Patent Application
20240026391
The present invention relates to thermophilic cells and methods for the microbial production of volatile compounds, including acetone, butanone and isopropanol. Also provided are nucleic acid constructs, vectors and host cells useful in such methods.
Acetone production via fermentation in Clostridium acetobutylicum was started at an industrial scale to meet the military needs during World War I. The technology spread rapidly and in the course of first half of the twentieth century it ranked in importance second only to ethanol fermentation. With the development of petrochemical industry acetone fermentation declined in the West, but some countries continued to use it up to 1980s and 90s.
Current concerns about the environmental impact of the use of petroleum and its depletion drive the search for alternative ways of chemicals production and has revived the interest in biological production of acetone. Acetone consumption in 2014 was 5.9 million tons and projected to grow up to 7.2 million tons by 2020 at a rate of about 3% annually (“Acetone market: global industry analysis and opportunity assessment, 2014-2020,” 2015). Today the vast majority of acetone is produced chemically via the cumene process. This is accompanied by significant environmental costs.
The native clostridial acetone pathway consists of three enzymatic steps starting from acetyl-CoA and acetate (
Representatives of the genus Geobacillus are also increasingly used as hosts for the production of chemicals (Bosma et al., 2013). Advantages of thermophilic production in Geobacillus include: 1) reduced risk of contamination by mesophiles; 2) higher reaction rates at elevated temperatures; 3) reduction of energy input for cooling of thermally pre-treated biomass; 4) ability of of geobacilli to utilize a wide range of carbon sources, including C6 and C5 sugars, and acetate. Additionally, acetone and other volatile compounds are evaporating at high temperatures and can be collected downstream, which facilitates their purification, at the same time reducing issues with product toxicity and product inhibition.
To date, most efforts on metabolic engineering have been focused on enhancing the production of Geobacillus' own fermentation by-products, notably ethanol. This was done by knocking out genes from competitive pathways and upregulating pathways, which led to increased fluxes towards ethanol (Cripps et al., 2009; Zhou et al., 2016).
One of the strategies to achieve higher acetone yields would be to construct alternative biosynthetic pathways.
There thus remains a need for cells and methods allowing biological production of acetone, butanone and isopropanol in an efficient, cost-effective and sustainable manner.
Herein is provided a method of producing one or more compounds selected from acetone, butanone and isopropanol, said method comprising the steps of:
Herein is also provided a thermophilic cell capable of producing acetone and/or butanone and optionally isopropanol, said cell being a bacterial cell or an archaeal cell and expressing:
Herein is also provided a nucleic acid construct for modifying a thermophilic cell selected from a thermophilic bacterial cell and a thermophilic archaeal cell, comprising:
Herein is also provided a vector comprising the nucleic acid construct disclosed herein.
Herein is also provided a thermophilic cell comprising the nucleic acid construct and/or the vector disclosed herein, wherein the thermophilic cell is a thermophilic bacterial cell or a thermophilic archaeal cell.
Herein is also provided a kit comprising the nucleic acid construct, the vector or the thermophilic cell described herein.
The present disclosure relates to methods for microbial production of volatile compounds, in particular acetone, butanone and isopropanol. By taking advantage of thermophilic cells for production, these compounds can be easily and continuously removed from a fermentation, such as a fermentation broth, which, in addition to rendering the process labour- and cost-efficient, also solves the problem of product inhibition and negative effects on growth associated to the toxicity of the product(s). Other advantages include reduced risks of contamination due to the relatively high production temperatures.
The term “thermophile” herein refers to microorganisms, in particular bacteria and archaea, that thrive best, or at least capable of growing, at temperatures above 42° C.
Functional variant: the term 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 half-life, 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 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.
“Identity”, “similarity” and “homology” with respect to a polynucleotide (or polypeptide) is 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/similarity/homology, and considering any conservative substitutions according to the NCIUB rules (hftp://www.chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, 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, similarity or homology. 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, homology or similarity to another sequence means that the sequence shares at least 70% identity, homology or similarity 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, homology or similarity.
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.
The term “3-oxoacyl-ACP synthase” (3-oxoacyl-[acyl-carrier-protein] synthase) and “acyl-CoA:acyl-CoA alkyltransferase” refers to the same enzymes of EC number 2.3.3.20. They catalyse the conversion of two molecules of acyl-CoA into one molecule of (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 3-oxoacyl-ACP synthase activity. For example, the potential 3-oxoacyl-ACP synthase 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 3-oxoacyl-ACP synthase retains 3-oxoacyl-ACP synthase activity.
Acetoacetate decarboxylase: The term 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 can be monitored, e.g. manometrically. Release of CO2 indicates that the tested enzyme has acetoacetate decarboxylase activity.
Acetate CoA-transferase (EC 2.8.3.8) is an enzyme that catalyzes the chemical reaction: acyl-CoA+an acetatefatty 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. 3-oxoacid CoA-transferase (EC 2.8.3.5) is an enzyme which catalyzes 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.
An acyl CoA:acetate/3-ketoacid CoA-transferase (EC 2.8.3.1) is an enzyme that catalyzes the chemical reaction 3-ketoacyl-CoA+fatty acid3-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.
An acyl-CoA thioesterase II (EC 3.1.2.-) is an enzyme, which catalyzes the chemical reaction of hydrolysis: 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.
Isopropanol dehydrogenase: isopropanol dehydrogenase (NADP+) (EC 1.1.1.80) is an enzyme that catalyzes 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.
Titer: the titer of a compound refers herein to the produced concentration of a compound. 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 culture medium. This means that, particularly for volatile compounds, the titer includes the portion of the compound which may have evaporated from the culture medium, and it is thus determined by collecting the produced compound from the fermentation broth and from potential off-gas from the fermenter.
Herein is provided a method of producing one or more compounds selected from acetone, butanone and isopropanol, said method comprising the steps of:
The cells employed in the context of the present disclosure are thermophilic cells, more specifically bacterial or archaeal cells. In particular, bacterial or archaeal 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 thermophilic cells, more specifically thermophilic bacterial cells or thermophilic archaeal cells, i.e. cells which are capable of growing at temperatures of 42° C. or more.
Herein is provided a thermophilic cell capable of producing acetone and/or butanone and optionally isopropanol, said cell being a bacterial cell or an archaeal cell and expressing:
The present invention takes advantage of thermophilic cells 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.
The thermophilic cells described herein have been engineered to produce volatile compounds, i.e. acetone, butanone and/or isopropanol. The thermophilic cells described therein preferably do not occur naturally. In some embodiments, the thermophilic 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.
In some embodiments, the thermophilic cell capable of producing acetone and/or butanone and optionally isopropanol is a bacterial cell or an archaeal cell and expresses:
In some embodiments, the thermophilic cell is a bacterial cell, i.e. a cell of a thermophilic bacterium. In other embodiments, the thermophilic cell is an archaeal cell, i.e. a cell of a thermophilic archaeon.
In some embodiments, the thermophilic cell belongs to a genus selected from Geobacillus, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Thermoclostridium, Anoxybacillus, Caldicellulosiruptor, Moorella, Thermus, Thermotoga, Pseudothermotoga, Chloroflexus, Anaerocellum, Rhodothermus, Sulfolobus, Thermococcus, Pyrococcus and Clostridium. In specific embodiments, the thermophilic cell is a Geobacillus cell, a Bacillus cell or a Clostridium cell.
In some embodiments, the thermophilic cell belongs to a species selected from Geobacillus thermoglucosidasius, Geobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharotyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudoethanolicus, Thermoanaerobacter brockii, Thermoanaerobacter kivui, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Clostridium thermocellum, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus smithii, Bacillus methanolicus, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Caldicellulosiruptor bescii, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, Moorella thermoacetica, Moorella thermoautotrophica, Thermus thermophilus, Thermus aquaticus, Thermotoga maritima, Pseudothermotoga lettingae, Pseudothermotoga thermarum, Chloroflexus aurantiacus, Anaerocellum thermophilum, Rhodothermus marinus, Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus solfataricus, Thermococcus barophilus, Thermococcus kodakarensis, Pyrococcus abyssi, and Pyrococcus furiosus. In specific embodiments, the cell is a Geobacillus thermoglucosidasius cell. In other embodiments, the cell is a Bacillus subtilis cell. In other embodiments, the cell is a Clostridium thermocellum cell.
In some embodiments, the thermophilic 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 thermophilic 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 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 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.
In particular, the thermophilic cell is preferably able to grow at temperatures where at least part of the acetone, butanone and/or isopropanol it produces evaporates, thereby facilitating recovery of the produced acetone, butanone and/or isopropanol. Thus, in some embodiments, the thermophilic cell is able to grow at temperatures equal to or greater than the boiling point of acetone, butanone and/or isopropanol. In some embodiments, the thermophilic cell is capable of growing at a temperature of 56° C. (boiling point of acetone) or more.
Herein are disclosed methods and cells useful for producing volatile compounds, in particular one or more compounds selected from acetone, butanone and isopropanol.
The thermophilic cells disclosed herein express the enzymes necessary to achieve production of said compounds.
Herein is thus provided a method of producing one or more compounds selected from acetone, butanone and isopropanol, said method comprising the steps of:
The thermophilic cell of the 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 thermophilic cell is incubated in order to obtain one specific compounds. For example, the thermophilic cell is capable of producing acetone from acetyl-CoA, which the cell may be able to synthesise and/or which may be provided to the cells. In other cases, the thermophilic cell is capable of producing butanone from propionyl-CoA and acetyl-CoA, which the cell may be able to synthesise and/or which may be provided to the cells. If the cell expresses an isopropanol dehydrogenase, it can convert the produced acetone to isopropanol. The thermophilic cell is thus versatile: by changing the incubation conditions, all three compounds (acetone, butanone and isopropanol) can be obtained.
The thermophilic cell may be able to synthesise acetyl-CoA, for example when provided with acetic acid in the medium, or acetyl-CoA can be 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 acetyl-CoA, which could be provided in the fermentation broth. The thermophilic cell may be able to synthesise propionyl-CoA, for example when provided with propionic acid in the 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 fermentation broth.
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:
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:
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:
Steps 1) to 3) above can be performed by the same enzymes independently of which volatile compound is to be produced. Production of isopropanol according to the present methods requires that the thermophilic cell expresses a further enzyme, which is not required for production of acetone or butanone, as detailed herein below.
Herein is thus provided a method of producing one or more compounds selected from acetone, butanone and isopropanol, said method comprising the steps of:
The thermophilic cell may be as described herein, in particular the cell may be a bacterial cell or an archaeal cell.
The first enzyme can be either an acetyl-CoA acetyltransferase, also termed thiolase, or a 3-oxoacyl-ACP synthase.
Thiolases catalyse either the conversion of:
Which reaction actually occurs in the thermophilic cell will depend on which substrates are present in the 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 broth with acetic acid may increase the titer. Propionyl-CoA may be provided in the fermentation. The 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 a corresponding non-engineered cell.
Reaction i. is required for production of acetone according to the present methods. Reaction ii. is required for the production of butanone according to the present methods.
In some embodiments the first enzyme is an acetyl-CoA acetyltransferase 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.
In some embodiments, the first enzyme is an enzyme of EC number 2.3.3.20, i.e. a 3-oxoacyl-ACP synthase (3-oxoacyl-[acyl-carrier-protein] synthase) or an acyl-CoA:acyl-CoA alkyltransferase. These catalyse the conversion of two molecules of acyl-CoA into one molecule of (2R)-2-alkyl-3-oxoalkanoate in the presence of water, thereby generating two molecules of coenzyme A (CoA).
The first enzyme is selected from the group consisting of 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), SVA_3859 (SEQ ID NO: 12), Despr_2661 (SEQ ID NO: 13), and functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. Preferably, the first enzyme is an acetyl-CoA acetyltransferase (EC 2.3.1.9) selected from GHH_c20420 as set forth in SEQ ID NO: 1, Slip 0499 as set forth in SEQ ID NO: 2, Caur 1461 as set forth in SEQ ID NO: 3, Slip_0479 as set forth in SEQ ID NO: 4, Slip_0880 as set forth in SEQ ID NO: 7, and Dde1 as set forth in SEQ ID NO: 59, and functional variants thereof having at least 70% identity, homology or similarity thereto.
In one embodiment, the first enzyme is GHH_c20420 (SEQ ID NO: 1), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_0499 (SEQ ID NO: 2), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Caur 1461 (SEQ ID NO: 3), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_0479 (SEQ ID NO: 4), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Tfu_1520 (SEQ ID NO: 5), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Tfu_0436 (SEQ ID NO: 6), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_0880 (SEQ ID NO: 7), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Tfu_2394 (SEQ ID NO: 8), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_1236 (SEQ ID NO: 9), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Caur 1540 (SEQ ID NO: 10), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Tfu_0253 (SEQ ID NO: 11), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is CHY_1604 (SEQ ID NO: 14), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is CHY_1288 (SEQ ID NO: 15), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_2085 (SEQ ID NO: 16), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Slip_0465 (SEQ ID NO: 17), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Dde1 (SEQ ID NO: 59) or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Rxy2 (SEQ ID NO: 60) or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is CHY_1355 (SEQ ID NO: 18), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is SVA_3859 (SEQ ID NO: 12), or a functional variant thereof having at least 70% homology, similarity or identity thereto. In another embodiment, the first enzyme is Despr_2661 (SEQ ID NO: 13), or a functional variant thereof having at least 70% homology, similarity or identity thereto.
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 3-oxoacyl-ACP synthases 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 3-oxoacyl-ACP synthase retains 3-oxoacyl-ACP synthase activity.
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 preferably comprise said domains. Functional variants may have been engineered as is otherwise known in the art.
The thermophilic cells employed in the present methods 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.
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. 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.
Some of the above enzymes can catalyse different reactions. The type of reaction that actually occurs in the thermophilic cell will depend on which substrates are present in the broth or on how the cell has been engineered, as the skilled person well knows.
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% homology, similarity or 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% homology, similarity or 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% homology, similarity or 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% homology, similarity or identity thereto; wherein Ghh2 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% homology, similarity or 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% homology, similarity or identity thereto; and wherein Tme 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% homology, similarity or 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% homology, similarity or identity thereto.
Preferably, the second enzyme is Tle2 or Dde2 (SEQ ID NO: 21) or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In some embodiments, the second enzyme is Tle2 or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. This enzyme 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% homology, similarity or 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% homology, similarity or identity thereto. In some embodiments, the second enzyme is a functional variant of Tle2 consisting of Tle2 subunit A and a functional variant of Tle2 subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 20, 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% homology, similarity or identity thereto. In other embodiments, the second enzyme is a functional variant of Tle2 consisting of Tle2 subunit B and a functional variant of Tle2 subunit A having at least 70% homology, similarity or identity to SEQ ID NO: 19, 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% homology, similarity or identity thereto. 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% homology, similarity or identity to SEQ ID NO: 19, 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% homology, similarity or identity thereto, and a functional variant of Tle2 subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 20, 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% homology, similarity or identity thereto.
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% homology, similarity or 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% homology, similarity or identity thereto.
In some embodiments, the second enzyme is Ghh2 or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. This enzyme 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 Tle2 or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. In some embodiments, the second enzyme is a functional variant of Ghh2 consisting of Ghh2 subunit A and a functional variant of Ghh2 subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 23, 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% homology, similarity or identity thereto. In other embodiments, the second enzyme is a functional variant of Ghh2 consisting of Ghh2 subunit B and a functional variant of Ghh2 subunit A having at least 70% homology, similarity or identity to SEQ ID NO: 22, 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% homology, similarity or identity thereto. 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% homology, similarity or identity to SEQ ID NO: 22, 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% homology, similarity or identity thereto, and a functional variant of Ghh2 subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 23, 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% homology, similarity or identity thereto.
In some embodiments, the second enzyme is Tme or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. This enzyme of EC number 2.8.3.8 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 Ghh2 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 Tme or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto. In some embodiments, the second enzyme is a functional variant of Tme consisting of Tme subunit A and a functional variant of Tme subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 25, 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% homology, similarity or identity thereto. In other embodiments, the second enzyme is a functional variant of Tme consisting of Tme subunit B and a functional variant of Tme subunit A having at least 70% homology, similarity or identity to SEQ ID NO: 24, 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% homology, similarity or identity thereto. In some embodiments, the second enzyme is a functional variant of Tme consisting of a functional variant of Tme subunit A having at least 70% homology, similarity or identity to SEQ ID NO: 24 and a functional variant of Tme subunit B having at least 70% homology, similarity or identity to SEQ ID NO: 25, 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% homology, similarity or identity to SEQ ID NO: 24 and SEQ ID NO: 25, respectively.
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% homology, similarity or 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% homology, similarity or identity thereto.
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% homology, similarity or 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% homology, similarity or identity thereto.
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 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.
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.
The thermophilic cells 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 thermophilic 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 thermophilic cell is incubated in conditions where 3-oxopentanoate is produced, e.g. if the broth comprises propionic acid, the enzyme can catalyse the decarboxylation of said 3-oxopentanoate to butanone.
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 for the present methods.
Thus in some embodiments, the acetoacetate decarboxylase is Cac as set forth in SEQ ID NO: 28, or a functional variant having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
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.
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.
Also provided herein are cells and methods for the production of isopropanol in a thermophilic cell. For this, the thermophilic 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) catalyzes the conversion of acetone to propan-2-ol. Thus a thermophilic 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.
In some embodiments, the isopropanol dehydrogenase is Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
The thermophilic cell is cultivated in a reactor, for example a bioreactor or a fermenter, as is known in the art. In the context of the present disclosure, the cell is cultivated at “high” temperatures, i.e. temperatures above the conventional 37° C. normally employed for bacterial cultivations. The advantage of performing the cultivation at higher temperatures is that this allows facilitated recovery of the produced volatile compounds. Preferably, the thermophilic 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, step b) 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.
The culture medium, or cultivation broth, comprises a fermentable carbon source as known in the art. In some embodiments, the 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 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.
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.
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. Pre-treatment 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) β-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 γ-valerolactone.
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.
Alternatively, the 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 Clostridium thermocellum, degrades the biomass and provides the necessary substrates for the other microorganism, which can produce the volatile compounds as described above.
In some embodiments, the medium comprises glucose, xylose, or a mixture thereof. For example, the medium may comprise between 0.1% and between 20% (w/vol) glucose, xylose, or mixture thereof. For example, the 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 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.
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 and Thermoanaerobacter kivui.
In embodiments where production of acetone is desired, the culture medium may advantageously further comprise acetic acid or acetate. In some embodiments, the medium comprises between 0.05% and 5% (w/vol) acetic acid or acetate. For example, the 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), such as between 2% and 4% (w/vol) such as 3% acetic acid or acetate or mixtures thereof. In some embodiments, the 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.
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 embodiments where butanone production is desired, the culture medium may advantageously further comprise propionic acid or propionate. In some embodiments, the medium comprises between 0.05% and 2% (w/vol) propionic acid or propionate. For example, the medium comprises between 0.05% and 2% (w/vol) acetic acid or acetate 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 medium comprises at least 0.05% (w/vol) propionic acid, propionate or mixtures thereof 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.
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.
The present thermophilic 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. Because 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 off-gas produced during the fermentation. In some embodiments, this is done by condensation.
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.
Methods for Production of Acetone, Butanone and/or Isopropanol
The present methods preferably allow production of acetone with a titer of 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.
In some embodiments, the method is for production of at least acetone, and the first enzyme is selected from the group consisting of 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) and Slip_0880 (SEQ ID NO: 7), or functional variants thereof having at least 70% homology, similarity or identity thereto, preferably Caur_1461 (SEQ ID NO: 3), Rxy2 (SEQ ID NO: 60), Slip_0880 (SEQ ID NO: 7) and Dde1 (SEQ ID NO: 59).
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY_1288 (SEQ ID NO: 15), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60)), Tle2 and Cac, or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY_1288 (SEQ ID NO: 15), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY 1288 (SEQ ID NO: 15), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Ghh2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY_1288 (SEQ ID NO: 15), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Tme and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY_1288 (SEQ ID NO: 15), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least acetone, said cell expressing:
In one embodiment, the thermophilic cell expresses: CHY_1288 (SEQ ID NO: 15), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1540 (SEQ ID NO: 10), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0880 (SEQ ID NO: 7), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
Preferably, the thermophilic cell can produce at least acetone, and expresses Cac as set forth in SEQ ID NO: 28 or a functional variant thereof having at least 70% identity or similarity thereto and one of the following combinations of first and second enzymes:
The present methods allow production of 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 0.2 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 0.75 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 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.
In some embodiments, the method is for production of at least butanone, and the first enzyme is selected from the group consisting of 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), Slip_0479 (SEQ ID NO: 4), Tfu_1520 (SEQ ID NO: 5), and Tfu_0436 (SEQ ID NO: 6), or functional variants thereof having at least 70% homology, similarity or identity thereto, preferably the first enzyme is GHH_c20420 (SEQ ID NO: 1), Slip_0499 (SEQ ID NO: 2), Caur_1461 (SEQ ID NO: 3) or Slip_0479 (SEQ ID NO: 4).
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2) Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Tle2 and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Dde2 (SEQ ID NO: 21) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Ghh2 and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Ghh2 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Tme and Cac (SEQ ID NO: 21), or functional variants thereof having at least 70% homology, similarity or identity thereto, wherein Tme 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% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Pth (SEQ ID NO: 26) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In some embodiments, the thermophilic cell is used for producing at least butanone, said cell expressing:
In one embodiment, the thermophilic cell expresses: GHH_c20420 (SEQ ID NO: 1), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0499 (SEQ ID NO: 2), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Caur_1461 (SEQ ID NO: 3), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Slip_0479 (SEQ ID NO: 4), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Dde1 (SEQ ID NO: 59), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Rxy2 (SEQ ID NO: 60), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_1520 (SEQ ID NO: 5), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
In another embodiment, the thermophilic cell expresses: Tfu_0436 (SEQ ID NO: 6), Rma (SEQ ID NO: 27) and Cac (SEQ ID NO: 28), or functional variants thereof having at least 70% homology, similarity or identity thereto.
Preferably, the thermophilic cell can produce at least butanone, and expresses Cac as set forth in SEQ ID NO: 28 or a functional variant thereof having at least 70% identity or similarity thereto and one of the following combinations of first and second enzymes:
The above thermophilic cells can produce acetone from acetyl-CoA, for example in the presence of a suitable substrate such as acetic acid, which may be synthesised by the cell, and/or butanone from propionyl-CoA, for example in the presence of a suitable substrate such as propionic acid. Supplementing the broth with acetic acid may increase the titer. Propionyl-CoA may be provided in the fermentation. The 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 a corresponding non-engineered cell. Any of the above thermophilic cells may in addition to the above express an isopropanol dehydrogenase, in particular Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% homology, similarity or identity thereto. This allows at least part of the acetone produced by the thermophilic cell (or provided to the cell) to be converted to isopropanol.
In some embodiments, at least isopropanol is produced. The isopropanol titer is preferably 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 0.2 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 0.75 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.
Preferably, the thermophilic cell can produce at least acetone and isopropanol, and expresses Cac as set forth in SEQ ID NO: 28 and Tbr (SEQ ID NO: 29) or functional variants thereof having at least 70% identity or similarity thereto and one of the following combinations of first and second enzymes:
In some embodiments, the thermophilic cell can produce at least butanone and isopropanol, and expresses Cac as set forth in SEQ ID NO: 28 and Tbr (SEQ ID NO: 29) or functional variants thereof having at least 70% identity or similarity thereto and one of the following combinations of first and second enzymes:
Useful thermophilic cells have been described in detail herein above. Once the skilled person has determined which enzymes to express in the thermophilic cells of the present disclosure, he/she will have no difficulty to do so.
The enzymes may be expressed by introducing nucleic acid sequences encoding each of them in the cell, for example on a plasmid, or by genomic integration. For example, genes can be inserted into a replicating plasmid, which is then transformed into the cell by means of electroporation. A gene in the same plasmid, which encodes an antibiotic resistance marker, will ensure that only the transformed cells survive in the medium with the respective antibiotic, but other selection systems may also be utilized. Genomic integration is achieved, for example, by using a plasmid, for example a temperature sensitive plasmid, carrying the genes of interest. In the right conditions under non-permissible temperatures, the plasmid will undergo double crossover by homologous recombination, leaving a seamless marker-free integration into the genomic DNA. Gene expression can be controlled as is known in the art, for example by using appropriate vectors, plasmids, promoters or codon-optimisation. For example, in embodiments where the thermophilic cell is a Geobacillus cell, in particular a Geobacillus thermoglucosiadus cell, the method described in Pogrebnyakov et al., 2017, may be employed.
Accordingly, the thermophilic cells disclosed herein may comprise one or more polynucleotides encoding the first enzyme, the second enzyme and the acetoacetate decarboxylase as described herein above, and optionally encoding the isopropanol dehydrogenase as described herein above. Each of said polynucleotides may encode a single enzyme, or it may encode several enzymes which then get expressed simultaneously.
Herein is thus also provided a nucleic acid construct for modifying a thermophilic cell selected from a thermophilic bacterial cell and a thermophilic archaeal cell, comprising:
In particular, herein is provided a nucleic acid construct for modifying a thermophilic cell selected from a thermophilic bacterial cell and a thermophilic archaeal cell, comprising:
Expression of each polynucleotide may be under the control of an inducible promoter, or under the control of a constitutive promoter.
Thus also disclosed herein is a nucleic acid construct for modifying a thermophilic cell, in particular a thermophilic bacterial cell or a thermophilic archaeal cell, which can be used to construct the thermophilic cells of the present disclosure, i.e. cells capable of producing acetone, butanone and/or isopropanol.
Said nucleic acid construct comprises or consists of:
The nucleic acid construct may comprise or consist of one or more polynucleotides. It will be understood that the term “nucleic acid constructs” may refer to one nucleic acid molecule, or to a plurality of nucleic acid molecules, comprising the relevant nucleic acid sequences. The nucleic acid construct may thus be one nucleic acid molecule, which may encode several enzymes, or it may be several nucleic acid molecules, each comprising one sequence encoding an enzyme. The relevant nucleic acid sequences may thus be comprised on one vector, or on several vectors. They may also be integrated in the genome, on one chromosome or even together in one location, or they may be integrated on different chromosomes. It is also possible to have some sequences on one or more vectors, and some integrated in the genome.
The nucleic acid construct comprises a polynucleotide encoding an acetyl-CoA acetyltransferase (EC 2.3.1.9) which is as described herein elsewhere. In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes GHH_c20420, such as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 30, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_0499, such as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 31, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Caur 1461, such as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 32 or SEQ ID NO: 63, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_0479, such as set forth in SEQ ID NO: 4, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 33, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Dde1, such as set forth in SEQ ID NO: 59, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 61, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Rxy2, such as set forth in SEQ ID NO: 60, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 62, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Tfu_1520, such as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 34, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Tfu_0436, such as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 35, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_0880, such as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 36, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Tfu_2394, such as set forth in SEQ ID NO: 8, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 37, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_1236, such as set forth in SEQ ID NO: 9, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 38, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Caur_1540, such as set forth in SEQ ID NO: 10, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 39, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Tfu_0253, such as set forth in SEQ ID NO: 11, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 40, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes CHY_1604, such as set forth in SEQ ID NO: 14, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 43, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes CHY_1288, such as set forth in SEQ ID NO: 15, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 44, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_2085, such as set forth in SEQ ID NO: 16, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 45, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Slip_0465, such as set forth in SEQ ID NO: 17, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 46, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Dde1, such as set forth in SEQ ID NO: 59, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 61, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Rxy2, such as set forth in SEQ ID NO: 60, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 62, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes CHY_1355, such as set forth in SEQ ID NO: 18, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 47, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetyl-CoA acetyltransferase activity.
In preferred embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes 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% homology, similarity or identity thereto. Thus in some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase comprises or consists of SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 39, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 63, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 36, or homologues thereof having at least 70% homology, similarity or identity thereto encoding enzymes which retain acetyl-CoA acetyltransferase activity. In particular embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Caur_1461 (SEQ ID NO: 3), Dde1 (SEQ ID NO: 59) or Slip_0880 (SEQ ID NO: 7), or a functional variant thereof having at least 70% homology, similarity or identity thereto. Thus in some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase comprises or consists of SEQ ID NO: 32, SEQ ID NO: 61 or SEQ ID NO: 36, or homologues thereof having at least 70% homology, similarity or identity thereto encoding enzymes which retain acetyl-CoA acetyltransferase activity.
In other preferred embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes 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), Slip_0479 (SEQ ID NO: 4), Tfu_1520 (SEQ ID NO: 5), or Tfu_0436 (SEQ ID NO: 6), or a functional variant thereof having at least 70% homology, similarity or identity thereto. Thus in some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase comprises or consists of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 63, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 35, or homologues thereof having at least 70% homology, similarity or identity thereto encoding enzymes which retain acetyl-CoA acetyltransferase activity. In particular embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase encodes Caur_1461 (SEQ ID NO: 3) or Dde1 (SEQ ID NO: 59), or a functional variant thereof having at least 70% homology, similarity or identity thereto. Thus in some embodiments, the polynucleotide encoding the acetyl-CoA acetyltransferase comprises or consists of SEQ ID NO: 32, SEQ ID NO: 63, SEQ ID NO: 33 or SEQ ID NO: 61, or homologues thereof having at least 70% homology, similarity or identity thereto encoding enzymes which retain acetyl-CoA acetyltransferase activity.
The nucleic acid construct further comprises a polynucleotide encoding 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. In some embodiments, the second enzyme is Tle2. This enzyme consists of two subunits; the polynucleotide encoding Tle2 thus preferably encodes both subunits A and B of Tle2 as set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 48 and SEQ ID NO: 49, or homologues thereof having at least 70% homology, similarity or identity thereto, which encode subunits which together retain acetyl-CoA transferase activity.
In some embodiments, the polynucleotide encoding the second enzyme encodes Dde2, such as set forth in SEQ ID NO: 21, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 50, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains 3-oxoacid transferase activity.
In some embodiments, the second enzyme is Ghh2. This enzyme consists of two subunits; the polynucleotide encoding Ghh2 thus preferably encodes both subunits A and B of Ghh2 as set forth in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 51 and SEQ ID NO: 52, or homologues thereof having at least 70% homology, similarity or identity thereto, which encode subunits which together retain 3-oxoacid transferase activity.
In some embodiments, the second enzyme is Tme. This enzyme consists of two subunits; the polynucleotide encoding Tme thus preferably encodes both subunits A and B of Tme as set forth in SEQ ID NO: 24 and SEQ ID NO: 25, respectively. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 53 and SEQ ID NO: 54, or homologues thereof having at least 70% homology, similarity or identity thereto, which encode subunits which together retain 3-oxoacid transferase activity.
In some embodiments, the polynucleotide encoding the second enzyme encodes Pth, such as set forth in SEQ ID NO: 26, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 26, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acyl CoA:acetate/3-ketoacid CoA-transferase activity.
In some embodiments, the polynucleotide encoding the second enzyme encodes Rma, such as set forth in SEQ ID NO: 27, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments, said polynucleotide comprises or consists of SEQ ID NO: 56, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acyl-CoA thioesterase activity.
In preferred embodiments, the polynucleotide encodes Tle2 or a functional variant thereof having at least 70% homology, similarity or identity thereto. Thus in preferred embodiments, the polynucleotide encoding the second enzyme comprises or consists of SEQ ID NO: 48 and SEQ ID NO: 49, or homologues thereof having at least 70% homology, similarity or identity thereto.
The nucleic acid construct further comprises a polynucleotide encoding an acetoacetate decarboxylase. Preferably, the acetoacetate decarboxylase is Cac, such as set forth in SEQ ID NO: 28, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments said polynucleotide comprises or consists of SEQ ID NO: 57, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains acetoacetate decarboxylase activity.
The nucleic acid construct may further comprise a polynucleotide encoding an isopropanol dehydrogenase (EC 1.1.1.80). Preferably, the isopropanol dehydrogenase is Tbr such as set forth in SEQ ID NO: 29, or a functional variant thereof having at least 70% homology, similarity or identity thereto. In some embodiments said polynucleotide comprises or consists of SEQ ID NO: 58, or a homologue thereof having at least 70% homology, similarity or identity thereto, which encodes an enzyme which retains isopropanol dehydrogenase activity.
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
In some embodiments, the nucleic acid construct comprises or consists of:
Any of the above described nucleic acid constructs may further comprise a polynucleotide encoding an isopropanol dehydrogenase, preferably Tbr (SEQ ID NO: 29) or a functional variant thereof having at least 70% homology, similarity or identity thereto.
The term “at least 70% homology, similarity or identity” in relation to a nucleic acid sequence is herein to be understood as referring to homologues of a given nucleic acid sequence having at least 70% homology, similarity or identity to said nucleic acid 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% homology, similarity or identity thereto, which still encode an enzyme retaining the activity of the enzyme encoded by said given nucleic acid sequence. How to test for the relevant activities has been described herein above.
The term “at least 70% homology, similarity or identity” in relation to a protein or enzyme is herein to be understood as referring to homologues of a given protein or enzyme having at least 70% homology, similarity or identity to said protein or enzyme, 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% homology, similarity or identity thereto, which preferably retain at least some of the activity of the original protein or enzyme. How to test for the relevant activities has been described herein above.
All nucleic acid sequences may have been codon-optimised for expression in the microorganism, as is known in the art.
It may be of interest to take advantage of inducible promoters. Thus in some embodiments, the nucleic acid constructs comprises one or more of the above nucleic acid sequences under the control of an inducible promoter. This allows more control of when the enzyme encoded by the sequence is actually expressed, and can be advantageous for example if production of one of the volatile compounds negatively affects cell growth. The skilled person will have no difficulty in identifying suitable inducible promoters. In other embodiments, the nucleic acid constructs are under the control of a constitutive promoter. Such constitutive promoters may be strong promoters.
In some embodiments, the nucleic acid construct is one or more vectors, for example integrative or replicative vectors, such as a plurality of vectors which together form the nucleic acid construct. Suitable vectors are known in the art and readily available to the skilled person. Accordingly, herein is also provided a vector comprising any of the above nucleic acid constructs.
The above nucleic acid constructs are useful for modifying a thermophilic cell, in particular a cell of a genus selected from: Geobacillus, Thermoanaerobacterium, Thermoanaerobacter, Caldanaerobacter, Bacillus, Thermoclostridium, Anoxybacillus, Caldicellulosiruptor, Moorella, Thermus, Thermotoga, Pseudothermotoga, Chloroflexus, Anaerocellum, Rhodothermus, Sulfolobus, Thermococcus, Pyrococcus and Clostridium. In some embodiments, the thermophilic cell is selected from Geobacillus thermoglucosidasius, Geobacillus toebii, Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus kaustophilus, Geobacillus thermoleovorans, Geobacillus thermocatenulatus, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharotyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter mathranii, Thermoanaerobacter pseudoethanolicus, Thermoanaerobacter brockii, Thermoanaerobacter kivui, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Clostridium thermocellum, Clostridium thermosuccinogenes, Thermoclostridium stercorarium, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus smithii, Bacillus methanolicus, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Caldicellulosiruptor bescii, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, Moorella thermoacetica, Moorella thermoautotrophica, Thermus thermophilus, Thermus aquaticus, Thermotoga maritima, Pseudothermotoga lettingae, Pseudothermotoga thermarum, Chloroflexus aurantiacus, Anaerocellum thermophilum, Rhodothermus marinus, Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus solfataricus, Thermococcus barophilus, Thermococcus kodakarensis, Pyrococcus abyssi, Pyrococcus furiosus, preferably the cell is a Geobacillus thermoglucosidasius cell, a Bacillus subtilis cell or a Clostridium thermocellum cell.
Also provided herein is a thermophilic cell comprising the nucleic acid construct described herein above.
Also provided herein is a vector or a system of vectors comprising the nucleic acid construct described herein above.
Also provided herein is a host cell comprising the nucleic acid construct or the vector described herein above. The host cell may be a prokaryote or a eukaryote. In a preferred embodiment, the cell is a prokaryote, such as a bacterial cell, for example Escherichia coli. The host cell may be the thermophilic cell which is capable of producing acetone, butanone and/or isopropanol as described herein.
Also provided herein is a kit comprising the nucleic acid construct, the vector or the thermophilic cell described herein above, and optionally instructions for use.
In some embodiments, the kit comprises the nucleic acid constructs and/or the vectors described herein, and may further comprise the thermophilic cell to be modified. The thermophilic cell may be any of the cells described herein above. The kit may further comprise reagents useful for modifying the yeast cell.
Bacterial strains and plasmids used in this study are listed in Table 1.
E. coli NEB5-alpha
G. thermoglucosidasius
G. thermoglucosidasius G11
G. thermoglucosidasius G12
G. thermoglucosidasius G13
G. thermoglucosidasius G14
G. thermoglucosidasius G22
G. thermoglucosidasius G23
G. thermoglucosidasius G24
G. thermoglucosidasius G25
G. thermoglucosidasius G27
G. thermoglucosidasius G28
G. thermoglucosidasius G29
G. thermoglucosidasius G30
G. thermoglucosidasius G31
G. thermoglucosidasius DDC
G. thermoglucosidasius G32
G. thermoglucosidasius G33
G. thermoglucosidasius G34
G. thermoglucosidasius G35
G. thermoglucosidasius G36
G. thermoglucosidasius G37
G. thermoglucosidasius G38
G. thermoglucosidasius CTC
G. thermoglucosidasius CTCl
G. thermoglucosidasius G51
G. thermoglucosidasius G52
G. thermoglucosidasius G53
G. thermoglucosidasius G54
G. thermoglucosidasius G55
G. thermoglucosidasius G56
G. thermoglucosidasius G58
G. thermoglucosidasius G59
G. thermoglucosidasius G60
G. thermoglucosidasius G61
G. thermoglucosidasius G62
G. thermoglucosidasius G63
G. thermoglucosidasius G64
G. thermoglucosidasius G65
G. thermoglucosidasius G66
G. thermoglucosidasius G67
G. thermoglucosidasius G68
G. thermoglucosidasius G69
G. thermoglucosidasius G70
G. thermoglucosidasius G71
G. thermoglucosidasius G72
G. thermoglucosidasius G73
G. thermoglucosidasius G74
G. thermoglucosidasius G75
G. thermoglucosidasius G76
G. thermoglucosidasius G77
G. thermoglucosidasius G79
G. thermoglucosidasius G80
G. thermoglucosidasius G81
G. thermoglucosidasius G82
G. thermoglucosidasius STC
Geobacillus shuttle vector
E. coli cells were grown in lysogeny broth (LB) with 100 μg/mL ampicillin or 6.25 μg/mL kanamycin added when needed. Geobacillus strains were grown in either of several media.
The mTGP (modified from Taylor et al., 2008) medium contained per liter: 17 g tryptone, 3 g soy peptone, 5 g NaCl, 2.5 g K2HPO4. After autoclavation, sterile solutions were added to final concentrations: 4 mL/L glycerol, 4 g/L sodium pyruvate, 0.59 mM MgSO4, 0.91 mM CaCl2 and 0.04 mM FeSO4. Tripticase soy agar (TSA) contained per liter: 15 g pancreatic digest of casein, 5 g papaic digest of soybean, 5 g NaCl, 15 g agar. SPY medium consisted of 16 g/l soy peptone, 10 g/l yeast extract, 5 g/l NaCl. Its pH was adjusted to 7.0 by adding 5M NaOH. When indicated, glycerol was added to the final concentration of 10 g/l.
Thermophile minimal medium (TMM) was adapted from Fong et al., 2006, with some modifications. It contained, per liter: Six salts solution (SSS), 930 mL; 1 M MOPS (pH 8.2), 40 mL; 1 mM FeSO4 in 0.4 M tricine, 10 mL; 0.132 M K2HPO4, 10 mL; 0.953 M NH4Cl, 10 mL; 1 M CaCl2, 0.5 mL; trace elements solution, 0.5 ml; Wolfe's vitamin solution, 10 mL. SSS contained, per 930 mL: 4.6 g NaCl, 1.35 g Na2SO4, 0.23 g KCl, 0.037 g KBr, 1.72 g MgCl2·6H2O, 0.83 g NaNO3. Trace elements solution contained, per liter: 1 g FeCl3·6H2O, 0.18 g ZnSO4·7H2O, 0.12 g CuCl2·2H2O, 0.12 g MnSO4·H2O, 0.18 g CoCl2·6H2O. Yeast extract in final concentration of 0.05% (w/v) was added when indicated. For Geobacillus spp. selection 12.5 μg/mL kanamycin was used.
Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega) according to producer's specifications. Plasmid extractions were performed using NucleoSpin® Plasmid EasyPure kit (Macherey-Nagel).
Primers used in this study are described in Table 2.
PCR of DNA fragments for USER cloning was performed with primers containing uracil using the Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific). Colony PCR was performed with Taq 2x Master Mix (New England Biolabs) in order to detect positive colonies. Reactions were done according to manufacturers' recommendations with elongation times and annealing temperatures adjusted for specific targets and primers. DNA cloning was performed using USER (uracil-specific excision reagent) technology (Cavaleiro et al., 2015).
PCR-amplified DNA fragments containing a primer-incorporated uracil close to both of their 5′-ends were mixed (purification after PCR was not necessary) and treated with DpnI enzyme (Thermo Fisher Scientific) for 30 min at 37° C. to digest template DNA. USER™ enzyme (New England Biolabs) was then added, and the mixture was incubated in three steps: 1) 37° C. for 15 min; 2) 12° C. for 15 min; 3) 10° C. for 10 min. It was then transferred on ice and mixed with chemically competent E. coli cells.
Transformation of E. coli
Chemically competent E. coli NEB5-alpha cells (New England Biolabs) were transformed according to manufacturer's recommendations.
Transformation of G. thermoglucosidasius
The procedure was based on the protocol described by Taylor et al., 2008, with some steps modified. G. thermoglucosidasius was grown overnight on a TSA agar plate at 60° C. A loopful of cells was inoculated into 50 mL of pre-warmed liquid SPY medium in a 250 ml flask and incubated at 60° C. and 250 rpm until the culture reached OD600 of approximately 2.0. Cells were cooled down on ice for 10 min and harvested by centrifugation at 2600 g for 10 min. They were washed four times (2600 g for 10 min) with freshly prepared ice-cold electroporation buffer. The buffer contained (per 100 mL) 9.1 g mannitol, 9.1 g sorbitol, 10 mL glycerol, and was sterilized by filtration. Buffer was added at the volumes of 25 ml, 15 ml, 15 ml and 10 ml for each consecutive step. After the last washing step, the cell pellet was dissolved in 2 mL of electroporation buffer, distributed in 60 μL aliquots and stored at −80° C. until further use.
For the transformation, an aliquot was thawed on ice and mixed with DNA. It was transferred into an electroporation cuvette with a 1 mm gap between electrodes (Bio-Rad) and subjected to a discharge using the Gene Pulser Xcel™ (Bio-Rad) under following conditions: 2.5 kV, 600 Ω, 10 μF. Time constants typically were 4-5 ms. Immediately after electroporation cells were dissolved in 1 mL of pre-heated SPY medium supplemented with glycerol and recovered at 52° C. for 4 hours at 200 rpm. Afterwards they were spun down and seeded on selective agar media plates.
Codon optimization was done using the online service of Integrated DNA Technologies, Inc. (https://eu.idtdna.com/CodonOpt) or Gene Designer from DNA 2.0 (Villalobos et al., 2006). DNA sequencing was performed by Eurofins Scientific (Luxembourg).
Strains expressing acetone pathways were grown in 2 ml of TMM with respective supplements in sterile 20 ml headspace vials. To prevent acetone loss the vials were kept closed during the whole time of culture growth until sampling for chromatography. After 20 hours of incubation cultures were frozen at −20° C. to stop growth and metabolic activity, and transferred for measurement.
Acetone concentrations were measured with analytical GC-MS (Bruker Scion 436 GC TQ) using BP20 capillary column (30 m, internal diameter 0.25 mm, film thickness 0.25 mm). Helium was used as a carrier. The inlet temperature was set to 250° C., and the oven temperature was programmed as follows: run at 37° C. for 5 min, then raised at a rate of 5° C./min until it reached 100° C., followed by the increase at 15° C./min up to 250° C., and finally hold for 3 min. Mass spectrometry was run using electron ionization method. Full scan mode was used with scan range from 35 to 400 amu. Injection volumes were from 1 μl to 5 μl in splitless mode for low acetone concentrations and split mode (1:1) for high concentrations.
For production of acetone in G. thermoglucosidasius, we initially tested the tolerance of G. thermoglucosidasius towards acetone. When grown in tightly sealed vessels with 1:10 medium to headspace volume ratio, this strain was found to tolerate at least 25 g/l of acetone (
Combinations of four thiolase variants and three oxoacid CoA-transferase variants were constructed together with acetoacetate decarboxylase from C. acetobutylicum as operons under the control of thiolase promoter from C. acetobutylicum. Strains of G. thermoglucosidasius carrying these operons were grown in: i) semi-defined medium with 1% glucose, and ii) nutritionally rich medium supplemented with 0.2% glucose. Among all combinations, Dde1-Dde2-Cac produced highest titers in semi-defined medium, whereas Cau-Tle2-Cac performed best in rich broth. Results are shown in Table 3.
The CTC strain was also tested in a 30 L fed-batch fermentation, fed with 2 g/L/h glucose, 1 g/L/h acetic acid, 1 g/L/h yeast extract. The CTC strain was grown in TMM medium, supplemented with 2% glucose, 0.2% acetic acid, 1% yeast extract. The CTC genes were integrated into the genome under the control of the strong promoter P3 (Pogrebnyakov et al., 2017). Up to 2.9 g/L acetone were achieved (
Both the STC strain (Slip_0880-Tle2-Cac) and the CTC strain (Caur_1461-Tle2-Cac) were also tested in a 1 L constant fed-batch fermentation. The strains were grown in TMM medium, supplemented with 2% glucose, 0.2% acetic acid, 1% yeast extract. The CTC and STC genes were integrated into the genome under the control of the strong promoter P3. The STC strain achieved a final acetone titer of 1.6 g/L, while the CTC strain achieved a final acetone titer of 1.1 g/L (
Two of the best performing enzyme combinations, Dde1-Dde2-Cac and Cau-Tle2-Cac, where Cau is a codon optimized version of Caur_1461, were overexpressed in G. thermoglucosidasius. Each operon was integrated into the chromosome of G. thermoglucosidasius, replacing either putative acetone carboxylase (AOT13_RS09545, AOT13_RS09550 and AOT13_RS09555) in case of Dde1-Dde2-Cac, or lactate dehydrogenase (AOT13_RS05985) in case of Cau-Tle2-Cac. A range of constitutive promoters with various levels of activity, from low to high (Pogrebnyakov et al., 2017), were integrated upstream of these operons to drive their expression, yielding G. thermoglucosidasius strains G31-G38 and DDC. Acetone titers from Dde1-Dde2-Cac increased with the increasing strength of the promoter (Table 3).
Overexpression of Cau-Tle2-Cac operon in G. thermoglucosidasius strain CTC also led to increase in acetone titers. Addition of sodium acetate and especially acetic acid further increases acetone titers up to two-fold (
Sugar composition of the substrate also affects acetone production by G. thermoglucosidasius. This species is able to utilize many pentose and hexose sugars, in particular glucose and xylose. G. thermoglucosidasius CTC was grown in the presence of a mixture of these monosaccharides and converted them into high yields of acetone (
Multiple variants of thiolases and acyl-CoA:acyl-CoA alkyltransferases from thermophilic organisms were screened for the production of butanone and acetone. They were expressed under the control of thiolase promoter from C. acetobutylicum in the strain of G. thermoglucosidasius carrying Tle2 and Cac genes in the chromosome under the control of medium strength promoter P7, created previously (Pogrebnyakov et al., 2017). The resulting strains were named G51-G82. They were grown in semi-defined TMM medium supplemented with 1% glucose and 0.2% propionic acid. In these conditions, most strains produced a mixture of butanone and acetone at different ratios and titers (Table 5). Thiolase variants, which contributed to the highest butanone production, were Caur_1461, GHH_c20420, Slip_0499 and Slip_0479. Expression of variant Slip_0880 resulted in the highest acetone titer with relatively low butanone amounts. Results are shown in
G. thermoglucosidasius strain CTC from previous example overexpresses Cau-Tle2-Cac operon, where Cau is a codon optimized version of Caur_1461, one of the best butanone producers in this example. G. thermoglucosidasius CTC was grown in TMM supplemented with 1% glucose and 0.1% to 0.3% propionic acid, and produced up to 0.43 g/l butanone (
One enzymatic step involving alcohol dehydrogenase is required to convert acetone into isopropanol. A specific isopropanol dehydrogenase from Thermoanaerobacter brockii has been identified previously (Hanai et al., 2007). Codon optimized version of this gene was integrated into the genome of G. thermoglucosidasius CTC downstream of Cac gene, yielding strain CTCI. G. thermoglucosidasius CTCI grown in TMM supplemented with 1% glucose produced 0.11 g/l isopropanol.
Clark, “Co-production of acetone and ethanol with molar ratio control enables production of improved gasoline or jet fuel blends,” Biotechnol Bioeng, vol. 9999, no. 10, pp. 1-9, 2016
R. Glenn, B. A. Neilan, and P. L. Rogers, “Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost,” Extremophiles, vol. 10, no. 5, pp. 363-372, 2006
Sulfurifustis variabilis, NCBI accession
Deferribacter desulfuricans SSM1, NCBI
thermopropionicum SI, NCBI accession
Rhodothermus marinus DSM 4252, NCBI
| Number | Date | Country | Kind |
|---|---|---|---|
| 20193767.9 | Sep 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/074134 | 9/1/2021 | WO |
