vol), such as at least 0.75% (w / vol), such as at least 1% (w / vol), such as at least 2.5% (w / vol), such as at least 5% (w / vol), such as at least 10% (w / vol), such as at least 15% (w / vol), such as 20% (w / vol) glucose, xylose, or mixtures thereof.

[0188] In some embodiments, the thermophilic cell is an acetogenic thermophilic cell, in particular an acetogenic bacterial cell, which has been engineered to produce acetone, butanone or isopropanol. Such cells are capable of converting carbon monoxide, carbon dioxide, hydrogen, or a mixture thereof into acetyl-CoA, which is a substrate or a co-substrate for the above compounds. For example, acetogenic species include Moorella thermoacetica, Moorella thermoautotrophica, Neomoorella thermoacetica and Thermoanaerobacter kivui. Within the context of the present disclosure, said thermophilic acetogenic bacterial cell is also sporulation-deficient. Preferably, the thermophilic acetogenic bacterial cell has been modified, such as engineered, to be sporulation-deficient. With respect to the methods disclosed herein, said carbon monoxide, carbon dioxide, hydrogen, or a mixture thereof is preferably provided in the step of cultivating said sporulation-deficient bacterial cell.

[0189] In embodiments where production of acetone is desired, the cultivation medium may advantageously further comprise acetyl-CoA, acetic acid or acetate. In some embodiments, the cultivation medium comprises between 0.05% and 5% (w / vol) acetic acid or acetate. For example, the cultivation medium comprises between 0.05% and 5% (w / vol) acetic acid or acetate or mixtures thereof, such as between 0.1% and 5% (w / vol), such as between 0.5% and 5% (w / vol), such as between 1% and 5% (w / vol), P6952PC00 such as between 2% and 4% (w / vol) such as 3% acetic acid or acetate or mixtures thereof. In some embodiments, the cultivation medium comprises at least 0.05% (w / vol) acetic acid or acetate or mixtures thereof, such as at least 0.1% (w / vol), such as at least 0.5%, such as at least 1% (w / vol), such as at least 2% (w / vol), such as at least 3% (w / vol), such as at least 4% (w / vol), such as 5% (w / vol) acetic acid or acetate or mixtures thereof.

[0190] As described herein above, the cell may also have been engineered to synthesise acetyl-CoA more efficiently to be used as a substrate, or it may be cultivated with a microorganism which is capable of producing acetyl-CoA from the fermentable carbon source. In some embodiments, the cultivation medium comprises acetyl-CoA and / or the bacterial cell may be capable of synthesising acetyl-CoA.

[0191] Thus, in embodiments where production of acetone and optionally isopropanol is desired, the bacterial cell may be capable of synthesising acetyl-CoA and / or the cultivation medium may comprise acetyl-CoA, acetic acid, or acetate.

[0192] In embodiments where butanone production is desired, the cultivation medium may advantageously further comprise propionic acid or propionate. Thus, in embodiments where butanone production is desired and wherein the sporulation-deficient bacterial cell is capable of producing said butanone, the cultivation medium may comprise propionic acid or propionate, and acetyl-CoA, acetate and / or acetic acid. In some embodiments, the cultivation medium comprises between 0.05% and 2% (w / vol) propionic acid or propionate. For example, the cultivation medium comprises between 0.05% and 2% (w / vol) propionic acid or propionate or mixtures thereof, such as between 0.1% and 2% (w / vol), such as between 0.5% and 2% (w / vol), such as between 1% and 2% (w / vol) propionic acid, propionate or mixtures thereof. In some embodiments, the cultivation medium comprises at least 0.05% (w / vol) propionic acid, propionate or mixtures thereof, such as at least 0.1% (w / vol), such as at least 0.5%, such as at least 1% (w / vol), such as 2% (w / vol) propionic acid, propionate or mixtures thereof.

[0193] As described herein above, the cell may also have been engineered to synthesise propionyl-CoA to be used as a substrate, or it may be cultivated with a microorganism which is capable of producing propionyl-CoA from the fermentable carbon source. P6952PC00

[0194] Titers

[0195] The present methods, taking advantage of any of the sporulation-deficient thermophilic bacterial cells described herein above in detail, can lead to increased titers and / or amounts of acetone, butanone and / or isopropanol compared to the amounts produced by a reference cell, i.e. a sporulation-proficient thermophilic cell otherwise identical to the sporulation-deficient cell and cultivated in the same conditions. The titers, amounts and improvements obtained or obtainable by the present methods have been described in detail in the section “Titers” herein above.

[0196] Cultivation broth and off-gasses

[0197] Disclosed herein is also a cultivation broth obtained or obtainable by a method described herein. Said cultivation broth obtained or obtainable by a method described herein may comprise acetone, butanone and / or isopropanol. The cultivation broth may also be termed fermentation liquid or fermentation broth. Disclosed herein is also acetone, butanone and / or isopropanol obtained or obtainable by a method described herein.

[0198] The present sporulation-deficient bacterial cells can be cultivated in a continuous fermentation set-up, as is known in the art. This can be particularly advantageous as it also allows continuous product recovery, thereby preventing feedback inhibition and product toxicity. When the cell is thermophilic and the cultivation is performed at higher temperatures than typically used for fermenting mesophilic cells, the volatile compounds will at least partly evaporate and can easily be recovered from the off-gas produced by the thermophilic cell. Thus in some embodiments, the methods disclosed herein further comprise recovering the one or more volatile compounds from the offgas produced during the fermentation. In some embodiments, this is done by condensation.

[0199] In this setup an off gas is continuously removed from the bioreactor and cooled down to the temperature below the boiling point of the compound of interest. This leads to it going from the gas to liquid phase, at which point it is collected. Alternatively, the off gas can be flushed through the solvent, such as water, which has a temperature below the boiling point of the compound of interest. This process produces a saturated solution of this chemical. Alternatively, the off gas can be passed through a filter, such as for example activated charcoal, which binds the product. P6952PC00

[0200] The temperature of the cultivation may depend on the volatile compound that is being produced. Skilled person is well capable of deciding on the optimal temperature or temperature range for the cultivation to ensure at least partly evaporation of the volatile compound.

[0201] Provided is a method for producing acetone, butanone, and / or isopropanol, comprising the steps of: i. providing a sporulation-deficient bacterial cell as described herein above; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining an off-gas comprising acetone, butanone, and / or isopropanol; and iii. optionally recovering said acetone, butanone, and / or isopropanol from said off-gas.

[0202] Also provided is a method for increasing the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell capable of producing acetone, butanone and / or isopropanol, said method comprising the steps of: i. providing said sporulation-proficient bacterial cell; ii. inactivating sporulation in said bacterial cell, thereby obtaining a sporulationdeficient bacterial cell capable of producing acetone, butanone and / or isopropanol; iii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining an off-gas; whereby said sporulation-deficient bacterial cell produces acetone, butanone and / or isopropanol with an increased titer compared to the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, such as the sporulation-proficient bacterial cell provided in step i., in the same conditions; and iv. optionally recovering the acetone, butanone and / or isopropanol from said off-gas.

[0203] Thus, provided herein is an off-gas obtained or obtainable by a method described herein. Said off-gas may be a mixture of different gasses. When said method is a method for producing acetone, butanone and / or isopropanol, it is preferred that least one of said gasses is acetone, butanone or isopropanol. Thus, when said method is a method for producing acetone, butanone and / or isopropanol, said off-gas preferably P6952PC00 comprises acetone, butanone and / or isopropanol, respectively. Herein provided is also, a cultivation broth obtained or obtainable by a method described herein.

[0204] Methods for producing acetone, isopropanol and butanone

[0205] With respect to both the acetone-, isopropanol- and the butanone-producing sporulation-deficient bacterial cells described in this section, said cells expresses Tle2. Said Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8, SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9, SEQ ID NO: 20), and optionally the functional variant of Tle2 comprises or consists of a subunit having at least 70% identity to Tle2 subunit A (EC 2.8.3.8, SEQ ID NO: 19) and another subunit having at least 70% identity to Tle2 subunit B (EC 2.8.3.9, SEQ ID NO: 20).

[0206] Acetone

[0207] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing an increased amount and / or increased titer of acetone compared to a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, said cell comprises i. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of- function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; ii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity; or iii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; wherein said sporulation-deficient bacterial cell is capable of producing acetone at a titer of at least 280 mg / L, or more.

[0208] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing acetone an increased amount and / or increased titer of acetone compared to P6952PC00 a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, said cell comprises: i. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of- function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; ii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity thereto; or iii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; wherein said sporulation-deficient bacterial cell is capable of producing acetone at a titer which is increased by at least 35%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0209] The acetone-producing sporulation-deficient bacterial cell described in this section is preferably of the species P. thermoglucosidasius.

[0210] The acetone-producing sporulation-deficient bacterial cell described in this section is in particular useful in a method for producing acetone. Thus, the methods disclosed herein can be performed using such acetone-producing sporulation-deficient bacterial cell.

[0211] Isopropanol

[0212] The produced acetone or a least a part of the produced acetone may be converted to isopropanol by said sporulation-deficient bacterial cell, for example by expressing Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto, in the sporulation-deficient bacterial cell.

[0213] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing isopropanol, said cell comprises: P6952PC00 i. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; ii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity thereto; or iii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; preferably wherein said sporulation-deficient bacterial cell is capable of producing isopropanol at a titer of at least 0.05 g / L, or more.

[0214] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing isopropanol, said cell comprises: i. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; ii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity thereto; or iii. Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28), Tbr (SEQ ID NO: 29), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; preferably wherein said sporulation-deficient bacterial cell is capable of producing isopropanol at a titer which is increased by at least 5 %, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions. P6952PC00

[0215] The isopropanol-producing sporulation-deficient bacterial cell described in this section is preferably of the species P. thermoglucosidasius.

[0216] The isopropanol-producing sporulation-deficient bacterial cell described in this section is in particular useful in a method for producing isopropanol. Thus, the methods disclosed herein can be performed using such isopropanol-producing sporulationdeficient bacterial cell.

[0217] Butanone

[0218] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing butanone, said cell comprises: i. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of- function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; ii. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity thereto; or iii. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; wherein said sporulation-deficient bacterial cell is capable of producing butanone at a titer of at least 560 mg / L, or more.

[0219] In some embodiments, wherein the sporulation-deficient bacterial cell is capable of producing butanone, said cell comprises: i. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65) and a loss-of- function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; P6952PC00 ii. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOA (SEQ ID NO: 65), such as a loss-of-function mutation of a polynucleotide encoding SpoOA (SEQ ID NO: 65), or functional variants thereof having at least 70% identity thereto; or iii. Caur_1461 (SEQ ID NO: 3), Tle2, Cac (SEQ ID NO: 28), and inactivation of SpoOB (SEQ ID NO: 67), such as a loss-of-function mutation of a polynucleotide encoding SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto; wherein said sporulation-deficient bacterial cell is capable of producing butanone at a titer which is increased by at least 30%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0220] The butanone-producing sporulation-deficient bacterial cell described in this section is preferably of the species P. thermoglucosidasius.

[0221] The butanone-producing sporulation-deficient bacterial cell described in this section is in particular useful in a method for producing butanone. Thus, the methods disclosed herein can be performed using such butanone-producing sporulation-deficient bacterial cell.

[0222] Examples

[0223] Example 1 - Materials and methods

[0224] Strains, plasmids, and media

[0225] The bacterial strains and plasmids included in this study, herein examples 2 to 4, are listed in Table 1.

[0226] P. thermoglucosidasius strains were routinely grown in SPY medium at 60 °C, 200 rpm. SPY contains, per litre: 16 g soy peptone, 10 g yeast extract, and 5 g NaCI, with pH adjusted to 6.8. In addition, P. thermoglucosidasius strains were plated on Trypticase Soy Agar (TSA) plates (Becton Dickinson, US) unless stated differently.

[0227] For growth in more controlled conditions, Thermophile Minimal Medium (TMM) was utilised. TMM is adapted from Fong et al. (Fong et al. 2006) and contains following sterile solutions, per litre: 930 mL Six Salts Solution (SSS), 40 mL of 1 M MOPS P6952PC00 solution (pH = 8.2), 10 mL of 1 mM FeSO4 in 0.4 M tricine, 10 mL of 0.132 M K2HPO4, 10 mL of 0.953 M NH4CI, 0.5 mL of 1 M CaCI2, 0.5 mL of trace elements solution, and 1mL of Wolfe’s vitamin solution, with the final pH adjusted to 6.5. SSS contains, per litre: 4.95 g NaCI, 1.45 g Na2SO4, 0.25 g KCI, 0.04 g KBr, 1.85 g MgCI2-6H2O, and 0.89 g NaNO3. The trace elements solution contains, per litre: 1 g FeCI3 6H2O, 0.18 g

[0228] ZnSO4-7H2O, 0.12 g CuCI2-2H2O, 0.12 g MnSO4 H2O, and 0.18 g CoCI2-6H2O. Finally, Wolfe’s vitamin solution contains, per litre: 10 mg Pyridoxine HCI, 5 mg Thiamine HCI, 5 mg Riboflavin, 5 mg Nicotinic acid, 5 mg Ca-D-(+)pantothenate, 5 mg p-Aminobenzoic acid, 5 mg Thioctic acid (Dithiolane Pentanoic acid), 2 mg Biotin, 2 mg Folic acid, and 0.1 mg Vitamin B12.

[0229] Table 1 : Strains and plasmids used in the present work. P6952PC00

[0230] Preparation of electrocompetent cells

[0231] The spoOA gene deletion was performed in both production strains through the use of integrative spoOA deletion plasmid pMM7 (Millgaard et al. 2023). Before transformation with the plasmid, the cells had to be made electrocompetent. This was accomplished through a protocol adapted from Taylor et al. 2008. The strains were initially inoculated from plate into shake flasks containing 50 mL of pre-heated SPY, after which the strains were cultured at 60 °C, 200 rpm. Once reaching an ODeoo of approximately 1.5, the strains were diluted to an ODeoo of around 0.5 in new shake flasks containing 30 mL of fresh pre-heated SPY. These flasks were subsequently cultured under the same conditions until they reached an ODeoo of around 1 .8. The flasks were left to cool down on ice for 10 min, after which they were spun down at 4,000 g and 4 °C in a pre-cooled centrifuge. The cells were then washed four times by spinning them down at 4,000 g and resuspending them in 15, 10, 10, and 5 mL of ice-cold electroporation buffer. The buffer consisted of 0.5 M mannitol, 0.5 M sorbitol, and 10% glycerol and was filter sterilised prior to use. Following the last wash step, the cells were resuspended in 1.5 mL of electroporation buffer and transferred as 60 pl aliquots into tubes pre-chilled on dry ice. The resulting competent cells were then stored at -80 °C.

[0232] Transformation with pMM7

[0233] For transformation, 60 pl of thawed electrocompetent cells were gently mixed with 2.5 pl of pMM7. The cell-plasmid mixtures were then transferred to ice-cold electroporation cuvettes. Using an electroporation machine, the cells were shocked with an exponential electric pulse with voltage of 2500 V, capacitance of 10 pF, and resistance of 600 Q. The cells were then quickly transferred to a tube containing 1 mL of preheated SPY supplied with 1 % glycerol, after which the cells were left to recover for 3 hours at 52 °C, 200 rpm. The cells were then spun down at 3,000 g and plated onto TSA plates containing 12.5 mg / L kanamycin. Finally, the plates were left to incubate overnight at 52 °C.

[0234] Deletion of spoOA

[0235] For deletion of the spoOA gene, cells carrying pMM7 were inoculated into 2 mL of SPY with 12.5 mg / L kanamycin and incubated overnight at 62 °C, 200 rpm. The following morning, the cultures were streaked onto TSA with 12.5 mg / L kanamycin, and the plates were left to incubate overnight at 60 °C. Colonies displaying green fluorescence were likely to have the pMM7 integrated in the spoOA genomic region. As such, P6952PC00 multiple green fluorescent colonies of each strain were selected for deletion of the spoOA. These colonies were inoculated in 2 mL SPY and left to incubate overnight at 60 °C, 200 rpm. For 2-5 days, the cultures were then routinely passaged to fresh preheated SPY during the morning and afternoon. On the afternoon of the final day, the cultures were seeded onto TSA plates and left to incubate overnight at 60 °C. The resulting colonies were examined under blue light, and non-fluorescent colonies were selected for screening of the spoOA knockout.

[0236] Screening of the strains was performed through colony PCR with the primer sets PNJ1245 / PNJ1246 (to test for the spoOA knockout) and PNJ1658 / PNJ1247 (to verify the presence of the production pathway). Primer sequences are listed in Table 2. Prior to colony PCR, the colonies were resuspended in 20 pl of 20 mM NaOH and heated to 95 °C for 10 min. Once cooled to room temperature, colony PCRs were then performed on the resuspensions using OneTaq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs, US), in accordance with manufacturers’ protocol. For both strains, a colony displaying the appropriate short band in the spoOA genomic region was selected (see Figure 1), and the knockout of the spoOA gene was later confirmed via sequencing.

[0237] Table 2. Primers used for AspoOA and AspoOB strain verification.

[0238] Microscopy

[0239] To determine the presence of spores in a culture, phase contrast microscopy was performed. Prior to microscopy, 1% agarose was used to prepare agar pads were P6952PC00 prepared on the microscopy slides. Following this, 5 pl of the culture samples was each transferred onto an agar pad and covered with a glass coverslip. Phase contrast microscopy was performed with a Leica DM4000 B microscope (Leica Microsystems, Germany) rigged with a 63x oil immersion objective. Images were captured with a Leica DFC300 FX camera (Leica Microsystems, Germany) utilising the Leica Application Suite software, version 4.12.0 (Leica Microsystems, Germany).

[0240] Heat assay

[0241] The purpose of this experiment was to determine the heat-resistance of nutrient- starved cultures of P. thermoglucosidasius wildtype and AspoOA cultures. To this end, the strains were each inoculated three times in tubes containing 2 mL of 3mL of TMM supplied with 3 g / L yeast extract. The tubes were then left to incubate for 24 hours at 60 °C, 200 rpm. Subsequently, the ODeoo of the cultures were measured and sample was collected for microscopy (see previous section). 500 pl of each culture was then transferred to new tubes, which were then heat-treated for 40 min at 100 °C, 1200 rpm. Following this, 100 pl of each heat-treated culture was inoculated into a tube containing 2900 pl of fresh pre-heated SPY. Additionally, 100 pl of non-treated culture was inoculated in the same manner to serve as controls. The tubes were incubated at 60 °C, 200 rpm. ODeoo samples were collected at 24-hour intervals.

[0242] GC-MS analysis

[0243] To determine the acetone and butanone production efficiency of the strains, GC-MS analysis was performed. Here, the desired production strains were initially inoculated from plate into 2 mL of TMM supplied with 2 g / L acetic acid, and 5 g / L yeast extract for acetone, and 10 g / L glucose, 2 g / L propionic acid and 2 g / L yeast extract for butanone. The cultures were grown overnight at 60 °C, 200 rpm. The following day, the cultures were spun down at 3,000 g, after which the cell pellets were resuspended in 2 mL fresh pre-heated medium of the same composition. The cultures were then transferred into autoclaved GC headspace vials with a total volume of 20 mL. To avoid loss of the volatile products through evaporation, the vials were closed tightly before leaving them to incubate for 20 hours at 60 °C, 200 rpm. Following this, the cultures were frozen at - 20 to prevent further growth and metabolism. The cultures were then transferred for measurement with analytical GC-MS. P6952PC00

[0244] Example 2 - Verification of sporulation negative phenotype

[0245] Due to the hardy nature of spores, sporulating strains can resist heat-treatments where non-sporulating strains would be unable to survive. To determine if the spoOA deletion had successfully supressed sporulation in the production strains, a heat resistance assay was therefore performed. Prior to heat treatment, microscopy analysis of the cultures revealed an abundance of spores in the wildtype production strains, while none were observed in the spoOA deletion strains. Following heat treatment, it was subsequently confirmed that the spoOA deletion strain cultures were not able to regrow, indicating that no heat-resistant spores were present. This is in accordance with previously described results(Millgaard et al. 2023).

[0246] Using the heat treatment assay, we also confirmed the that deleting spoOA in both acetone and butanone production strains lead to non-sporulating phenotype (Figure 3).

[0247] Example 3 - Measurement of acetone and butanone production

[0248] Through the use of GC-MS analysis, the concentrations of acetone, isopropanol and butanone were measured in 2 mL batch cultures with the wildtype and sporulation- supressed production strains.

[0249] Based on these measurements, it was concluded that both strains displayed a statistically significant increase in the titers of their respective products once the spoOA gene had been deleted. The sporulation-negative acetone production strain (STC AspoOA) was observed to produce up to 0.35 g / L compared to 0.27 g / L in a sporulating background. The sporulation-negative butanone strain (CTC AspoOA) produced up towards 0.77 g / L, while sporulating parent produced up to 0.55 g / L (Figure 2 and Table 3, and Figure 4 for STC AspoOA). Compared to the sporulating parent strains, this represents an improvement of 37% and 31% from the acetone and butanone strains, respectively, with p value for both less than 0.05, representing statistically significant differences (see Figure 2). In addition, strain STC AspoOA produced 0.12 g / L isopropanol, indicating that one or more native alcohol dehydrogenases can convert produced acetone into this compound.

[0250] From these results, it can be concluded that the derived non-sporulating P. thermoglucosidasius strains are in particular well-suited as hosts for microbial P6952PC00 production. In addition, the lack of spores will make the sterilisation requirements less strict and costly, thereby further improving their eligibility as production hosts.

[0251] Table 3. Average production titers of acetone and butanone in sporulating and non- sporulating backgrounds. NA: not analysed

[0252] Example 4 - Deletion of spoOB, another sporulation regulator, leads to increased acetone production

[0253] To confirm that the increase in acetone titer is the result of sporulation negative phenotype, and not just the deletion of spoOA gene itself, we performed a knockout of spoOB gene in the acetone production strain STC. Primer sequences used to test for the spoOB knockout are listed in Table 2. The SpoOB protein is part of a signal transduction system (phosphorelay) to the SpoOA, and we found that deletion of the spoOB gene also lead to reduced ability to sporulate. In addition, this type of mutation results in an increased acetone titer from 209 mg / L for P. thermoglucosidasius STC to 316 mg / L for STC AspoOB (an increase of 51%) (Figure 4), when cultivated in medium containing 7.5 g / L acetate and 2 g / L yeast extract (pH 6.5).

[0254] Two other genes regulating the sporulation, either spoOF or sigF were also individually deleted in the acetone production strain STC. However, these mutations resulted in decreased acetone production, although they led to sporulation-deficient phenotype (data not shown).

[0255] Sequence overview P6952PC00 P6952PC00 P6952PC00 P6952PC00

[0256] References

[0257] Fong et al. (2006). Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost. Extremophiles 10: 363-372. https : / / do i . org / 10.1007 / s00792-006-0507-2

[0258] Millgaard et al. (2023). An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator. AMB Express 13. https: / / doi.org / 10.1186 / s13568-023-01544- 9

[0259] Najar and Thakur (2020). A systematic review of the genera Geobacillus and Parageobacillus: Their evolution, current taxonomic status and major applications. Microbiol (United Kingdom) 166:800-816. https: / / doi.Org / 10.1099 / mic.0.000945 Pogrebnyakov and Nielsen (2021). Methods and cells for production of volatile compounds. WO2022049125A1

[0260] Taylor et al. (2008). Development of a versatile shuttle vector for gene expression in Geobacillus spp. Plasmid 60:45-52. https: / / doi.Org / 10.1016 / j.plasmid.2008.04.001

[0261] Items

[0262] 1. A sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising; i. a first enzyme selected from an acetyl-CoA acetyltransferase and an enzyme of EC number 2.3.3.20; ii. a second enzyme selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, an acyl CoA:acetate / 3-ketoacid CoA-transferase, and an acyl-CoA thioesterase II, and iii. an acetoacetate decarboxylase (EC 4.1.1.4), whereby said sporulation-deficient bacterial is capable of converting acetyl-

[0263] CoA to acetone, thereby producing acetone, and / or whereby said sporulation-deficient bacterial is capable of converting acetyl- CoA and propionyl-CoA to butanone, thereby producing butanone; and iv. optionally an isopropanol dehydrogenase (EC 1.1.1.80), whereby said sporulation-deficient bacterial cell is capable of converting acetone to isopropanol, thereby producing isopropanol, whereby said sporulation-deficient bacterial cell is capable of producing acetone, butanone, and / or isopropanol, P6952PC00 and wherein said sporulation-deficient bacterial cell is capable of producing an increased amount and / or increased titer of acetone, butanone, and / or isopropanol compared to a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions.

[0264] 2. The sporulation-deficient bacterial cell according to item 1 , wherein the bacterial cell is a thermophilic bacterial cell, preferably the bacterial cell is a Parageobacillus cell, such as a P. thermoglucosidasius cell.

[0265] 3. The sporulation-deficient bacterial cell according to any one of the preceding items, comprising a modification for inactivating sporulation.

[0266] 4. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said modification for inactivating sporulation is a modification of: i. one or more polynucleotides involved in sporulation; ii. one or more polynucleotides encoding one or more polypeptides involved in sporulation; iii. one or more polynucleotides regulating expression, such as transcription and / or translation, of one or more polypeptides involved in sporulation; and / or iv. one or more polypeptides involved in sporulation.

[0267] 5. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said modification is a loss-of-function modification, such as a loss-of-function mutation in one or more polynucleotides involved in sporulation and / or in one or more polynucleotides encoding one or more polypeptides involved in sporulation.

[0268] 6. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said modification is not an inactivation of a polynucleotide encoding a stage 0 sporulation protein F, such as SpoOF as set forth in SEQ ID NO: 69 or a functional variant thereof having at least 70% identity thereto. P6952PC00

[0269] 7. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein A.

[0270] 8. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said stage 0 sporulation protein A is as set forth in SEQ ID NO: 65 (SpoOA) or a functional variant thereof having at least 70% identity thereto.

[0271] 9. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the polynucleotide encoding SpoOA is spoOA as set forth in SEQ ID NO: 64 or a homologue thereof having at least 70% identity thereto.

[0272] 10. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein B.

[0273] 11. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said stage 0 sporulation protein B is as set forth in SEQ ID NO: 67 (SpoOB) or a functional variant thereof having at least 70% identity thereto.

[0274] 12. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the polynucleotide encoding SpoOB is spoOB as set forth in SEQ ID NO: 66 or a homologue thereof having at least 70% identity thereto.

[0275] 13. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the first enzyme is selected from: i. an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Tfu_1520 (SEQ ID NO: 5), Tfu_0436 (SEQ ID NO: 6), Slip_0880 (SEQ ID NO: 7), Tfu_2394 (SEQ ID NO: 8), Slip_1236 (SEQ ID NO: 9), Caur_1540 (SEQ ID NO: 10), Tfu_0253 (SEQ ID NO: 11), CHY_1604 (SEQ ID NO: 14), CHY_1288 (SEQ ID NO: 15), Slip_2085 (SEQ ID NO: 16), Slip_0465 (SEQ ID NO: 17), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60) and CHY_1355 P6952PC00

[0276] (SEQ ID NO: 18), or functional variants thereof having at least 70% identity thereto; and ii. an enzyme of EC number 2.3.3.20 selected from the acyl-CoA:acyl-CoA alkyltransferase SVA_3859 (SEQ ID NO: 12) and the acyl-CoA:acyl- CoA alkyltransferase Despr_2661 (SEQ ID NO: 13); and functional variants thereof having at least 70% identity thereto.

[0277] 14. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the second enzyme is selected from Tle2, Dde2 (EC 2.8.3.5) (SEQ ID NO: 21), Ghh2 (EC 2.8.3.5), Tme (EC 2.8.3.8), Pth (EC 2.8.3.1) (SEQ ID NO: 26), Rma (EC 3.1.2.-) (SEQ ID NO: 27), and functional variants thereof having at least 70% identity thereto, wherein: i. Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), and optionally wherein the functional variant of Tle2 comprises or consists of a subunit having at least 70% identity to Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19 and another subunit having at least 70% identity to Tle2 subunit B (EC 2.8.3.9) as set forth in SEQ ID NO: 20; ii. Ghh2 comprises or consists of Ghh2 subunit A (SEQ ID NO: 22) and Ghh2 subunit B (SEQ ID NO: 23), and optionally wherein the functional variant of Ghh2 comprises or consists of a subunit having at least 70% identity to Ghh2 subunit A as set forth in SEQ ID NO: 22 and another subunit having at least 70% identity to Ghh2 subunit B as set forth in SEQ ID NO: 23; and iii. Tme comprises or consists of Tme subunit A (SEQ ID NO: 24) and Tme subunit B (SEQ ID NO: 25), and optionally wherein the functional variant of Tme comprises or consists of a subunit having at least 70% identity to T me subunit A as set forth in SEQ I D NO: 24 and another subunit having at least 70% identity to Tme subunit B as set forth in SEQ ID NO: 25.

[0278] 15. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the acetoacetate decarboxylase is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto. P6952PC00

[0279] 16. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the isopropanol dehydrogenase is Tbr (SEQ ID NO: 29), or a functional variant thereof having at least 70% identity thereto.

[0280] 17. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is selected from a substitution in, a deletion of, and an insertion in a polynucleotide or a part thereof, said loss-of- function mutation resulting in loss-of-function of said polynucleotide, optionally wherein said polynucleotide encodes SpoOA (SEQ ID NO: 65) or a functional variant thereof having at least 70% identity thereto.

[0281] 18. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is selected from a substitution in, a deletion of, and an insertion in a polynucleotide or a part thereof, said loss-of- function mutation resulting in loss-of-function of said polynucleotide, optionally wherein said polynucleotide comprises or consists of spoOA (SEQ ID NO: 64) or a homologue thereof having at least 70% identity thereto.

[0282] 19. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is a full-length deletion of a polynucleotide encoding SpoOA (SEQ ID NO: 65) or a functional variant thereof having at least 70% identity thereto, for example wherein the gene encoding SpoOA (SEQ ID NO: 65) is spoOA (SEQ ID NO: 64) or a homologue thereof having at least 70% identity thereto.

[0283] 20. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is selected from a substitution in, a deletion of, and an insertion in a polynucleotide or a part thereof, said loss-of- function mutation resulting in loss-of-function of said polynucleotide, optionally wherein said polynucleotide encodes SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto.

[0284] 21. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is selected from a substitution in, a deletion of, and an insertion in a polynucleotide or a part thereof, said loss-of- P6952PC00 function mutation resulting in loss-of-function of said polynucleotide, optionally wherein said polynucleotide comprises or consists of spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto.

[0285] 22. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the loss-of-function mutation is a full-length deletion of a polynucleotide encoding SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto, for example wherein the gene encoding SpoOB (SEQ ID NO: 67) is spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto.

[0286] 23. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said polynucleotide is a gene.

[0287] 24. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is an acetogenic cell.

[0288] 25. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell has an optimal growth temperature of between 42°C and 80°C, such as between 50°C and 75°C, for example of about 60°C.

[0289] 26. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell belongs to the phylum Bacillota.

[0290] 27. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell belongs to the class Bacilli.

[0291] 28. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell belongs to the order Bacillales.

[0292] 29. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell belongs to the family Anoxybacillaceae or Bacillaceae, preferably wherein the bacterial cell belongs to the family Anoxybacillaceae. P6952PC00

[0293] 30. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of a genus selected from the group consisting of Parageobacillus, Geobacillus, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Acetivibrio, Heyndrickxia, Thermoclostridium, Anoxybacillus, Moorella, and Clostridium.

[0294] 31. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of a genus selected from the group consisting of Parageobacillus, Geobacillus, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Acetivibrio, Heyndrickxia, Thermoclostridium, Anoxybacillus, and Clostridium.

[0295] 32. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of a genus selected from the group consisting of Anoxybacillus, Bacillus, Geobacillus, Heyndrickxia and Parageobacillus.

[0296] 33. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of a species selected from the group consisting of Parageobacillus thermoglucosidasius, Parageobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Acetivibrio thermocellus, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Heyndrickxia coagulans, Bacillus subtilis, Bacillus licheniformis, Bacillus smithii, Bacillus methanolicus, Anoxybacillus flavithermus, Bacillus flavothermus, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Moorella thermoacetica, Moorella thermoautotrophica, and Neomoorella thermoacetica, preferably the bacterial cell is a Parageobacillus thermoglucosidasius cell, a Bacillus subtilis cell or a Acetivibrio thermocellus cell, optionally wherein the cell is a Parageobacillus thermoglucosidasius DSM2542 cell. P6952PC00

[0297] 34. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the cell belongs to a species selected from the group consisting of Parageobacillus thermoglucosidasius, Parageobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Acetivibrio thermocellus, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Heyndrickxia coagulans, Bacillus subtilis, Bacillus licheniformis, Bacillus smithii, Bacillus methanolicus, Anoxybacillus flavithermus, Bacillus flavothermus, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, and Anoxybacillus gonensis.

[0298] 35. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the cell belongs to a species selected from the group consisting of Anoxybacillus gonensis, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, Anoxybacillus flavithermus, Bacillus flavothermus, Bacillus licheniformis, Bacillus methanolicus, Bacillus smithii, Bacillus subtilis, Geobacillus kaustophilus, Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoleovorans, Heyndrickxia coagulans, Parageobacillus thermoglucosidasius, and Parageobacillus toebii

[0299] 36. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is capable of synthesising acetyl-CoA and / or when acetyl-CoA, acetic acid, and / or acetate is provided to the bacterial cell said bacterial cell is capable of producing acetone, and optionally isopropanol.

[0300] 37. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is capable of synthesising acetyl-CoA and / or when acetyl-CoA, acetic acid, acetate, propionic acid, and / or propionate is provided to the bacterial cell said bacterial cell is capable of producing butanone. P6952PC00

[0301] 38. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell comprises one or more polynucleotides encoding the first enzyme, the second enzyme, the acetoacetate decarboxylase, and optionally the isopropanol dehydrogenase.

[0302] 39. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the one or more polynucleotides are codon optimised for expression in the bacterial cell.

[0303] 40. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the one or more polynucleotides are comprised within a vector or are integrated in the genome of the bacterial cell.

[0304] 41. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the one or more polynucleotides are under the control of an inducible promoter or under the control of a constitutive promoter.

[0305] 42. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is a non-natural cell.

[0306] 43. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is capable of producing at least acetone and wherein the first enzyme is CHY_1288 (SEQ ID NO: 15), CHY_1355 (SEQ ID NO: 18), Caur_1540 (SEQ ID NO: 10), GHH_c20420 (SEQ ID NO: 1), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), or Slip_0880 (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto, preferably wherein the first enzyme is Caur_1461 (SEQ ID NO: 3), Rxy2 (SEQ ID NO: 60), Slip_0880 (SEQ ID NO: 7), or Dde1 (SEQ ID NO: 59).

[0307] 44. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the first enzyme is an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Slip_0880 (SEQ ID P6952PC00

[0308] NO: 7), and Dde1 (SEQ ID NO: 59), or functional variants thereof having at least 70% identity thereto.

[0309] 45. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the second enzyme is selected from Tle2 and Dde2 (EC 2.8.3.5) (SEQ ID NO: 21), or functional variants thereof having at least 70% identity thereto, wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), or functional variants thereof having at least 70% identity thereto.

[0310] 46. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the first enzyme is Caur_1461 (SEQ ID NO: 3), Slip_0880 (SEQ ID NO: 7), or Dde1 (SEQ ID NO: 59), or a functional variant thereof having at least 70% identity thereto.

[0311] 47. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the first enzyme is GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), or Slip_0479 (SEQ ID NO: 4), or a functional variant thereof having at least 70% identity thereto.

[0312] 48. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell comprises Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto, wherein the bacterial cell further comprises: i) Dde1 (SEQ ID NO: 59) and Dde2 (SEQ ID NO: 21), or functional variants thereof having at least 70% identity thereto, whereby at least acetone is produced; ii) Caur_1461 (SEQ ID NO: 3) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least acetone and / or butanone is produced; iii) Slip_0880 (SEQ ID NO: 7) and Tle2; or functional variants thereof having at least 70% identity thereto, whereby at least acetone is produced; P6952PC00 iv) GHH_c20420 (SEQ ID NO: 1) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; v) Slip_0499 (SEQ ID NO: 2) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; or vi) Slip_0479 (SEQ ID NO: 4) and Tle2, or functional variants thereof having at least 70% identity thereto, whereby at least butanone is produced; wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), or functional variants thereof having at least 70% identity thereto.

[0313] 49. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said increased amount of acetone, butanone, and / or isopropanol is an increased titer of produced acetone, butanone, and / or isopropanol by said sporulation-deficient bacterial cell compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulationdeficient bacterial cell.

[0314] 50. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said increased amount of acetone, butanone, and / or isopropanol is an increased titer of produced acetone, butanone, and / or isopropanol by said sporulation-deficient bacterial cell compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulationdeficient bacterial cell, when cultivated in similar conditions, such as in the same and / or identical conditions.

[0315] 51. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said sporulation-deficient bacterial cell is capable of producing acetone with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at P6952PC00 least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.37 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least

[0316] 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0317] 52. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the titer of acetone is increased by at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more, compared to the titer produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell.

[0318] 53. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said sporulation-deficient bacterial cell is capable of producing butanone with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.3 g / L, such as at least 0.4 g / L, such as at least 0.5 g / L, such as at least 0.55 g / L, such as at least 0.560 g / L, such as at least 0.7 g / L, such as at least 0.73 g / L, such as at least 0.75 g / L, such as at least 1.0 g / L, such as at least 2.0 g / L, such as at least 3.0 g / L, such as at least 4.0 g / L, such as at least 5.0 g / L, such as at least 7.5 g / L, such as at least 10.0 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more. P6952PC00

[0319] 54. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the titer of butanone is increased by at least 30%, such as at least 31%, such as at least 32%, such as at least 33%, such as at least 34%, such as at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more compared to the titer of butanone produced by a sporulation-proficient bacterial cell otherwise identical to said sporulationdeficient bacterial cell.

[0320] 55. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein said sporulation-deficient bacterial cell is capable of producing isopropanol with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0321] 56. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the titer of isopropanol is increased by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, P6952PC00 such as at least 30%, such as at least 31%, such as at least 32%, such as at least 33%, such as at least 34%, such as at least 35%, such as at least 36%, such as at least 37%, such as at least 38%, such as at least 39%, such as at least 40%, such as at least 42%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, or more compared to the titer of isopropanol produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell.

[0322] 57. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7) ; or ii. Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell is a P. thermoglucosidasius cell.

[0323] 58. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is a P. thermoglucosidasius cell, wherein said bacterial cell comprises an inactivation of SpoOA (SEQ ID NO: 65) or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7) ; or ii. Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto.

[0324] 59. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is a P. thermoglucosidasius cell, wherein said bacterial cell comprises an inactivation of SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto. P6952PC00

[0325] 60. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is a P. thermoglucosidasius cell, wherein said bacterial cell comprises an inactivation of SpoOA (SEQ ID NO: 65) and an inactivation of SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto.

[0326] 61. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOA (SEQ ID NO: 65), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0327] 62. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOB (SEQ ID NO: 67), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0328] 63. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67), or functional variants thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or P6952PC00 ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0329] 64. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOA (SEQ ID NO: 64) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0330] 65. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0331] 66. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOA (SEQ ID NO: 64) and spoOB (SEQ ID NO: 66) or homologues thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

[0332] 67. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of the species P. thermoglucosidasius, and wherein said bacterial cell comprises: i. an inactivation of SpoOA (SEQ ID NO: 65); P6952PC00 ii. an inactivation of SpoOB (SEQ ID NO: 67); or iii. an inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67); or a functional variant thereof having at least 70% identity thereto, and wherein the bacterial cell comprises Slip_0880 (SEQ ID NO: 7), Tle2 and Cac (SEQ ID NO: 28), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell is capable of producing acetone with a titer of least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, or more.

[0333] 68. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of the species P. thermoglucosidasius, and wherein said bacterial cell comprises: i. loss-of-function SpoOA (SEQ ID NO: 65); ii. loss-of-function SpoOB (SEQ ID NO: 67); iii. loss-function SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67); iv. an inactivation of SpoOA (SEQ ID NO: 65); v. an inactivation of SpoOB (SEQ ID NO: 67); or vi. an inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67); or a functional variant thereof having at least 70% identity thereto, and wherein the bacterial cell comprises Slip_0880 (SEQ ID NO: 7), Tle2, Cac (SEQ ID NO: 28) and Tbr (SEQ ID NO: 29), or a functional variant thereof having at least 70% identity thereto, P6952PC00 preferably wherein the bacterial cell is capable of producing isopropanol with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, or more.

[0334] 69. The sporulation-deficient bacterial cell according to any one of the preceding items, wherein the bacterial cell is of the species P. thermoglucosidasius, and wherein said bacterial cell comprises: i. loss-of-function SpoOA (SEQ ID NO: 65); ii. loss-of-function SpoOB (SEQ ID NO: 67); iii. loss-function SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67); iv. an inactivation of SpoOA (SEQ ID NO: 65); v. an inactivation of SpoOB (SEQ ID NO: 67); or vi. an inactivation of SpoOA (SEQ ID NO: 65) and SpoOB (SEQ ID NO: 67); or a functional variant thereof having at least 70% identity thereto, and wherein the bacterial cell comprises Caur_1461 (SEQ ID NO: 3), Tle2 and Cac (SEQ ID NO: 28), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell is capable of producing butanone with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.3 g / L, such as at least 0.4 g / L, such as at least 0.5 g / L, such as at least 0.560 g / L, such as at least 0.7 g / L, such as at least 0.73 g / L, such as at least P6952PC00

[0335] 0.75 g / L, such as at least 1.0 g / L, such as at least 2.0 g / L, such as at least 3.0 g / L, such as at least 4.0 g / L, such as at least 5.0 g / L, such as at least 7.5 g / L, such as at least 10.0 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0336] 70. The sporulation-deficient bacterial cell according to any one of items 57 to 69, wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), and optionally wherein the functional variant of Tle2 comprises or consists of a subunit having at least 70% identity to Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19 and another subunit having at least 70% identity to Tle2 subunit B (EC 2.8.3.9) as set forth in SEQ ID NO: 20.

[0337] 71. A method of producing acetone, butanone, and / or isopropanol, comprising: i. providing a sporulation-deficient bacterial cell as set forth in any one of items 1 to 70; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth comprising acetone, butanone, and / or isopropanol produced by said sporulation-deficient bacterial cell; and iii. optionally recovering the cultivation broth or the acetone, butanone, and / or isopropanol.

[0338] 72. A method for increasing the titer of acetone, butanone, and / or isopropanol produced by a sporulation-proficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising: i. providing said sporulation-proficient bacterial cell; ii. inactivating sporulation in said bacterial cell, thereby obtaining a sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol; iii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth; whereby said sporulation-deficient bacterial cell produces acetone, butanone, and / or isopropanol with an increased titer compared to the titer of P6952PC00 acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, such as the sporulation-proficient bacterial cell provided in step i., in the same conditions; and iv. optionally recovering the cultivation broth or the acetone, butanone, and / or isopropanol.

[0339] 73. The method according to item 72, wherein said sporulation-deficient bacterial cell is a sporulation-deficient bacterial cell as set forth in any one of items 1 to 70.

[0340] 74. The method according to any one of the preceding items, wherein the step of cultivating said sporulation-deficient bacterial cell is performed at a temperature of between 42 and 80 °C, such as between 50 and 75 °C, for example at 60 °C.

[0341] 75. The method according to any one of the preceding items, wherein the step of cultivating said sporulation-deficient bacterial cell comprises a continuous fermentation.

[0342] 76. The method according to any one of the preceding items, wherein said step of recovering acetone, butanone, and / or isopropanol comprises recovering said compounds from the off-gas produced during the step of cultivating said sporulation-deficient bacterial cell, such as by condensation.

[0343] 77. The method according to any one of the preceding items, wherein the cultivation medium comprises a fermentable substrate comprising a carbon source, such as a carbohydrate, for example glucose, xylose, or a mixture thereof, or such as a biomass hydrolysate.

[0344] 78. The method according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is an acetogenic cell, and wherein the bacterial cell is provided with carbon monoxide, carbon dioxide, hydrogen, or a mixture thereof, preferably wherein said carbon monoxide, carbon dioxide, hydrogen, or a mixture thereof is provided in the step of cultivating said sporulation-deficient bacterial cell. P6952PC00

[0345] 79. The method according to any one of the preceding items, wherein the cultivation medium comprises acetyl-CoA, acetic acid, or acetate.

[0346] 80. The method according to any one of the preceding items, wherein the cultivation medium comprises propionic acid or propionate.

[0347] 81. The method according to any one of the preceding items, wherein the bacterial cell is capable of producing acetone and optionally isopropanol, and wherein the bacterial cell is capable of synthesising acetyl-CoA and / or wherein the cultivation medium comprises acetyl-CoA, acetic acid, or acetate.

[0348] 82. The method according to any one of the preceding items, wherein at least acetone is produced and wherein the first enzyme is CHY_1288 (SEQ ID NO: 15), CHY_1355 (SEQ ID NO: 18), Caur_1540 (SEQ ID NO: 10), GHH_c20420 (SEQ ID NO: 1), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), or Slip_0880 (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto, preferably Caur_1461 (SEQ ID NO: 3), Rxy2 (SEQ ID NO: 60), Slip_0880 (SEQ ID NO: 7), or Dde1 (SEQ ID NO: 59), or a functional variant thereof having at least 70% identity thereto.

[0349] 83. The method according to any one of the preceding items, wherein acetone is produced with a titer of 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such P6952PC00 as at least 1.8 g / L, such as at least 1 .9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0350] 84. The method according to any one of the preceding items, wherein the sporulation-deficient bacterial cell comprises Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto, whereby at least part of the produced acetone is converted to isopropanol.

[0351] 85. The method according to any one of the preceding items, wherein isopropanol is produced with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.3 g / L, such as at least 0.4 g / L, such as at least 0.5 g / L, such as at least 0.560 g / L, such as at least 0.7 g / L, such as at least 0.73 g / L, such as at least 0.75 g / L, such as at least 1.0 g / L, such as at least 2.0 g / L, such as at least 3.0 g / L, such as at least 4.0 g / L, such as at least 5.0 g / L, such as at least 7.5 g / L, such as at least 10.0 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0352] 86. The method according to any one of the preceding items, wherein the sporulation-deficient bacterial cell is capable of producing butanone, and wherein the cultivation medium comprises propionic acid / propionate, acetyl- CoA, acetate and / or acetic acid.

[0353] 87. The method according to any one of the preceding items, wherein at least butanone is produced, and wherein the first enzyme is GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), Tfu_1520 (SEQ ID NO: 5), or Tfu_0436 (SEQ ID NO: 6), or a functional variant thereof having at least 70% identity thereto, preferably wherein the first enzyme is P6952PC00

[0354] GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60), or Slip_0479 (SEQ ID NO: 4).

[0355] 88. The method according to any one of the preceding items, wherein butanone is produced, wherein the cultivation medium comprises propionic acid or propionate, and wherein the first enzyme is GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59), or Slip_0479 (SEQ ID NO: 4), or a functional variant thereof having at least 70% identity thereto.

[0356] 89. The method according to any one of the preceding items, wherein butanone is produced with a titer of at least 0.05 g / L, such as at least 0.075 g / L, such as at least 0.1 g / L, such as at least 0.12 g / L, such as at least 0.14 g / L, such as at least 0.16 g / L, such as at least 0.175 g / L, such as at least 0.19 g / L, such as at least 0.2 g / L, such as at least 0.28 g / L, such as at least 0.3 g / L, such as at least 0.305 g / L, such as at least 0.310 g / L, such as at least 0.315 g / L, such as at least 0.320 g / L, such as at least 0.330 g / L, such as at least 0.335 g / L, such as at least 0.340 g / L, such as at least 0.35 g / L, such as at least 0.355 g / L, such as at least 0.360 g / L, such as at least 0.365 g / L, such as at least 0.380 g / L, such as at least 0.4 g / L, such as at least 0.45 g / L, such as at least 0.5 g / L, such as at least 0.6 g / L, such as at least 0.7 g / L, such as at least 0.75 g / L, such as at least 0.8 g / L, such as at least 0.9 g / L, such as at least 1.0 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.3 g / L, such as at least 1.4 g / L, such as at least 1.5 g / L, such as at least 1.6 g / L, such as at least 1.7 g / L, such as at least 1.8 g / L, such as at least 1.9 g / L, such as at least 2.0 g / L, such as at least 5 g / L, such as at least 7.5 g / L, such as at least 10 g / L, such as at least 12.5 g / L, such as at least 15 g / L, such as at least 20 g / L, such as at least 25 g / L, such as at least 50 g / L, such as at least 75 g / L, such as at least 100 g / L, such as at least 150 g / L, such as at least 250 g / L, or more.

[0357] 90. A cultivation broth obtained or obtainable by the method of any one of items 71 to 89.

[0358] 91. An off-gas obtained or obtainable by the method of any one of items 71 to 89. P6952PC00

[0359] 92. Acetone, butanone and / or isopropanol obtained or obtainable by the method of any one of items 71 to 89.

Claims

P6952PC00Claims1. A sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising; i. a first enzyme selected from acetyl-CoA acetyltransferase and an enzyme of EC number 2.3.3.20; ii. a second enzyme selected from an acetate CoA transferase, a 3-oxoacid CoA transferase, an acyl CoA:acetate / 3-ketoacid CoA-transferase, and an acyl-CoA thioesterase II, and iii. an acetoacetate decarboxylase (EC 4.1.1.4), whereby said sporulation-deficient bacterial is capable of converting acetyl- CoA to acetone, thereby producing acetone, and / or whereby said sporulation-deficient bacterial is capable of converting acetyl- CoA and propionyl-CoA to butanone, thereby producing butanone; and iv. optionally an isopropanol dehydrogenase (EC 1.1.1.80), whereby said sporulation-deficient bacterial cell is capable of converting acetone to isopropanol, thereby producing isopropanol, whereby said sporulation-deficient bacterial cell is capable of producing acetone, butanone, and / or isopropanol, and wherein said sporulation-deficient bacterial cell is capable of producing an increased amount and / or increased titer of acetone, butanone, and / or isopropanol compared to a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, preferably when cultivated in the same conditions, wherein the cell is a thermophilic bacterial cell of a genus selected from the group consisting of Anoxybacillus, Bacillus, Geobacillus, Heyndrickxia and Parageobacillus, optionally wherein the cell belongs to a species selected from the group consisting of Anoxybacillus gonensis, Anoxybacillus ayderensis, Anoxybacillus kamchatkensis, Anoxybacillus flavithermus, Bacillus flavothermus, Bacillus licheniformis, Bacillus methanolicus, Bacillus smithii, Bacillus subtilis, Geobacillus kaustophilus, Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoleovorans, Heyndrickxia coagulans, Parageobacillus thermoglucosidasius, and Parageobacillus toebii.P6952PC002. The sporulation-deficient bacterial cell according to claim 1 , wherein the bacterial cell is a Parageobacillus cell, such as a P. thermoglucosidasius cell.

3. The sporulation-deficient bacterial cell according to any one of the preceding claims, comprising a modification for inactivating sporulation, optionally wherein said modification for inactivating sporulation is a modification of: i. one or more polynucleotides involved in sporulation; ii. one or more polynucleotides encoding one or more polypeptides involved in sporulation; iii. one or more polynucleotides regulating expression, such as transcription and / or translation, of one or more polypeptides involved in sporulation; and / or iv. one or more polypeptides involved in sporulation, preferably wherein said modification is not an inactivation of a polynucleotide encoding a stage 0 sporulation protein F, such as SpoOF as set forth in SEQ ID NO: 69 or a functional variant thereof having at least 70% identity thereto, optionally wherein said polynucleotide is a gene.

4. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein said modification is a loss-of-function modification, such as a loss-of-function mutation in one or more polynucleotides involved in sporulation and / or in one or more polynucleotides encoding one or more polypeptides involved in sporulation.

5. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein said modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein A, optionally wherein said stage 0 sporulation protein A is as set forth in SEQ ID NO: 65 (SpoOA) or a functional variant thereof having at least 70% identity thereto, further optionally wherein the polynucleotide encoding SpoOA is spoOA as set forth in SEQ ID NO: 64 or a homologue thereof having at least 70% identity thereto.P6952PC006. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein said modification is an inactivation of a polynucleotide encoding a stage 0 sporulation protein B, optionally, wherein said stage 0 sporulation protein B is as set forth in SEQ ID NO: 67 (SpoOB) or a functional variant thereof having at least 70% identity thereto, further optionally wherein the polynucleotide encoding SpoOB is spoOB as set forth in SEQ ID NO: 66 or a homologue thereof having at least 70% identity thereto.

7. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein the first enzyme is selected from: i. an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3), Slip_0479 (SEQ ID NO: 4), Tfu_1520 (SEQ ID NO: 5), Tfu_0436 (SEQ ID NO: 6), Slip_0880 (SEQ ID NO: 7), Tfu_2394 (SEQ ID NO: 8), Slip_1236 (SEQ ID NO: 9), Caur_1540 (SEQ ID NO: 10), Tfu_0253 (SEQ ID NO: 11), CHY_1604 (SEQ ID NO: 14), CHY_1288 (SEQ ID NO: 15), Slip_2085 (SEQ ID NO: 16), Slip_0465 (SEQ ID NO: 17), Dde1 (SEQ ID NO: 59), Rxy2 (SEQ ID NO: 60) and CHY_1355 (SEQ ID NO: 18), and functional variants thereof having at least 70% identity thereto; ii. an enzyme of EC number 2.3.3.20 selected from the acyl-CoA:acyl-CoA alkyltransferase SVA_3859 (SEQ ID NO: 12) and the acyl-CoA:acyl-CoA alkyltransferase Despr_2661 (SEQ ID NO: 13); and functional variants thereof having at least 70% identity thereto, and / or wherein the second enzyme is selected from Tle2, Dde2 (EC 2.8.3.5) (SEQ ID NO: 21), Ghh2 (EC 2.8.3.5), Tme (EC 2.8.3.8), Pth (EC 2.8.3.1) (SEQ ID NO: 26), Rma (EC 3.1.2.-) (SEQ ID NO: 27), and functional variants thereof having at least 70% identity thereto, wherein: a. Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), and optionally wherein the functional variant of Tle2 comprises or consists of a subunit having at least 70% identity to Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19P6952PC00 and another subunit having at least 70% identity to Tle2 subunit B (EC2.8.3.9) as set forth in SEQ ID NO: 20; b. Ghh2 comprises or consists of Ghh2 subunit A (SEQ ID NO: 22) and Ghh2 subunit B (SEQ ID NO: 23), and optionally wherein the functional variant of Ghh2 comprises or consists of a subunit having at least 70% identity to Ghh2 subunit A as set forth in SEQ ID NO: 22 and another subunit having at least 70% identity to Ghh2 subunit B as set forth in SEQ ID NO: 23; and c. Tme comprises or consists of Tme subunit A (SEQ ID NO: 24) and Tme subunit B (SEQ ID NO: 25), and optionally wherein the functional variant of Tme comprises or consists of a subunit having at least 70% identity to Tme subunit A as set forth in SEQ ID NO: 24 and another subunit having at least 70% identity to Tme subunit B as set forth in SEQ ID NO: 25, and / or wherein the acetoacetate decarboxylase is Cac (SEQ ID NO: 28) or a functional variant thereof having at least 70% identity thereto, and / or wherein the isopropanol dehydrogenase is Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% identity thereto.

8. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein the loss-of-function mutation is selected from a substitution in, a deletion of, and an insertion in a polynucleotide or a part thereof, said loss-of- function mutation resulting in loss-of-function of said polynucleotide, optionally wherein said polynucleotide encodes SpoOA (SEQ ID NO: 65) and / or SpoOB (SEQ ID NO: 67), or a functional variant thereof having at least 70% identity thereto, further optionally wherein said polynucleotide comprises or consists of spoOA (SEQ ID NO: 64) and / or spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto.

9. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein the loss-of-function mutation is a full-length deletion of a polynucleotide encoding SpoOA (SEQ ID NO: 65) or a functional variant thereof having at least 70% identity thereto, for example wherein the gene encoding SpoOA (SEQ ID NO: 65) is spoOA (SEQ ID NO: 64) or a homologue thereofP6952PC00 having at least 70% identity thereto, and / or wherein the loss-of-function mutation is a full-length deletion of a polynucleotide encoding SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto, for example wherein the gene encoding SpoOB (SEQ ID NO: 67) comprises or consists of spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto.

10. The sporulation-deficient bacterial cell according to any one of the preceding claims, wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell is a P. thermoglucosidasius cell.11 . A method of producing acetone, butanone, and / or isopropanol, comprising: i. providing a sporulation-deficient bacterial cell as set forth in any one of claims 1 to 10; ii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth comprising acetone, butanone, and / or isopropanol produced by said sporulation-deficient bacterial cell; and iii. optionally recovering the cultivation broth or the acetone, butanone, and / or isopropanol.

12. The method according to claim 11 , wherein: i. acetone is produced with a titer of at least 0.3 g / L, or more; ii. isopropanol is produced with a titer of at least 0.1 g / L, or more; and / or iii. butanone is produced with a titer of at least 0.6 g / L, or more.

13. A method for increasing the titer of acetone, butanone, and / or isopropanol produced by a sporulation-proficient bacterial cell capable of producing acetone, butanone, and / or isopropanol, comprising: i. providing said sporulation-proficient bacterial cell, wherein the sporulation-proficient bacterial cell is a thermophilicP6952PC00 bacterial cell of a genus selected from the group consisting of Anoxybacillus, Bacillus, Geobacillus, Heyndrickxia and Parageobacillus', ii. inactivating sporulation in said bacterial cell, thereby obtaining a sporulation-deficient bacterial cell capable of producing acetone, butanone, and / or isopropanol; iii. cultivating said sporulation-deficient bacterial cell in a cultivation medium, thereby obtaining a cultivation broth; whereby said sporulation-deficient bacterial cell produces acetone, butanone, and / or isopropanol with an increased titer compared to the titer of acetone, butanone and / or isopropanol produced by a sporulation-proficient bacterial cell otherwise identical to said sporulation-deficient bacterial cell, such as the sporulation-proficient bacterial cell provided in step i., in the same conditions; and iv. optionally recovering the cultivation broth or the acetone, butanone, and / or isopropanol, further optionally wherein the sporulation-deficient cell is as defined in any one of claims 1 to 10.

14. The method according to any one of claims 11 to 13, wherein: i. said bacterial cell comprises an inactivation of SpoOA (SEQ ID NO: 65) or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: a) Slip_0880 (SEQ ID NO: 7); or b) Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto; or ii. said bacterial cell comprises an inactivation of SpoOB (SEQ ID NO: 67) or a functional variant thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: a) Slip_0880 (SEQ ID NO: 7); or b) Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto; or iii. said bacterial cell comprises an inactivation of SpoOA (SEQ ID NO: 65) and an inactivation of SpoOB (SEQ ID NO: 67) or a functional variantP6952PC00 thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: a) Slip_0880 (SEQ ID NO: 7); or b) Caur_1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto.

15. The sporulation-deficient bacterial cell and / or the method according to any one the preceding claims, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOA (SEQ ID NO: 64) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

16. The sporulation-deficient bacterial cell and / or the method according to any one the preceding claims, wherein the sporulation-deficient bacterial cell is a P. thermoglucosidasius cell comprising a loss-of-function mutation in spoOB (SEQ ID NO: 66) or a homologue thereof having at least 70% identity thereto, further wherein the bacterial cell comprises Tle2, Cac (SEQ ID NO: 28), and one of: i. Slip_0880 (SEQ ID NO: 7); or ii. Caur_1461 (SEQ ID NO: 3), or functional variants thereof having at least 70% identity thereto.

17. The sporulation-deficient bacterial cell and / or the method according to any one the preceding claims, wherein Tle2 comprises or consists of Tle2 subunit A (EC 2.8.3.8) (SEQ ID NO: 19) and Tle2 subunit B (EC 2.8.3.9) (SEQ ID NO: 20), and optionally wherein the functional variant of Tle2 comprises or consists of a subunit having at least 70% identity to Tle2 subunit A (EC 2.8.3.8) as set forth in SEQ ID NO: 19 and another subunit having at least 70% identity to Tle2 subunit B (EC 2.8.3.9) as set forth in SEQ ID NO: 20.

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